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Fabrication and evaluation of evanescent wave absorption based polyaniline-

cladding modified fiber optic urea biosensor

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DOI: 10.1016/j.yofte.2017.11.002

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 Optical Fiber Technology 40 (2018) 8–12

Contents lists available at ScienceDirect

Optical Fiber Technology

journal homepage: www.elsevier.com/locate/yofte

Regular Articles

Fabrication and evaluation of evanescent wave absorption based MARK

polyaniline-cladding modified fiber optic urea biosensor
⁎
S.N. Botewad, V.G. Pahurkar, G.G. Muley
Department of Physics, Sant Gadge Baba Amravati University, Amravati, Maharashtra 444602, India

A R T I C L E I N F O A B S T R A C T

Keywords: The fabrication and study of cladding modified intrinsic fiber optic urea biosensor has been reported in the
Optical fiber present investigation. A simple cladding modification technique was used to construct the sensor by uncladding
Biosensor the small portion from optical fiber. Further bare core was decorated by supportive porous, chemically and
Polyaniline optically sensitive matrix material polyaniline (PANI) as an active cladding for enzyme residency. Enzyme-
Cladding modification
urease (Urs) was cross-linked on the active cladding region via glutaraldehyde solution. Confirmation of the
Urease
prepared PANI in proper form determined by ultraviolet-visible and Fourier transform infrared spectroscopic
Cross-linking
techniques. X-ray diffraction technique was employed for nature and compatibility examination of PANI. Sensor
parameters such as sensitivity, selectivity, stability and lower detection limit have been analyzed by absorption
variation study in evanescent wave field. The response of prepared sensor was studied towards urea in the wide
concentration range 100 nM–100 mM and confirmed its lowest detection limit as 100 nM. The stability of sensor
was found 28 days with little variation in response. The fabricated sensor has not shown any response towards
interference species like glucose, ascorbic acid, L-alanine, L-arginine and their combination with urea solution
and hence found selective for urea solution only.

1. Introduction related to the urea biosensors have been cumulatively reviewed by

A simple, rapid, reliable, reproducible, sensitive and handheld de-
vice for urea estimation is in demand for medical as well as other
various fields because widespread existence of urea everywhere [1,2].
Increase or decrease in the permissible level of urea concentration in
human body causes various dangerous diseases such as congestive
heart, urinary tract obstruction, gastrointestinal disorders, chronic or
acute renal failure, burns, dehydration, starvation, shock, malnutrition,
little dietary protein in the diet, liver diseases, hepatic failure, nephritic
syndrome and cachexia [3,4]. In 1969 Guilbault et al. [5] were in-
troduced a potentiometric enzyme electrode biosensor first time for
urea determination. Nevertheless, environmental monitoring, health
diagnosis, agriculture, food science, fishery industry, milk industry are
waiting for a precise, fast and affordable instrument for urea detection
[6–9]. For the fabrication of a biosensor, the deposition of enzyme on
the electrode surface can improve the sensitivity, stability, response
time and selectivity of biosensor. It can be achieved by selecting a
specific immobilization technique [10]. Urea biosensors utilize enzyme-
Urease (Urs) as the recognition element and reported as a highly effi-
cient catalyst for the hydrolysis of urea, with a rate approximately 1014
times the rate of the non-catalyzed reaction [11]. Various useful aspects
Dhawan et al. [12] and Singh et al. [13]. Amperometric [14–18], po-
tentiometric [19–21], conductometric [22–24], colorimetric [25,26]
and optical [27–29] biosensors are useful for the urea determination.
The optical fiber based sensors are found more prominent, effective
and convenient for chemical and biosensing purpose. Small and com-
pact size, highly sensitive, reliable, fast response toward analyte, ability
to be multiplexed, real time and parallel detection, remote sensing
capability, immunity to electromagnetic interference, non-conducting
and intrinsically safe for patients, etc., features make optical fiber
sensor as an excellent candidate for biosensing [30,31]. Between two
main extrinsic and intrinsic fiber optic types of sensors, cladding
modified intrinsic fiber optic sensors are more advantageous because of
their large dynamic range, high sensitivity, superior integration into
other structures as well as convenient to prepare [32–35]. For the
cladding modification, conducting polymers (CPs) such as polyaniline
(PANI), polypyrrole, polythiophene and polyacetylene are being widely
used because of their ability to provide stable and porous matrix for the
immobilization of biomolecules. Moreover, catalytic reaction of en-
zyme-biomolecules on the sensing surface causes a change in redox and
protonation state of CPs which directly useful for sensing response. This
catalytic reaction may cause change in the microenvironment,

⁎
Corresponding author.
E-mail address: gajananmuley@sgbau.ac.in (G.G. Muley).

http://dx.doi.org/10.1016/j.yofte.2017.11.002
Received 16 November 2016; Received in revised form 19 March 2017; Accepted 5 November 2017
1068-5200/ © 2017 Elsevier Inc. All rights reserved.
S.N. Botewad et al.
surrounding the sensing element and triggers the sensing mechanism
[36,37]. PANI has interesting electrochemical, electronic, optical,
electro-optical as well as thermal and environmental properties for
biosensor applications. It acts as an effective mediator for electron
transfer in chemical and biochemical reactions. It makes a favorable
porous suitable matrix for immobilization of biomolecules and easy to
synthesize [37].
Present paper reports the fabrication of cladding modified fiber
optic intrinsic urea biosensor (FOIUB) and its sensing response study.
The cladding modification was achieved by chemically sensitive PANI
matrix. Further enzyme-Urs was immobilized onto the PANI supporting
matrix via cross-linking technique. Prepared biosensor offers relatively
long self life, broad detection range, high sensitivity and specific se-
lectivity towards urea. In the present paper we are reporting con-
siderably lowest detection of urea as 100 nM.
2. Experimental
2.1. Materials and methods
For the PANI synthesis, aniline (monomer) and ferric chloride
(oxidant) were purchased from Fisher Scientific, USA. The enzyme-Urs
from jack beans, with activity 380 unit’s mg−1, was procured from
Sisco Research Laboratories (SRL), India. Analyte-urea, cross-linking
agent glutaraldehyde solution (25%), glucose, ascorbic acid, L-arginine
and L-alanine were purchased from sdFine chemicals, India. For the
preparation of buffer solution, potassium dihydrogen orthophosphate
and sodium hydroxide were also purchased from sdFine chemicals,
India. All the synthesis processes were carried out in freshly prepared
double distilled water and in 0.1 M phosphate buffer solution (pH 7.4).
All supplementary chemicals were of analytical grades and used as re-
ceived without any further purification. The stock solutions of Urs in
proportion 1 mg/3ml and urea were prepared in phosphate buffer (pH
7.4) and kept at temperature 4 °C for 24 h before use. The different
concentrations of urea solution were freshly prepared at each time in
the phosphate buffer of pH 7.4.
2.2. Characterizations
Deposited PANI film was characterized by powder X-ray dif-
fractometer (XRD) (Mini Flex II, Rigaku, Japan) with CuKα radiations
of wavelength 1.5406 Å to identify its morphology. The functional
groups were confirmed by Fourier transform infrared spectroscopy (FT-
IR) using α-ATR-IR-spectrophotometer (Brucker, Japan). Ultraviolet-
visible (UV-vis) portable spectrophotometer BLACK-Comet-SR (Stellar
Net, USA) in the spectral range 200–1100 nm was used for recording
UV-vis spectrum of synthesized film as well as for the examination of
sensing response of prepared sensor.
2.3. Cladding modification
Multimode optical fiber (40 cm long) of core/cladding dimensions
530/500 μm (1030 μm) of plastic cladded silica core was used for sensor
fabrication. The SMA905 connectors were connected to both the ends of
optical fiber using adhesive. Afterward both ends were furnished as
well as polished using the fine polish papers of 1200 and 0.3 μm
roughness respectively. Furnishing and polishing helps for enhancing
the light gathering capacity of optical fiber [30]. Further, 2 cm cladding
portion was removed from optical fiber with the help of stripper and
surgical blade. Bare core surface was gently cleaned with hydrofluoric
acid (HF) and distilled water. The active cladding was achieved by
smoothly depositing uniform layer of PANI and kept it to settle.
Synthesis of PANI was carried out by simple oxidative polymerization
method using 0.2 M aniline monomer solution and 0.05 M FeCl3 oxi-
dant solution in 30 min reaction time. After dried the modified portion,
it cleans with double distilled water to prepare the hydrophobic base
9
Optical Fiber Technology 40 (2018) 8–12
Fig. 1. UV-vis spectrum of PANI matrix.
for enzyme-Urs immobilization. Then using 1% glutaraldehyde solution
as a homo-bi-functional cross-linking agent enzyme-Urs were im-
mobilized on PANI modified sensing element. Sensing element was
dried for 30 min at room temperature in clean environment and washed
2–3 times with phosphate buffer solution before it was used for sensing
experiment. For unwanted effect of external light on the recorded
sensing response, the experiments were carried out in dark room. The
experimental arrangement adopted is reported in our previous article
[38].
3. Results and discussion
3.1. UV-vis study
Fig. 1 shows the UV-vis spectrum of PANI film used to deposit as an
active cladding of sensing element. The spectrum elaborates the dif-
ferent oxidation states of PANI. Three oxidation states occurs in PANI
viz. reduced form leucoemeraldine, fully oxidized pernigraniline state
and half oxidized emeraldine state. For the three different oxidation
states in PANI, the nitrogen atom is responsible [39]. In present in-
vestigation PANI shows two characteristic peaks at ∼227 and 280 nm.
The absorption peaks below 300 nm correspond to π–π∗ transitions in
the benzenoid ring distorted by the presence of amine groups which is
characteristic of the leucoemeraldine form and pernigraniline form of
PANI (π–π∗ transition in the quinoid ring) [40]. These two peaks ensure
the synthesized PANI matrix is in proper form.
3.2. FT-IR study
Fig. 2 pictures the FT-IR spectrum of PANI along with its functional
group frequencies and shows all the main characteristic peaks of PANI.
The absorption peak at 713 cm−1 corresponds to the mono substitution
of benzene ring and 1095 cm−1 stands for the CeH plane bending vi-
bration [41]. The absorption peaks at 1237 and 1338 cm−1 may con-
firms the CeN stretching of primary aromatic amines. The strong ab-
sorption peak at 1720 cm−1 can be assigned to C]NH stretching. The
peak at 1961 cm−1 may be attributed to the CeN bending and peak at
2103 cm−1 is of aromatic isonitrile N^C stretching. The absorption
peak at 2363 cm−1 may be due to aliphatic nitrile C^N stretching. The
absorption peaks at 2936 and 3067 cm−1 may corresponds to NeH
stretching with hydrogen bonded amino groups and free OeH
stretching vibration respectively. The absorption peak at 3429 cm−1
may be assigned to asymmetrical and symmetrical stretching vibrations
of NH2 group [42]. Thus, the FT-IR spectrum shows all expected
S.N. Botewad et al.
Fig. 2. FT-IR spectrum of PANI matrix.
functional groups of PANI and confirms the formation of PANI film.
3.3. XRD analysis
The typical diffuse XRD pattern of PANI is shown in Fig. 3. There is
no one characteristic peak of used oxidant is present. Although, as is
evident in PANI, broad diffraction peaks occur between 20 and 40° due
to the parallel and perpendicular periodicity of the polymer (PANI)
chain. In between the PANI peak diffracted at various angles in XRD
pattern shows low crystallinity or amorphous nature of the conductive
polymer-PANI due to the repetition of benzenoid and quinoid rings in
its chains [43]. Such amorphous nature of PANI deposited on optical
fiber core made porous surface and permits the enzyme immobilization
on the sensing element with high enzyme storing capacity.
3.4. Sensor response
3.4.1. Sensitivity in the form of absorbance
Analyte-urea in solutions with concentrations ranging from 100 nM
Fig. 3. XRD pattern of PANI matrix.
10
Optical Fiber Technology 40 (2018) 8–12
Fig. 4a. Sensing response in terms of absorption for 100 nM–100 mM urea solutions.
to 100 mM limits were used to study the sensing response of prepared
FOIUB. The response of the sensor was observed in the form of absor-
bance between the spectral range 245–260 nm as shown in Fig. 4a. The
exposure time for each concentration of urea solution was ∼120 s
throughout the experiment. The sensor shows absorbance at ∼250 nm
in increasing order with increase in concentration of urea solutions
prepared all in phosphate buffer of pH 7.4. Basically spectra of pure
urea and phosphate buffer solution of pH 7.4 are reported as optically
transparent with lower cutoffs at ∼205 and 200 nm, respectively [44].
Moreover, the absorption spectrum of pure PANI matrix in Fig. 1 shows
the absorption peaks at ∼227 and 280 nm. Here, when various con-
centrations of urea exposed to FOIUB’s sensing element, it shows the
absorption peaks at ∼250 nm. Thus, the peak at ∼250 nm may be at-
tributed due to the combine effect of PANI matrix and a cultivated
product from enzyme-Urs and urea reaction around sensing portion.
The reaction around modified cladding surface of FOIUB may enforce
π–π∗ transitions and leads to increase in absorption at ∼250 nm with
increasing concentration of urea [39]. From the results it can con-
veniently be said that the prepared biosensor exhibits excellent re-
sponse towards urea solutions ranging from 100 nM to 100 mM with
very good result reproducibility. Fig. 4b represents the variation in
Fig. 4b. Variation in absorbance for various urea concentrations measured at ∼250 nm.
S.N. Botewad et al. Optical Fiber Technology 40 (2018) 8–12

Fig. 5. (a) Absorbance spectra of interfering species on UV-vis spectrophotometer, (b) response towards interfering species using sensor.

absorption at a wavelength ∼250 nm for urea solutions of used con-

centrations. It clears that the variation in the response is not linear, but
it follows exponential associate fit to the original experimental data.
However, the lowest detection limit of the present sensor can indirectly
be considered as 100 nM. The sensing reaction (hydrolysis) takes place
between enzyme-Urs and analyte-urea of various concentrations at the
sensing element portion can be expressed in Eq. (1) [36] as;
Urease
NH2 CONH2 (Urea) + 2H2 O+ H+⎯⎯⎯⎯⎯⎯→2NH+4 + HCO−3 (1)

3.4.2. Selectivity
Fig. 5(a) and (b) shows the UV-vis absorbance spectra and se-
lectivity measurement of prepared FOIUB toward interfering species-
glucose, ascorbic acid, L-alanine, L-arginine and their combination with
urea prepared in pH 7.4 phosphate buffer solution [45] and each of
them has the concentration 1 mM. Fig. 5(a) shows all the interfering
species including urea are near about transparent in nature. Likewise,
from the absorbance spectra of FOIUB in Fig. 5(b), it was identified that
there was no any response of the sensor for glucose, ascorbic acid, L-
alanine, L-argenine and their combine solutions with urea. But the
sensor shows the huge response in case of urea solution centered at Fig. 6. Stability curve measurement for 100 mM urea solution.

∼250 nm. While a little change due other interfering species may be
due the change in refractive index of sensing element portion [46] and
in case of combine solution, the response shows the presence of urea in
it. Therefore it can be concluded that the sensor-FOIUB shows the
variation in sensitivity in the form absorbance is not only due to the
change in refractive index of the solutions and it is due to the reaction
between enzyme-Urs and urea in local vicinity of sensing element. The
present sensor is selective towards urea and does not respond to the
other interfering species [44].
forth for the sensing, decrease in response may be possible due to the
minute changes in conditions and due to regular addition and removal
of various concentrations of urea solutions every time in sensing cell.
This time can also be increase by providing a more suitable/porous
material, changing enzyme immobilization method or other ambient
conditions.
4. Conclusions

3.4.3. Sensor stability
Fig. 6 shows the response of the FOIUB for the stability measure-
ment towards same 1 mM urea solution throughout the experiment
prepared in phosphate buffer solution (pH 7.4). The sensing response of
the sensor was taken continuously for 30 days with the interval of
2 days in the form of absorbance. After each measurement the sensor
was kept at 4 °C and tried to maintain the same ambient conditions
during measurement. It was found that the sensor has approximately
same response for 28 days with slight decrease in every next measure-
ment, if once prepared. It indicates that the present sensor can be stable
up to 28 days. The mechanism for deterioration on the performance of
the sensor may be due to the loss or leaching of enzyme-Urs from
modified portion of sensing element. Moreover, when the sensor put
In this study, optical biosensor has been successfully developed for
urea detection using PANI as a supporting matrix. The sensor para-
meters such as sensitivity, selectivity, stability and lower detection limit
have been determined by using absorption measurement. The sensor
has shown variation in absorbance at ∼250 nm with various con-
centrations of urea solution and found highly sensitive towards it. The
prepared sensor especially selective for urea only that was confirmed by
comparing with interference species like glucose, ascorbic acid, L-ala-
nine, L-arginine and their combine solution with urea. The stability
study shows that the sensor is stable up to 28 days under appropriate
conditions. The prepared sensor shows lower detection limit up to
100 nM and precisely gives the best choice for practical approach.
Therefore, the prepared sensor can be applied as a promising device for

11
S.N. Botewad et al.
biomedical, agriculture, fishery industries, and milk industries for ac-
curate and specific urea determination due to its lower detection limit
with wide range, specific selectivity, quick response and high sensi-
tivity.
Acknowledgements
The authors acknowledge financial support by Defense Research
and Development Organization (DRDO), New Delhi, India (ERIP/ER/
1003856/M/01/1293) and Head, Department of Physics, Dr.
Babasaheb Ambedkar M. University, Aurangabad for extending FT-IR
facility.
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