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Bioelectrode based chitosan-nano copper oxide for application to lipase

biosensor

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Journal of Applied Pharmaceutical Research 2017, 5 (1): 30 – 39

Journal of Applied Pharmaceutical Research (JAPTRonline)

ISSN No. 2348 – 0335 www.japtronline.com

BIOELECTRODE BASED CHITOSAN-NANO COPPER OXIDE FOR APPLICATION TO

LIPASE BIOSENSOR
A. B. Bodade, M. A. Taiwade, G. N. Chaudhari*
Nanotechnology Research Laboratory, Department of Chemistry, Shri Shivaji Science College, Amravati M.S-444603 India

Article Information ABSTRACT

Received: 14th December 2016 Chitosan (Chit)-nanocrystalline CuO composite prepared from Chitosan and CuO nanoparticles
Revised: 22nd December 2016 by a spin coating method. CuO nanoparticles (45 nm) synthesized by Sol-gel citrate method and
Accepted: 28th December 2016 characterized by X-Ray diffraction (XRD), Raman spectroscopy, UV-visible spectroscopy,
Fourier transform spectroscopy (FTIR) and Scanning electron microscopy (SEM). The
Keywords electrochemical studies revels that these Chit-nano CuO electrode provide favorable condition
Chitosan (Chit), nano-CuO, for immobilization of enzyme lipase [LIP] specific enzyme for triglyceride detection, resulting
bioelectrode, Lipase [LIP], cyclic in enhanced electron transfer at the interface. The prepared bioelctrode (LIP/Chit-nano CuO/Au
voltammetry (CV), triglyceride [TG] bioelectrode) is utilized for triglyceride [TG] sensing using cyclic voltammetry (CV) with
biosensor hexacyanoferrate as mediator. The electrochemical response studies shows on improved sensing
performance of bioelectrode exhibit high sensitivity, low detection limit and good linearity of
tributyrin concentration with fast response time. The low value of Michallis-Menten constant
indicates high affinity of LIP towards the analyte (tributyrin). The Redox behavior of nano CuO
makes an efficient matrix with chitosan for triglyceride [TG] biosensor.

INTRODUCTION indium tin oxide are some commonly used working electrode
Triglyceride (TG) can be generated by an esterification process in these biosensors. In order to detect tributyrin concentration
of three hydroxyl (-OH) group of glycerol with three molecule with enhanced sensitivity and specificity, surface patterning of
of fatty acid that produce an ester as product[1].Lipase electrode with nanoparticles [9] is required. Recently use of
triglyceride ester hydrolase has many industrial applications, lipase as catalyst to produce biodiesel by transporting
because it catalyses the hydrolysis of triacylglycerol into triglyceride into fatty acid, alkyl acid have been reported [10].
glycerol and fatty acid [2]. Triglyceride acts is important role in However, free lipase is not favored in industrial development
metabolism as an energy source and also as a dietary fat because it is difficult to recover for reuse and it has low
transporter. Cardiovascular diseases are major cause of global stability .These drawback can be overcome by immobilization
death [3], Triglyceride (TG) determination is crucial since its on various supports for instant Chitosan is polysaccharide
concentration could lead to hyperlipidemia [4, 5].Abnormal carrying amino group useful. Chitosan supports have been used
triglyceride concentration in blood leds to chronic obstructive for lipase immobilization that permits to retain initial activity.
pulmonary diseases which includes bronchitis, Nanostructured metal oxide have been widely used in biosensor
[6]
bronchopneumia, Sinusitis larystic etc. , Therefore, ensuring application as they provide a large surface to volume ratio, high
the level of triglyceride in our body in normal range women catalytic activity, high electron conductivity due to small band
(35-135 mg/ dL) and men (40-160 mg/ dL) is a need[7, 8]. In the gap and the strong adsorption ability[11]. Among the various
recent past, electrochemical nanobiosensors have been used for methods available for determination of tributyrin (TG) [12],
rapid determination of tributyrin in blood due to its high biosensors are comparatively more simple, sensitive, rapid and
sensitivity and specificity. Gold, glassy carbon electrode and possible at beside the patient. Three types of TG biosensor

*For Correspondence: cgnroa@yahoo.com

©2017 The authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY NC), which permits
unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from
the authors or the publishers.
Chaudhari et. al Bioelectrode based chitosan-nano copper oxide for application to lipase biosensor

have been reported: (i) DO metric TG biosensor which stirring at room temperature for 3hrs, and then sol gel (figure 1)
measured Dissolved oxygen consumed in enzyme reaction [13] synthesized CuO nanoparticles were dissolved in 20 ml of
(ii) electrochemical biosensor which measured electron under chitosan solution and stirring for 30 minutes at room
high potential [14] and (iii) potentiometric TG biosensor which temperature. Finally, viscous solution of Chit-nano CuO was
measure the voltage [15], However electrochemical biosensor obtained. Then Chit-nano CuO solution was pour onto the
are more common in use as these are unaffected by surface of Au plate by spin coating technique and then, the
environment factors and easy to operate. The most common Chit-nano CuO/Au electrode was kept too dry, finally dried
device used for TG determination is an enzymatic Chit-nanoCuO/Au electrode was rinsed repeatedly with 50 mM
amperometric triglyceride biosensor. Most triglyceride phosphate buffer.
biosensor reported till date are based on multienzyme [16]
however, there are works that used single enzyme (lipase) for
triglyceride [17, 18].this methods is more preferable than
multienzyme (lipase, glycerol, kinase-α-glycerophosphate
oxidase and peroxidase) because single enzymatic method are
less time consuming and inexpensive.

EXPERIEMENTAL
Materials:
Lipase (Aspergillus Niger, Himedia), Chitosan (Chit, Sigma
Aldrich), Copper nitrate [Cu (NO3)2.3H2O], Citric acid, ethanol,
potassium chloride [KCl], Potassium fericynide
3-
[K3[Fe (CN)6] , Sodium dihydrogen phosphate [NaH2PO4], Fig.1. Sol-gel citrate method for synthesis of Copper oxide
Sodium monohydrogen phosphate [Na2HPO4] and all other Nanoparticles
chemicals were analytical grade. Aqueous solution and buffer
were prepared in Mili Q.Water. Fabrication of LIP/Chit-nano CuO/Au Bioelectrode
For preparation of bio electrode, enzyme Lipase (LIP) solutions
Synthesis of Copper oxide Nanoparticles were freshly prepared in phosphate buffer [50Mm], (pH.-7.0):
Copper oxide (CuO) nanoparticles (45nm) were synthesized by solution prepared by adjusting the proportion of monobasic
sol-gel citrate method 19 is shown in figure.1 by precursor using sodium phosphate and dibasic sodium phosphate solution of
a copper nitrate (Cu (NO3)2.3H2O), Citric acid (C6H8O7), and 0.05M Concentration by dissolving 1 mg of LIP [21]. Then,
ethanol (C2H5OH,>99% pure).Copper nitrate and citric acid lipase was immobilized onto surface of Chit-nano CuO/Au
were dissolved in ethanol with 1:1:2 proportion and stirred at electrode by physical adsorption method and kept in humid
80°C for 3 hrs to get homogeneous solution was further chamber for about 4hrs.The fabricated bio electrode (LIP/Chit-
heated at about 120°C for 12hrs in heating vessel to form the nano CuO/Au electrode) was then washed with phosphate
gel precursor. solution, then the The prepared product was buffer to remove any unbound enzyme and stored at 4°C when
subjected to 3hrs heat treatment (calcinated) at 350°C in a not in use.
muffle furnace The dried powder then calcinated at 650°C in
order to improve the crystallinity and sensitivity of the RESULT AND DISCUSSION
material. The structural analysis of synthesized nano CuO has been done
using X-Ray diffractometer; Fourier transform infrared (FTIR)
Preparation of Chit-nano CuO/ Au electrode studies have been performed to confirm the bonding in nano
Chitosan (Chit), a natural copolymer having β-1, 4, CuO. The morphological analyses have been conducted using
glucosamine and N-acetyl glucosamine units is a biodegradable scanning electron microscopy (SEM).The electrochemical
polysaccharide exhibiting bioactive amino group [20]. Chitosan analysis was done by using Electrochemical impedance
(200 mg) was dissolved in 100 ml of 0.2M acetic acid by spectroscopy (EIS) and Cyclic Voltametry (CV).

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Chaudhari et. al Bioelectrode based chitosan-nano copper oxide for application to lipase biosensor

X-Ray Diffraction shows obviously that pure single phase CuO nanoparticles are
Crystalline natures of prepared CuO nanoparticles were formed in the above experimental condition.
identified from their corresponding powder XRD pattern. The
XRD pattern (Figure.2.) of nano CuO Particles exhibit
reflection plane (2θ= 32.20, 35.10, 38, 48, 52.72.12, 58, 62, 66,
66.10, 72, and 75), 110, 002, 111, 202, 020, 202, 113, 311, 113,
311, 004 which have been assigned to the monoclinic
hexagonal crystalline system of CuO (Std JCPDS File No:
048-1548 and 800-1917). The broad and well resolved
diffraction peaks reveal the nano scale dimension of the plane
along [111] plane. No characteristics peaks of any impurities
were detected suggesting that high quality of CuO
nanoparticles was prepared. The crystalline Size was calculated Fig.3 Raman spectra of synthesized CuO nanoparticles
using Debye Scherer’s formula
𝐷 = K λ/ βCos θ UV Spectroscopy
Where, D is crystalline size, K is usually taken as 0.94, known Optical characterization of the sample was recorded on UV
as Brags constant usually taken as 0.89, λ is the wavelength of Visible absorption spectrophotometer shown in fig.4.In order to
X-Ray radiation (0.15418nm) for Cu-Kα, β is a full width half determine the band gap energy of CuO.Eg value of CuO
maximum of a diffraction peak measured at 2Θ. according to the following equation,
Eg= hv freq
The nanocrystalline size of CuO Nanoparticles was found to be Where, Eg is low gap energy is Planck’s constant. vfreq is
45 nm. The peaks with high intensities and narrow full width frequency of emitted radiation .The band gap of nano CuO is
half maximum shows that crystallinity of the prepared material calculated to be 3.42 eV, which is higher than the reported
is good. value of CuO [23]. The increasing in band gap may be due to the
quantum size effect of sample [24].

Fig.2 X-Ray diffraction pattern of synthesized CuO Fig. 4 UV Spectroscopy of synthesized CuO nanoparticle
nanoparticles
FTIR Study
Raman spectra Fourier transform infrared Spectroscopy [FTIR] is a technique
Raman spectra of CuO nanoparticles are shown in figure.3. It used to measure vibrational frequencies of bond in the
can be seen at three Raman peaks at 282, 330 & 616cm-1. molecule. FTIR spectroscopy analysis of CuO nanoparticles
Synthesized CuO has monoclinic structure with space group was scanned at room temperature in the range 4000-400 cm-1.
C2h6. In Raman spectra the peak at 282cm-1 to the Ag modes Figure.5 Shows FTIR spectra of CuO nanoparticles. The
and peak at 330 and 616cm-1 to the Bg modes which is observed peaks at 453,494,609 Cm-1 corresponds to the
consistent with previous work [22]. Raman active modes characteristics stretching vibration of Cu-O bond in the CuO.
(Au+2Bu) occurred in raman observed spectra. This result There is a Sharp peak observed at 609 cm-1 in the spectrum on

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Chaudhari et. al Bioelectrode based chitosan-nano copper oxide for application to lipase biosensor

CuO nanoparticles, which is a characteristic of Cu-O bond

formation. Moreover, no other IR active mode was observed in
the range of 605 to 660 cm-1, which totally rules out the
existence of another phase, i.e., Cu2O [25]. The broad absorption
peak at around 3466 cm-1 is caused by the adsorbed water
molecule since the nanocrystalline materials exhibit a high
surface to volume ratio absorb moisture. Thus, the pure phase
CuO with monoclinic structure is also confirmed from the
Fourier transform infrared Spectroscopy analysis.
Scanning electron microscopy
Scanning electron microscopy (SEM) have been used to
investigate surface morphology of prepared material. Figure.6. Fig.5 FTIR spectra of nanocrystalline CuO nanoparticles
[A], [B] shows SEM of CuO nanoparticles at different
magnification [A], [B], which shows globular morphology.

Fig.6. Scanning electron microscopy of Synthesized CuO nanoparticles

Figure.7 [A-D] shows Scanning electron microscopy (SEM) area has also reduced and maximum absorption of enzyme
morphology of [A] Chit-CuO/Au electrode [B] Lipase/Chit- occur. This shows perfect immobilization of enzyme.
CuO/Au bio electrode respectively at different magnification
from [A] to [D]. The SEM images of Chit-CuO/ Au electrode ELECTROCHEMICAL STUDY:
exhibit cotton globes like structure and also shows polymer is The electrochemical studies have been carried out by dipping
embedded in CuO nanoparticles. The revealed feature confirms the electrodes in KCl (0.1M) containing 5mM [Fe (CN)6]3-
the uniform pores are open and extend through the surface into using electrochemical impedance spectroscopy (EIS) and
the bulk which is believed to play important role in enzyme Cyclic voltammetry (CV) at scan rate 10 mV/s.
immobilization. Chit-CuO/Au bio electrode at higher
magnification images [B] surface shows nanosized distribution Electrochemical impedance spectroscopy (EIS)
of CuO nanoparticles provide high surface area of Electrochemical impedance spectroscopy (EIS) technique
nanostructured and polymer matrix which can lead greater measure impedance of electrode surface as a function of
amount of an immobilized enzyme on the surface. After frequency due to variation in the interfacial properties of the
immobilization of enzyme lipase image [C] and [D] which interface of the electrode. EIS is an effective and non
shows morphology and perfect immobilization, as shape destructive tool to investigate the process of modification with
changes from cotton globes like structure to a crystal like respective to enzyme on the surface of electrode.Nyquist plot
structure. As the structure has varied, the corresponding surface includes a semicircle region observed at higher frequency

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Chaudhari et. al Bioelectrode based chitosan-nano copper oxide for application to lipase biosensor

corresponding to electron transfer limited process on Z’ axes is Electron transfer kinetics parameter (Ke) of chit-nano CuO/Au
followed by straight line at 45°C two real axes at lower electrode and LIP/Chit-nano CuO/Au bioelectrode can be
frequency revealing the diffusion limited process. calculated from following equation,
RT
ke = …………. [1]
n 2 F 2 A Rct C
The complex impedance may be represented as a sum of real
Where, R is the gas constant (8.314Jmol-1k-1), T is temperature
(Z’) and imaginary (Z”) components that originate from the
(300 K), n is the number of electron transfer (1), F is faraday
resistance and capacitance of the cell including the ohmic
constant (96485 J mol-1K-1), A is area of electrode (1cm2) and
resistance of the solution (Rs), Warburg impedance (Zw),
C is the concentration of redox species in the electrolyte
capacity of electric double layer (Cdl) and surface electron
solution (5Mm), Heterogeneous electron transfer (Ke) values
resistance (RCT) [26] and it appears when the current flows
for chit-nano CuO/Au electrode and LIP/Chit-nano CuO/Au
through LCR circuit (equivalent to randle circuit).
bioelectrode found to be 0.00166 Cm.s-1 and 0.00115 Cm.s-1.
High value for bioelectrode shows indicating a faster electron
exchange between the redox species.

Cyclic voltammetry
In cyclic voltammetry, Current at the working electrode is
plotted versus the applied voltage (i.e., the working electrode's
potential) to give the cyclic voltammogram trace. Cyclic
voltammetry is generally used to study the electrochemical
properties of an analyte in solution. A standard CV experiment
employs a cell fitted with three electrodes: reference electrode,
working electrode, and the counter electrode. This combination
is sometimes referred to as a three-electrode setup, Ag/AgCl as
reference electrode, platinum as counter electrode, and
LIP/Chit-nano CuO/Au bioelectrode as working electrode. The
Electrolyte is usually added to the sample solution to ensure
sufficient conductivity. The solvent, electrolyte, and material
composition of the working electrode will determine the
potential range that can be accessed during the experiment.
Cyclic voltammogram of Chit-nano CuO/Au electrode and
LIP/Chit-nano CuO/Au bioelectrode have been recorded at
Fig. 7 Scanning electron microscopy of [A] Chit-CuO/ Au scan rate of 10mV/s using three electrode cell in a KCl solution
electrode [B] Lipase/Chit-CuO/Au bioelectrode containing K3[Fe(CN)6]3- as a mediator in potential range -0.2
to 0.6 V. Well defined Oxidation Reduction peaks are
From figure. 8[A], RCT value of electrode depends upon observed.
dielectric characteristics of the electrode/electrolyte interface.
The Figure shows Nyquist plot ,after spin coating of Chit- The Figure.8. [B] Shows cathodic peak current at 0.00166 mA,
nano-CuO onto the Au electrode, Rct value decreases to 32.13 0.00132mA for Chit-nano CuO/Au electrode and LIP/Chit-
KΩ,this decrease in Rct may be due to nanosized structure of nano CuO/Au bioelectrode. The enhancement of cathodic peak
nano CuO Particles that provide increased electro active current of Chit-nano CuO/Au electrode due to conducting
surface leading to higher rate of electron transfer moreover, nature of CuO nanoparticles and formation of a network
after immobilization of lipase onto Chit-nano CuO/Au between the positively charged CuO nanoparticles and the
electrode ,electron transfer by negatively charged redox marker hydroxylamine group of CH. After immobilization of enzyme
[Fe(CN)6]3- may perhaps be hindrance resulting in increased on nanoparticles the magnitude of current is found to be
Rct value 46.42 KΩ.

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Chaudhari et. al Bioelectrode based chitosan-nano copper oxide for application to lipase biosensor

decreased because of the inherent non conducting nature of ferro/ferri molecules in PBS electrolyte.
enzyme which acts as barriers for electron transport due to

Fig.8. [A] Electrochemical impedance (EIS) and [B] Cyclic voltammetry (CV) study of [a] CHIT-CuO/ Au electrode [b]
Lipase/CHIT-CuO/Au Bio electrode.

The CV investigation at various scan rate (10-70mV/S) have Ic (A) =0.003(mA.V-1.S-1) +0.004(mA)* scan rate (m V /s),
been performed for Lipase/Chit-CuO/Au bio electrode as R2=0.99………………(4)
shown in Figure 9.(A), indicating as we move towards higher Ia (A) = -0.264 (mA.V-1.S-1) – 0.027(mA)* scan rate (mV /s),
scan rate peak current increases. The cathodic and anodic peak R2=0.99…………… (5)
current found to be proportional to the square root of scan rate
over the range (10-70mV/S) suggesting that it has controlled The diffusion coefficient value (D) of redox probe [Fe(CN)6]3-
/4
diffusion process (Figure 9.[B]), inset the figure (Figure 7.A), - From electrolytic solution to corresponding to electrode
Linear sweep voltammetry of cathodic peak was carried out it surface has been calculated using Randles Sevcik equation [27]
perfectly indicate the current is proportional to current with which is given as,
increases in scan rate Figure 9. [C]), Peak current of IP = 2.69 ∗ 105 n3/2 . A. D1/2 . C. V ………… (6)
bioelectrode proportional to scan rate according to equation (2) Where, Ip is peak current of corresponding electrode, n is
and (3). number of electron involved or electron stiochiometry (1), A is
Where, R is the gas constant (8.314Jmol-1k-1), T is temperature surface area of electrode (1cm2), C is concentration (5Mm),v is
(300 K), n is the number of electron transfer (1), F is faraday scan rate (10 mV/s). The D value of Chit-nano CuO/Au
constant (96485 J mol-1K-1), A is area of electrode (1cm2) and electrode and LIP/Chit-nano CuO/Au bioelectrode was found
C is the concentration of redox species in the electrolyte to be 1.536*10-12 and 0.980*10-12 cm2s-1 respectively. The
solution (5Mm), Heterogeneous electron transfer (Ke) values higher value of D for Chit-nano CuO/Au electrode may have
for chit-nano CuO/Au electrode and LIP/Chit-nano CuO/Au been due to better conductive nature of electrode compared to
bioelectrode found to be 0.00166 Cm.s-1 and 0.00115 Cm.s-1. that of LIP/Chit-nano CuO/Au bioelectrode. The surface
High value for bioelectrode shows indicating a faster electron concentration of Lipase/Chit-CuO/Au bio electrode (1.55×10-
3
exchange between the redox species. mol/cm2) estimated from plot of Ip vs. Scan rate (ῠ1/2)
Ic= 0.00166mA (mV/s) * Scan rate………….. (2) n 2 F2 I∗ A V
IPC = …………………………. (7)
4RT
Ia = 0.00132mA (mV/s) * Scan rate…………. (3)
The reversibility of electron transfer kinetics did not depend on
V; it also depends on standard heterogeneous electron transfer
Redox peak current show linear behaviour with square root of
rate constant (ks) with Laviron model as given in equation.1.
scan rate ((Figure 9.B) revelling diffusion controlled process
Ks = mnFv/RT ………………………… (8)
and follow equation (4) and (5)

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Chaudhari et. al Bioelectrode based chitosan-nano copper oxide for application to lipase biosensor

Where, m is peak to peak separation of potential (V). The transfer between the electrode surface and redox species. The
estimated value of Ks for Chit-nano CuO/Au electrode and value of Ks found to be 5.327 and 5.227 S-1
LIP/Chit-nano CuO/Au bioelectrode indicated a fast electron

Fig.9.[A] Cyclic voltamogram of Lipase/Chit-CuO/Au Bio electrode at different scan rate (10-70 mV/S) and Linear sweep
voltammetry inside [B]Redox current with respect to square root of scan rate of (10-70mV/s) [C] Linear sweep voltammetry of
Cathodic peak current of Lipase/Chit-CuO/Au Bio-electrode(inset figure. [A] at scan rate (10-70mV/s)

Biosensing studies verses Current. The obtained sensitivity is much higher

Figure.10. [A] represent the CV studies of Lipase/CHIT- compared to other [28], with regression Coefficient (R2=0.99)
CuO/Au bio electrode carried out at different concentration of response time was found to be 2s revels the enhanced catalytic
tributyrin (0.25-1.50mg/Ml) immersing the electrolyte in KCl activity of enzyme on chit-nano CuO/Au electrode due to high
(0.1M) containing [Fe (CN)6]3-/4-.it has been observed that there charge transfer property of matrix. Detection limit (LOD)
is a linear increase in the peak current with increase in analyte calculated for linear region using expression 3ϭ/sensitivity [29] is
concentration indicating an increased in the charge transfer to found to be 0.13 mg/mL where standard deviation for blank.
the working electrode figure 10[B]. The mechanism involved The shelf life of bioelctrode has been monitored by measuring
in the detection of triglyceride using of Lipase/CHIT-CuO/Au the electrochemical current response with regular interval of
bio electrode is shown in the figure.10[C]. LIP helps in the one week; it is observed that current response decreases about
hydrolysis of tributyrin (triglyceride) which results in the 4weeks. The reveal an affinity of enzyme (Lipase) for the
production of fatty acid along with glycerol molecule. substrate (tributyrin), enzyme substrate kinetics parameters has
Sensitivity of prepared. Lipase/CHIT-CuO/Au bio electrode been observed. The value of apparent Michaelis-Menten
towards tributyrin is found to be 0.0006 mA (mg/mL) which is constant (Km) for Lipase/CHIT-CuO/Au bio electrode
calculated from the slope of the plot of tributyrin concentration calculated by using Line weaver–Burke plot (1/I versus 1/[C])

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Chaudhari et. al Bioelectrode based chitosan-nano copper oxide for application to lipase biosensor

and Km value has been found to be 1.76 mg/mL.This low value the low value of km indicates enhanced affinity of lipase from
shows that a strong affinity between enzyme and substrate and Aspergillus Niger towards tributyrin.

Fig.10 [A] Biosensing study of Lipase/CHIT-CuO/Au Bio electrode as a function of tributyrin concentration[0.25-1.50mg/mL [B]
Calibration curve between current response and different concentration of tributyrin in KCl containing 5Mm [Fe(CN)6]3-[C]
mechanism involved in the detection of triglyceride using of Lipase/CHIT-CuO/Au bio electrode

CONCLUSION of 0.0006 mA/mg mL with linear regression coefficient as 0.94

CuO nanoparticles (45 nm) successfully synthesized by Sol-gel and standard deviation as 0.0004 mA/mg mL−1. The low Km
citrate method and characterized by, X-Ray diffraction value obtained as 1.76 mg/mL indicates high affinity of
(XRD), Raman spectra, Fourier transform spectroscopy Lipase/Chit-CuO/Au bio electrode bioelectrode for tributyrin
(FTIR), UV spectra, and Scanning electron microscopy
(SEM).A successful immobilization of enzyme [Lipase] on the ACKNOWLEDGEMENT
functionalized surface of Chitosan-CuO /Au electrode via We gratefully acknowledge for financial support from the
physical adsorption was accomplished. Scanning electron Major Research project (MRP) [No.43-224/2014(SR)],
microscopy of Chit-CuO/Au electrode and Lipase/Chit- University Grant Commission (UGC), New Delhi, and also
CuO/Au bio electrode shows perfect immobilization of enzyme Principal, Shri Shivaji Science College Amravati his kind
(Lipase).The electrochemical studies revealed that well defined cooperation during this research work
Oxidation Reduction peaks are observed and also
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