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A comprehensive review on thin film-based nano-biosensor for uric acid

determination: arthritis diagnosis

Article in World Review of Science Technology and Sustainable Development · January 2018
DOI: 10.1504/WRSTSD.2018.10013891

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52 World Review of Science, Technology and Sust. Development, Vol. 14, No. 1, 2018

A comprehensive review on thin film-based

nano-biosensor for uric acid determination:
arthritis diagnosis

Panchatchram Parthasarathy* and

S. Vivekanandan
School of Electrical Engineering,
VIT University,
Vellore, Tamilnadu, India
Email: arjunsarathii@gmail.com
Email: svivekanandan@vit.ac.in
*Corresponding author

Abstract: This article aims to provide a survey on the development of thin

film-based nano-biosensor over the decade, which includes sensing principles,
electrode types and various thin film matrices. The theoretical and experimental
results suggest that the thin film-based sensor are suitable for highly sensitive
detection of low concentration bio molecules and can be used for early
diagnosis and screening of arthritis, kidney diseases and other cardiovascular
diseases. This review concludes that enzyme immobilisation and surface
functionalisation methods were used to increase sensitivity of the biosensor and
new combination of polymer nano-composites with metal oxide thin films
results in increased efficiency due to its high biocompatibility.

Keywords: uric acid; immobilisation; enzymes; biocompatibility; thin films;

nanomaterials.

Reference to this paper should be made as follows: Parthasarathy, P. and

Vivekanandan, S. (2018) ‘A comprehensive review on thin film-based
nano-biosensor for uric acid determination: arthritis diagnosis’, World Review
of Science, Technology and Sustainable Development, Vol. 14, No. 1,
pp.52–71.

Biographical notes: Panchatchram Parthasarathy is currently pursuing his PhD

in VIT University. His research interest mainly focused on sensor development
for some real time application in environmental and medical fields. He obtained
his MTech in Sensor System Technology from the VIT University, India
in 2016, and his Bachelor’s in Electronics and Instrumentation Engineering
in 2013. His research interest is centre around the development of the sensor for
medical, industries and environmental applications and some interest in
nano-bio materials took my career research towards the development of sensors
for medical applications.

S. Vivekanandan is working as an Associate Professor in the School of

Electrical Engineering, VIT University. He received his PhD degree from the
Vellore Institute of Technology (VIT University). His current research
interests include instrumentation development for bio-medical applications;
telemedicine-based instruments and worked as a PI in various funded projects
and having more than ten years in teaching and research field.

Copyright © 2018 Inderscience Enterprises Ltd.

 A comprehensive review on thin film-based nano-biosensor 53

1 Introduction

Arthritis – India’s main cause of disability – a complex disease that affects millions of
people of all ages and ethnic groups which may leads to peoples immobility. India may
become the osteoarthritis capital of the world with over 60 million cases by 2025
(Arthritis Foundation, 2017). Doctors say osteoarthritis is the most prevalent form of
arthritis in India, affecting over 15 million adults every year. “In the last few decades,
Indians in the age-group of 30 to 50 years are falling prey to osteoarthritis and it
continues to have serious impact on the lives of elderly people,” said Mudit Khanna,
consultant Orthopedics at the Wockhardt Hospital (Lee et al., 2017).
There are different types of arthritis and related conditions but the common thread is
that it attacks connective tissues and joints and, making everyday life challenging. While
estimates vary by location, about one in every 100 children has been diagnosed with
juvenile idiopathic arthritis. The estimates are higher when other childhood forms of
arthritis and other rheumatic conditions are included due to uric acid (UA) abnormality
(Sobti and Sudhakar, 2017).
UA – an end product from purine derivatives in human metabolism is a chief
nitrogenous component of biological fluids such as urine and blood serum (Kannan et al.,
2016). With balanced production and excretion it undergoes no further metabolism in
human and is excreted by kidneys and intestinal tract (Dawson and Walters, 2013.
The normal level of UA in serum is between 240 and 520 μM in urinary excretion
and for blood is 2.4–7.0 mg/dl (Falasca, 2006). Abnormal UA level in biological fluids is
a marker of several disorders such as GOUT (Merriman, 2015; Nakagawa et al., 2006),
renal disease (Hahn et al., 2017) and leach-Nyhan syndrome (Nyhan, 1997). An
excessive amount of UA in serum (hyperuricemia) has been found to be associated with
hypertension (Nakagawa et al., 206), metabolic syndrome and cardiovascular diseases
(Dawson and Walter, 2013; Erden and Kilic, 2013). Consequently, fast and reliable
determination of UA in biological fluids is routinely required for diagnosis and treatment.
The first method was introduced by Offer is based on chemical oxidation of UA to
allotoin, which reduces phosphotungstic acid to a tungsten blue chromophoric compound
in 1894 but it has interference effect (Erden and Kirlic, 2013). In 1942 (Movlaee et al.,
2017), Bulger and Johns introduced a method based on the use of uricase enzyme though
it is commercially available its application is limited due to the cost of uricase enzyme.
Alternative methods such as chemiluminescence (Wu et al., 2006), fluorescence (Erden
and Kilic, 2013; Afrasiabi et al., 2016), spectrophotometry (Zhao et al., 2009),
HPLC-mass spectrometry (Weiss and Lunte, 2000), ion-chromatography (Revin and
John, 2012), high performance liquid chromatography and calorimetry (Zhuang et al.,
2017) have been reported. However these methods are usually expensive, laborious and
complex to perform. Therefore, there is a great interest in developing an inexpensive,
simple and rapid method for UA determination for arthritis diagnosis.
It is a growing requirement to develop new methodologies for easy, selective,
accurate and reliable quantification of the important metabolites to be used as an index
for the state of health in these years. Rapid growth in the development of new materials
and improvements in sensing techniques have led to the evolution of advance bio-sensors
and have explored the production of novel nanoscale metal-oxides, carbon nanotubes and
nanocomposites.
54 P. Parthasarathy and S. Vivekanandan

Novel analytical device based on nanostructured metal-oxides are cost effective,

highly sensitive due to large surface to volume ratio of the nanostructure and additionally
show excellent selectivity when coupled to bio-recognition molecules with simple design
(Grieshaber et al., 2008). Various matrices including metal-nanopartilces show high
isoelectric point, excellent bio-compatibility and easy synthetic procedure for
nanostructures that enables reliable immobilisation of uricase.
This article provides a comprehensive review of the research activities that focus on
several important thin films, metal-oxides nanostructures and nanocomposites in addition
to the application of thin film metal-oxides to UA sensing.

2 Electrochemical bio-sensors

Electrochemistry implies the transfer of charge from an electrode to another phase;

sample can be of solid or liquid. During this process chemical changes takes place at the
electrode and the charge is conducted through the bulk of the sample phase. Both the
electrode reactions and charge transport can be modulated chemically and serve as the
basis of the sensing process (Bhawana dn Pundir, 2010).
In bio-sensing, the measurement of electrical properties for extracting information
from biological systems is normally electrochemical in nature, whereby a
bio-electrochemical component serves as the main transduction element. Although
bio-sensing devices employ a variety of recognition elements, electrochemical detection
techniques use predominantly enzymes which are mostly due to their specific binding
capabilities and bio-catalytic activities (Elekes et al., 1995).
The electrochemical method is robust and user friendly technique, well suited for the
detection of bio-molecules such as UA, ascorbic acid (AA), dopamine (DA) and nitrite
are classified as potentiometric, conductometric and amperometric based on the
transduction modes. The different principle always requires a specific design for
electrochemical cell.

2.1 Amperometric biosensor

It is based on the transfer of electrons generated by an oxido-reductase enzyme to an
electrode surface. Amperometric biosensors typically rely on an enzyme system that
converts electrochemically non-active analytes into products that can be oxidised or
reduced at a working electrode which is maintained at a specific potential with reference
electrode. The electrochemical reaction occurs at the surface, giving rise to current which
is directly related to the bulk substrate concentration.
Amperometric enzyme electrodes for UA determination are generally based on the
determination of enzymatically generated H2O2 or the consumption of O2 during the
enzymatic reaction (Erickson and Wilding, 1993; Wolfbeis et al., 2000). H2O2 detection
has received considerable interest, as it can sense directly either by its reduction or
oxidation.
Uricase and horseradish peroxidise (HRP) enzymes are widely used in amperometric
UA biosensors. Mostly oxygen and peroxide-based systems are classified as ‘first
generation bio-sensors’, whereas mediated systems are ‘second generation sensors’
(Rocchitta et al., 2016). Conducting polymers have been used for the development of
‘third generation sensors’ (Zhou et al., 2012) and they can act as electron transferring
 A comprehensive review on thin film-based nano-biosensor 55

agents as well as immobilisation matrices and its comparisons are well reported (Mahshid
et al., 2012) (Table 1).
Table 1 comparison of three generations of biosensors

First generation biosensor Second generation biosensor Third generation biosensor

It mainly rely on enzymes
namely oxidases and
dehydrogeneases
Mediator less biosensor
Limits its application due
to low concentration of
dissolved oxygen
Not suitable for use under
ischemic conditions
Fouling and interference
effect affects the sensor
performances
Oxygen dependence
molecules
Possible to work at low Based on bio-electro catalysis
potential
Mediator biosensor Mediator less biosensor
Use of immobilised mediators Use of polymers improves the
affects the stability sensor performance
Possible to work at low Low response time
potential
No such effects No such effects
Avoiding O2 dependence and Independent of oxygen and
impact of interfering cofactor concentrations

Zhou et al. developed an electrochemical sensor based on polyaniline-Agcl hybrid

materials which has good electro catalytic properties for detection of DA and UA
simultaneously. The oxidation over potential of DA decreases drastically and its peak
current increases significantly with the said hybrid materials for the concentration range
of 0.7 to 6.0 μM and detection limit of 5 × 10–8 (Kimmel et al., 2012).
In an another study, combinations of Au/Pt/Pd/TiO2 nano-tubes electrode by
anodising method gives a high sensitivity for individual detection of DA as well as
simultaneous detection of DA and UA using 0.1 M phosphate buffer solution (pH 7.00)
as the base solution. The detection limit was 3 × 10–8 M for DA with good reproducibility
and stability.
However, bare electrodes like glassy carbon and other metal electrodes fail to detect
the bio-molecules simultaneously due to overlapping of their voltammetric signals and
fouling effects (Kimmel et al., 2012).
Compared with bare electrodes, mediator-modified and mediator-less electrodes have
the advantages of low over potential, lower detection limit, less interference effects.
Therefore, the sensitivity and selectivity of the mediator-modified and mediator-less
electrodes can be improved.
It is clear that choosing an electrode with enhanced material properties, chemical
stability as well as biocompatibility plays an important role. Meanwhile, other factors
such as the electrical conductivity, mechanical properties, toxicity, cost, can be
considered.

2.2 Electrode substrates

The basis of electrochemical analysis mainly depends on the reaction of sensing materials
on an electrode surface. Since the working electrode substrates can strongly influence the
efficiency, the properties of electrode substrates are of great significance for successful
electrochemical analysis (Johnson and Lacourse, 1990).
56 P. Parthasarathy and S. Vivekanandan

Piyush et al. (Lakshmi et al., 2006) developed a UA sensor based on mercury drop
electrodes which fails to detect the interferents adsorbed on the surface of sensor
electrode is the major cause for lowering the sensitivity, detection limits. In another
study, Greygory et al. developed an electrode with increased anodic potential which leads
to lack of specificity, reproducibility and formation of intermetallic compounds results in
reduced stability. Detailed comparisons of various electrodes are tabulated (March et al.,
2015) (Table 2).
Therefore, choosing appropriate electrode substrate plays a high impact for successful
electrochemical analysis. To overcome the limitations of electrode substrate, chemically
modified electrodes (CME) have been developed.
Table 2 Comparison of electrodes

Metal electrodes Mercury electrodes Carbon electrodes

It favours electron transfer
kinetics
Wide anodic potential
Low hydrogen voltage tends to
restricts cathodic potential
Increased background current
Kinematics of electrode
reaction problem can be solved
by using pulse potential cycle
(Lakshmi et al., 2006)
Copper and nickel provides
stable response for bio-sensing
than Pt and Au electrodes
(March et al., 2015)
Classic electrode Easily renewable for electron
exchange
Low anodic potential Broad potential window
High hydrogen voltage In normal range
extends cathodic potential
Low background current and Low background current and
highly Toxic chemically inert
Self-renewing Self-renewing
Dropping mercury electrode Commonly used carbon
were used electrodes are GCE,
pyrolytic graphite electrode,
carbon paste electrode, and
carbon fibre electrode and
carbon composites.

2.3 Chemically modified electrodes

In many research works, enzymes under investigation are usually immobilised onto the
surface of an electrode. However, this immobilisation procedure may denaturalise most
enzymes with the conformational change, which may affect further analysis. Therefore,
electrodes are not ideal interfaces to obtain direct electrochemistry; thus, CMEs are
developed to improve the situation. CMEs emerged in 1973 when Lane and Hubbard
modified various olefin compounds on clean platinum electrode through chemisorption,
which significantly changed the electrochemical response of the electrode (Lane and
Hubbard, 1973).
Since then, CMEs have been developing rapidly to investigate the direct
electrochemistry of proteins, enzymes and the mechanisms of redox reactions. The
fabrication of CMEs is to immobilise molecules with specific functions on the ordinary
electrode surface by chemical or physical methods. The different types CME are
conducting polymers (Ates, 2013; Zen et al., 1996), metal-oxide thin films (Kannan et al.,
 A comprehensive review on thin film-based nano-biosensor 57

2016; Kumar et al., 2005), metal-oxide nanoparticles (Komathi et al., 2010; Valentini
et al., 2003) and hybrid and carbon-based material modified electrodes (Li et al., 2013;
Rouhallahi et al., 2007; Lane and Hubbard, 1973).

2.3.1 Conducting polymers

Since the discovery of high conductivity in doped polyacetylene in 1977 (Chiang et al.,
1977), numerous well-characterised and ordered conducting polymers have been
exploited; thus, many conducting polymer–modified electrodes have been prepared for
the analysis purpose.
Later Muguruma (2007) conducted a review on plasma-polymerised thin films (PPF)
for biosensors for UA and glucose determination. The review mainly focuses on the
development of PPF’s used for biosensor design (Figure 1) with extremely thin surface
(< 1micro meter) and PPF-based enzyme immobilisation methods. The result shows good
adhesion to the substrate and chemical stability because of highly branched cross-linked
structure of the polymer.

Figure 1 PPF-based method for enzyme immobilisation (see online version for colours)

Wang et al. (2007) linear relationship is obtained over the concentration range of 5–150
μM by using polystyrene and polymaleimidostyrene coated over a gold electrode. The
concentration of UA was determined at a negative potential based on the decrease in the
reduction current of oxygen and interferents of L-AA also shows better interferent
abilities because of thin compact films.
Arslan (2008) developed a biosensor based on uricase immobilised polyaniline-
polypyrrole film in the working range of 2.5 × 10–6 – 8.5 × 10–5 carried out by the
oxidation of enzymatically generated H2O2 at 0.4V. A few common substances found in
serum or urine was studied for any possible interfering effects on the analysis of UA.
Known concentrations of AA, glucose, bilirubin, urea, paracetamol (acetaminophen),
were added to 4.0 × 10–5 M UA solution and the results are compared (Table 3). It has
been shown that glucose, urea, bilirubin and AA have been no interfering effects on the
analysis of UA. But interference of paracetamol (31%) in the analysis of UA has been
observed (Arslan, 2008).
58 P. Parthasarathy and S. Vivekanandan

Table 3 Interfering substances on the amperometric response of the biosensor

Interfering substance Concentration Interference effects of substance

–4
Ascorbic acid 1.0 ×10 NIL
–3
Glucose 5.0 ×10 NIL
Bilirubin 1.0 ×10–5 NIL
–4
Urea 1.0 ×10 NIL
Acetaminophen 1.0 ×10–4 31%

Thakur and Sawant (2013) showed polyaniline/prussian-blue (PANI-PB) modified

electrode possesses maximum response towards H2O2 sensing by cyclic voltammetry
than PANI modified electrode in the low potential range. It shows linear response for UA
concentration range between 10–160 μM with good sensitivity of 160 μA mM–1 cm–2.
The low working potential range of this composite enables the elimination of interfering
effects caused by analytes present in the real sample.
A new material with the combination of poly functionalised reduced garphene oxide
and Au nanoparticles mixed with polyoxometalates clusters had been developed for
better transmission of electric charges in the electrode. These sensors showed good
analytical performances and lower detection limit. The AFM images of the composite
film showed low surface roughness, which indicates it is suitable for the fabrication of
large-area and thin-film electronic devices. In addition, the thickness of the composite
film was estimated to be about 3 μm (Figure 2) from the cross-section observations (Bai
et al., 2017).

Figure 2 Top (a) cross-sectional SEM image (b) SEM images of the
{PEI/[P2W16V2-Au/PDDA-rGO]8} composite film on the ITO substrate

The various combinations of conductive polymers with graphene (Grp), carbon

nanotubes (CNT’s) and metal nanoparticles are tabulated (Prakash et al., 2013) (Table 4).
These types of metal-nanoparticles-polymer thin film, carbon nanotubes and
graphene-based electrochemical biosensor electrodes offer high sensitivity, a resistance to
thermal implications and a low cost. However certain limitations in its working
mechanism, time consuming and stability are biggest difficulty in these types of
electrochemical sensors.
 A comprehensive review on thin film-based nano-biosensor 59

Table 4 Methods for fabrication and their target analytes

Modified electrodes Fabrication methods Target analytes

AgNPs/PEDOT-PSS/Au Ex-Situ Ganodermaboninens
(Dutse et al., 2012)
AuNPs-PDDA/PANI/GCE In-Situ Dopamine
(Prakash et al., 2009)
Ag-PANI-silica/ITO γ-radiolysis H2O2 (Kim et al., 2010)
Pt-CNT CHIT/PSS/Au Self-assembled Cholesterol
ChOx/CT-SnO2/ITO Sol-gel Cholesterol
(Ansari et al., 2008)
Cu-SPEEK/GCE SPHINER H2O2 (Muraviev et al., 2006)
CuNPs-PANI/GCE Electrochemical Ascorbic acid (Xi et al., 2010)
PAMAM-AuNP/GOx/ITO Layer by layer Glucose
(Crespilhpo et al., 2006)
Nafion/PAMAM-Au//GOx MWCNT Ultrasound radiated Glucose (Wei et al., 2010)
poly-thionine with CNT’s Electrochemical Uric acid and glucose
method (Ghica and Brett, 2014)

2.3.2 Hybrid and carbon-based material modified electrode

Hybrid materials based on graphene, with fast electron transportation, high thermal
conductivity, excellent mechanical properties and biocompatibility, can avoid the
problems associated with polymer metal nanoparticles and CNTs and also leads to
potential applicability in electrochemical biosensor (Zhu et al., 2012).
Zhou et al. (2012) developed an electrochemical biosensor based on new hybrid
material. He used polyaniline/AgCl hybrid material for the determination of UA and DA
in the presence of high concentration of AA. The electrochemical test showed that the
hybrid material have a strong synergetic effect, ability to improve current, fast electron
transfer kinetics and obviously decrease over the potentials. Wang et al. (2010)
have developed a glassy carbon electrode (GCE) modified with poly (sodium
styreneulfonate) – graphene material. The result shows with linear range of 3–300 μM
and good selectivity because of its high electrical conductivity.
Ghanbari and Moloudi (2016) introduced a biosensor with nano-composites of
novel materials. ZnO (flower like)/polyaniline nano-fibre/reduced graphene oxide
nano-composite was coated over the GCE to detect the UA content. The modified
electrode shows linear response in simultaneous determination of both DA and UA. The
result proved that the modified nano-composites electrode could be utilised for the
voltammetric determination of DA and UA individually and simultaneously with low
detection limits, good repeatability and reproducibility.
Later, Shi et al. (2017) showed enhanced electrochemical responses of UA sensor
using Au nanoparticles. The morphology of the Au nanoparticles deposited on GCE
surface was characterised and nanoparticles were found to be well-dispersed spheres with
the average diameter approaching 26.1 nm. The results showed linear relationship
between peak current and UA concentration.
Luoa et al. (2006) is the first to report the utilisation of uricase/Ir-c electrode for the
determination of UA by using chronoamperometric method. The high sensitivity was
achieved due to the reduction of H2O2 oxidation potential on the electrode. The
60 P. Parthasarathy and S. Vivekanandan

fabrication was done by screen printing technique and the result shows sensitivity of
around 16.60 μA mM–1 over the concentration range of 0.1–0.8 mM with low detection
limit. The pristine graphene-based electrochemical sensor was developed by Qi et al.
(2015) to detect UA, DA and AA by liquid phase exfoliation of graphene. The result
shows linear range with high detection limits and low sensitivity when compared with
others types of electrode materials.

2.3.3 Metal oxide thin film and nanoparticles-based electrodes

Nanomaterials possess unique geometric, mechanical, electronic and chemical properties
and have some properties different from macroscopic materials, such as quantum effect,
surface effect, small size effect, etc. (Balzani, 2005)
These properties have greatly prompted a broad range of applications of
nanomaterials in medicine, electronics, biomaterials, environmental science, energy
production and biosensors. Nanomaterials-modified electrodes have many advantages
over the traditional material–modified electrodes. Firstly, nanomaterials offer huge
specific surface area for the immobilisation of more functional molecules on the
electrodes.
Secondly, some semiconductor nanomaterials may act as promoters of
electrochemical communications, accelerating the electron transfer rate. Thirdly, some
biocompatible nanomaterials can help proteins or cells maintain their activities on the
electrode for a long period. Therefore, a variety of nanomaterials have been synthesised
and characterised for electrochemical analysis for UA determination. The thin film types,
deposition techniques and other parameters also discussed (Table 5) (Vailiev, 2002;
Wang, 2005; Wang et al., 2007; Zhang and Huang, 2012; Zhou et al., 2006; Zhu et al.,
2011).
The doping is important aspect to increase the sensitivity and selectivity of the
bio-electrode. The surface modified and aligned mesoporous nano-fibre allowed
enhanced loading of enzyme molecules in the material surface. The high aspect ratio
and surface area of nano-materials shows excellent response resulting in improved
detection limits. Mondal et al. (2014) conducted experiments on TiO2 nanofibre based on
bio-functionalisation for bio sensing applications. The improved detection limit,
voltammetric and catalytic response were attained for the proposed design fabricated by
using electro-spinning method.
Yu et al. (2016) developed a boron doped diamond electrode to increase the grain size
and result shows that there is a decrease in grain size with increase in boron doping and
also the peak separation decreased due to decrease in the Fermi level with high sensitivity
and low detection limit.
Rashdan (2013) investigated and provided an overview on synthesis and
characterisation of nanoparticles for biomedical application, including cane therapy and
diagnosis, anti-bacterial treatment, etc. The study mainly focussed on several metals
(mainly Au, Ag) and oxides (mainly Fe3O4) at the nanoscale range and results are very
promising. Charbgoo et al. (2017) describes about the several properties of cerium oxide
nanoparticles (CNP) such as catalytic activity, electrochemical activity, high surface area
and oxygen transfer ability. The advantages and disadvantages of CNP’s-based
biosensors that functions through various detection modes regarding the detection of
small organic chemicals, metal-ions and biomarkers was discussed (Table 7). It can work
also as a catalyser instead of enzymes in an enzyme free approach.
 Table 5

Thin film type and deposition Uric acid Immobilise

Methods used Sensitivity Responses
techniques concentration range d enzyme
Metal electrodes (Au, Cr, Cu, Conductometr i-c type 0.1 mM to 5 mM 50 μs/mM Uricase Fabrication is easier than field effect
Ni) vacuum deposition transistor (FET) type and no complexity
(Sul’gao et al., 1994)
Nickel oxide (NiO) thin film Physical adsorption, electrochemical 0.05 to 1.0 mM 1,278.48 μA/mM Uricase Increased responses due to nanoporous
matrix by sputtering method analysis by CV and UV-V is morphology and high electron transport
spectrometry property (Arora et al., 2011)
Zinc oxide (ZnO) nanowires by Potentiometric type with CV 1 to 650 μM 32 mV/decade Uricase Range extended up to 1,000 μM by using
chemical synthesis analysis nafion membrane (Ali et al., 2011)
ZnO nanoflakes by Potentiometric type with CV 500 nM to 1.5 mM ~66 mV/deca de Uricase Increase sensitivity and stability by using
hydrothermal method analysis nanoflakes (Ali et al., 2012)
Copper oxide (CuO) thin film Amperometric – electrochemical 0.05 to 1.0 mM 2.7 mA/mM Uricase No external mediator used (Jindal et al.,
by pulsed laser deposition type and CV analysis 2012)
techniques
ZnO by pulsed laser deposition Amperometric – electrochemical 5 to 200 mg/dl 36 μA mM–1 cm–2 Uricase Increased oxygen pressure in PLD
(PLD) method type and CV analysis method while preparing electrodes
enhanced the sensitivity response (Batra
Studies on thin film-based bio-electrodes for UA sensing

et al., 2013)
Amperometric type 0 to 1.0 mM 0.2 mA/mM Uricase Increased shelf life – 20 weeks
A comprehensive review on thin film-based nano-biosensor

Tin oxide (SnO2) by sputtering

(Arora et al., 2011)
61
62 P. Parthasarathy and S. Vivekanandan

Table 6 Studies on thin film electrode by doping

Thin
Sensitivity
film Dopant Methods Concentration Responses
range
type
ZnO Nitrogen Bio-sensing 0 to 1.0 mM 1.38 N doping offers
thin doping response by CV mA/mM good
film (ZnO : N) and electro-catalytic
electrochemical activity and
impedance promotes direct
spectroscopy transfer of
electron from
active sites to
the electrode
(Jindal et al.,
2014)
ZnO Iron doping Amperometric- 0.05 to 1.0 298.33 μA Excellent
thin electrochemical mM mM–1cm–2 selectivity and
film type and CV reproducibility
analysis (Arora nad
Gupta, 2012)

Table 7 advantages and disadvantages of CNP-based biosensors

Types of sensor Advantages Disadvantages

CNP’s-based 1 Fast electron transfer rate 1 Possibility of CNP’s
electrochemical sensors 2 Continuous detection of agglomeration while they
target even in hypoxia are not immobilised on the
condition surface
2 pH sensitive
3 Non-enzymatic detection
also possible
4 Low cost
5 High sensitivity
6 High surface area
7 High stable
CNP’s-based 1 Easy operation 1 pH sensitive
colorimetric sensors 2 Rapid detection
3 Highly stable
CNP’s-based 1 High sensitivity 1 Limited sensitivity
fluorescence sensors 2 Used for in-vivo detection 2 Less stable
CNP’s-based 1 High sensitivity 1 pH sensitive
chemoluminscent sensors 2 Simple optical setup

Among different kinds of CNP’s-based biosensors, electrochemical type shows good

performances due to its rapid responses and high sensitivity. It is used for the continuous
detection of biomarkers even in hypoxia condition which was a big challenge in the
detection field based on oxidising reactions (Charbgoo et al., 2017). Thin films and
nanoparticles-based sensors have proven its potential as a biomaterial for bio-sensing
applications and have advantages over the traditional material-modified electrodes having
 A comprehensive review on thin film-based nano-biosensor 63

specific surface area for the immobilisation of more functional molecules on the
electrodes and possess some properties like fast electron transfer rate and
biocompatibility.

3 Immobilisation methods

Effective immobilisation approaches can preserve enzymatic activity and improve

biosensor stability. Different strategies have been used to improve the performance of
biosensors. In addition to improving biosensor stability immobilisation can also be used
to improve sensitivity, selectivity, response time, as well as operational and storage
stability. Various immobilisation strategies (Figure 3) include entrapment, cross-linking,
physical adsorption and covalent linking is discussed (Sirisha et al., 2016; Sassolas et al.,
2012).

Figure 3 Various methods of enzyme immobilisation (see online version for colours)

3.1 Entrapment
The entrapment method of immobilisation is based on restricting an enzyme within the
layers of matrix or membrane. It is done in such a way that matrix retains the desired bio
molecules while allowing the penetration of the substrate (Livage et al., 2001). When a
polymeric gel is prepared in a solution containing bio molecules, they get trapped within
a gel matrix. The use of sol-gels for enzyme immobilisation in biosensors started in the
1990s (Reetz et al., 2000) which results as a stable material where enzyme activity is
64 P. Parthasarathy and S. Vivekanandan

preserved and biosensor sensitivity is enhanced. Sol-gel immobilisation is conducted at

low temperature so it is compatible with bio molecules. Currently, there are sol-gel
methods for generating nanomaterials that look promising for biosensor applications.
Gill ad Ballesteros (2000) estimate galatose in milk and is found to be linear in the
range of 0.5-3.0 dl–1 in this method of developing galatose biosensor. In this method
some exchange occurs between the ion present in the matrix and the bio molecules to be
immobilised resulting in the ionic binding of bio molecules. Therefore, these methods
yields immobilised enzymes with higher activities in most cases.

3.2 Covalent binding

In recent times, the most intensely studied immobilisation techniques is the formation of
covalent bonds between the bio molecules and the support matrix (Arya et al., 2006).
Covalent method of immobilisation is usually applicable in the most of the cases using
different functional groups of the matrices capable of covalent coupling or being
activated to give such groups.
Zhou et al. reported the development of an amperometric sensor based on covalent
immobilisation on a boron doped diamond electrode to detect phenolic compounds. The
results showed the linear response of 1–200 μM and sensitivity of about 232.5 mA M–1
cm–2 (yu et al., 2012).
Even though the binding force between an enzyme and the carrier is so strong that no
leakage of enzyme occurs, even in the presence of substrate or solution of high ionic
strength but it limits its application by having complex immobilisation techniques.

3.3 Cross linking

Cross linking of bio molecules with a matrix occurs by use of some compounds having
two or more functional groups which are capable of binding two different materials under
different conditions. Intermolecular cross-linking of bio molecules has been achieved by
using bi-functional or multifunctional reagents (Lee et al., 2009; Vidal et al., 2006).
Singh et al. (2007) used cross linking technique via gultaraldehyde for fabricating
biosensor. The fabricated biosensor showed an apparent bio molecule affinity (Km) value
of 75 mg/dl with sensitivity of 0.042 μA mg dl–1.

4 Physical adsorption

This method of immobilisation is one of the oldest and simplest for binding bio
molecules on to the surface of a matrix by physisorption. Hence, the method causes little
or no conformational change of the enzyme or destruction of its active centre.
Arora et al. (2006) used the physical adsorption technique to immobilise the uricase
on to the surface of RF sputtered NiO thin film substrate. The sensing and
characterisation was carried out by cyclic voltammentry and UV Visible spectrometry
techniques. The developed NiO matrix with nanoporous morphology shows high
 A comprehensive review on thin film-based nano-biosensor 65

sensitivity and good linearity. In this binding technique weak binging force such as
hydrogen bonds, multiple salt linkage, Vander wall’s force and formation of electron
transition complexes are involved. Because of such weak bonds, sometimes desorption of
a bio molecule is observed as a result of change in temperature, pH, ionic strength
(Kriech and Conboy, 2005).
However, it has the disadvantage that the adsorbed enzyme may leak from the matrix
during use due to weak binding force between the matrix and bio molecules (Sharma
et al., 2007). A major advantage of this method is no reagents and only minimum active
steps are required. The advantages and disadvantages of different immobilisation
methods were tabulated (Sirisha et al., 2016) (Table 8).
Table 8 Advantages and disadvantages of various methods of immobilisation

Method Advantages Disadvantages

Physical No biocatalyst modification Binding forces are susceptible
adsorption Matrix can be regenerated to change in pH, ionic
strength
Low cost
Entrapment Only physical confinement are allowed High diffusion barrier
Low cost
Covalent Loss of biocatalyst is low Harsh treatment of bio-
binding Moderate cost catalyst by toxic chemicals

Cross Low diffusional resistance Matrix cannot be regenerated

linking Stable under adverse conditions Harsh treatment by toxic
chemicals

5 Conclusions

UA is a primary end product of purine metabolism in humans and abnormal levels of UA

in biological fluids are symptoms of several conditions. Therefore, the demand for the
continuous analysis of UA is increasing. Electrochemical sensors based on the oxidation
of UA seem to be a good approach for UA determination. However, these sensors
generally suffer from varying degrees of interference from AA or DA which can be
oxidised at a potential close to that of UA. Incorporation of enzymes in electrochemical
sensors can be a solution to this problem. Metal oxide thin films and nano-structured
materials had proven its potential as a biomaterial for bio-sensing applications. The thin
film and nanoparticles as a biosensor matrix material highlights the various procedures
that can be employed for the formation and the applicability of various structures in bio-
molecule immobilisation. UA can be efficiently determined using thin films/nanoparticles
based amperometric biosensors. In this review recent work in the development of
amperometric UA biosensors with an emphasis on the detection mechanisms, types of
immobilisation techniques and the different types of electrodes used are described in
detail.
66 P. Parthasarathy and S. Vivekanandan

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