One-step green synthesis of silver nanoparticle-modified reduced graphene oxide nanocomposite for H2O2 sensing applications

Journal Pre-proof One-step green synthesis of silver nanoparticle-modified reduced graphene oxide nanocomposite for H2O2 sensing applications Pedro Salazar, Iñigo Fernández, Miriam C. Rodríguez, Alberto Hernández Creus, José Luis González-Mora PII:

S1572-6657(19)30906-3

DOI:

https://doi.org/10.1016/j.jelechem.2019.113638

Reference:

JEAC 113638

To appear in:

Journal of Electroanalytical Chemistry

Received Date: 5 August 2019 Revised Date:

7 November 2019

Accepted Date: 8 November 2019

Please cite this article as: P. Salazar, Iñ. Fernández, M.C. Rodríguez, Alberto.Herná. Creus, José.Luis. González-Mora, One-step green synthesis of silver nanoparticle-modified reduced graphene oxide nanocomposite for H2O2 sensing applications, Journal of Electroanalytical Chemistry (2019), doi: https://doi.org/10.1016/j.jelechem.2019.113638. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

One-step green synthesis of silver nanoparticle-modified reduced graphene oxide nanocomposite for H2O2 sensing applications Pedro Salazar a, *, Iñigo Fernández a, Miriam C. Rodríguez b, Alberto Hernández Creus b , José Luis González-Mora a a) Laboratory of Sensors, Biosensors and Advanced Materials, Faculty of Health Sciences, University of La Laguna, Campus de Ofra s/n, 38071 La Laguna, Tenerife, Spain b) Área de Química Física, Departamento de Química, Facultad de Ciencias, Universidad de La Laguna (ULL)Instituto de Materiales y Nanotecnología (IMN) 38200 La Laguna (Tenerife), España *Corresponding author

Abstract In the present study, silver nanoparticles-modified reduced graphene oxide nanocomposite (rGox/AgNPs) was obtained using a green synthesis method for reducing both Ag+ cations and graphene oxide sheets with tea extract. Tea polyphenols are highly reactive and water-soluble reducing agents allowing the correct reduction of precursors. Nanocomposite was conveniently analyzed using different techniques such as ultraviolet-visible and Fourier-transform infrared spectroscopies, X-ray diffraction, atomic force and transmission electron microscopies. Glassy carbon electrodes were conveniently modified with rGox/AgNPs nanocomposite and electrochemical tests were carried out to study the electrocatalytic properties of the GCE/rGox/AgNPs sensor against H2O2 reduction. The main analytical factors such as selectivity, sensitivity, time of response and stability, limit of quantification and detection were also studied. Our sensor had a sensitivity of about 236 µA mM-1cm-2 (R2= 0.999) in the range of concentrations from 0.002 to 20 mM H2O2, a fast response (~2 s) and a limit of detection (S/N = 3) of about 0.73 µM. The selectivity of the sensor was tested against different biological interferences including dopamine, glutamate, glucose and ascorbic

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acid with promising results. In addition, sensor response was evaluated for 7 months without significant loss of sensitivity. Finally, the viability of using the GCE/rGox/AgNPs electrode for real samples was positively confirmed in different commercial disinfectant solutions and spiked samples (commercial milk and urine). Keywords: reduced graphene oxide, electroanalysis, non-enzymatic sensor, hydrogen peroxide, silver nanoparticles. 1. Introduction Hydrogen peroxide (H2O2) is an extensively used oxidizing agent in industrial applications (pharmaceutical, agro-alimentary, textile, waste treatment, etc.)[1-5]. Moreover, it acts as a messenger in relevant signaling processes[6, 7], as an indicator of oxidative stress in cells[8, 9] and it is produced as a by-product of several oxidoreductase enzymes used in first-generation biosensors[5, 10-13] and laboratory bioassays[14]. Due to the great interest of this molecule, great effort has gone into finding more operative analytical procedures for its selective and quantitative detection, especially in complex biological matrixes. Among the different analytical approaches such as chemiluminescence, fluorescence, spectrophotometry and electrochemical methods[5, 15], the latter offers a rapid, simple and low cost method for the determination of H2O2 with high enough sensitivity, selectivity and low detection limit[16, 17]. In this context, enzyme-based electrochemical biosensors, based on peroxidase enzyme (HRP), have become popular due to their high sensitivity and selectivity against most common interferences and their easy implementation. Nevertheless, HRP-based biosensors have some limitations because of their thermal and pH instability, complex immobilization procedures, high initial cost and low temporal stability of the HRP enzyme[1, 15, 18, 19]. As a result of these problems, new alternatives (such as non-enzymatic sensors) with better sensitivity, selectivity, stability 2

and reproducibility properties have recently been proposed[5]. In this regard, carbonbased nanomaterials such as carbon nanotubes, graphene-derived materials, metalorganic frameworks (MOFs), 3D carbon aerogels and N-doped porous carbon nanosheets have received considerable attention because of their high surface/volume ratio, chemical stability and enhanced electronic and electrocatalytic activity. Nowadays, such materials are widely employed in industrial (transport, remediation, electronic, energy, desalination) and biomedical (bioimaging, tissue engineering, drug delivery, cancer treatment) applications [5, 17, 20-28] constituting a revolution in the design of novel devices and applications. Among different carbon-based materials, graphene presents some advantage (e.g. against CNTs) such as low cost production, ease of processing and safety, and more important, it is free from the contamination of transition metals [29]. Moreover, graphene-related materials decorated with metallic nanoparticles (NPs) have become increasingly important since hybrid nanocomposites can show synergic effects, improving the electrocatalytic properties of such materials when they are individually compared against each constituent [30, 31]. AgNPsmodified graphenic composites have been reported with different methods and morphologies due to their excellent electrocatalytic properties for H2O2 sensing. Therefore, Tian el al.[32], reported the synthesis of AgNPs anchored sulphur doped graphene using NaBH4 and prepared by a microwave-assisted method. In addition, Liu et al.[33], reported the chemical reduction of graphene oxide by hydrazine hydrate in the presence of poly[(2-ethyldimethylammonioethyl methacrylate ethyl sulfate)-co-(1vinylpyrrolidone)]

(PQ11),

and

the

latter

modification

with

AgNPs

(AgNP/PQ11/graphene). However, the use of strong reducing agents such as hydrazine and its derivatives, NaBH4 and hydroquinone may produce toxic, explosive and dangerous products that 3

hinder its handling in larger scale production, and more importantly, there is a high impact on the environment[34-36]. In order to solve this problem, new aqueous, facile, low-cost, non-toxic and eco-friendly approaches have recently been reported in the literature using green reductant agents such as polyphenols, polyallyamine, ascorbic acid, potassium hydroxide, citric acid, dextran, etc.[34-39]. Based on this approach, Yang et al.[40], reported the hydrothermal synthesis of AgNPs and the latter modification of graphene oxide (AgNPs-TWEENGO) using Tween 80 as stabilizing and reducing agent. Moreover, Ag nanoparticlesdecorated reduced graphene oxide (AgNPs-rGO) was successfully fabricated by hydrothermal treatment of the mixture of graphene oxide and AgNO3 solution under strong alkaline conditions[41]. Under a similar approach other authors have reported the synthesis of Ag nanowires[42] and Ag nanorods[43] decorated graphene sheets using ethylene glycol as mild reducing agent. Other interesting approach for obtaining Aggraphenic hybrid structures have been reported by Nia et al.[44]. These authors developed a H2O2 sensor using reduced graphene oxide decorated with both polypyrrole nanofibers and AgNPs. On the other hand, natural polyphenols (contained in green tea, for detail see Scheme 1 in the supporting information) have been used to obtain both rGox and metallic NPs due to their biocompatibility and biodegradability [34, 36, 38, 39]. In addition, tea polyphenols (TPs), whose content is about 10-15%, are rich in catechol and pyrogallol groups, making TPs highly reactive and water-soluble[34]. Here, a simple one-step strategy is presented to obtain silver nanoparticle-modified reduced graphene oxide nanocomposites (rGox/AgNPs) using an eco-friendly method. Although green tea extract has been used to obtain rGox and Ag NPs separately[34, 36,

4

38, 39] or to obtain rGox/AgNPs nanocomposites using a multistep approach[36], the one-step method has not been studied to the best of our knowledge. Different techniques such as X-ray diffraction, FTIR, AFM, electrochemical impedance (EIS), constant potential amperometry (CPA) and cyclic voltammetry (CV) are used to describe the chemical nature, the oxidation state and the electrocatalytic properties of the rGox/AgNPs nanocomposite. Finally, rGox/AgNPs nanocomposite is used to develop an electrochemical sensor for detecting H2O2, where the main analytical parameters (sensitivity, selectivity, reusability, stability, etc.) and the sensor response in complex real samples (antiseptic solutions, urine and milk) demonstrated the reliability of this sensor for H2O2 detection. 2. Materials and methods 2.1 Materials Graphite flakes, H2O2 standard solution (30%w/w), all reagents (reagent grade) such as AgNO3, HCl, KMnO4, H3PO4, H2SO4 interference species, etc. were purchased from Sigma-Aldrich and used as received without further purification. Commercial green tea, antiseptics solutions and commercial milk were obtained from local markets. Urine samples were donated by volunteers. 2.2 Green synthesis of silver nanoparticle-modified reduced graphene oxide nanocomposite (rGox/AgNPs) Graphene oxide (Gox) was synthesized under soft conditions according to previous works with some modifications[45, 46]. In a typical procedure, 1 g of graphite powder was dispersed in 55 mL of (H2SO4:H3PO4) (49:6 mL) under moderate agitation. Subsequently, 5 g KMnO4 was slowly added (producing a light exothermic reaction) and the reaction was left for three days to allow the soft oxidation reaction of graphite. The solution turned from dark purplish green to dark brown, confirming the formation

5

of graphite oxide. After 3 days, 10 mL H2O2 (30%) was slowly added to stop the oxidation reaction and stirred until gas evolution ceased. The graphite oxide was purified using the centrifugation method (centrifugation force = 15.000 g)[45]. Therefore, graphite oxide, the work solution was washed with 1 M HCl and deionized (DI) water (3 times each) to eliminate the excess of manganese salt and acid. During the washing step with DI water, the reaction product was exfoliated. This exfoliation was confirmed by the formation of a brown graphene oxide (Gox) gel during this stage[45]. Finally, Gox gel was dried at 60ºC overnight. The one-step green synthesis of the silver nanoparticle-modified reduced graphene oxide nanocomposite (rGox/AgNPs) was obtained as follows: 2 green tea bags (ca. 3.4 g) were added to 100 mL of DI water and boiled at 100 ºC for 15 minutes. Once the tea extract was cold, it was filtered through a 0.44 µm cellulose membrane (solution A). Separately, 50 mL of a solution containing 100 mg Gox with 2 mM AgNO3 was sonicated for 1 h (solution B). Subsequently, 50 ml of solution A and solution B were mixed in a flask reactor under moderate agitation (ca. 800 rpm), heated at 90 ºC during 1h and then left stirring overnight at room temperature. The obtained black powder (rGox/AgNPs sheets) was washed and centrifuged three times with DI water to eliminate the excess of tea phenols and dried at 60 ºC overnight. 2.3 Material characterization X-ray diffraction patterns were recorded with a Philips Panalytical X’Pert powder diffractometer with CuKα (λ=1.540 Å) radiation in the 2θ range from 5º to 80º. Transmission electron microscopy images were obtained using a TEM 200 kV JEOL 2100 microscope. FTIR spectra were recorded with a Varian 670-IR spectrophotometer in the range 400-4000 cm-1. UV-Vis spectra were obtained with a Thermo Scientific Evolution 300 spectrophotometer. AFM images were acquired in Peak-Force mode 6

using a Nanoscope V Multimode microscope from Bruker. AFM tips (Scan Asyst HR; 0.4 N/m) were acquired from Bruker. 2.4 Electrochemical characterization Electrochemical

measurements

(CV,

CPA)

and

electrochemical

impedance

spectroscopy (EIS) analysis were performed with a PalmSens4 potentiostat and PSTrace5 software (PalmSens). For all electrochemical measurements, an Ag/AgCl (3 M KCl) and a Pt wire were used as reference and counter electrodes, respectively. Electrochemical tests were done in 0.1 M phosphate buffer solution (PBS) at pH 7. EIS analysis was performed in PBS containing different H2O2 concentrations. For EIS measurements the stabilization potential was fixed at -0.5 V against the reference electrode while the signal amplitude was 10 mV in a frequency range from 0.1 to 105 Hz. 3. Results and discussion 3.1 Material characterization of the rGox/AgNPs nanocomposite Graphene oxide (Gox) was obtained using the simplified Hummer’s method, without controlling the temperature during the chemical oxidation of the graphite[45]. Although such a method requires 3 days for the adequate oxidation of the graphite flakes, it is a safe and non-tedious, does not need special care and the mixing and washing steps are straightforward. The solution turned from dark purplish green to dark brown during the oxidation reaction confirming the formation of graphite oxide. The graphite oxide was exfoliated and formed a brown Gox gel during the washing and centrifugation step. Finally, Gox gel was dried at 60 ºC overnight. Atomic force microscopy (AFM) images for Gox confirmed the correct oxidation and exfoliation of the graphite flakes, obtaining

7

Gox sheets of single (thickness: ~ 0.9 nm) and double layers (thickness: ~ 1.8 nm) (for details see Fig. S1, supporting information). Physicochemical properties, chemical structure and performance of Gox and rGox/AgNPs nanocomposite were obtained using different techniques such as XRD, TEM, AFM, FTIR and UV-Vis spectroscopies. Fig. 1A shows the UV-Vis spectra for the Gox and rGox/AgNPs composite dispersed in H2O. The UV-Vis spectrum of the Gox solution has a band (ca. 229 nm) and a shoulder (ca. 300 nm) which can be assigned to the π → π* transition of aromatic C–C bonds and the n → π* transition related to C=O bonds, respectively[47, 48]. Furthermore, in the case of the rGox/AgNPs composite the peak at 229 nm red shifted to 258 nm after the reduction reaction with tea extract, due to the restoration of the electronic conjugation (C=C bonds) within the graphene sheets[47, 48]. Besides which, the peak at ca. 410 nm in rGox/AgNPs sample was ascribed to the plasmon resonance absorption of the AgNPs and confirmed the correct formation of the hybrid composite[36, 38, 49]. Fig. 1B shows the FTIR spectra for the Gox and the rGox/AgNPs nanocomposite. The Gox spectrum confirmed the presence of oxygen containing groups after the oxidation reaction, where the main peaks were found at 1050, 1,216, 1630, 1735, and 3426 cm-1. The peak at 1050 cm-1 was specific to stretching vibrations from the C–O–C bonds of alkoxy and epoxy groups. The peak found at 1216 cm-1 was ascribed to C–OH bonds of carboxyl groups[50], meanwhile the peak found at 1630 cm-1 was specific to C=C bonds related with the vibrations of unoxidized graphene structure[51]. Finally, the peaks at 1735 cm-1 and 3426 cm-1 were related to C=O bonds (found in carbonyl moieties and carboxylic acid)[52] and stretching of –OH groups, respectively[51]. After reduction, reduced graphene oxide (rGOx) displayed lower peak intensities related to alkoxy and hydroxyl groups, meanwhile C=C phenol ring stretching found at ca. 1630 8

cm-1 remained. In addition, new bands related to alliphatic -CH bonds appeared at 2923 and 2847 cm-1 and confirmed the convenient reduction of the Gox sheets[51, 53, 54].

Figure 1

To understand the atomic structures and confirm the chemical modification of the graphite flakes, X-ray diffractograms were performed during different synthesis stages and the results are shown in Fig. 2A. The diffactogram for graphite flakes displayed a strong and sharp peak at 26.5º corresponding to (002) planes and an inter-layer spacing of 0.336 nm. After the chemical oxidation of the pristine graphite, the diffraction peak of Gox shifted to a lower angle (ca. 8.7º), corresponding to (001) planes and an interlayer spacing of 1.01 nm. Such an increase in the inter-layer spacing can be justified by the intercalation of water molecules and the formation of oxide functional groups on the carbon basal plane during the chemical oxidation reaction [35, 45]. It is noteworthy that after the chemical reduction of the Gox sheets with the green tea extract, the diffraction peak, corresponding to (001) planes, almost disappeared and confirmed the reduction of the Gox sheets. The presence of this residual peak in the rGox/AgNPs composite can be attributed to unreacted oxide functional groups that remain in the C basal planes (common observed in chemical reduced graphene composites). Finally, the nature of such NPs was studied by comparing the experimental diffractogram with different XRD patterns. The rGox/AgNPs nanocomposite has diffraction peaks at 38.2º, 44.4º, 64.4º, 77.5º and 81.5º that were specific to (111), (200), (220), (311) and (222) planes of the metallic Ag (JCPDS. No 01-087-0717). The broadening of Bragg’s diffraction peaks confirmed the formation of AgNPs. The mean crystallite size (D) was determined from 9

the Debye-Scherrer’s equation using the main diffraction plane (111) at 38.3º according to (Eq. 1): D=0.94 λ / β cos θ (1) where λ is the X-ray wavelength, β is the full width at half maximum (FWHM) and θ is the diffraction angle. Finally, the calculated average size for the AgNPs was ca. 25 nm, and confirmed the nanoscale dimensions of the AgNPs.

Figure 2

TEM and AFM images were obtained to confirm the nature of the nanocomposite. AgNPs appeared well-distributed on the rGox sheets, as shown in Fig. 3A. Fig. 3B and 3C show the TEM image and the size distribution for AgNPs respectively. These results confirmed the quasi-spherical shape of the AgNPs with an average size of about 24.6 ± 11.1 nm (mean ± SD, n = 200) and a size range distribution from 5 to 60 nm. Moreover, Fig. S2 shows the AFM image for the AgNPs-modified rGox nanocomposite with AgNPs size in good agreement with the TEM and XRD analysis described above. The chemical composition of the nanocomposite was analyzed by energy dispersive X-ray (EDX) detection. Fig. 2B shows the typical optical absorption peak at ca. 3 keV for Ag, which confirmed the presence and the purity of the AgNPs on the graphene sheets. Finally, the selected-area electron diffraction (SAED) patterns for single AgNPs were obtained (Fig. 3D) and confirmed the monocrystalline nature of the AgNPs.

10

Figure 3

3.2 Electrochemical characterization and sensing properties of the rGox/AgNPs nanocomposite Glassy carbon electrodes (GCEs, ϕ: 3 mm) were used as working electrodes for the electrochemical characterization of the rGox/AgNPs nanocomposite. GCEs were first polished with 0.05 mm alumina slurry, rinsed thoroughly with DI water and sonicated in DI water, acetone and finally dried under an N2 stream. After that, the rGox/AgNPs nanocomposite was dispersed in DMF (1mg/mL) and sonicated for 3 hours. Finally, the working electrode was casted with 10 µL of the dispersion described above and dried at room temperature overnight. Fig. 4 shows the CVs for the bare GCE and GCE/rGox/AgNPs electrodes obtained in 0.1 M PBS (pH= 7, scan rate 100 mV s-1). For comparative purposes, the CV for the rGox-modified electrode without AgNPs (GCE/rGox) is also shown. As expected, the bare GCE did not show significant redox peaks in the studied range of potential. The modification of the GCE with rGox resulted in a rise of the capacitive current associated with the increase of the electroactive surface of the sensor, whereas the GCE/rGox/AgNPs electrode showed two welldefined redox peaks at the potentials of ca. 0.05 V and 0.4 V, confirming the reduction and oxidation of the AgNPs found in the rGox/AgNPs nanocomposite. Figure 4

The electrocatalytic behavior of the GCE/rGox/AgNPs electrode was investigated against the H2O2 reduction using CV. Hence, the CVs before and after the addition of 6

11

mM H2O2 in 0.1 M PBS are shown in Fig. 5. The electrochemical response for H2O2 at the GCE/rGox/AgNPs electrode surface may be ascribed due to the direct 2H+ and 2e− involved in the reduction reaction [55], leading to the formation of H2O according to Eqs.2 and 3: H2O2 + H+ + e− → OH(ad) + H2O OH(ad) + H+ + e− → H2O

(2)

(3)

Furthermore, the CV response for the bare GCE and GCE/rGox electrodes are shown for comparative purposes. As shown in Fig. 5 the reduction current increased in all electrodes with the addition of 6 mM H2O2. Nevertheless, GCE response against H2O2 (Fig. 5A) showed an extremely weak cathodic response for H2O2 reduction. The incorporation of rGox in the next tested configuration (GCE/rGox) showed and enhancement of the cathodic current(Fig. 5B), confirming the excellent electrocatalytic activity ascribed previously for graphenic materials and attributed to the high density of edge-plane-like defective sites on rGox[29]. Finally, the GCE/rGox/AgNPs electrode (Fig. 5C) displayed a notable increase in the reduction current when it is compared against the GCE/rGox electrode, indicating a better electrocatalytic activity of the rGox/AgNPs nanocomposite against H2O2. Such result may be justified due to the excellent electrocatalytic activity of the AgNPs and the amplification of the electron transfer kinetic ascribed to graphene. Figure 5

Sensor response showed a linear response against H2O2 concentration as can be seen in Fig. S3. The dependence of the cathodic and anodic currents on the square root of the

12

scan rate and the scan rate were also studied. Fig. S4 shows the CVs recorded for the sensor here (GCE/rGox/AgNPs) in 0.1 M PBS containing 1 mM H2O2 at different scan rates. Both the anodic and the cathodic currents increased linearly against the scan rate (see Fig. S4B), which is characteristic of a non-diffusion controlled process, suggesting that the kinetic boundaries are mostly related with the charge propagation in the nanocomposite. EIS experiments were performed to better understand the electrochemical behavior of the electrode. EIS is a non-destructive technique for evaluating both the electrical interfacial and electrochemical characteristics of sensor devices, and it even allows the study of different surface modifications during sensor assemble or changes of impedance during absorption or electrochemical reactions that occur on the sensor surface. In order to do this, the GCE/rGox/AgNPs electrode was introduced in a conventional three-electrode electrochemical cell as described in section 2.4. A Nysquit plot was used to model the equivalent electrical circuit and to obtain the impedance of the sensor at different H2O2 concentrations. Fig. S5 shows the Nyquist plot for different H2O2 concentrations. Nyquist plot usually has a straight line (at low frequencies) and a semicircular region (at high frequencies). The linear zone represents a diffusion-limited process and the semicircle region is associated to a charge-transfer limited process. Therefore, the semicircle diameter in the Nyquist plot is related to the charge-transfer resistance, Rct, at the electrode surface. The data here shows that, in the range of frequencies studied, the main component affecting the sensor response is specific to the electron transfer reaction of the H2O2 on the electrode interface, which is in good agreement with data reported above (current response toward scan rate experiments, Fig S4). In addition, Fig. S5 shows that the Rct value was lower at higher concentrations of H2O2, indicating that H2O2 improves the electric conducting properties and facilitates 13

the electron transfer reaction on the surface of the GCE/rGox/AgNPs electrode [56, 57], constituting a suitable media for quick electron transferring reaction with promising sensing properties. H2O2 sensing properties for the GCE/rGox/AgNPs electrode were evaluated using CPA. In addition, working potential and the effect of the pH value of the electrolyte solution (Fig. S6) were studied in detail. All experiments were carried out under stirring conditions at ca. 800 rpm. The results showed a very low response towards H2O2 at higher potentials (-0.1 V), while the best analytical signal was obtained in the range of potential from -0.4 to -0.6 V. These data agree with the CV data above and with the results reported by other authors[16, 58]. On the other hand, the effect of the pH solution value regarding the electrochemical sensing properties of the rGox/AgNPs nanocomposite was evaluated in the range of pH from 3 to 11. The sensor had a relatively low response in acidic mediums, which may be attributable to the instability of the metallic AgNPs in acidic conditions. After which, the signal current rapidly increased from pH 5 to 8, and then slightly decreased again when the pH changed from pH 8 to 11. Therefore, pH 8 and -0.4 V were selected for improving the electrochemical response toward H2O2 to obtain the better analytical performance of the sensor. 3.3 Electrochemical sensing properties of GCE/rGox/AgNPs electrode against H2O2 detection Under optimized experimental conditions the sensitivity of the GCE/rGox/AgNPs electrode was evaluated under stirring conditions (ca. 800 rpm). The sensor always had a fast response of ca. 2 s (Fig. S7A), with two different linear range responses at different H2O2 concentrations (Fig. 6). The first one up to a value of 20 mM, yielded a sensitivity of ca. 236 µA mM-1cm-2 (R2= 0.999) and the second one, from 30 to 160

14

mM, displayed a sensitivity of ca. 67 µA mM-1cm-2 (R2= 0.995). The limits of detection (LOD) (S/N = 3) and quantification (LOQ) (S/N = 10) obtained for the GCE/rGox/AgNPs electrode were 0.73 and 2.45 µM, respectively. The reproducibility (RSD: 3.6%; n= 3) and repeatability (RSD: 5.6%; n=5) of the sensor were also determined from the slope of the first linear concentration range.

Figure 6

Table 1 demonstrates the performance of the GCE/rGox/AgNPs electrode compared against other electrodes based on Ag-modified graphene nanocomposites in term of sensitivity, LOD and linear range. As observed, our sensor presents excellent sensitivity and low LOD with an adequate linear range for analytical purpose.

Table 1

Short and long term sensor stabilities were investigated for different periods. Short stability was analyzed after adding 1 mM H2O2. The obtained results showed a stable response (ca. 98% of the initial response) after 30 min (Fig. S7B). The long term stability of the rGox/AgNPs nanocomposite was evaluated for 7 months. In order to do this, the nanocomposite (dispersed in DMF) was conserved at room temperature without special conditions. Different sensors were assembled and calibrated in the concentration range of 0 to 10 mM H2O2 during this period and its sensitivity was evaluated. The

15

sensor did not show significant loss of response (Fig. 7A) during this period, supporting the good stability of the rGox/AgNPs nanocomposite for future analytical applications. The influence of common interference species that could affect the H2O2 detection in biological matrixes, was also investigated. Fig. 7B shows that when ascorbic acid, dopamine, glutamate and glucose (100 µM) were added to the working solution, the GCE/rGox/AgNPs electrode did not display any current response. However, when 100 and 200 µM H2O2 aliquots were added the sensor response had a strong amperometric current, confirming the good discrimination of the sensor against H2O2.

Figure 7 3.4 Determination of H2O2 in urine samples and commercial disinfectant solutions and milk Five different disinfectant solutions (obtained in local markets) in the range from 3 to 6 %w/w H2O2 were analyzed to demonstrate the future application of the GCE/rGox/AgNPs electrode in real applications. Fig. S8 shows the raw current data comparing the current response for the standard solution (3 %w/w) and a commercial sample (sample B) labelled with a concentration of about 4.9 %w/w. Table 2 displays the concentration labelled from manufacturers, the mean value obtained with the sensor, the coefficient of variations (CV%) and the recoveries obtained. The good results reported confirm that the GCE/rGox/AgNPs electrode can be used for quality control applications in commercial disinfectant solutions.

Table 2 16

However, other more complex matrixes have been explored due to the great interest in H2O2 detection in agroalimentary and biomedical applications. H2O2 can be used for activing natural lactoperoxidase (LP) enzymes found in fresh raw milk and has been used to preserve raw milk quality in countries where it is not possible to use mechanical refrigeration [59]. Although the use of H2O2 improves the quality of raw dairy products, in some countries, where refrigeration is available such as the United States, the addition of H2O2 to milk is not allowed (with some exceptions e.g., during the preparation of modified whey and certain applications prior to cheese-making)[60]. Therefore, H2O2 analysis is a useful method to detect adulterated milk in agro-food laboratories. Table 3 shows the recoveries (n=3) for different H2O2 amounts added to commercial milk acquired in local markets. Such recoveries, around 100%, demonstrate the reliability of the GCE/rGox/AgNPs electrode for H2O2 in this matrix.

Table 3

Another noteworthy application of the sensor is the quantification of H2O2 in biological fluids. The level of H2O2 in urine has been suggested as a potential biomarker of whole body oxidative stress[61], and more interestingly, it is well known that coffee beverages may increase the level of H2O2 in urine after ingestion in healthy persons[62]. Therefore, and as a probe of concept, Table 3 shows the recoveries obtained for different H2O2 additions in urine samples taken from for healthy volunteers. Furthermore, recovery values were close to 100% (raw data can be seen in Fig. S9)

17

confirming the good analytical properties of the sensor for use in biomedical applications too.

Conclusions In summary, one-step green synthesis of silver nanoparticle-modified reduced graphene oxide nanocomposite has been demonstrated to be a good method to develop a novel hydrogen peroxide sensor. Such a methodology provides an eco-friendly and simple method to develop future hybrid nanocomposites with other metal nanoparticles for other interesting analytical applications. The hybrid nanocomposite was thoroughly studied examined with a wide range of techniques, confirming its chemical composition and its good electrocatalytic properties. Moreover, it has good electroanalytical properties (sensitivity, selectivity, stability, LOD). Finally, its application in commercial samples (milk and antiseptic solutions) and biological fluid (urine) confirmed the ability of the GCE/rGox/AgNPs electrode to sense H2O2 in quality control analysis.

Acknowledgments The support by Agustín de Betancourt program (Cabildo de Tenerife) is gratefully acknowledged. The authors would like to acknowledge SEGAI of the ULL for the XRD, AFM and TEM measurements.

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Figure and table captions Figure 1. (a) UV-Vis and (b) FTIR spectra for graphene oxide (Gox) sheets and AgNPs-modified reduced graphene oxide (rGox/AgNPs) nanocomposite. Figure 2. (a) XRD diffractogram for graphite, graphene oxide (Gox) and AgNPsmodified reduced graphene oxide (rGox/AgNPs). (b) EDX analysis of the of rGoxAgNPs nanocomposite. Figure 3. TEM images of the rGox-AgNPs nanocomposite with different magnifications (a, b,). (c) Size distribution of the AgNPs. (d) Selected area electron diffraction pattern for the AgNPs. Figure 4. Cyclic voltammograms obtained in 0.1 M PBS (pH 7) for GCE, GCE/rGox and GCE/rGox/AgNPs electrodes. Reference electrode: Ag/AgCl (3 M); scan rate:0.1 V s-1. Figure 5 Cyclic voltammograms before and after adding 6 mM H2O2 for (a) GCE, (b) GCE/rGox and (c) GCE/rGox/AgNPs electrodes in 0.1 M PBS (pH 7). Scan rate: 0.1 V s-1 against Ag/AgCl (3M) reference electrode. Figure 6 Calibration curves obtained for the GCE/rGox/AgNPs electrode in 0.1 M PBS under optimized conditions (applied potential: -0.4 V against Ag/AgCl (3 M) and pH 8). Stirring rate: 800 rpm.

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Figure 7 (a) Control chart of the rGox/AgNPs nanocomposite. The measurements correspond to the sensitivity of the GCE/rGox/AgNPs electrode obtained in the H2O2 concentration range of 1 to 10 mM. The lower and upper control lines were fixed at 95% and 105% of the initial value. (b) Raw amperometric response of the GCE/rGox/AgNPs electrode for H2O2 and interference additions at -0.4 V against Ag/AgCl (3 M) in 0.1 M PBS (pH 8). Studied interferences: glucose (gluc), glutamate (glut), ascorbic acid (AA) and dopamine (DA). Table 1 Electrode performance for some silver-graphene modified electrodes Table 2 Determination of H2O2 concentration in commercial antiseptic solutions. Table 3 Recoveries obtained for H2O2 additions in commercial milk and urine samples (n=3).

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Table 1 Performance for some silver-graphene modified electrodes reported in the literature Electrode S (µAmM-1cm-2 ) LOD (µM) Ag/S/RGO 428.57 0.14 AgNP/PQ11/graphene/GCE 57.14 28 AgNPs-TWEEN-GO 10.65 8.7 AgNPs–rGO/GCE 142.85 1.8 Ag NWs-graphene 12.37 1 Ag NRs-rGO 2.5 µA/mM 2.04 PpyNFs-AgNPs-rGO-2/GCE 0.736 µA/mM 1.099 GCE/rGox/AgNPs 236 0.73 GCE/rGox/AgNPs 67

Linear range (mM) 0.1–136.5 0.1–40 0.02–23.1 0.1–60 0.01–34.3 0.1–70 0.1–5 0.002–20 30–160

Reference [32] [33] [40] [41] [42] [43] [44] This work This work

Table 2 Determination of H2O2 concentration in commercial antiseptic solutions. [H2O2] (%w/w) CV (%) Recovery [H2O2] (%w/w) Sample labelled found (n=3) (%) 3 A 2.97 7.91 99.00 4.9 B 4.94 3.71 100.86 4.9 C 5.08 7.08 103.77 5.1 D 5.16 5.67 98.60 5.5 E 5.42 4.33 101.21

Table 3 Recoveries obtained for H2O2 additions in commercial milk and urine samples (n=3). Matrix [H2O2] added (mM) [H2O2] found (mM) Recovery (%) Milk Milk Milk Urine Urine Urine

5 10 20 5 10 20

4.97 9.72 19.56 5.03 10.24 19.71

99.41 97.20 97.78 100.71 102.38 98.56

Research highlights

1. Silver nanoparticles-modified reduced graphene oxide (rGox/AgNPs) is obtained. 2. rGox/AgNPs nanocomposite is analyzed using different techniques. 3. GCE/rGox/AgNPs electrode is used in H2O2 sensing applications. 4. The main analytical parameters and its application in real samples are presented. 5. rGox/AgNPs nanocomposite has excellent stability over 7 months.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: