Electrochemical enzymeless detection of superoxide employing naringin–copper decorated electrodes

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Electrochemical Enzymeless Detection of Superoxide Employing Naringin-Copper Decorated Electrodes Sasya Madhurantakam, Stalin Selvaraj, Noel Nesakumar, Swaminathan Sethuraman, John Bosco Balaguru Rayappan, Uma Maheswari Krishnan www.elsevier.com/locate/bios

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Biosensors and Bioelectronics

Received date: 15 January 2014 Revised date: 10 March 2014 Accepted date: 12 March 2014 Cite this article as: Sasya Madhurantakam, Stalin Selvaraj, Noel Nesakumar, Swaminathan Sethuraman, John Bosco Balaguru Rayappan, Uma Maheswari Krishnan, Electrochemical Enzymeless Detection of Superoxide Employing Naringin-Copper Decorated Electrodes, Biosensors and Bioelectronics, http://dx. doi.org/10.1016/j.bios.2014.03.029 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 galley proof before it is published in its final citable 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.

Electrochemical Enzymeless Detection of Superoxide Employing Naringin-Copper Decorated Electrodes Sasya Madhurantakam1,2, **, Stalin Selvaraj1,2, **, Noel Nesakumar1, Swaminathan Sethuraman1,2, John Bosco Balaguru Rayappan1,3, Uma Maheswari Krishnan1,2* 1

Centre for Nanotechnology & Advanced Biomaterials (CeNTAB)

2

School of Chemical & Biotechnology

3

School of Electrical & Electronics Engineering

SASTRA University, Thanjavur – 613 401 ** - Both the authors have contributed equally *Corresponding Author Prof. Uma Maheswari Krishnan Ph. D. Deakin Indo–Australia Chair Professor Associate Dean for the Departments of Chemistry, Bioengineering & Pharmacy Centre for Nanotechnology & Advanced Biomaterials (CeNTAB) School of Chemical & Biotechnology SASTRA University, Thanjavur – 613 401 TamilNadu, India Ph.: (+91) 4362 264101 Ext: 3677 Fax: (+91) 4362 264120 E–mail: [email protected] Abstract Flavonoid-metal ion complexes are a new class of molecules that have generated considerable interest due to their superior anti-oxidant and pharmacological acticities. The metal ion present in these complexes can participate in redox reactions by toggling between different oxidation states. This property can be invaluable for sensing applications. But, the use of flavonoid-metal ion complexes as sensors remains an unexplored facet. The present work attempts to develop a nonenzymatic superoxide sensor using naringin-copper complex. Detection of superoxide has been 1   

mainly based on enzymes and cytochromes. However, these sensors are limited by their poor structural stability and high cost. The naringin-copper based non-enzymatic sensor exhibits good sensitivity over a range of 0.2 µM to 4.2 µM with a response time of < 1s. The performance of the sensor is not affected by pH and common interferents. Keywords: Naringin-copper, superoxide, electrochemical sensor, platinum electrode, nonenzymatic sensor

1.Introduction Reactive oxygen species (ROS) that comprise superoxide anions, hydroxyl radicals, hydrogen peroxide and nitric oxide radicals, have been implicated in many diseases such as inflammation, ageing, cardiovascular diseases and neurodegenerative disorders (Uttara et al. 2009). Quantification of ROS can therefore serve as a risk indicator for many oxidative stress-induced diseases. Superoxide anion is a product of the metabolic reations in the biological system(Kim et al. 2012; Xu et al. 2013). The superoxide radicals are transformed by the cellular enzymes to hydrogen peroxide and to hydroxyl radicals through the Haber-Weiss reaction(Kehrer 2000). The ROS react with macromolecules leading to disruption of the metabolic activities of the cell and cause cell destruction through lipid peroxidation, hypoxia and DNA damage(Alfadda and Sallam 2012; Brieger et al. 2012; Datta et al. 2000). However, detection of superoxide anion poses a major challenge due to its extremely short half life and instability causing its frequent disproportionation to oxygen and H2O2 (Beissenhirtz et al. 2003; Thandavan et al. 2013). Spectrophotometry, chemiluminesence and electron spin resonance spectroscopy are the major methods that have been employed for the detection of superoxide(Tarpey and Fridovich 2001). But, these methods are expensive, time-consuming and are not portable. The development of enzyme-based biosensors offer a better alternate strategy for both identificaton and quantification of superoxide anion in a highly specific manner (Thandavan et al. 2013). Superoxide dismutase (SOD), an enzyme that catalyses the dismuation of O2·- to O2 and H2O2 through a redox reaction has been widely employed for detection of superoxide. Superoxide dismutase enzymes with Mn, Cu/Zn and Fe co-factors have been employed for the sensing applications(Greenwald 1990; 2   

Thandavan et al. 2013). These metal ions serve as redox centres and toggle between different oxidation states during their reaction with the superoxide anion(Greenwald 1990). A ping-pong mechanism has been suggested to explain the superoxide scavenging action of metal ion containing centres (Patel 2009). Mox + O2.- → Mred + O2 Mred + O2.- + 2H+ → Mox + H2O2 where Mox is the oxidized form of the redox active metal centre and Mred is the reduced form of the redox active metal centre. The use of nano-interfaces has served to improve the sensitivity and response time of the biosensors (Greenwald 1990). However, enzyme-based sensors suffer from several drawbacks – the chief among them being poor enzyme stability and reusability(Kim et al. 2012). In addition, development of SOD enzyme-based sensors is further hindered by its high cost (Chesney et al. 1998). Several attempts to develop enzymeless sensors are available in literature. Till date, much emphasis has been laid on the development of enzyme-free glucose sensors. Copper nanoparticles, calixarenes, copper wires integrated with carbon nanotubes and graphene sheets have been employed for enzyme-free detection of glucose(Liu et al. 2013; Mu et al. 2011; Park et al. 2006). A scan of literature reveals a very few reports on non-enzymatic superoxide sensors. Some of the enzymeless superoxide sensors include those based on platinum nanoparticles incorporated into thiol functionalized multi-walled carbon nanotubes (Kim et al. 2012), polymeric porphyrin iron complexes (Yuasa et al. 2005) and hemin modified electrode (Chen et al. 2000). Thus, the field remains wide open for development of novel non-toxic, low cost non-enzymatic sensors for quantification of superoxide. Flavonoids belong to a class of polyphenols that possess excellent anti-cancer, anti-oxidant, antiallergic, anti-viral and anti-microbial properties (Havsteen 2002; Selvaraj et al. 2013). Apart from their ability to scavenge free radicals(Havsteen 2002), flavonoids also possess metal chelating ability(De Souza and De Giovani 2004). It has been reported that formation of flavonoid-metal ion complexes can augment the anti-oxidant property of the parent flavonoid. Many unique pharmacological properties have been attributed to the flavonoid-metal ion 3   

complexes. These include insulin mimetic, anti-microbial and SOD mimetic activities(De Souza and De Giovani 2004; Kostyuk et al. 2004; Uivarosi et al. 2010). Complexes of rutin with copper and iron, vanadyl complex of hesperitin and copper complex of curcumin have been demonstrated to scavenge superoxide anions and it has been suggested that they might transform the superoxide anion akin to SOD (Barik et al. 2005; Kostyuk et al. 2004). The metal ion in the complex may function in a similar manner to the metal ion co-factor in SOD(Kostyuk et al. 2004). However, no attempts have been made till date to utilize this property of flavonoid-metal ion complexes towards quantification of superoxide. The present work aims to develop for the first time an enzymeless biosensor based on naringin-copper complex and investigate its sensing characteristics. Copper ion is an abundant species present in biological systems that is involved in many transcriptional events and reversible redox reactions(Leary and Winge 2007). It is reported that copper ions possess several medicinal, anti-inflammatory and anti-oxidant properties(Leary and Winge 2007). However, beyond a critical level, copper ion concentrations can result in adverse effects(Leary and Winge 2007). The complexation of copper ions with a biologically active organic ligand has been suggested to mitigate its toxic effects while enhancing its beneficial effects(González-Álvarez et al. 2005; Starosta et al. 2013) Naringin is a type of flavanone glycoside derived from the aglycone naringenin. Naringin-copper has been demonstrated to exhibit anti-cancer and anti-oxidant properties (Wang et al. 2012a). The central metal ion in naringin-copper can participate in redox reactions by alternating between the oxidized and reduced states. This property can be explored for biosensing applications towards quantification of superoxide which forms the crux of this work. 2.Materials and methods Naringin was purchased from M/s Sigma–Aldrich Ltd (USA). Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, sodium chloride, dimethyl sulfoxide, copper(II) acetate were purchased from Merck (India). All solutions used in this experiment were prepared using double distilled water.

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2.1Preparation of naringin-copper complex Naringin-copper complex was synthesized using a one-pot room temperature procedure adapted from a protocol reported earlier for the synthesis of chrysin-copper complex(Selvaraj et al. 2011). Briefly, 0.1 M naringin solution in ethanol was mixed with 0.1 M copper acetate solution. The mixture was stirred for 6 h at room temperature and the pale green coloured precipitate obtained was filtered, dried and characterized.

2.2 Characterization of the naringin-copper complex The elemental analysis of the complex was carried out using C,H, N and S analyser (Elementar Vario EL 3, Germany). EPR spectrum (EMS PUlus Bruker, Germany) of the complex was recorded at room temperature to confirm complexation of copper with the ligand. Copper content in the complex was quantified using atomic absorption spectroscopy (AAS, AA Analyst 400HGA 900- AS-800, Perkin Elmer, USA). The electronic spectra of the free ligand (naringin) and its copper complex was recorded between 190-780 nm using UV-visible spectrophotometry (Lambda 25, Perkin Elmer, USA) to determine the geometry of the copper complex. FTIR spectra of naringin and its copper complex was recorded between 4000-400 cm-1 averaging 20 scans (Spectrum 100, Perkin Elmer, USA) to identify the coordinating sites in naringin.The distribution of naringin-copper complex on the surface of the electrode is observed using FESEM JSM 6701F, JEOL, JAPAN. 2.3 Generation of superoxide Superoxide was produced in situ using a method proposed by Hyland et al.(Hyland and Auclair 1981; Hyland et al. 1983) 5 mM of NaOH and 0.1% (v/v) DMSO in water was used to produce superoxide anions. DMSO reacts with dissolved oxygen in the medium and NaOH to generate superoxide anion radical and hydroxyl radical. Generally superoxide radicals disappear rapidly which can be prevented by the remaining DMSO present in the system. The superoxide anions produced by this method exhibit long term stability (Haseloff et al. 1989). The mechanism of superoxide formation is as follows:(Qiao et al. 2001) 5   

(CH3)2SO → CH3SOCH2.+ H2O O2+OH- → O2.- + OH. OH. + (CH3)2SO → CH3SOOH + CH3. CH3. + O2 → CH3O2. 2CH3O2. → CH3OOCH3 + O2. Net reaction: 2OH. + 3O2 + 2(CH3)2SO → 2CH3SOOH + CH3OOCH3 + 2O2.

Concentration of

O2·-

was determined using its absorbance at 271 nm and the molar

absorptivity of O2·- in DMSO which is 2006 M cm-1 (Di et al. 2004; Di et al. 2007). 2.4 Preparation of naringin-copper decorated platinum electrode The working electrode was prepared by polishing the platinum electrode using 1.0, 0.3, 0.05 micron alumina powder respectively. The polished electrode was then ultrasonicated in ethanol, acetone and de-ionized water separately for ten minutes each. The electrode surface was coated using 3 µL of naringin-copper (N-Cu) solution dispersed in 0.05% nafionic solution and allowed to air-dry for 20 min. The modified electrode is denoted as N-Cu/Pt and used for further electrochemical studies. 2.5 Electrochemical studies Electrochemical studies were performed using an electrochemical analyser (CHI600C, CH Instruments, USA) employing a three-electrode system comprising a platinum wire counter electrode, Ag/AgCl (saturated, 0.1 M KCl) reference electrode and N-Cu/Pt as the working electrode with the dimension of 2 mm diameter. Cyclic voltammograms were recorded in phosphate buffer at 298 K and a scan rate of 0.01 Vs-1. The amperometric patterns were recorded at time intervals of 100 s at an applied potential of 0.123 V. Each step in the amperometric pattern corresponds to the introduction of 0.2

M of superoxide anions.

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3. Results and Discussions 3.1 Characterization of naringin-copper complex The key features that confirm the formation of the naringin-copper complex are summarized in Table S1. The UV –Visible spectra for naringin showed prominent bands at 286 nm (Band II) and 327 nm (Band I) that are characteristic of the core flavonoid framework of naringin. The band I exhibited a significant shift to 344 nm in the case of the naringin-copper complex indicating the coordination of copper with naringin (Pereira et al. 2007; Selvaraj et al. 2011). The vibrational frequency data recorded in FTIR spectroscopy reveals that the vibration stretching for the carbonyl group (C=O) of naringin at 1654 cm-1 is shifted to 1614 cm-1 in the case of naringincopper (Pereira et al. 2007). This indicates the coordination of the metal ion with 4-keto group in the complex. The pronounced shift of the vibration band due to aromatic C=C stretching from 1540 cm-1 to 1504 cm-1 in the naringin-copper complex suggests changes in the aromatic double bonds owing to the complexation of naringin with copper which can be correlated with the previous report on chrysin-copper complex (Selvaraj et al. 2011). The EPR spectrum of the complex shows the absence of any multiplets that may arise due to the presence of uncomplexed copper ions suggesting that the copper ion is present only in the complexed state. The values of g ╧

are 2.017 and g‫ װ‬2.117. As g‫>װ‬g ╧> 2, it can be inferred that the complex possesses elongated

axial symmetry(Arab Ahmadi et al. 2013; Selvaraj et al. 2012) and the unpaired spinning electron in the Cu(II) ion is present in the dx2 – y2 orbital.

This result is in agreement with the

other reports indicating coordination of Cu2+ ion to the carbonyl oxygen atom(Selvaraj et al. 2012). The dx2 – y2 ground state is indicative of a square planar geometry for the complex(Barik et al. 2005; Selvaraj et al. 2012). The elemental analysis of the naringin-copper complex showed a C, H and Cu content that were in good agreement with the theoretical values obtained for a 1:2 metal-ligand ratio. Based on these results, the proposed structure of the naringin-copper complex is presented in Figure 1.

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3.2 Electrochemical studies of N-Cu/Pt 3.2.1 Cyclic voltammetry Figure 2 shows the cyclic voltammogram of the unmodified working electrode and the N-Cu coated working electrode. It is observed that when the potential was varied from -0.4 V to +0.4 V, the bare electrode did not show any characteristic redox peak, while the N-Cu/Pt electrode shows a significant reduction peak at a potential of +0.123 V. This can be attributed to the redox shuttling of copper ion present in the naringin-copper complex (1). Cu2+ + e-

Cu+ ------------------(1)

Generally, the standard reduction potential of Ag/AgCl is 0.2223 V and the standard reduction potential of Cu2+ is 0.16 V(Vs SHE). Since, Ag/AgCl is used as a reference electrode, E⁰ shifted to -0.08839 V that is E⁰ = (0.16-0.2223) V = -0.08839 V. In this case the same trend was observed. This is confirmed the reduction of Cu2+ to Cu+ in the anodic process and thereby enzyme (SOD) mimicking nature of Cu2+ was proved. It is also confirms the single electron transfer in the anodic end. Scan rate studies were carried by increasing the scan rate from 0.01 Vs-1 to 0.1Vs-1. Figure 3 shows the cyclic voltammograms of N-Cu/Pt electrode recorded in the presence of 0.2 µM superoxide anions in phosphate buffer (pH 7.4) for increasing scan rates from 0.01 Vs-1 to 0.1Vs-1. A progressive increase in the reduction peak is observed with increasing scan rates indicating an increase in the flux towards the electrode. The plot of peak current against the scan rate shown in the inset is linear suggesting electron transfer processes occur at electrode surface (Yang et al. 2012). Figure 4 shows the cyclic voltammetric patterns obtained in phosphate buffer using the N-Cu/Pt electrode for increasing concentrations of superoxide at a scan rate of 0.01Vs-1. A progressive increase in the peak current is observed with increasing concentrations of superoxide anions between the range 0.2 µM to 2 µM. A plot of peak current against the concentration indicates a

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linear trend. This finding is concurrent with the previous report for superoxide sensor (Wang et al. 2012b). 3.2.2 Amperometric studies Figure 5 shows the amperometric pattern obtained for successive addition of superoxide anions to phosphate buffer (pH 7.4) at 298 K and 0.123V. It is observed from Figure 5 that a stepwise decrease in the current occurs for successive addition of 0.2 µM of superoxide anion. The slight decrease in the current in between the addition of superoxide which may be attributed to the poor conductivity of the material or poor adherence between electrode and naringin-copper. Such phenomenon has also been reported by other groups. The change in current on introduction of superoxide may be attributed to the following mechanism occurring at the electrode-electrolyte interface: The Cu2+ present in the naringin-copper interacts with the superoxide anion resulting in its reduction to the cuprous form (Cu+) while the superoxide gets transformed to oxygen. The release of oxygen causes a decrease in the currents. This finding is also in agreement with the previous report on superoxide dismutase mimicking activity of four coordinated copper (II) complexes (Patel 2009) (Scheme S1). The plot of current vs concentration of superoxide exhibits an excellent linear trend between the concentration range of 0.2 µM to 2.8 µM. Amperometric steps were discernible up to a maximum concentration of 4.2 µM but a slight deviation from the linearity (Figure 5 inset). The surface coverage of the electrode by the electroactive naringin-copper is calculated from the following relationship: Surface coverage (Γ) =

Q nFA

where ‘Q’ is the charge, ‘n’ refers to the number of electrons transferred, ‘F’ denotes the Faraday constant and ‘A’ is the area of the electrode The surface coverage of the electrode by N-Cu was found to be 11.68 nM/cm2. The changes in the current on addition of superoxide until a concentration of 2.8 µM is directly proportional to 9   

the number of electroactive species on the electrode surface. Beyond this concentration, a change in the slope is observed that suggests the saturation limit of N-Cu has been attained beyond which there is a decrease in the rate of current change on addition of further quantities of superoxide. The sensor exhibits a rapid response to the addition of superoxide which is evident from the very low response time of <1s. This quick response time is superior to several reports available in literature that have ranged from 3-20 s (Kim et al. 2012). The limit of detection and limit of quantification for this non-enzymatic sensor was determined from the following equations: Limit of Detection = 3.3 × SD/ S Limt of Quantification = 10× SD/ S. Where, ‘SD’ represents standard deviation and ‘S’ represents Sensitivity. The limit of detection (LOD) was found to be 150 nM and the limit of quantification (LOQ) was determined to be 453 nM (Figure 5B). Figure S1 shows the relationship between the concentration of superoxide anion (µM) and net current (µA). When the concentration is increased beyond 4.2 µM, a plateau is observed which resembles Michaelis Menten kinetics for enzymatic sensors. In enzymology, cooperativity is defined as a phenomenon where binding of a substrate to the enzyme influences further binding of the substrate molecules. Similarly, in the case of enzyme-less sensors, the initial binding of an analyte to the sensing element may influence the further binding of the analyte molecules. This cooperativity can be determined using the following equation: I = I max ( Sn / Kmn + Sn) Here, n = cooperative active site or Hill coefficient S = superoxide concentration 10   

where A positive cooperativity results if the n (Hill coefficient) values are greater than 1 indicating that the analyte binding will enhance the binding of more analyte molecules with the sensing element while n values below 1 indicate negative cooperativity where the analyte binding will hinder further binding of analyte molecules. In the present study, the n value for the binding of superoxide with the naringin-copper decorated electrode was determined to be 1.05±0.235. As the n value is close to 1, it suggests a non-cooperative interaction where binding of superoxide radicals do not influence further binding of other superoxide radicals with naringin-copper (Glass 2000) at the electrode surface. We also calculated KM (Michaelis-Menten constant) and Imax value for the naringin-copper decorated platinum working electrode using the electrochemical version of Lineweaver-Burk equation to elucidate the binding efficiency between superoxide and naringin-copper (Wang et al. 2012b). It was found that KM value was low i.e 2.46 ±0.76 µM indicating high binding affinity of the analyte and naringin-copper. The Imax was found to be 8.43±0.055 µA. These parameters are superior than that reported for an enzyme-based superoxide biosensor using sodium alginate sol-gel film (Wang et al. 2012b) and comparable with other superoxide dismutase-based sensors detected using fluorescence probes (Gomes et al. 2005). The sensitivity of the sensor was calculated using the following formula: Theoretical sensitivity =

Im ax KM

The sensitivity was found to be 3.41 µA/µM which is higher than the practical sensitivity of 1.54 µA/μM. A comparison of the sensor characteristics of several superoxide sensors reported in

recent times and the novel enzymeless superoxide sensor based on naringin-copper is shown in Table 2.

It is observed from the Table S2 that the naringin-copper enzyme-less sensor reported in the present work has the fastest response time among enzymatic and non-enzymatic sensors. The detection range is comparable to several superoxide sensors reported in literature. However, the 11   

detection range may be further improved by increasing the loading of naringin-copper complex and surface area of the electrode. The catalytic efficiency of N-Cu towards superoxide was found to be good. Biosensor Efficiency of N-Cu/ Pt working electrode was calculated using following formula: Biosensor efficiency = theoretical sensitivity/ practical sensitivity. The efficiency of the designed biosensor was found to be 2.038 % that was comparable with other superoxide sensors reported earlier (Thandavan et al. 2013; Wang et al. 2012b). The catalytic efficiency of N-Cu was found to be 2.46 µA/µM which can be comparable with other non-enzymatic superoxide sensors reported in the literature (Wang et al. 2012b). 3.2.3 Influence of interferents Interference studies were carried out for the common interferents like uric acid, ascorbic acid, citric acid, H2O2 and the results are shown in Figure S2. It is observed that the sensor exhibits good specificity towards superoxide and no appreciable change in the current occurred on addition of 0.2 µM ascorbic acid or 0.2 µM uric acid. Addition of 0.2 µM hydrogen peroxide also did not alter the current. These are the normal potential interfering molecules in the biological systems. 3.2.4 Influence of different pH on Naringin-copper sensor The effect of different pH on the electrode stability and sensitivity was evaluated at pH 4, 7 and 10 using cyclic voltammetry in the presence of 0.2 µM superoxide and the results are presented in Figure 6. It is observed that there is no significant shift in the reduction potential at all three pH studied. This confirms the stability of naringin-copper decorated working electrode in different pH conditions unlike enzyme-based sensors reported earlier where a strong pH dependency was reported (Wang et al. 2012b).

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4. Conclusion The sensing capability of a flavonoid-metal ion complex has been investigated for the first time. The naringin-copper complex exhibits good sensitivity towards superoxide over a range of 0.2 µM to 4.2 µM. Cyclic voltammetric experiments reveals that the increase in the reduction current with increase in the scanrate can be attributed to a surface controlled process.The response time of <1 s and LOD of 150 nM makes it on par with enzyme-based sensors.This study has opened up new vistas for exploration of other flavonoid-metal ion complexes as sensing elements. The simple synthesis of the plant-derived flavonoid-metal ion complexes make them low-cost non-enzymatic options for sensing applications that are not limited by structural instability which plagues enzymatic sensors. Acknowledgement The authors wish to acknowledge the financial support from the Department of Science & Technology, Government of India, under the Grant SR/SO/BB–35/2004, DST/TSG/PT/2008/28 and SM thankful to DST inspire fellowship (DST/IF/120812) and the infrastructural support from SASTRA University. References Alfadda, A.A., Sallam, R.M., 2012. Reactive oxygen species in health and disease. J Biomed Biotechnol 2012, 936486. Arab Ahmadi, R., Hasanvand, F., Bruno, G., Amiri Rudbari, H., Amani, S., 2013. Synthesis, Spectroscopy, and Magnetic Characterization of Copper(II) and Cobalt(II) Complexes with 2Amino-5-bromopyridine as Ligand. ISRN Inorganic Chemistry 2013, 7. Barik, A., Mishra, B., Shen, L., Mohan, H., Kadam, R.M., Dutta, S., Zhang, H.-Y., Priyadarsini, K.I., 2005. Evaluation of a new copper(II)–curcumin complex as superoxide dismutase mimic and its free radical reactions. Free Radical Biology and Medicine 39(6), 811-822. Beissenhirtz, M., Scheller, F., Lisdat, F., 2003. Immobilized Cytochrome c Sensor in Organic/Aqueous Media for the Characterization of Hydrophilic and Hydrophobic Antioxidants. Electroanalysis 15(18), 1425-1435. 13   

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List of figures Figure 1: Proposed structure of naringin-copper complex. Figure 2: Cyclic voltammograms recorded in PBS at 298 K and scan rate 0.01Vs-1 using: A) bare electrode B) N-Cu/Pt electrode. Inset: Scanning electron micrograph of naringin-copper coated electrode. Figure 3 Cyclic voltammograms obtained using the N-Cu/Pt electrode in the presence of 0.2 μM superoxide at various scan rates in phosphate buffer at 298 K; Inset : Plot of peak current against scan rate. Values are expressed as mean±Standard Deviation. n=3. Figure 4 Cyclic voltammograms obtained after addition of different concentrations of superoxide anion at 298 K in phosphate buffer (pH 7.4) at a scan rate of 0.01 Vs-1; Inset : Plot of peak current against concentration of superoxide anion. Values are expressed as mean±Standard Deviation. n=3. Figure 5 Amperometric pattern obtained at 0.123 V in phosphate buffer (pH 7.4) for successive additions of superoxide anions; Inset: Plot of peak current against concentration of superoxide anion. Values are expressed as mean±Standard Deviation. n=3. Figure 6 Influence of different pH on the stability of naringin-copper decorated platinum electrode in the presence 0.2 µM of superoxide.

18   

Fig 1

19   

Fig 2

20   

Fig 3

Current (µA)

4 R² = 0.9895

3 2 1 0 0

21   

0.05 Scan rate (Vs-1)

0.1

Current (µA)

Fig 4

4 3

R² = 0.9253

2 1 0 0

22   

0.5 1 1.5 Superoxide (µM)

2

Fig 5  

4.50

Net Current (μΑ)

3.75

2 R = 0.974

3.00 2.25 1.50 0.75 0.00 0.45

0.90

1.35

1.80

Superoxide (μΜ)

23   

2.25

2.70

Fig 6

Highlights Synthesis and characterization of naringin-copper complex Superoxide sensing using naringin copper Elucidation of sensitivity and reliability of developed superoxide sensor Interference studies carried out using potential interferents

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