Author's Accepted Manuscript

Electrochemical Enzymeless Detection ofSuperoxide Employing Naringin-Copper De-corated Electrodes

Sasya Madhurantakam, Stalin Selvaraj, NoelNesakumar, Swaminathan Sethuraman, JohnBosco Balaguru Rayappan, Uma MaheswariKrishnan

PII: S0956-5663(14)00210-3DOI: http://dx.doi.org/10.1016/j.bios.2014.03.029Reference: BIOS6657

To appear in: Biosensors and Bioelectronics

Received date: 15 January 2014Revised date: 10 March 2014Accepted date: 12 March 2014

Cite this article as: Sasya Madhurantakam, Stalin Selvaraj, Noel Nesakumar,Swaminathan Sethuraman, John Bosco Balaguru Rayappan, Uma MaheswariKrishnan, Electrochemical Enzymeless Detection of Superoxide EmployingNaringin-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 forpublication. As a service to our customers we are providing this early version ofthe manuscript. The manuscript will undergo copyediting, typesetting, andreview 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 whichcould affect the content, and all legal disclaimers that apply to the journalpertain.

www.elsevier.com/locate/bios

1 

 

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*

1Centre for Nanotechnology & Advanced Biomaterials (CeNTAB) 2School of Chemical & Biotechnology 3School 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: umakrishnan@sastra.edu

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 non-

enzymatic superoxide sensor using naringin-copper complex. Detection of superoxide has been

2 

 

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, non-

enzymatic 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;

3 

 

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, anti-

allergic, 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

4 

 

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.

5 

 

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 400-

HGA 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 FE-

SEM 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)

6 

 

(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.

7 

 

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 naringin-

copper (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 g2.117 װ. As gװ>g ╧> 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.

8 

 

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

9 

 

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) = nFAQ

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

10 

 

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

11 

 

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 =M

ax

KmI

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

12 

 

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).

13 

 

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 2-

Amino-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.

14 

 

Brieger, K., Schiavone, S., Miller, F.J., Jr., Krause, K.H., 2012. Reactive oxygen species: from

health to disease. Swiss Med Wkly 142, w13659.

Chen, J., Wollenberger, U., Lisdat, F., Ge, B., Scheller, F.W., 2000. Superoxide sensor based on

hemin modified electrode. Sensors and Actuators B: Chemical 70(1–3), 115-120.

Chesney, A., R. Bryce, M., S. Batsanov, A., A. K. Howard, J., M. Goldenberg, L., 1998.

Selective electrochemical magnesium and calcium sensors based on non-macrocyclic nitrogen-

containing ferrocene ligands. Chemical Communications(6), 677-678.

Datta, K., Sinha, S., Chattopadhyay, P., 2000. Reactive oxygen species in health and disease.

Natl Med J India 13(6), 304-310.

De Souza, R.F., De Giovani, W.F., 2004. Antioxidant properties of complexes of flavonoids with

metal ions. Redox Rep 9(2), 97-104.

Deng, Z., Rui, Q., Yin, X., Liu, H., Tian, Y., 2008. In Vivo Detection of Superoxide Anion in

Bean Sprout Based on ZnO Nanodisks with Facilitated Activity for Direct Electron Transfer of

Superoxide Dismutase. Analytical Chemistry 80(15), 5839-5846.

Di, J., Bi, S., Zhang, M., 2004. Third-generation superoxide anion sensor based on superoxide

dismutase directly immobilized by sol–gel thin film on gold electrode. Biosensors and

Bioelectronics 19(11), 1479-1486.

Di, J., Peng, S., Shen, C., Gao, Y., Tu, Y., 2007. One-step method embedding superoxide

dismutase and gold nanoparticles in silica sol–gel network in the presence of cysteine for

construction of third-generation biosensor. Biosensors and Bioelectronics 23(1), 88-94.

Glass, T.E., 2000. Cooperative chemical sensing with bis-tritylacetylenes: Pinwheel receptors

with metal ion recognition properties. Journal of the American Chemical Society 122(18), 4522-

4523.

Gomes, A., Fernandes, E., Lima, J.L.F.C., 2005. Fluorescence probes used for detection of

reactive oxygen species. Journal of Biochemical and Biophysical Methods 65(2–3), 45-80.

15 

 

González-Álvarez, M., Alzuet, G., García-Giménez, J.L., Macías, B., Borrás, J., 2005. Biological

Activity of Flavonoids Copper Complexes. Zeitschrift für anorganische und allgemeine Chemie

631(11), 2181-2187.

Greenwald, R.A., 1990. Superoxide dismutase and catalase as therapeutic agents for human

diseases a critical review. Free Radical Biology and Medicine 8(2), 201-209.

Haseloff, R.F., Ebert, B., Damerau, W., 1989. Superoxide generation in alkaline dimethyl

sulphoxide. Anal Chim Acta 218(0), 179-184.

Havsteen, B.H., 2002. The biochemistry and medical significance of the flavonoids. Pharmacol

Ther 96(2-3), 67-202.

Hyland, K., Auclair, C., 1981. The formation of superoxide radical anions by a reaction between

O< sub> 2</sub>, OH< sup>−</sup> and dimethyl sulfoxide. Biochemical and biophysical

research communications 102(1), 531-537.

Hyland, K., Voisin, E., Banoun, H., Auclair, C., 1983. Superoxide dismutase assay using alkaline

dimethylsulfoxide as superoxide anion-generating system. Analytical Biochemistry 135(2), 280-

287.

Kehrer, J.P., 2000. The Haber–Weiss reaction and mechanisms of toxicity. Toxicology 149(1),

43-50.

Kim, S.K., Kim, D., You, J.-M., Han, H.S., Jeon, S., 2012. Non-enzymatic superoxide anion

radical sensor based on Pt nanoparticles covalently bonded to thiolated MWCNTs.

Electrochimica Acta 81(0), 31-36.

Kostyuk, V.A., Potapovich, A.I., Strigunova, E.N., Kostyuk, T.V., Afanas'ev, I.B., 2004.

Experimental evidence that flavonoid metal complexes may act as mimics of superoxide

dismutase. Archives of Biochemistry and Biophysics 428(2), 204-208.

Leary, S.C., Winge, D.R., 2007. The Janus face of copper: its expanding roles in biology and the

pathophysiology of disease. Meeting on Copper and Related Metals in Biology. EMBO Rep

8(3), 224-227.

16 

 

Liu, M., Liu, R., Chen, W., 2013. Graphene wrapped Cu2O nanocubes: non-enzymatic

electrochemical sensors for the detection of glucose and hydrogen peroxide with enhanced

stability. Biosens Bioelectron 45, 206-212.

Mu, Y., Jia, D., He, Y., Miao, Y., Wu, H.L., 2011. Nano nickel oxide modified non-enzymatic

glucose sensors with enhanced sensitivity through an electrochemical process strategy at high

potential. Biosens Bioelectron 26(6), 2948-2952.

Park, S., Boo, H., Chung, T.D., 2006. Electrochemical non-enzymatic glucose sensors. Anal

Chim Acta 556(1), 46-57.

Patel, R.N., 2009. Synthesis, characterization and superoxide dismutase activity of some four

coordinated copper (II) complexes. Indian Journal of chemistry 48A, 1370-1377.

Pereira, R., Andrades, N.E., Paulino, N., Sawaya, A.C., Eberlin, M.N., Marcucci, M.C., Favero,

G.M., Novak, E.M., Bydlowski, S.P., 2007. Synthesis and characterization of a metal complex

containing naringin and Cu, and its antioxidant, antimicrobial, antiinflammatory and tumor cell

cytotoxicity. Molecules 12(7), 1352-1366.

Qiao, X., Chen, S., Tan, L., Zheng, H., Ding, Y., Ping, Z., 2001. Investigation of formation of

superoxide anion radical in DMSO by ESR: Part 1. Influence of Fe2+ and Cu2+. Magnetic

Resonance in Chemistry 39(4), 207-211.

Rajesh, S., Kanugula, A.K., Bhargava, K., Ilavazhagan, G., Kotamraju, S., Karunakaran, C.,

2010. Simultaneous electrochemical determination of superoxide anion radical and nitrite using

Cu,ZnSOD immobilized on carbon nanotube in polypyrrole matrix. Biosensors and

Bioelectronics 26(2), 689-695.

Selvaraj, S., Krishnaswamy, S., Devashya, V., Sethuraman, S., Krishnan, U.M., 2011.

Investigations on Membrane Perturbation by Chrysin and Its Copper Complex Using Self-

Assembled Lipid Bilayers. Langmuir 27(21), 13374-13382.

Selvaraj, S., Krishnaswamy, S., Devashya, V., Sethuraman, S., Krishnan, U.M., 2012. Membrane

fluidization & eryptotic properties of hesperidin-copper complex. RSC Advances 2(29), 11138-

11146.

17 

 

Selvaraj, S., Krishnaswamy, S., Devashya, V., Sethuraman, S., Krishnan, U.M., 2013.

Flavonoid–Metal Ion Complexes: A Novel Class of Therapeutic Agents. Medicinal Research

Reviews, n/a-n/a.

Starosta, R., Bykowska, A., Kyzioł, A., Płotek, M., Florek, M., Król, J., Jeżowska-Bojczuk, M.,

2013. Copper(I) (Pseudo)Halide Complexes with Neocuproine and Aminomethylphosphines

Derived from Morpholine and Thiomorpholine – In Vitro Cytotoxic and Antimicrobial Activity

and the Interactions with DNA and Serum Albumins. Chemical Biology & Drug Design, n/a-n/a.

Tarpey, M.M., Fridovich, I., 2001. Methods of Detection of Vascular Reactive Species: Nitric

Oxide, Superoxide, Hydrogen Peroxide, and Peroxynitrite. Circulation Research 89(3), 224-236.

Thandavan, K., Gandhi, S., Sethuraman, S., Rayappan, J.B.B., Krishnan, U.M., 2013. A novel

nano-interfaced superoxide biosensor. Sensors and Actuators B: Chemical 176(0), 884-892.

Uivarosi, V., Barbuceanu, S.F., Aldea, V., Arama, C.-C., Badea, M., Olar, R., Marinescu, D.,

2010. Synthesis, Spectral and Thermal Studies of New Rutin Vanadyl Complexes. Molecules

15(3), 1578-1589.

Uttara, B., Singh, A.V., Zamboni, P., Mahajan, R.T., 2009. Oxidative stress and

neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic

options. Curr Neuropharmacol 7(1), 65-74.

Wang, D., Gao, K., Li, X., Shen, X., Zhang, X., Ma, C., Qin, C., Zhang, L., 2012a. Long-term

naringin consumption reverses a glucose uptake defect and improves cognitive deficits in a

mouse model of Alzheimer's disease. Pharmacology Biochemistry and Behavior 102(1), 13-20.

Wang, X., Han, M., Bao, J., Tu, W., Dai, Z., 2012b. A superoxide anion biosensor based on

direct electron transfer of superoxide dismutase on sodium alginate sol–gel film and its

application to monitoring of living cells. Anal Chim Acta 717(0), 61-66.

Xu, F., Deng, M., Li, G., Chen, S., Wang, L., 2013. Electrochemical behavior of cuprous oxide–

reduced graphene oxide nanocomposites and their application in nonenzymatic hydrogen

peroxide sensing. Electrochimica Acta 88(0), 59-65.

18 

 

Yang, Y., Zhou, J., Wu, L., Zhang, X., Chen, J., 2012. A superoxide anion electrochemical

sensor based on the direct electrochemistry of superoxide dismutase assembled layer-by-layer at

the L-cysteine modified gold electrode. Indian Journal of chemistry 51A, 1057 - 1063.

Yuasa, M., Oyaizu, K., Yamaguchi, A., Ishikawa, M., Eguchi, K., Kobayashi, T., Toyoda, Y.,

Tsutsui, S., 2005. Electrochemical sensor for superoxide anion radical using polymeric iron

porphyrin complexes containing axial 1-methylimidazole ligand as cytochrome c mimics.

Polymers for Advanced Technologies 16(4), 287-292.

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.

19 

 

Fig 1

20 

 

Fig 2

21 

 

Fig 3

R² = 0.9895

0

1

2

3

4

0 0.05 0.1

Cur

rent

(µA

)

Scan rate (Vs-1)

22 

 

Fig 4

R² = 0.9253

01234

0 0.5 1 1.5 2

Cur

rent

(µA

)

Superoxide (µM)

23 

 

Fig 5

 

0.45 0.90 1.35 1.80 2.25 2.700.00

0.75

1.50

2.25

3.00

3.75

4.50

Net

Cur

rent

(μΑ

)

Superoxide (μΜ)

R2 = 0.974

24 

 

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