Nitrogen-Doped Graphene Supported α-Co(OH)2 for Sensitive Determination of Adrenaline

For a happy and healthy life, there should have a balance of flight-to-flight hormones, i.e., adrenaline (AD). The necessity of determination of AD is inevitable for the diagnosis of associated diseases with it. For this purpose, N-doped graphene supported α-Co(OH)2 (denoted as NrGO/α-Co(OH)2) was synthesized via a hydrothermal process, where α-Co(OH)2 acted as an active site and NrGO provided a better defective surface for immobilized α-Co(OH)2 nanoparticles. The as-prepared nanocomposite altered the electronic configuration due to its defective nature, which played an important role to increase the stability, surface area and electron transfer capability. As a result, NrGO/α-Co(OH)2 demonstrated remarkable analytical performances toward AD with a lower limit of detection (14.7 nM), wide linear range (0.5–800 μM), and good sensitivity (115.983 μA mM−1 cm−2). The CA response time was obtained as 2.2 s. The proposed sensor showed precious selectivity during AD detection in presence of coexisting biomolecules such as DA, AA, UA, TY, 5-HT, and NE, and 50-fold excess of common ions such as Na+, K+, Ca2+, Mn2+, Fe2+, CO3 2−, and SO4 2−. Furthermore, it also provided long-term stability, good reproducibility and repeatability with practical feasibility in the urine sample. Importantly, the effect of pH was studied in detail on AD oxidation.

Adrenaline (4-[(1 R)−1-hydroxy-2-(methylamino)ethyl]benzene-1,2-diol; AD) is a catecholamine neurotransmitter, 1,2 and the adrenal glands and certain neurons usually secrete AD in physiological stress or hypoglycemia. 1,3 AD plays an important role in the regulation of the sympathetic nervous system, and its imbalance concentration is responsible for heart disease, thyroid hormone diseases, schizophrenic disease, Parkinson's disease, Huntington's disease, and Alzheimer's disease. 3 There is rising demand for AD injections to treat sudden cardiac arrest and asthma, available cost-effective generic AD products globally for myocardial infection, glaucoma, severe anaphylaxis, emphysema and hypertensive disease. [3][4][5] According to B 10 Space, the global AD market size is expected to reach USD 4.78 billion in 2028. 4 Owing to the significant importance of AD in physiochemical activities, medicine and life science, accurate concentration determination is highly desirable for the diagnosis of associated diseases and drug quality control. Among different analytical detection techniques, the electrochemical system exhibited the advantages of high signaling amplification, easy fabrication, simple operation, low cost, and rapid detection. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] It has been reported that due to sluggish electrode kinetics and high overpotential, the conventional electrodes provided negligible response toward electrochemical oxidation of AD. 3,6 Furthermore, in physiological samples AD usually coexist with other biomolecules such as dopamine (DA), ascorbic acid (AA), and Uric acid (UA). [7][8][9] The presence of other interfering substances could lead to poor sensitivity and reproducibility due to cross-interference and electrode fouling in the electrochemical determination of AD. 14 A large number of research has been carried out inorder to improve the efficiency of modified electrodes for sensitive and selective determination of AD. 9,11,13 Among them carbonaceous, metal, and composite nanomaterials have taken lots of attention such as MgO/SWCNTs, NiO/graphite paste, metal oxide doped phthalocyanine/MWCNT, oxidized graphene nanoribbons, CeO 2 /GCE, MIP modified electrode, graphene oxide/ZnO nano Composite, Au-Pd/ Graphene, graphene and MOF. 9 Still, there is a necessity for depth research, and chemically modified electrodes could be the solving route to overcoming the above problems. 3,15 One of the graphene derivatives, graphene oxide (GO), has come to the forefront in electrochemical applications during recent years due to its unique two-dimensional structure with the availability of a large number of electrochemically favourable carbon edges, and remarkable mechanical, thermal and electronic properties which hindrance the fouling effect. 9,16,17 Due to π-π interaction, the firm self-stacking tendency of graphene faces the aggregation problem during the synthesis process of reduced graphene oxide (rGO), which decreases its surface area, electrical conductivity and solubility compared to GO. 18,19 Surface modification of the rGO substrate could reduce the aggregation problem. Doping heteroatom (such as N, B, P, S etc.) on rGO effectively enhances the electron density, increases the band gap, and accelerates the electron transfer rate. 20,21 Nitrogen-doped reduced graphene oxide (NrGO) has been extensively investigated in electrocatalysis owing to its large surface area, high electrical conductivity, and N-related active sites. 22,23 It has been reported that PDA-functionalized rGO enhanced its dispersion and compatibility. 24,25 Additionally, the presence of two amino groups (−NH 2 ) in benzene rings of Ortho phenylenediamine(o-PDA) donate electrons which break the π-π bond by reacting with functional groups of rGO layers, 26,27 enhancing the surface-tovolume ratio, thermal stability, and accelerated the electron transfer rate as well as increase the electrochemical performances. [28][29][30] Among various metal oxide/hydroxide nanoparticles (NPs), cobalt oxide/hydroxide has arisen as an efficient catalyst for electrochemical application due to its exceptional physical and chemical properties. 31 Particularly, Co(OH) 2 has been extensively explored due to its availability, cheap cost, environmentally friendly, enormous surface-to-volume ratio, small particle size, layered structure with large interlayer spacing, and high catalytic efficiency. 32 Co(OH) 2 has two different crystallographic polymorphs: α and β. 33 α-Co(OH) 2 has larger interlayer spacing (>7.0 A°) than that of β-form (4.6 A°), 34 and it provides superior electrochemical performances compared to β-Co(OH) 2 . 35 Advanced materials technologies contain remarkable physiochemical characteristics as well as new technologies based on novel materials. 36 Furthermore, they decrease the cost and fabrication time of the prepared sensors and develop the gateway between academic research and the commercialization of these sensors. 37 These types of sensors are capable to perform qualitative and quantitative analyses in very complex matrices. 38 In this study, α-Co(OH) 2 z E-mail: swjeon3380@naver.com decorated o-PDA functionalized rGO (denoted as NrGO/α-Co(OH) 2 ) was synthesized via a simple hydrothermal process for the sensitive determination of AD. α phase of Co(OH) 2 , and N-doping on the rGO surface was confirmed by Xray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), respectively.
The study demonstrated that NrGO/α-Co(OH) 2 exhibited significant improvement in the detection of AD owing to its defective nature. Furthermore, a lower limit of detection (LOD), wide linear range, selectivity, long-term stability, reproducibility, repeatability and practical applicability confirmed the proposed sensor as a potential candidate for the diagnosis of AD.

Synthesis
of NrGO/α-Co(OH) 2 .-In this study, NrGO/α-Co(OH) 2 was synthesized via a simple hydrothermal process. Firstly, 1 mg ml −1 GO suspension was prepared in a round-bottomed flask by 1 h ultrasonication. Then 30 mg o-PDA solution (1:1, w/v) was added slowly into GO suspension under magnetic stirring to form a homogeneous solution, after 20 min of continuous stirring, 1% NaBH 4 (450 μl) was added slowly (6 drops min −1 ) into the above dispersion. After the solution was cooled at room temperature, 15 ml of 10 mM Co(NO₃) 2 ·6H₂O was added into NrGO solution. The mixture was then refluxed for 6 h at 110°C, and the obtained black solution was kept under magnetic stirring for 12 h. After that, the black solution was centrifuged several times with distilled water and ethanol, and the final product, NrGO/α-Co(OH) 2 , was obtained by drying in a vacuum oven at 60°C for 24 h (Scheme 1). NrGO and rGO/α-Co(OH) 2 were synthesized following the same procedure without adding Co(NO₃) 2 ·6H₂O and o-PDA, respectively.
to NrGO due to more surface defects accordingly to graphene lattice integrity. 43 And the lower I D /I G of NrGO/α-Co(OH) 2 than that of NrGO, due to the reduction of sp 3 to sp 2 carbon and smaller sp 2 domain size of carbon atoms. 16 Figure 3a shows the survey XPS spectra of NrGO, rGO/α-Co(OH) 2 , and NrGO/α-Co(OH) 2 with their identifiable elemental and augur peaks. The elemental peaks at ∼285.08 eV, and ∼531.08 eV, and the augur peak at ∼979.08 eV, confirmed the presence of C, O, and O KLL, respectively in all samples. 44 NrGO and NrGO/α-Co(OH) 2 exhibited a peak at ∼400.08 eV due to the presence of N. rGO/α-Co(OH) 2 and NrGO/α-Co(OH) 2 demonstrated conjugated peaks in the range of 780.08-796.08 eV, which indicated the presence of Co NPs. Figure 3b shows the high-resolution C1s spectrum of NrGO and NrGO/α-Co(OH) 2 . NrGO demonstrated four peaks at ∼285.28 eV, 286.58 eV, 288.98 eV, and 292.28 eV corresponding to C-C/C=C, C-O/ C-N, O-C=O, and π-π * , respectively. Whereas, the π-π * peak disappeared and the intensity of C-O/C-N decreased in NrGO/α-Co(OH) 2 , due to the π-π interaction between α-Co(OH) 2 and NrGO. 31 Figure 3c shows the high-resolution O1s spectrum of NrGO and NrGO/α-Co(OH) 2 . NrGO demonstrated three peaks at ∼531.08 eV, ∼531.88 eV, and ∼532.78 eV corresponding to C-OH, C-O, and H 2 O, respectively. Whereas an additional peak at ∼530.08 eV was observed due to metal oxides (Co-O) and peak position of C-OH and C-O shifted to lower binding energy compared to NrGO in NrGO/α-Co(OH) 2 . The existence of C-OH and Co-O, suggested the presence of Co(OH) 2 in the prepared nanocomposite. 22,31 Figure 3d shows the high-resolution N1s spectrum of NrGO and NrGO/α-Co(OH) 2 . NrGO exhibited two peaks at ∼400.08 and 401.88 eV corresponding to =NH− and N-H, respectively. Both of the peaks slightly shifted to lower binding energy in NrGO/α-Co(OH) 2 compared to NrGO, which indicated the different electronic configurations of N in NrGO/α-Co(OH) 2 due to the electron density difference. 16 Figure 3e shows the core level Co2pspectrum of rGO/α-Co(OH) 2 and NrGO/α-Co(OH) 2 . Both of them consist of two major peaks (Co2p 3/2 and Co2p 1/2 ) and two satellite peaks. The spin energy difference between Co2p 3/2 and Co2p 1/2 was obtained as 16.10 eV for both of them, which confirmed the existence of Co(OH) 2 in rGO/α-Co(OH) 2 and NrGO/α-Co(OH) 2 . 31,45 Furthermore, Co2p spectrum of NrGO/α-Co(OH) 2 slightly shifted to lower binding energy compared to rGO/α-Co(OH) 2 , which indicated the different electronic configuration due to N-doping. 18 Morphology analysis.-Transmission electron microscopy (TEM) images were analyzed to study the surface morphology of GO, rGO, NrGO, rGO/α-Co(OH) 2 , and NrGO/α-Co(OH) 2 . TEM image of GO shows a few layer structures with wrinkles and defective surfaces (Fig. S1a (available online at stacks.iop.org/ ECSA/1/046501/mmedia)). However, NrGO reveals a cloud-like structure with many wrinkles (Fig. S1c), whereas RGO displays comparably translucent and smooth images (Fig. S1b). In Fig. 4a, the TEM image of rGO/α-Co(OH) 2 shows a blackish appearance of coagulated Co(OH) 2 NPs on the rGO surface, while Fig. 4b exhibited a homogeneous distribution of numerous Co(OH) 2 NPs with no serious aggregation on the NrGO surface. The magnified TEM image of NrGO/α-Co(OH) 2 demonstrated that the Co(OH) 2 NPs were spherical shaped (Fig. 4c), and high-resolution TEM (HRTEM) image of NrGO/α-Co(OH) 2 revealed the lattice d-spacing of 1.55 A°corresponding to (110) plane of α-Co(OH) 2 (Fig. 4d). 46    Figure 5a shows all electrodes provided well-defined redox peaks due to their redox reactions, which indicated the charge transfer ability of all electrodes in both scans. Obviously, bare GCE showed the lowest redox peaks due to its sluggish charge transfer kinetics. Between rGO and NrGO, NrGO exhibited higher redox peaks which suggested that N-doping increased the charge transfer ability. Similarly, NrGO/α-Co(OH) 2 demonstrated higher redox peaks than that of rGO/α-Co(OH) 2 , which confirmed that α-Co(OH) 2 decorated N-doped rGO surface is suitable for facile charge transfer as well as a better catalyst for enhanced electrochemical performances. The electrical conductivity of bare GCE, α-Co(OH) 2 , rGO/α-Co(OH) 2 , and NrGO/α-Co(OH) 2 was monitored by electrochemical impedance spectroscopy (EIS) in 0.1 M KCl containing 5.0 mM [Fe(CN) 6 ] 4−/3− . Figure 5b shows the Nyquist plots of α-Co(OH) 2 , rGO/α-Co(OH) 2 , and NrGO/α-Co(OH) 2 , all of them consist of semicircle diameter with a straight line. From the semicircle diameter, the charge-transfer resistance (R ct ) could be calculated easily, 47 and the R ct value of bare GCE (Fig. 5b inset) was significantly greater than other modified electrodes. The obtained R ct of α-Co(OH) 2 , rGO/α-Co(OH) 2 , and NrGO/α-Co(OH) 2 was 166.38 Ω, 44.93 Ω, and 30.31 Ω, respectively, which indicated that the electrical conductivity of α-Co(OH) 2 increased after attachment with rGO, and its improved more after Ndoping on rGO surface.

Electrochemical behaviors of AD on fabricated electrodes.-
The electrochemical behavior of the bare GCE, NrGO, rGO/α-Co(OH) 2 , and NrGO/α-Co(OH) 2 modified electrodes was investigated by CV at a scan rate of 50 mV s −1 in 0.1 M PBS (pH 7.4) in absence and presence of AD. There was no noteworthy peak for NrGO/α-Co(OH) 2 /GCE in absence of AD. But it exhibited distinguishable AD redox peaks in presence of 200 μM AD: one strong peak at 0.224 V (vs Ag/AgCl) and a tiny peak at −0.169 V (vs Ag/AgCl) due to oxidation of AD to adrenochrome, and a reversible peak at −0.234 V (vs Ag/AgCl) due to reduction back to AD (Fig. 6a). Similarly, bare GCE showed redox peaks towards 200 μM AD, but no significant response in absence of AD (Fig. 6a inset). Figure 6b demonstrated that both rGO/α-Co(OH) 2 and NrGO/α-Co(OH) 2 provided two anodic peaks and one cathodic peak corresponding to CoO 2 and Co(OH) 2 formation, respectively. 48 Whereas NrGO showed only one anodic peak, which confirmed that α-Co(OH) 2 is the active site of the as-prepared nanocomposite. 31 When AD was introduced into the PBS (pH 7.4) system, Co(OH) 2 converted to CoO 2 due to the presence of OH − ions according to the following reactions: The higher peak current and lower potential of NrGO/α-Co(OH) 2 compared to rGO/α-Co(OH) 2 , suggested that N-doping provide faster electron transfer kinetics and enhanced the electrochemical performances of the proposed sensor.   Where R 2 is the correlation coefficient. Figure 7b showed the DPV curves of different concentrations of AD (0 to 750 μM). Similarly, I pa increased gradually with the subsequent addition of C AD from 10 to 750 μM according to the following linear fit:   This indicated that NrGO/α-Co(OH) 2 /GCE could detect AD in a large range of scan rates.
pH optimization of the experimental conditions.-The effects of pH of NrGO/α-Co(OH) 2 /GCE towards 200 μM AD were conducted by CV at a scan rate of 50 mV s −1 in 0.1 M PBS (pH 6.0 to 9.0). Figure 9a shows the CV curves of different pH. The plot of I pa vs pH (Fig. 9b) demonstrated that I pa increased with the increment of pH from 6.0 to 7.4, then decreased from 7.4 to 9.0. pH 7.4 was selected for determining AD in this study since the highest I pa was observed there. Furthermore, E pa gradually shifted to negative potential with the increases of pH, which indicated the active participation of protons in the electrochemical reaction. 49 From the plot of E pa vs pH, the obtained slope value was −55.6 mV pH −1 (Fig. 9c), which was almost equal to the Nernst's value of −59.0 mV pH −1 and indicated that the equal number of electron and proton of AD was involved during the electrochemical reactions. 50 Quantitative determination of AD at NrGO/α-Co(OH) 2 /GCE .-Choronoamperometric (CA) was employed to determine the limit    The first injection of AD was 0.5 μM at 48 s, and the current response was observed within 2.2 s (Fig. 10a, inset). With the time being, the subsequent addition of AD increased and the CA curve demonstrated a remarkable current response due to the faster electron transfer capacity of NrGO/α-Co(OH) 2 /GCE. Figure Table I. represents the comparison in electrochemical parameters of NrGO/α-Co(OH) 2 /GCE with other previously reported modified sensors for the determination of AD, which implied the proposed modified electrode as one of the best sensors.
Interference study and real sample analysis.-Selectivity of NrGO/α-Co(OH) 2 /GCE towards 150 μM AD was investigated by CA curve in the presence of 50-fold excess of common ions such as Na + , K + , Ca 2+ , Mn 2+ , Fe 2+ , CO 3 2− , SO 4 2− , and 500 μM coexist biomolecules such as dopamine (DA), ascorbic acid (AA), uric acid (UA), tyrosine (TY), serotonin (5-HT), and Norepinephrine (NE) in   Figure 11a illustrated that a significant current response was observed for each addition of AD but there was no noteworthy response for other interfering substances. Furthermore, there was an almost equal current response for injection of every 150 μM AD, which indicated that NrGO/α-Co(OH) 2 /GCE could determine AD without the influence of other interfering substances.
The practical application of NrGO/α-Co(OH) 2 /GCE for different concentrations of AD was verified by DPV curves in presence of the urine sample in 0.1 M PBS (pH 7.4) (Fig. 11b). Urine sample was preserved in a refrigerator for 24 h after 20 min centrifugation, then 50 μl of urine sample was added in 3950 μl of 0.1 M PBS (pH 7.4) for real sample analysis. The obtained I pa values of real sample for 25, 50 and 100 μM AD were measured and compared in calibration curves of I pa vs C AD (Fig. 11b inset). The recovery range was 95.56%-106.55%, which was summarized in Table II.
Stability, reproducibility and repeatability.-Effects of the stability, reproducibility, and repeatability of NrGO/α-Co(OH) 2 /GCE towards 200 μM AD were examined by CV in 0.1 M PBS (pH 7.4). After 30 d, the I p change was 88.89% of the initial I p . As seen in Fig. S2a, first 15 d the I p decreased gradually, but last 15 d it decreased dramatically. The obtained relative standard deviation (RSD) was 2.44% when NrGO/α-Co(OH) 2 modified five different GCEs were used for reproducibility (Fig. S2b). RSD was obtained as 1.99% for fifteen consecutive measurements of the identical electrode.

Conclusions
The NrGO/α-Co(OH) 2 was synthesized via a simple hydrothermal process, in which α-Co(OH) 2 acted as an active site and NrGO decreased the aggregation of rGO. As a result, NrGO provided a better defective surface for immobilized α-Co(OH) 2 NPs and increase the stability, surface area and electrical conductivity of NrGO/α-Co(OH) 2 . Furthermore, α phase of Co(OH) 2 demonstrated synergistic electrochemical performances due to its significant physicochemical characterizations. The as-prepared catalyst provided a lower LOD, wider linear range and better sensitivity for the sensitive determination of AD. The proposed catalyst showed good storage stability, reproducibility, and repeatability, and also no significant interference in the presence of possible coexisting substances. The practical feasibility in the urine samples confirmed NrGO/α-Co(OH) 2 could be used as an effective sensor for the diagnosis of associated diseases of AD and drug quality control.