ORIGINAL PAPER

Cobalt oxide magnetic nanoparticles–chitosan nanocomposite basedelectrochemical urea biosensor

A Ali1,2, M Israr-Qadir1, Z Wazir2*, M Tufail2, Z H Ibupoto1, S Jamil-Rana1, M Atif3,4,

S A Khan5 and M Willander1

1Department of Science and Technology, Linkoping University, Norrkoping, Sweden

2Department of Basic Sciences, Riphah International University, Islamabad, Pakistan

3Physics and Astronomy Department, King Saud University, Riyadh, Saudi Arabia

4National Institute of Lasers and Optronics, Islamabad, Pakistan

5National Centre for Physics, Islamabad, Pakistan

Received: 05 June 2014 / Accepted: 29 August 2014

Abstract: In this study, a potentiometric urea biosensor has been fabricated on glass filter paper through the immobi-

lization of urease enzyme onto chitosan/cobalt oxide (CS/Co3O4) nanocomposite. A copper wire with diameter of 500 lm

is attached with nanoparticles to extract the voltage output signal. The shape and dimensions of Co3O4 magnetic nano-

particles are investigated by scanning electron microscopy and the average diameter is approximately 80–100 nm.

Structural quality of Co3O4 nanoparticles is confirmed from X-ray powder diffraction measurements, while the Raman

spectroscopy has been used to understand the chemical bonding between different atoms. The magnetic measurement has

confirmed that Co3O4 nanoparticles show ferromagnetic behavior, which could be attributed to the uncompensated surface

spins and/or finite size effects. The ferromagnetic order of Co3O4 nanoparticles is raised with increasing the decomposition

temperature. A physical adsorption method is adopted to immobilize the surface of CS/Co3O4 nanocomposite. Potentio-

metric sensitivity curve has been measured over the concentration range between 1 9 10-4 and 8 9 10-2 M of urea

electrolyte solution revealing that the fabricated biosensor holds good sensing ability with a linear slope curve of *45 mV/

decade. In addition, the presented biosensor shows good reusability, selectivity, reproducibility and resistance against

interferers along with the stable output response of *12 s.

Keywords: Potentiometric biosensors; Metal oxide; Nanoparticles and urea sensing

PACS Nos.: 87.85.fk; 81.07.Nb; 07.55.Db; 75.50.Xx

1. Introduction

Magnetic nanoparticles based research is playing a tre-

mendous role in the diagnostic, therapeutic as well as for the

clinical applications. These nanoparticles are also used for

microbial, anti-microbial treatments due to their high

quantum yield. Cobalt oxide is p-type semiconducting

material with a direct band gap of *2.4 eV. The nano-

particles of Co3O4 exhibit excellent properties and are used

widely in rechargeable lithium ion batteries, gas sensors,

adsorbent, ceramics, electro-chromic devices and drug

delivery [1–7].The Co3O4 magnetic nanoparticles are not

only used as electro-catalyst for oxidation of H2O2 and As

[8, 9] but also as a support for immobilization of haemo-

globin and heterogeneous catalysis [10, 11]. Immobilization

of enzymes onto the solid electrode surfaces is a key step to

design, fabricate and enhance the performance of biosen-

sors. The immobilized solid surfaces with highly smaller

amount of enzymes can retain almost similar activity for a

longer period of time. The urea estimation is very important

in monitoring the kidney functions as well as disorders

associated with it. Moreover, nature contains huge amount

of widely distributed urea, its analysis is important not only*Corresponding author, E-mail: zafar_wazir@yahoo.com

Indian J Phys

DOI 10.1007/s12648-014-0594-3

� 2014 IACS

in the clinical and agricultural chemistry but also as non-

protein nitrogen in cow milk [12–14]. The normal physio-

logical level of blood serum urea is 15–40 mg/dl [15]. The

higher levels of blood urea and urine can increase the risk of

kidney failure, urinary tract obstruction and loss of water,

shocks, burns and gastrointestinal bleeding. Beside higher

levels of blood urea and urine, the lower levels are also

responsible for hepatic failure, cachexia and nephritic

syndrome. Detection of blood urea concentration in clinical

laboratories is vital for routine analysis.

Numerous urea biosensors have been developed through

the immobilization of urease on various solid state matrices

because urease plays an important role in the enzymatic

biosensors to fulfill the demand of urea detection. The most

widely investigated methods for urea determination are

colorimetric and spectrometric in the literature [16, 17],

where the urea concentrations have been estimated utilizing

NH4? or HCO3- as sensitive electrodes [18–20]. Some

other results about the preparation of urea biosensors using

composite of graphene lipid membranes and bi-layer lipid

membranes have also been reported recently [21, 22].

However, it is still highly desirable to introduce new

exciting materials for the enhancement of the working

activity of the biosensors using a facile and easy way to

design the enzymatic biosensors, which can attain the

higher bio-catalytic activity. Now days, nanoscale materials

have attracted a huge attention in order to develop the

nanodevices in the recognition of bioactive stuff in the

fields of biological, medical and electronic applications

[23–25]. To date, a number of metal oxide nanoparticles;

such as, manganese oxide, zirconium oxide, titanium oxide,

cerium oxide, zinc oxide, tin oxide, tungsten oxide, iridium

oxide, nickel oxide and iron oxide have been employed for

the fabrication of the amperometric biosensors [26–29]. The

present study reveals structural, morphological character-

izations of Co3O4 magnetic nanoparticles and their use for

the miniaturization of electrochemical urea biosensor uti-

lizing facile fabrication method. The potentiometric urea

sensing measurements have been performed through a

simple two electrode experimental set-up [30, 31].

2. Experimental details

Urease (E.C.3.5.1.5 from jack Bean 100 l/mg) and urea

(ACS reagent 99.9 %) were commercially purchased from

Sigma Aldrich. Phosphate buffer solution (PBS) of 10 mM

concentration was prepared from Na2HPO4 and KH2PO4

(Sigma Aldrich) with sodium chloride concentration of

0.1315 mM and pH was adjusted to 7.4. A stock solution of

100 mM of urea was prepared in BPS. A standard low

concentration solution of urea was prepared prior to the

measurements. This suspension was dispensed on a copper

wire mounted on a glass fiber filter. For the fabrication of a

CS modified electrode based on Co3O4 magnetic nano-

particles, following steps were followed. The CS sol–gel

was prepared in 1 % acetic acid and 1 M hydrochloric acid

(HCl) solutions and kept on stirring for 24 h. Cobalt oxide

magnetic nanoparticles were mixed with deionized water

and stirred for 1 h. Finally, the Co3O4 magnetic nanopar-

ticles were suspended in the sol–gel of CS. The Co3O4

magnetic nanoparticles based biosensing electrode was

prepared by drop-wise dispersion of sol–gel solution on the

suspended copper wire. Then the Co3O4 magnetic nano-

particles based electrode was immobilized with urease

enzyme using simple physical adsorption method. The

urease enzyme solution was prepared in PBS of pH 7.4

with activity of 2 mg/ml. The cell assembly consisted of

the following elements: the immobilized CS/Co3O4 nano-

composite based biosensing electrode as working electrode

and Ag/AgCl as a reference electrode.

A pH meter (Model 215, Denver Instrument) was used

to measure the potentiometric output voltage in the

experiments. The particle size of the as-synthesized Co3O4

magnetic nanoparticles was recorded using high resolution

field-emission scanning electron microscope (FESEM)

(JEOL JSM-6301F). The structure of the said magnetic

nanoparticles was checked by X-ray diffraction (XRD)

using X-ray diffractometer [(Bragg–Brentano) h–2h dif-

fractometer]. To confirm the main chemical composition of

the said magnetic nanoparticles, Raman spectroscopy by a

Shamrock SR-303i-A from Andor Technology, USA was

used. The magnetic properties of Co3O4 nanoparticles was

measured by using Vibrating sample magnetometer (VSM)

model Lake Shore, new 7400 series, USA.

3. Results and discussion

It can be seen from FESEM image shown in Fig. 1 that as-

grown nanoparticles are of regular shape and their average

particle size is in the range of 80–100 nm, representing

better morphological aspects compared to previously

reported results [32–36]. Figure 2 shows a representative

X-rays powder diffraction (XRD) pattern showing all dif-

fraction peaks of the Co3O4 magnetic nanoparticle sample.

The XRD pattern can be readily indexed to the cubic spinel

Co3O4 having the lattice parameter a = 8.072 A and

revealing high consistency with the JCPDS card No.

76-1802. It can be clearly observed from the XRD dif-

fraction pattern that synthesized Co3O4 nanoparticles have

high purity and good crystal quality. Moreover, broadening

of the diffraction peaks confirms the small particle size and

high crystalline nature of the material. Figure 3 shows the

results of the Raman scattering measurements, which is a

highly sensitive tool for microstructural investigation of

A Ali et al.

nanostructures. The three Raman peaks located at around

450, 500 and 700 cm-1 positions correspond to the active

modes i.e. A1g, Eg and F2g, which confirm the nano-

crystalline nature of the Co3O4 magnetic nanoparticles as

well as the presence of chains of covalent bonding.

Moreover, the purity level of the presented Co3O4 nano-

particles is highly consistent with the previously reported

results for pure Co3O4 nanomaterial [37].

The magnetic properties of Co3O4 nanoparticles prepared

at temperature 200 �C have been measured at same tem-

perature. As shown in Fig. 4, the magnetization curve for the

Co3O4 nanoparticles display higher ferromagnetic properties

with saturation magnetization value of 0.37 emu g-1 at the

applied field of 15 kOe. The measurement has been con-

ducted on a bulk sample in order to prove the ferromagnetic

behavior of the nanoparticles [38]. The explanation of fer-

romagnetic behavior of the nanoparticles is as follows: bulk

Co3O4 has a normal spinel structure with antiferromagnetic

exchange between ions, which occupy the tetrahedral and

octahedral sites [39]. The experimental results show zero net

magnetization to the complete compensation of sublattice

magnetizations. Therefore this change from an antiferro-

magnetic state for bulk Co3O4 to a weakly ferromagnetic

state for the Co3O4 nanoparticles is attributed due to the

uncompensated surface spins and/or finite size effects [39–

42]. Hence the magnetic properties of Co3O4 nanoparticles

are strongly dependent on size and the shape of their parti-

cles, magnetization direction and crystallinity. Raman

spectroscopy is used to characterize materials and find the

crystallographic orientation of a sample. It typically employs

laser as a source of monochromatic light, which is scattered

by phonons or other excitations from the system. This results

in a shift in the energy of the laser phonons, which in turn

yields information specifically about the chemical bonds and

the symmetry of molecules.

Figure 5 shows the schematic illustration to carry out

potentiometric urea biosensing measurements, where

working electrode (urea sensing bioelectrode) comprises

Co3O4 nanoparticle, urease and Cu wire along with the

working electrode of Ag/AgCl, used as reference

electrode.

Fig. 1 FESEM image of the nanocrystalline cobalt oxide

Fig. 2 XRD pattern of cobalt oxide nanoparticles

Fig. 3 Raman spectrum of Raman shift showing various active

modes

-15000 -10000 -5000 0 5000 10000 15000

-0.45

-0.30

-0.15

0.00

0.15

0.30

0.45

Mag

netiz

atio

n (e

mu/

g)

Applied field (Oe)

Fig. 4 M–H loop for Co3O4 magnetic nanoparticle

Cobalt oxide magnetic nanoparticles–chitosan nanocomposite

The detection principle of urea molecules is based on

hydrolysis of urea catalyzed by urease, which releases the

ammonia NH3 and CO2 gases as product of the chemical

reaction, [43] as given below:

NH2ð Þ2CO + H2O! 2NH3 þ CO2 ð1Þ

The generation of electromotive force (EMF) response

laid behind the reaction between ammonia and water

through acid–base chemistry, which results into production

of the charged ions in the urea test solution. Referring to

Eq. (1) the acid base chemistry is expressed below. In the

water:

NH3 þ H2O$ NHþ4 þ OH� ð2Þ

CO2 þ H2O$ H2CO3 ð3Þ

H2CO3 þ H2O$ H2CO3 þ H3Oþ ð4Þ

CO2 reacts with water to form weak acid so CO2 is

acidic in nature. NH3 reacts with water to form NH4? ions

and is basic due to presence of OH- ions. Chitosan due to

presence of less –NH2 (amine) group, (depending upon

degree of deacetylation) is acidic and act as selective layer

for NH3 ions. So, easier interaction of acid and base occurs.

A rapid hydrolysis reaction of the urea into ammonia and

carbon dioxide takes place with urease immobilized nano-

surfaces of the CS/Co3O4 nanocomposite mounted on glass

fiber filter with copper wire, when the working electrode

encounters with the urea test solution. According to the

mechanism mentioned above, the increase in EMF values

could be accredited to the accumulation of ammonium ions

on the surface of the working electrode because the potential

value of the reference electrode (Ag/AgCl) has a constant

value of 222.34 mV at room temperature as reported earlier

[44]. The potentiometric EMF response is measured for the

wide range of urea concentrations varying from 1 9 10-4 to

8 9 10-2 M as shown in Fig. 6. It has been noticed that the

sensitivity curve reveals a linear rapid increase in the sen-

sitivity of the biosensor with the elevated concentrations of

urea molecules in the urea test solution and a linear rela-

tionship for the sensitivity (45 mV/decade) against the log-

arithmic values of urea concentration has been achieved

from urease immobilized Co3O4 nanocomposite.

The physiological investigations related to the stability

and performance of the biosensor in the acidic, basic and

neutral pH environments have been tested for the urea test

solutions. The pH range has been selected from 3 to 11 and

the maximum sensitivity value from the presented bio-

sensor based on Co3O4 nanocomposite has been achieved

at around physiological pH value of 7 as shown in Fig. 7. It

Fig. 5 Schematic illustration of urea sensing bioelectrode comprised

of Co3O4 nanoparticle, urease, and Cu wire along with reference

electrode

Fig. 6 Sensitivity response curve measured for logarithmic concen-

tration range from 0.1 to 80 mM

Fig. 7 Sensitivity response of the biosensor measured at the pH

values range of 3–11

A Ali et al.

is well known that extremely high or low pH values result

in complete loss of activity of urease. However, the reac-

tivity of urease shows maximum activity and stability at pH

7, which further leads towards the rapid hydrolysis of urea.

Moreover, the selectivity of the presented biosensing

electrode is inspected through the addition of variety of

interferers’ e.g. uric acid and glucose etc., to the urea test

solution. The measured EMF values from working elec-

trode are independent from the volume of urea electrolyte

solution utilized for the experiments and from the area of

working electrode dipped for the detection of urea mole-

cules. A series of consecutive experiments have been

performed over the period of couple of weeks for the

detected range of urea concentrations and it is observed

that the fabricated biosensing electrode shows good

reproducibility and repeatability with almost similar EMF

response values. Figure 8 reveals that the fabricated bio-

sensing electrode holds quick stable response of *12 s for

the urea electrolyte solution with the concentration of

40 mM. Moreover, the chemical reaction activity of the

fabricated biosensor is investigated for the period of

approximately 1 month and it has been verified that bio-

sensing electrode reserves *85 % of its initial capacity

reflecting a strong binding and compatibility between

urease enzyme and Co3O4 nanocomposite.

4. Conclusions

A potentiometric biosensor based on CS/Co3O4 nanocom-

posite has been developed with the conjugation of urease

enzyme by utilizing physical adsorption method. The FE-

SEM, XRD and Raman spectroscopic measurements show

the nanoscale dimensions, pure crystalline nature and

existence of the covalent bonding in the material. The

magnetic measurement of Co3O4 nanoparticles reveals the

ferromagnetic behavior, which is attributed due to the

uncompensated surface spins and/or finite size effects. It

has been found that by increasing the decomposition tem-

perature the ferromagnetic order of the Co3O4 nanoparti-

cles is increased. The presented biosensor shows

substantial advantages; i.e. eco-friendly, facile, highly

sensitive and selective and reproducible; in addition, the

EMF response is independent from the influence of the

other interferers introduced in the solution. The parameters,

e.g. small size and large surface area of CS/Co3O4 nano-

composites play a paramount role in order to produce

excellent micro-environmental conditions to detect the urea

molecules. Moreover, the physiological investigations

show that the prepared biosensor works really well around

neutral biological pH values.

Acknowledgments We are thankful to International Research

Support Initiative Programme (IRSIP) offered by Higher Education

Commission of Pakistan and Riphah Academy of Research &Edu-

cation (RARE), Riphah International University, Islamabad, Pakistan.

This project is supported by King Saud University, Deanship of

Scientific Research, College of Science Research Center.

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