Graphene oxide composite as efficient electrode material for dissolved oxygen sensors

Accepted Manuscript Title: CoTRP/Graphene oxide composite as efficient electrode material for dissolved oxygen sensors Author: Lucas P.H. Saravia Anandhakumar Sukeri Josue M. Gonc¸alves Juan S. A. Aguirre Bruno B.N.S. Brand˜ao Tiago A. Matias Marcelo Nakamura Koiti Araki Henrique E. Toma Mauro Bertotti PII: DOI: Reference:

S0013-4686(16)32521-X http://dx.doi.org/doi:10.1016/j.electacta.2016.11.159 EA 28449

To appear in:

Electrochimica Acta

Received date: Revised date: Accepted date:

25-7-2016 22-11-2016 27-11-2016

Please cite this article as: Lucas P.H.Saravia, Anandhakumar Sukeri, Josue M.Gonc¸alves, Aguirre Juan S.A., Bruno B.N.S.Brand˜ao, Tiago A.Matias, Marcelo Nakamura, Koiti Araki, Henrique E.Toma, Mauro Bertotti, CoTRP/Graphene oxide composite as efficient electrode material for dissolved oxygen sensors, Electrochimica Acta http://dx.doi.org/10.1016/j.electacta.2016.11.159 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

CoTRP/Graphene oxide composite as efficient electrode material for dissolved oxygen sensors Lucas P. H. Saravia, Anandhakumar Sukeri, Josue M. Gonçalves, Juan S. Aguirre A., Bruno B. N. S. Brandão, Tiago A. Matias, Marcelo Nakamura, Koiti Araki, Henrique E. Toma and Mauro Bertotti* Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, 05508-000, São Paulo, SP, Brazil Corresponding author: Mauro Bertotti, e-mail: [email protected]

Graphical Abstract

A novel CoTRP/Graphene oxide composite modified electrode was fabricated for oxygen reduction reaction via a direct four electron reduction pathway at very low overpotetnial (0.05 V) in aqueous solution.

Highlights 1. Graphene oxide (GO) was modified with a Co porphyrin (CoTRP) film 2. Highly efficient and stable electrocatalyst for ORR in aqueous solution. 3. The results of electrochemical measurements indicated that oxygen was reduced to H2O via 4-electron transfer. 4. The CoTRP/4GO modified electrode was used as probe for dissolved oxygen (DO) sensing. 5. The CoTRP/4GO modified electrode exhibited higher electrocatalytic activity for DO reduction with good stability.

Abstract The preparation of the trifluoromethanesulfonate salt of [tetrakis-bisdimethylbipyridine chlororuthenium(II)]-5,10,15,20-tetrapyridylporphyrinate cobalt(II) complex (CoTRP) and its composites with graphene oxide (GO) is described. The CoTRP molecules found to lie flat on the GO surface, generating nanostructures that are more or less dispersible in aqueous media, depending on the relative amounts of CoTRP and GO. This feature can be useful to tune the electrocatalytic activity of the composites in the oxygen reduction reaction (ORR) in neutral media. The material was characterized by spectroscopy (Raman, UV-visible, FT-IR) and microscopy (TEM, AFM) techniques where the results confirmed that CoTRP is strongly supported on GO through electrostatic interactions. Further, the electrocatalytic activity of CoTRP/GO modified electrode was evaluated for oxygen reduction reaction (ORR), and observe a very low overpotential (0.05 V) via a four-electron reduction pathway of dioxygen. Besides, the reduction peak potential was dramatically shifted to positive potentials, by 0.88 V in

relation to the glassy carbon electrode and the electrode performance allows its use as effective sensor probe for continuous monitoring of dissolved oxygen (DO). Additionally, analytical parameters such as sensitivity, selectivity, reproducibility, lower detection limit (LOD) and stability were evaluated in order to prove the suitability of the developed platform for dissolved oxygen sensing.

Keywords: Nanocomposite; graphene oxide; oxygen reduction reaction; cobalt porphyrin; four-electron reduction.

1.

Introduction

Oxygen is essential for sustaining animal life in our planet and is a potential electron acceptor in fuel cells owing to its high reduction potential and clean reaction generating water as the only product [1]. However, the poor kinetics of the oxygen reduction reaction (ORR) hinders its use in neutral media. In addition, controlling the mechanism of ORR is very important for several technological applications, especially in fuel cells since their efficiency is strongly dependent on the number of electrons transferred to oxygen (2e- or 4e-). Platinum (Pt) and other noble metals has been extensively employed as 4e- ORR catalyst, but the high cost, scarcity and propensity of poisoning by CO have limited more extensive application, fueling the search for alternative electrocatalytic materials. In this context, since the seminal reports on the properties of cobalt phthalocyanine and porphyrins [2]. Also, several other molecular mediators such as DNA [3], anthraquinone [4,5], poly(methylene blue) [6], nanostructure peptide [7], cobalt complexes [8-12], iron(II) tetrasulfonated phthalocyanine and iron(II) tetra-(Nmethyl-pyridyl) porphyrin [13], magnesium (II) phthalocyanine[14], nickel Salen [15],

vitamin B-12 [16], ABTS-laccase [17], ZnO doped RuO2-SE [18], diamino-obenzoquinone [19], indigo tetrasulfonate [20] and eugenol [21], have been described. The continuous measurement of oxygen concentration is essential in many medical procedures and applications, technological processes, environmental monitoring and analysis. Several electrochemical sensors based on ORR mediators have been proposed for monitoring oxygen dissolved in water [13, 22-29], but none of them exhibited the robustness, reproducibility and sensitivity requirements to compete with Clark electrodes. In this context, recently we described a sensor based on CoTPP modified glassy carbon electrodes for monitoring the oxygen consumption in biological samples [30] by a bielectronic process. However, cobalt porphyrins are particularly interesting because the four electron transfer mechanism for ORR can be activated [31] improving the sensitivity and limit of detection. In fact, enhanced catalytic and electrocatalytic properties have been reported for supramolecular species obtained by coordinating ruthenium complexes to the periphery of cobalt meso-tetra(4-pyridyl)porphyrin (CoTPyP) [30-35]. In addition, the covalent, electrostatic and hydrophobic interaction of cobalt porphyrins with the surface of carbon materials such as HOPG, CNTs and graphene can shift the Co(III/II) redox potential to more positive values and activate the four electron mechanism of ORR [36-39]. In this work, we demonstrate that the electrostatic interaction [40,41] of [tetrakis-bisdimethylbipyridylchlororuthenium(II)]5,10,15,20-tetrapyridylporphyrinatecobalt complex (CoTRP) with graphene oxide (GO), generates a stable nanocomposite materials, whose electrocatalytic ORR mechanism can be tuned by the CoTRP:GO ratio, exhibiting potential use in electrochemical sensors for monitoring the dissolved oxygen.

2.

Experimental methods

2.1. Reagents and Apparatus Analytical grade chemicals were used throughout this work, and the aqueous solutions were prepared using 18.2 MΩ cm resistivity water (Millipore). Potassium nitrate (KNO3), sulfuric acid (H2SO4), potassium permanganate (KMnO4), sodium nitrate (NaNO3), hydrochloric acid (HCl), ethanol (CH3CH2OH), tetrapyridylporphyrin (TPyP), cobalt (II) acetate (Co(Ac)2), methanol (CH3OH) and dimethylformamide (DMF) were purchased from Sigma-Aldrich and used as received. Cyclic voltammetry and amperometry data were recorded using a PalmSens portable Potentiostat (Netherlands). Experiments were carried out in a conventional three-electrode electrochemical cell containing a glassy carbon (GC) electrode (bare or modified) (3 mm diameter), a Ag/AgCl (saturated KCl) and a platinum foil, which served as working, reference and counter electrodes, respectively. Rotating disc electrode (RDE) experiments were carried out using an analytical rotator (AFMSRX, Pine Instrument Company) and an Autolab (Metrohm Pensalab) PGSTAT 302 N potentiostat/galvanostat. A glassy carbon (GC) electrode (bare or modified) (7.5 mm diameter) was used as working electrode in RDE experiments. Dissolved oxygen concentration was measured using an oximeter (DM-4P). All measurements were performed at ambient temperature (25 ± 1 0C). UV-vis absorption spectra recorded on a Hewlett Packard 8453A diode-ray spectrophotometer, in 190 to 1100 nm range. UVVisible absorption spectroelectrochemistry was carried out using a custom-built electrochemical cell consisting of a gold mini-grid-working electrode, an Ag/AgCl (saturated KCl) reference, and a platinum wire auxiliary electrode mounted inside a

quartz cuvette with a path length of 25 μm. The potentials were controlled by an EG & G PAR 173 potentiostat. Raman spectra were recorded in a WITec 300R Alpha confocal microscopy, equipped with a 488 nm Ar laser (power = 0.06 mW·cm-2), 100X objective (0.80 NA), 1800 lines mm-1 grating, and integration time of 60 s. Raman images were obtained mapping point-by-point a 20x20 µm surface (400 points) with a piezo‐driven XYZ table and the spectra of pure materials were used as standards for image deconvolution. Cosmic rays were removed and the base line was subtracted from each crude spectrum. Transmission electron microscopy (TEM) images were obtained in a JEOL JEM2100 field emission equipment with an acceleration voltage of 200 kV. Samples were prepared on carbon-coated copper grids (TedPella) by dispersing 5 µL of a sample suspension prepared in water. Scanning Probe Microscopy (SPM) images were obtained using a PicoSPM I equipment, with a PicoScan 2100 controller, and silicon cantilevers, under the attractive regime with amplitude set point around 6.5 V. The tip radius was less than 10 nm.

2.2.

Preparation of graphene oxide (GO) Graphene oxide (GO) was prepared by a modified Hummers’ method [42,43].

Briefly, concentrated H2SO4 (115 mL) was added to a mixture of graphite flakes (5.0 g) and NaNO3 (2.5 g). KMnO4 (15.0 g) was added slowly and the reaction mixture heated to 50 °C while stirring for 6 h. The reaction was cooled to room temperature and kept in a water bath while 400 mL of water and then 20 mL of an aqueous H2O2 solution (30% v/v) were added. The mixture was centrifuged (5000 rpm for 10 min) and the supernatant decanted away. The remaining solid material was washed with 800 mL of a 30% HCl solution and 400 mL of ethanol for at least three times. Finally, the solid

product re-suspended in water, precipitated with 400 mL of ether and filtered out with a PTFE membrane with a 0.45 µm pore size. The obtained solid product dried overnight in a desiccator under vacuum at room temperature. A 2.0 mg mL-1 graphene oxide (GO) suspension was prepared in pH 10 water and used throughout.

2.3.

Synthesis of CoTRP porphyrin CoTPyP was prepared by refluxing TPyP (100 mg, 0.16 mmol) and cobalt (II)

acetate (100 mg, 0.40 mmol) in DMF for four hours, in nitrogen atmosphere. The purple precipitate was filtered and washed with DMF and then with methanol. The UV-vis spectrum of as prepared sample exhibited the Soret band at 413 nm and the Q band at 500 nm, characteristic of a Co (II) porphyrin. Elemental analysis calc.(exp.): C% 71.11 (70.53), N% 16.59 (16.15), H% 3.58 (3.78). [Ru(dmbpy)2Cl2] was prepared refluxing 809.8 mg (19.1 mmol) of LiCl, 500 mg (1.91 mmol) of RuCl3·3H2O and 703.8 mg (3.82 mmol) of 4,4’-dimethyl-2,2’bipyridine in 30 mL of DMF for 8 hours, in the dark, under nitrogen atmosphere. The solution was concentrated in a flash evaporator, precipitated in acetone, filtered out and washed with cold water and diethylether. Elemental analysis calc. (exp.) for RuC24H24N4Cl2: C% 53.34(53.18), N% 10.37(10.17), H% 4.48(5.06). CoTRP was synthesized [33] by refluxing [Ru(dmbpy)2Cl2] and CoTPyP in glacial acetic acid in a 4.2:1 ratio, for one hour. The solution was dried in a rotary evaporator, redissolved in methanol and refluxed for an additional hour. The solvent was removed in a flash evaporator, the solid redissolved in DMF and the product precipitated out with a saturated LiTFMS aqueous solution. The dark brown solid was filtered off and washed several times with cold water and dried under vacuum. Calc. (exp.) for {[Ru(dmbpy)2Cl]4CoTPyP}(TFMS)5·4H2O: C% 48.21(47.82), N%

9.57(9.75), H% 3.67(3.84). 1H NMR (500 MHz, METHANOL-d4) δ ppm 2.86 (s, 24 H) 3.00 (s, 24 H); 7.15 (d, J=5.49 Hz, 8 H) 7.98 (s, 8 H) 8.26 - 8.66 (m, 56 H) which is in agreement with the 72 protons expected in the aromatic region for CoTRP. Methyl protons appeared as singlets at 2.86 and 3.00 ppm, corresponding to fourty-eight protons; pyridyl protons were assigned at 7.98 ppm for the position 3, and a doublet at 7.15 ppm for the position 5 of the aromatic ring. The pyrrole and pyridine protons from the porphyrin yield a wide signal between 8.26-8.66 ppm.

2.4. Preparation of CoTRP/nGO composites The graphene oxide/porphyrin composites were prepared by mixing 10 mL of an aqueous CoTRP solution (1.0 mg mL-1) and a graphene oxide suspension (2.0 mg mL-1) in different proportions. Accordingly, 5, 10, 20 and 30 mL of graphene oxide suspension were added to 10 mL of porphyrin solution, filtered out and washed with water generating the composites denoted as CoTRP/GO, CoTRP/2GO, CoTRP/4GO and CoTRP/6GO, respectively, where n = 1 to 6 refers to the mass ratio of GO relative to the {[Ru(dmbpy)2Cl]4CoTPyP}(TFMS)5·4H2O complex.

2.5. Preparation of CoTRP/nGO modified GC electrodes The GC electrode was well-polished with a polishing cloth using 0.05 μm alumina powder and cleaned in a ultrasound bath for 5 min. Afterwards, 50 μL of a CoTRP/nGO dispersion (CoTRP/GO, CoTRP/2GO, CoTRP/4GO and CoTRP/6GO) were drop casted on the GC surface and allowed to dry open to air for 4 hours.

3.

Results and discussion

3.1. UV-Visible and spectroelectrochemical characterization

The electronic absorption spectrum of CoTRP (Fig. 1), exhibited typical bands of the [RuCl(dmbpy)2]+ and [CoTPyP] moieties. The band at 292 nm was attributed to the pπ→pπ* transition characteristic of the dmbpy ligands, [33] whereas the bands at 348 and 490 nm (shoulder of Soret band) were ascribed to the Ru(dπ)→pπ*dmbpy MLCT2 and MLCT1 transitions associated with the four peripheral ruthenium complexes. The Soret band at 434 nm is broadened at the higher wavelength side due to the contribution of the ruthenium complexes MLCT bands, whereas the Q1-0 and Q0-0 bands at 550 and 595 nm are typical of CoIIITPyP moiety [33], consistent with the oxidation of the cobalt ion to Co(III) during the preparation of the CoTRP complex.

The electrochemical behavior of CoTRP (Fig. 2A) was examined in water and two redox processes were noticed in the -1.25 to 1.00 V range. The reversible wave at E1/2 = +0.71 V was assigned to the RuIII/RuII process in the four independent sites, as previously reported for structurally similar tetraruthenated porphyrins.[33] This process is spectroelectrochemically supported by the disappearance of the MLCT bands at 348 and 490 nm and the decrease of the ligand pπ→pπ* transition at 292 nm (Fig S1). The wave associated to the CoIII/CoII process in the CoTPyP moiety did not appear in the CVs, because of its slow heterogeneous electron-transfer kinetics, but the process was clearly revealed by spectroelectrochemistry.[33] As a matter of fact, spectral changes characteristic of the conversion of CoIIITRP to CoIITRP appeared in the 0.15 to -0.32 V range, shifting the Soret band from 434 to 414 nm and the Q1-0 band from 550 to 530 nm, while the intensity of the Q0-0 band decreased, generating well-defined isosbestic points, as shown in Fig. 2B. The redox potential of the CoIII/IITPyP process was estimated from the Nernst plot considering the Soret band as E1/2= -0.03 V. The redox

process at E1/2= -0.76 V in the cyclic voltammogram was assigned to the CoII/ITPyP process. [33]

3.2. AFM Characterization The CoTRP/nGO composites were prepared by interacting the graphene oxide suspension with CoTRP. Since CoTRP is a pentacationic species, it can bind electrostatically to the negatively charged sites present at the GO surface and edges, imparting an overall positive charge high enough to keep the nanostructures in suspension. In fact, micrometric and sub-micrometric plate like structures displaying only 1.45 nm thick (estimated from the height profile shown in Fig. 3) were found in the AFM images, indicating single GO sheet fragments decorated with CoTRP, consistent with CoTRP/1GO (Fig. 3A-3B). However, as the proportion of GO increases in the CoTRP/2GO to CoTRP/6GO composites, the net positive charge on the platelets should decrease, facilitating their stacking. Accordingly, the nanocomposite with the highest graphene oxide content (1:6 m/m) exhibited multilayer stacks of CoTRP-GO structure, consistent with a series of 1.45 nm high steps (Fig. 3C-3D). The ruthenated porphyrin is a square molecule with about 2.0 nm side, where the thickness (~1.1 nm), is mainly defined by the bulky peripheral ruthenium complexes. The central cobalt porphyrin ring of CoTRP molecules lying flat on GO surface (0.9 nm, Fig. S2) should be "floating" or not strongly π-stacked, as observed for the basic porphyrin derivatives such as CoTPyP and CoTPP. Accordingly, our synthetic procedure should favor the rapid binding of positively charged CoTRP on the negatively charged GO sheets, and its concentration on the surface should be low enough to minimize the possibility of two of them be superimposed during the stacking process.

3.3. TEM and EDS analysis TEM images corroborate the AFM results, as the 1:1 composite typically exhibited isolated monolayers of CoTRP/GO sheet (Fig. 4A-4B), whereas CoTRP/4GO appears as stacks of few layers. The presence of the CoTRP complex on the surface of GO was confirmed by EDS analysis, that showed ruthenium and cobalt signals always coupled with carbon signals (Fig. 4E). The Si signal comes from impurities present in graphite, whereas S and K probably incorporated in the graphite oxidation process. As expected, the amount of CoTRP present in the CoTRP/4GO composite was significantly low, precluding the detection of ruthenium and cobalt by EDS spectroscopy.

3.4. Raman characterization Graphene oxide and the supramolecular complex were characterized by Raman spectroscopy, while the CoTRP/4GO composite was studied by confocal Raman microscopy (Fig. 5). Graphene oxide showed an intense and symmetric D band at 1353 cm-1 (FWHM = 98 cm-1) and a G band at 1608 cm-1 (FWHM = 69 cm-1). The ratio ID/IG = 0.77 indicates high concentration of structural defects caused by oxidation of graphene sheets and incorporation of sp³ (tetrahedral) (Fig. 5A) carbon sites. The presence of G’, D’ and D+G (2700, 2950, 3220 cm-1) bands is commonly found in this type of material, where the G' (or 2D) band is related to the structural organization in the two-dimensional graphene plane (Fig. S3). The CoTRP complex shows peaks that can be assigned to the bipy ligands, pyridine bridge and porphyrin core vibrational modes at 1020 cm-1 v(py), 1213 cm-1 δ(py), 1316 cm-1 vNC, 1369 cm-1 v(C-N)+ v(CC), 1479 cm-1 v(C-C), 1544 cm-1 v(C-C), 1606cm-1 v(py).

Confocal Raman microscopy is also a very important technique for characterization of composite materials, since it allows the chemical mapping of the material based on the typical Raman spectra embedded in every pixel. Graphene oxide and CoTRP exhibit typical spectral profiles and peaks that can be found in the composites, particularly the CoTRP/4GO composite, as shown in Fig. 5A. In fact, this spectrum resembles the sum of the components spectral features, indicating that the interaction between them is relatively weak and confirming the relatively large distance separating the GO and the CoTPyP π-systems. Furthermore, the distribution of CoTRP on GO was found to be homogeneous throughout the surface (Fig. 5B-5D). The good co-localization and distribution is confirmed in Fig. 5E, which shows the Raman cross section profile corresponding to a vertical line drawn in Fig. 5D.

3.5. Electrochemical behavior of CoTRP/nGO modified electrodes Cyclic voltammograms of the CoTRP/4GO modified electrode in 0.1 mol L-1 KNO3 solution as a function of scan rate (0.02 to 0.20 V s-1) are shown in Fig. 6A. The intense pair of waves around 0.9 V can be easily assigned to the RuIII/RuII redox couple associated with CoTRP, whereas the very small pair of waves at 0.6 V can be assigned to the RuIII/RuII couple of the [Ru(dmbpy)2Cl2] complex present as impurity. The waves associated with the CoIII/CoII process generally are not observed in the voltammograms because of the slow heterogeneous electron transfer process, but can be revealed by spectroelectrochemistry, as discussed previously. The anodic current density is linearly dependent on the potential scan rate, as expected for surface bound redox materials (Fig. 6B). The amount of CoTRP/4GO immobilized on the electrode surface was determined by using the equation

Г = jp4RT/n2F2υ and the average value was found to be equal to 51.3 x 10-11 mol cm-2 of Ru sites (calculated from the RuII/RuIII wave), that corresponds to 11.8 x 10-11 mol cm-2 of CoTRP sites. Unfortunately, also with the modified electrode no wave was observed in the CVs that could be associated with the cobalt porphyrin site. The surface concentration is relatively high considering a monolayer, but one should remember that our film is constituted by an multilayer instead.

3.6. Electrocatalytic reduction of dioxygen at CoTRP/nGO electrodes Typical cyclic voltammograms of dioxygen dissolved in 0.1 mol L-1 KNO3 solution (pH = 7.0) using a bare GC electrode and GC electrodes modified with GO, CoTRP and CoTRP/4GO are shown in Fig. 7A. A broad irreversible reduction wave appeared at -0.82 V when the electrolyte solution was saturated with oxygen for GC electrode. However, the peak shifted respectively to -0.60 V, -0.28 and 0.05 V when GC electrodes modified with GO, CoTRP and CoTRP/GO were used instead. These results indicate that GO exhibits catalytic activity for electroreduction of oxygen, but a more pronounced effect is noticed with CoTRP, and CoTRP/GO has the highest activity among them all. Hence, a synergic effect is clearly observed in the nanocomposite material, significantly enhancing the electrocatalytic activity of CoTRP towards the reduction of oxygen. The influence of the proportion of CoTRP and GO on the electrocatalytic activity was also investigated, as shown in Fig. 7B. Note that the O2 electrocatalytic reduction wave shifted from about -0.23 V (curve a’), in the composite with the largest relative amount of CoTRP (1:1), to 0.05 V (curve b’), as the amount of GO in the CoTRP/nGO composite increased to 1:4 (Fig. 7B).

The reduction potential for O2 is almost the same by comparing results obtained with GC electrodes modified with the CoTRP and GO composite (1:1) (-0.23 V, curve a’, Fig. 7B) and pure CoTRP (-0.28 V, curve c, Fig. 7A), indicating that the activation of the CoTRP molecule by GO may depend on the interaction with specific sites on the surface. The best performance for ORR was achieved for the 1:4 instead of the 1:6 composite, because π-π interactions between the graphene sheets tend to induce aggregation, sterically hindering the cobalt porphyrin site and decreasing the catalytic activity of the nanocomposite [25]. This effect and the decrease of the number of available cobalt porphyrin sites should account for the significant decrease of the electrocatalytic peak current density in the 1:6 as compared to the 1:4 composite material. Accordingly, the electrocatalytic reduction should involve the binding of O2 to one of cobalt(II) porphyrin axial positions leading to the activation of the two electron or four electron oxygen reduction mechanism.

In order to shed light on the mechanism of electroreduction of oxygen catalyzed by the CoTRP/4GO modifier, a careful study was carried out by RDE in O2 saturated 0.1 mol L-1 KNO3 solution. This technique allows controlling the mass transport rate and access the heterogeneous electron transfer rate constant. The RDE voltammograms obtained under hydrodynamic conditions (from 400 to 3600 rpm) shown in Fig. 8A indicate that the limiting electrocatalytic current density is a linear function of the square root of rotation rate according to the Levich plot (Fig. 8B), confirming the occurrence of a mass-transport controlled reaction. Thus, at potential values around

-0.10 and 0.0 V the current density is controlled by the rate of O2 diffusion from the bulk solution to the electrode surface. In the potential range from +0.10 V to -0.10 V, both diffusion and kinetics play simultaneously a role on the overall electrode process. In this situation, the measured current density is conveniently analyzed by plotting the reciprocal of current density as a function of the reciprocal of the square root of the rotation rate. The linearity of such plots at different potential values indicates that the electron transfer process follows the Koutecky-Levich equation j-1 = jk-1 + (0.62nFD2/3ω1/2 ν -1/6C0)-1 [44], in which j-1 is the measured current density, ω is the angular velocity of the electrode, D is the diffusion coefficient of oxygen in KNO3 solution (1.9×10–5 cm2 s–1), n is the overall number of electrons transferred in the ORR, F is the Faraday constant, C0 is the bulk concentration of O2 (1.24×10–3 mol L–1) and ν is the kinematic viscosity of the electrolyte (0.01 cm2 s–1 in 0.1 mol L-1 KNO3). The number of electrons transferred per O2 molecule (n) at different potentials was calculated from the slope of the straight lines shown in Fig. 8C. For comparison purpose, the curves corresponding to ORR involving 2 and 4 electrons were included in the figure. Clearly the reduction process is by four electron transfer, at potentials more negative than 0.06 V, suggesting that GO is interacting in someway with the prophyrin ring transferring electron density and activating the cobalt porphyrin. At potentials more positive than 0.06 V, the rate of electron transfer from the electrode to the CoTRP center seems to be limiting the reaction to the two electron transfer process. The activity of the developed CoTRP/4GO composite electrode was compared with other graphenebased electrocatalysts reported in the literature in terms of onset potential and number of electrons involved in the oxygen reduction reaction. Results presented in Table S1

demonstrated that the proposed platform has a more positive onset potential for ORR in a 4-electron process.

3.7. Analytical applications The analytical performance of CoTRP/4GO modified electrodes was examined by continuous measurements of dissolved oxygen concentration upon bubbling the gas in deaerated 0.1 mol L-1 KNO3 electrolyte solution. The actual oxygen concentration was simultaneously monitored with a standardized oximeter. The amperometric responses at the selected potential of -0.05 V presented in Fig. 9A confirm the linear current density increase as a function of dissolved oxygen concentration (Fig. 9B). The limit of detection (LOD) of 0.19 mg L-1 (5.8 x 10-6 mol L-1) was calculated using the equation LOD = 3SD/m, where SD is the standard deviation and m is the slope of the calibration plot. The LOD for the CoTRP/4GO modified electrode is reasonably comparable to some reported values (Table S2).

A fast cycling of oxygen concentration between 0 and 100% saturation was employed to evaluate the dynamic response of the sensor. A typical response time curve recorded when switching from oxygen saturated to argon saturated supporting electrolyte solution is presented in Fig. 10. The results confirmed that the CoTRP/4GO modified electrode responds rapidly to changes in the dissolved oxygen concentration and gives reproducible responses over a long period of time. Furthermore, the potential interference of physiological substances such as glucose, urea and ascorbic acid (each, 1 mmol L-1) [45] was also carefully evaluated and no significant change in the amperometric signal was noticed for glucose and urea. However, a slight variation in current density was witnessed after ascorbic acid addition (Fig. S4). Therefore, these

results demonstrated the suitability of continuous monitoring and excellent selectivity of the CoTRP/4GO modified electrode for dissolved oxygen sensing. Finally, the stability and reproducibility of the developed sensor platform were investigated. A stable steady state current for oxygen reduction over a period of time (1000 sec) at an applied potential of -0.05 V (Fig S5A) was noticed, indicating the sensor response stability. To assess the reproducibility, ten different CoTRP/4GO modified GC electrodes were prepared and the response towards ORR was evaluated by cyclic voltammetry. The relative standard deviation (RSD) among peak current measurements was found to be 2.8 %. (Fig S5B). Consequently, these obtained results evidently suggest that the proposed sensor is highly stable, reproducible and suitable for oxygen sensing in aqueous system.

4.

Conclusions CoTRP/4GO, synthetized by interacting [tetrakis-bisdimethylbipyridine chloro

ruthenium (II)]-5,10,15,20-tetrapyridylporfirinate cobalt(II) with graphene oxide (GO) dispersion was found to be an stable and very active electrocatalyst for the four electron reduction of dioxygen to water in neutral 0.1 mol L-1 KNO3 aqueous solution. The mechanism of ORR was confirmed by rotating disk electrode (RDE) voltammetry. More interestingly, that material also showed to form stable enough films on glassy carbon electrode surface delivering fast and reproducible responses, suitable for preparation of sensors with good sensitivity for continuous monitoring of dissolved oxygen in water at room temperature, at -0.05 V, with no or small influence of typical biological interfering compounds such as glucose, urea and ascorbic acid. This attractive platform can be exploited to fabricate microelectrochemical sensors for realtime localized monitoring of the respiratory activity in single cells by using SECM

(Scanning Electrochemical Microscopy) and work is in progress to develop such miniaturized device.

Acknowledgements The authors thank FAPESP, CNPq and CAPES for financial support and one of the authors (SAK) gratefully acknowledges the Sao Paulo Research Foundation (FAPESP) for the award of a Postdoctoral Fellowship grant [2014-15215-5].

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Fig. 1. UV-Vis spectrum of CoTRP, GO and CoTRP/4GO in aqueous solution (A) and wire frame structure of the CoTRP complex (B).

Fig 2. Cyclic voltammogram of 1.0 mmol L-1 CoTRP in 0.10 mol L-1 KNO3 aqueous solution, scan rate= 0.1 V s-1 (A). Spectroelectrochemical changes associated with the reduction of the cobalt center of CoIII/IITRP in 0.10 mol L-1 KNO3 aqueous solution (B).

Fig. 3. AFM images and cross-section analyses displaying the height profile of CoTRP/GO (top A and B) and CoTRP/4GO film (bottom C and D).

Fig. 4. TEM images of CoTRP/GO (A and B) and CoTRP/4GO (C and D). EDS spectrum at the edge of CoTRP/GO in (B), 1: vacuum and 2: composite (E).

Fig. 5. Raman spectra of CoTRP, GO and the Co-TRP/4GO nanocomposite (A). Confocal Raman images of the nanocomposite CoTRP/4GO generated from CoTRP spectra (B), from GO (C), and the convoluted image (D). Raman profile cross section profile along the vertical green line indicated in “D” considering the contribution of CoTRP (blue line) and GO (red line) (E).

A

B

Fig. 6. Cyclic voltammograms of a CoTRP/4GO modified electrode in 0.1 mol L-1 KNO3 electrolyte solution as a function of the scan rate (A) and corresponding ip vs υ plot (B).

Fig. 7. Cyclic voltammograms recorded in an O2-saturated 0.1 mol L-1 KNO3 solution with GC (a), GO/GC (b), CoTRP/GC (c) and CoTRP/4GO electrodes (d) (A) and with CoTRP/nGO modified electrodes prepared with composites containing different proportions between porphyrin and GO: CoTRP/GO (a’), CoTRP/4GO (b’), CoTRP/6GO (c’) (B). Scan rate: 0.1 V s-1.

B

A

C B

Fig. 8. Rotating disk electrode (RDE) voltammograms recorded with a CoTRP/4GO modified electrode in O2-saturated 0.1 mol L-1 KNO3 solution (A). Corresponding Levich (B) and Koutecky-Levich (C) plots at different potentials and theoretical curves for 2- and 4-electron processes (solid green and black lines, respectively).

Fig. 9. Amperometric responses recorded at -0.05 V in a 0.1 mol L-1 KNO3 solution with a CoTRP/4GO modified electrode at various concentrations of dissolved oxygen (0.00 to 21.24 mg L-1) (A). Corresponding calibration plot (B).

Fig. 10. Amperometric response measured with the CoTRP/4GO modified electrode in 0.1 mol L-1 KNO3 solution upon bubbling of argon and oxygen at E= -0.05 V.