Electrocatalytic oxidation of Epinephrine and Norepinephrine at metal oxide doped phthalocyanine/MWCNT composite sensor

Glassy carbon electrode (GCE) was modified with metal oxides (MO = Fe3O4, ZnO) nanoparticles doped phthalocyanine (Pc) and functionalized MWCNTs, and the electrocatalytic properties were studied. Successful synthesis of the metal oxide nanoparticles and the MO/Pc/MWCNT composite were confirmed using FTIR, Raman and SEM techniques. The electrodes were characterized using cyclic voltammetry (CV) technique. The electrocatalytic behaviour of the electrode towards epinephrine (EP) and norepinephrine (NE) oxidation was investigated using CV and DPV. Result showed that GCE-MWCNT/Fe3O4/2,3-Nc, GCE-MWCNT/Fe3O429H,31H-Pc, GCE-MWCNT/ZnO/2,3-Nc and GCE-MWCNT/ZnO/29H,31H-Pc electrodes gave enhanced EP and NE current response. Stability study indicated that the four GCE-MWCNT/MO/Pc modified electrodes were stable against electrode fouling effect with the percentage NE current drop of 5.56–5.88% after 20 scans. GCE-MWCNT/Fe3O4/29H,31H-Pc gave the lowest limit of detection (4.6 μM) towards EP while MWCNT/ZnO/29H,31H-Pc gave the lowest limit of detection (1.7 μM) towards NE. The limit of detection and sensitivity of the electrodes compared well with literature. Electrocatalytic oxidation of EP and NE on GCE-MWCNT/MO/Pc electrodes was diffusion controlled with some adsorption of electro-oxidation reaction intermediates products. The electrodes were found to be electrochemically stable, reusable and can be used for the analysis of EP and NE in real life samples.

Determination of the level of EP is important for diagnosis of Parkinson's disease, among other mental disorders 17 . It has been used as a common healthcare medicine, for instance EP drugs are used to treat anaphylactic shock, bronchial asthma and organic heart disease 18 . Therefore, a quantitative determination of EP at physiological pH in our body fluids has become increasingly significant.
Carbon nanotubes (CNTs) are an important class of materials due to their unique electronic, mechanical, and structural characteristics. The physical and catalytic properties make CNTs ideal for use as chemical sensors and for electrochemical detection 18 . However their electrochemical properties are sensitive when chemically doped with various molecules 19 . It has also been reported that the application of pristine CNTs is limited due to their poor solubility in both organic and inorganic solvents 20 and also the CNTs walls are not reactive 19 ; therefore it is of best interest to attach functional groups such as -COOH, − OH or − C = O to their surface to optimize their use and increase their solubility 21 . The mechanically strong, small size and chemically stable nature of CNTs is very appealing for sensing applications 22 . Furthermore, CNT based platforms were cited as biocompatible sensors because of the similarity in size with analytes such as cells, proteins and even DNA; they have shown a significant development because of their promising applications in nanoelectronics as well as in highly sensitive biosensors 18 . The first electrochemical application of CNTs was carried out by Britto, where multi-walled carbon nanotubes (MWCNTs) were used to study the electrochemical oxidation of DA 23 . The results obtained from the study showed a two electron transfer redox reversible process and the oxidation of DA showed a low potential with a faster rate than that observed for graphite electrodes. These remarkable results suggest that MWCNTs possess properties such as high electrical conductivity, larger surface-active groups to-volume ratio, chemical stability and significant mechanical strength, as a consequence, MWCNTs can serve as excellent substrates for the development of biosensor devices 24 . From 1996 the application CNTs in electrochemistry increased severely towards detection of biological analytes and gases using sensors or biosensors.
There is currently an interest in the use of metal oxide nanoparticles (NPs), metal-doped metal oxides, metal oxide-CNTs nanocomposites, and metal oxide-polymer composites 25 in electrochemistry to improve the performance of electrochemical detection of biological and environmental analytes 1 . Analytical devices based on nanostructured metal oxides are cost-effective, highly sensitive due to the large surface-to-volume ratio of the nanostructure and additionally show excellent selectivity 25 . Metal and metal oxides NPs have been used to modify electrodes for use as electrocatalysts and biosensors; hence they play an important role in diagnostic devices 26 . Shan et al. 27 investigated Fe 3 O 4 /chitosan modified electrode as a feasible sensor for detection of H 2 O 2 . He found that there was limited interference, prompt response, good reproducibility and the electrode showed long term stability, therefore he suggested that these might be beneficial for future environmental and biological applications towards detection of H 2 O 2 . In addition Adekunle et al. 28 conducted a study on the voltammetric detection of DA using easily prepared nano-scaled iron oxide (Fe 2 O 3 ) catalyst supported on MWCNTs modified pyrolytic graphite electrode.
Phthalocyanines (Pcs) and their derivatives are well-known blue-green organic semiconductor materials that belong to the family of aromatic heterocyclic conjugated molecules, with alternating single-double bond structures 29 . They are made of delocalised π -electrons system 30 , where π -electron delocalisation and interactions with central metal atom on metal phthalocyanines MPcs determine the redox properties 31 . Their thermal and chemical stability are important properties that make them molecules to be incorporated into electrochemical sensors. Furthermore the possibility of incorporating about 70 different metal atoms into their ring 32 , and also being able to have variation of the substituents in the side chain, can allow production of peculiar and optimized thin films having different degrees of sensitivity, selectivity and stability 33 . Wang 34 reported that among the metal substituted Pc, lead-Pc is the most interesting one in terms of reproducibility, fast response recovery and excellent gas sensitivity. Pcs are capable of binding nonspecifically with various analytes through van der Waal forces, hydrogen bonding, and coordination interactions with the central metal 35 .
This study explored the unique electrocatalytic properties of GCE-MWCNT/MO/Pc modified electrodes towards the oxidation of epinephrine (EP) and Norepinephrine (NP). In this work, we have shown that the fabrication of GCE-MWCNT/Fe 3 O 4 /2,3-Nc, GCE-MWCNT/Fe 3 O 4 29H,31H-Pc, GCE-MWCNT/ZnO/2,3-Nc and GCE-MWCNT/ZnO/29H,31H-Pc modified electrodes is simple and successful. We also demonstrated that the nanocomposite modified electrodes enhanced EP and NP oxidation current compared with other electrode studied. This study showed that GCE-MWCNT/ZnO/29H,31H-Pc was the best electrodes towards EP and GCE-MWCNT/ZnO/29H,31H-Pc was the best towards NP in terms of limit of detection. The two electrodes have also provided well-defined voltammograms for simultaneous detection of EP or NP without interference from amino acid (AA).

Experimental
Materials and reagents. Glassy carbon electrode (3 mm diameter) was purchased from CH Instrument USA. Polishing pads were obtained from Buehler, IL, USA and Alumina micro powder (1.0, 0.3 and 0.05 μ m alumina slurries) was used for polishing the glassy carbon electrode (GCE). Pristine multi-walled carbon nanotubes (95% purity, 10-20 nm); Iron(III) chloride (FeCl 3 ), zinc nitrate hexahydrate (Zn(NO 3 ) 2 .6H 2 O), 29H,31H-Phthalocyanine (Pc), 2,3-Naphthalocyanine (Nc), (± )-Epinephrine hydrochloride, L-Norepinephrine hydrochloride, dopamine hydrochloride, ascorbic acid and other reagents are of analytical grade and obtained from Sigma-Aldrich, Merck chemicals and LABCHEM respectively. Ultra-pure water of resistivity 18.2 MΩ was obtained from a Milli-Q Water System (Millipore Corp., Bedford, MA, USA) and was used throughout for the preparation of solutions. A phosphate buffer solution (PBS) of 7.0 was prepared with appropriate amounts of NaH 2 PO 4 .2H 2 O, Na 2 HPO 4 .2H 2 O, and H 3 PO 4 , and adjusted with 0.1 M H 3 PO 4 or NaOH. Prepared solutions were purged with pure nitrogen to eliminate oxygen and prevent any form of external oxidation during every electrochemical experiment.     solution to the alkaline aqueous solution was kept stirred for two hours at 70 °C. The material formed was filtered and washed several times with deionised water. The washed sample was dried at 65 °C in the oven for 24 hours to obtain the dry powder 36 .

Synthesis of iron oxide (Fe 3 O 4 ) nanoparticles.
A mixture of FeCl 3 stock solutions (30 mL) 2.0 M, Na 2 SO 3 stock solution (20 mL) 1.0 M and concentrated ammonia (50.8 mL), diluted to a total volume of 800 mL was used. Fe 3+ and SO 3 2− were mixed; the colour of the solution changed from light yellow to red, indicating formation of a complex ion. The solution was quickly poured into the diluted ammonia solution under vigorous stirring, and the colour changed from red to yellow again. A black precipitate was formed and stirring continued for 30 minutes. After the reaction, the beaker containing the suspension was placed on a permanent magnet. The supernatant liquid was decanted and fresh deionised water was added into the beaker. This procedure was repeated several times until most of the ions in the suspensions were removed. The dry powder was obtained by filtration and drying at room temperature 37 .
Preparation of MWCNT/metal oxide/phthalocyanine hybrids. Fe 3 O 4 nanoparticles (2.5 mg) was dissolved in 1 mL DMF along with 2.0 mg of either of phthalocyanine, 2,3-Nc (naphthalocyanine) or 29H,31H-Pc; the solution was allowed to mix with the aid of ultrasonication for 1 hr. 2.0 mg of MWCNTs was dispersed in the DMF solution containing 2.5 mg of Fe 3 O 4 nanoparticles and 2.0 mg of phthalocyanine. After ultrasonication, the Fe 3 O 4 /phthalocyanine hybrids were allowed to adsorb onto MWCNTs by spontaneous adsorption and sonication for 5h to give MWCNT/Fe 3 O 4 /phthalocyanine hybrids 24 . MWCNT/ZnO/phthalocyanine hybrids were prepared using the same procedure. Equipment and procedure. Scanning electron microscopy (SEM) experiment was performed using Zeiss Ultra Plus 55 HRSEM (Germany). FTIR experiments were carried out using Fourier transformed infrared spectrophotometer (Agilent Technology, Cary 600 series FTIR spectrometer, USA), while the Raman spectra were obtained using Xplora Horiba Raman Spectrometer (Olympus BX41 microscope, UK).
Electrochemical experiments were carried out using an Autolab Potentiostat PGSTAT 302 (Eco Chemie, Utrecht, and The Netherlands) driven by the GPES software version 4.9. A Ag|AgCl in saturated KCl and platinum wire were used as reference and counter electrodes respectively. A bench top Crison pH meter, Basic 20+ model, was used for pH measurements. All experiments were performed at 25 ± 1 °C while the solutions were de-aerated before every electrochemical experiment.
Electrode modification procedure. Electrode modification was carried out using the drop-dry method.
The glassy carbon electrode was cleaned by gentle polishing in aqueous slurry of alumina nanopowder on a silicon carbide-emery paper followed by a mirror finish on a Buehler felt pad. The electrode was further sonicated in double distilled water, and then absolute ethanol for 2 min to remove residual alumina particles that were trapped on the surface and dried at room temperature. Separate suspensions of the individual metal oxide nanoparticles, phthalocyanines and MWCNT were prepared by dissolving 5 mg of each in 1 mL of DMF and sonicated for 1 hr. Similarly, a composite of the catalyst was prepared by mixing 5 mg each of MWCNT with 5 mg either of the metal oxide (MO) nanoparticles (ZnO or Fe 3 O 4 ) in 1 mL DMF, or by mixing 5 mg each of the three materials (phthalocyanines, MWCNT and MO) in 1 mL DMF 24,38

Results and Discussion
Microscopic and spectroscopic results. FTIR and Raman spectroscopy results. Figure 1 show the FTIR spectra of synthesized nanoparticles and functionalized MWCNT. The FTIR spectra of Fe 3 O 4 nanoparticles (Fig. 1a) showed absorption peak at 549 cm −1 which is attributed to the Fe-O stretching vibrations. The peak observed at 1649 cm −1 is related to the vibration of adsorbed water molecules, the H-OH group 40 and the absorption peak at 3401 cm −1 is associated with of OH stretching vibration which also suggests the presence of some ferric hydroxide in Fe 3 O 4 41 . Figure 1b shows the FTIR spectra of ZnO NPs, with Zn-O absorption band at 556 cm −1 , and the peak at 3437 cm −1 indicating the presence of -OH stretching due to atmospheric moisture 42 . Other observed absorption peaks might be attributed to intramolecular hydrogen bonds 43 . The FTIR spectrum of functionalized MWCNT which identifies the functional groups attached to the surface and the sidewalls of MWCNTs is shown in Fig. 1c. The absorption peak observed at 1625 cm −1 correlates with stretching vibration of the C = C or C-O group attached to the surface of MWCNTs, and the absorption band at 1705 cm −1 is due to the C = O of the COOH groups. The peak at 2320 cm −1 arises from the graphitic component of MWCNTs 44 . The absorption band around 3423 cm −1 is associated with the O-H stretch of the terminal carboxylic group 45 .
Raman spectroscopy is a good technique used to study the low energy elementary excitation in materials. It is also used to characterize structural, electronic, vibrational and magnetic properties of carbon nanotubes 45 . The Raman spectra in Fig. 2 showed the D-band of functionalised and pristine MWCNTs at 1364 cm −1 and 1346 cm −1 respectively. This D-band is known to be associated with disorder-induced of CNTs 46 which is due to the finite or nanosized graphitic planes and other forms of carbon 47 . The observed large intensity of the D-band peak as compared to the G-band intensity in CNTs indicates the presence of amorphous carbon 48 . Furthermore the D band in MWCNTs is attributed to defects in the tubes walls, vacancies, heptagon-pentagon pairs, kinks and heteroatoms 47,49 . The two G-bands observed appeared at 1598 cm −1 and 1587 cm −1 for functionalised and pristine MWCNTs respectively which represent E 2g tangential stretching mode of ordered crystalline graphitic and in-plane vibrations of sp 2 hybridized carbon 45,46 . The D'-band on the shoulder of the G-band, which is not clearly visible on these spectra, occurred at 1631 cm −1 and 1628 cm −1 for functionalised and pristine MWCNTs respectively. This band is attributed to double resonance feature induced by disorder and defects 47 . The intensity ratio of the D-to-G band (I D /I G ) was 0.85 and 0.8 for functionalised and pristine MWCNTs. The intensity ratio was calculated to evaluate the degree of disorder of walls of MWCNTs and the results obtained for the density ratio of functionalized is higher than that of pristine MWCNTs suggesting that functionalized MWCNTs walls had a higher degree of disorder. Theodore 45 mentioned that an increase in I D /I G indicates a higher degree of disorder, hence the G-band on functionalised MWCNTs is less affected by defects as compared to the D-band, therefore functionalised MWCNTs has functional groups attached to the surface and the walls, it is thus expected of functionalised MWCNTs to have a higher degree of disorder. , showed a tangled-net like structure of MWCNTs embedding the metal oxides in the pores and around its wall. The ZnO nanorods were almost homogeneously distributed in the Pc and the MWCNT forming a more conducting film. This may probably be due to robust ionic and intermolecular interaction between the MO nanoparticles, π -π electrons of the MWCNT and the π -π electrons or lone pair of electron on the nitrogen atoms of the Pc molecule 50 . On the other hand, the SEM pictures of MWCNT/ZnO/29H,31H-Pc (c) and MWCNT/Fe 3 O 4 /29H,31H-Pc (d) composite indicated that the MO nanoparticles formed an aggregate around the MWCNT probably due to less intermolecular π -π interactions between MO NPs, Pc/Nc and MWCNT. It should be noted that 29H,31H-Pc has less π -π electron system compare with Nc. More evidence of their clear distribution on the GCE is shown in supplementary TEM image Figure (S1).

Electrocatalytic oxidation of epinephrine (EP) and norepinephrine (NE). The electrocatalytic
behavior of EP and NE at the bare and modified electrodes was investigated in pH 7.2 PBS containing 0.14 mM of EP and 0.12 mM NE using CV as shown in Figs 5 and 6. Although broad EP and NP peaks were observed at these electrodes, due to the obstruction of flow of electrons, they gave low current response as compared to the bare electrode. No oxidation peak was observed for EP and NE at GCE-Fe 3 O 4, probably due to the passive oxide layers which are obstructive to the flow of electrons. It is well known that MWCNT have high electrical conductivity and large surface area, hence electrodes modified with MWCNT conveyed an enhanced current response, which might be due to the possibility of π -π interaction between the aromatic ring of EP and NE and the MWCNT 51 .
However electrodes modified with MWCNT/Fe 3 O 4 /2,3-Nc, MWCNT/Fe 3 O 4 /29H,31H-Pc, MWCNT/ ZnO/2,3-Nc and MWCNT/ZnO/29H,31H-Pc showed higher EP oxidation current. The EP peak current at MWCNT/Fe 3 O 4 /29H,31H-Pc electrode was 36 times higher than of the bare electrode and that at MWCNT/ ZnO/29H,31H-Pc was 29 times higher. Similarly, MWCNT/ZnO/2,3-Nc and MWCNT/Fe 3 O 4 /2,3-Nc gave NE current response that are 24 and 23 times higher than that at the bare electrode. The large oxidation currents and low oxidation potentials exhibited by these electrodes suggest that the nanocomposites have enhanced electrocatalytic behaviour towards oxidation of EP and NE respectively compared to the bare GCE. The increase in oxidation peak current values can be correlated with the enhanced adsorption capability of EP in the phthalocyanine layer 52 . Furthermore it has been reported that a sharper and well-defined peak and a considerable current increase is due to fast electron transfer 53 . This result agreed with other reports where the enhancement of the chemical sensors was as a result of the carbon nanotubes acting as an electrical conducting nanowire between the modifier and the base electrode [54][55][56][57] . All fabricated electrodes showed an electrochemically irreversible process  (Figs 7 and 8). It was found that the peak current of the first cycle was more than the peak current of the second cycle and after the third cycle there was a minimal decrease in current response. This behavior was observed with all the four modified electrodes. The percentage EP current drop obtained was 5.14%, 5.55%, 5.55% and 5.55% for MWCNT/Fe 3 O 4 /2,3-Nc, MWCNT/Fe 3 O 4 /29H,31H-Pc, MWCNT/ZnO/2,3-Nc and MWCNT/ ZnO/29H,31H-Pc modified electrodes respectively. Similarly, the percentage NE current drop are 5.56%, 5.88%, 5.56% and 5.56% for MWCNT/Fe 3 O 4 /2,3-Nc, MWCNT/Fe 3 O 4 /29H,31H-Pc, MWCNT/ZnO/2,3-Nc and MWCNT/ZnO/29H,31H-Pc modified electrodes respectively. Thus the anodic peak currents remained almost stable, indicating excellent electrodes stability during repeated CV scans. These results suggest that there is some level of adsorption of EP and NP at electrodes surface 31 . The adsorptive nature of electrodes might be due to the porous structured layer of the CNTs on the electrodes surface, The RSD obtained for MWCNT/Fe 3 O 4 /2,3-Nc, MWCNT/Fe 3 O 4 /29H,31H-Pc, MWCNT/ZnO/2,3-Nc and MWCNT/ZnO/29H,31H-Pc modified electrodes was 15%, 16%, 2.04% and 5.19% respectively, indicating that these electrodes are relatively stable and are not subjected to serious surface fouling. The oxidation peaks observed in Fig. 7(a,b) might be attributed to oxidation of Fe(II) of Fe 3 O 4 to Fe(III) and the oxidation peak in Fig. 7(c) might be attributed to the oxidation of ZnO to Zn(OH) 2 . GCE-MWCNT/ZnO/2,3-Nc showed the lowest RSD value as compared to other electrodes, indicating that it was the most stable electrode in determination of EP. Similarly MWCNT/ZnO/29H,31H-Pc modified electrodes is most stable towards NE oxidation having the lowest RSD values of 2.05%.  (Figs 9a,c and 10a,c) and NE (0.12 mM) (not shown) using cyclic voltammetry. Figure 9(b,d) are peak current vs. square root of scan rate plots of MWCNT/Fe 3 O 4 /2,3-Nc and MWCNT/Fe 3 O 4 /29H,31H-Pc electrodes respectively. Similarly, Fig. 10(b,d) are peak current vs. square root of scan rate plots of MWCNT/ZnO/2,3-Nc and MWCNT/ZnO/29H,31H-Pc electrodes respectively. The plots of oxidation peak current vs. scan rate (ν ) showed a linear relationship, indicating that the oxidation peak current increased linearly with the increasing scan rate. GCE-MWCNT/Fe 3 O 4 /2,3-Nc (a) showed the EP peak current up to 300 mVs −1 although the scan rate was studied at the range of 25-1000 mVs −1 . Similar results were obtained in NE (not shown). The electrode could not further detect EP and NE over increasing scan rate, indicating saturation of the EP and NE at the electrode surface caused by adsorption of the porous catalyst film 58 . No cathodic peak currents were observed, meaning reaction processes were irreversible. The electrodes gave a negative intercepts from the current versus square root of scan rate plot, indicating an adsorption process 58 . From the plot of peak current I p versus the square root of scan rate, the surface coverage of EP was calculated using Laviron equation (Eq. 1).
where n is the number of electron transfer, R, T and F are the molar gas constant, absolute temperature, and Faraday constant, respectively, ν is the scan rate and Γ is the surface coverage. Assuming that n ≈ 2 and other parameters have their usual meaning 59 where Ep is the peak potential (V), ν is the scan rate and b represents the Tafel value.
Concentration study. The effect of concentration of EP was investigated using differential pulse voltam-  63 mentioned that the decrease in sensitivity might be due to kinetic limitations. In addition it has been mentioned that high sensitivity won't necessarily equate to a low limit of detection, but a decrease in matrix interference (increasing selectivity) and high background noise. MWCNT/ZnO/29H,31H-Pc modified electrode also gave the lowest limit of detection towards NE. The limit of detection obtained in this work compared with others reported in literature 57,64 .    Fig. 16, the four electrodes showed two well distinguished oxidations peaks of AA and EP and at AA concentration which is 30-fold excess of EP, no significant interference of EP signal due to AA was observed. Similar results were obtained for NE (Fig. 17). The difference between peak potentials of EP and NE from AA is summarized in Tables 1 and 2. The anodic peak signals of AA and EP were independent from each other; therefore the modified electrodes were able to adequately identify the two analytes. The simultaneous determination of AA and EP using the four modified electrodes was achieved, indicating that the developed sensors can be suitably used to determine EP in biological fluids. However MWCNT/Fe 3 O 4 /2,3-Nc, MWCNT/ZnO/2,3-Nc and MWCNT/ZnO/29H,31H-Pc modified electrodes showed big potential peak separation as compared to MWCNT/ Fe 3 O 4 /29H,31H-Pc modified electrode. The simultaneous determination of AA and NE was achieved with only three electrodes, suggesting that those electrodes can be suitably used to determine NE in biological fluids. The enhanced current response of these electrodes was attributed to the synergy between the MWCNTs, MO NPs and the phthalocyanines. All the four electrodes showed a good stability with low current drop (5-10%). The catalysis of EP was more favoured on GCE-MWCNT/Fe 3 O 4 /29H,31H-Pc electrode while that of NE was more favored on MWCNT/ZnO/29H,31H-Pc electrode in terms of current response and onset potential for catalysis compared to other electrodes investigated. These two electrodes also gave the lowest limit of detection towards EP and NE respectively. Aside the fact that the modified electrodes gave comparable limit of detection in the micro molar range with literature, they have also demonstrated excellent resistant to electrode fouling effect as demonstrated by very low (5%) EP and NE current drop after long scans and effective separation of the analyte signals from that of AA at potential as large as 200 mV. The electrodes could conveniently detect EP and NE in the presence of AA with a wide potential separation of about 200 mV. The electrodes were found to be electrochemically stable, re-usable and can thus be used for the analysis of EP and NE in real biological samples.  Table 2. Cyclic voltammetric of binary mixture of NE and AA obtained for modified electrodes with constant concentration of NE (0.12 mM) and increasing concentration of AA.