Review On Biosensor Technologies

INTERNATIONAL
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International Journal of JOURNAL
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in Engineering RESEARCH
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6480(Print), ISSN 0976 6499(Online),
Volume 6, Issue 2, February
(2015), pp. 36-62 IAEME
ISSN 0976 - 6480 (Print)
ISSN 0976 - 6499 (Online)
Volume 6, Issue 2, February (2015), pp. 36-62
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IJARET
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REVIEW ON BIOSENSOR TECHNOLOGIES

TANU BHARDWAJ
Department of Instrumentation,
Shaheed Rajguru College of Applied Sciences for Women, University of Delhi,
Vasundhara Enclave, New Delhi-110096
ABSTRACT
This review revives concepts of construction and operation of biosensors. Combination of
suitable immobilization technique with effective transducer gives rise to an efficient biosensor.
Hence, various immobilization techniques are compared to understand which one can lead
manufacturing of an efficient biosensor. Along with, various transduction methods are also briefed.
Amongst all kinds of biosensors, electrochemical biosensors are known to be superior to many
tedious, costly and complicated techniques; therefore, the manuscript mainly focuses on different
electrochemical techniques employed in biosensing. Types of electrochemical biosensors,
voltammetric, potentiometric and impedimetric have been detailed out and explained with critical
analysis of the work done before. Moreover, voltammetric technique has been described
outstandingly in this review with illustrative examples and figures. Afterwards, with a summarized
history of electrochemical biosensors, future prospects have been described to present the predicted
life after a few years with these biosensors. Together with recent advancements in biosensors due to
nanomaterials, present trends of electrochemical biosensors are also illustrated in the form
of their applications in diversified fields, such as pharmaceutical industry, clinical sciences, military
applications, food industry and environmental sciences etc. Besides, 52 years of progress in the area
of biosensors, somehow, research in electrochemical biosensors is not translated to the
commercialization in the market. Various measures to commercialize biosensors at a high pace are
discussed in the end to minimize this wide gap.
Keywords: Biosensor,
Impedimetric
Immobilization,
Electrochemical,
Voltammetric,
Potentiometric,
INTRODUCTION
Formal birth ceremony of biosensor technology was conducted when Leland C. Clark
developed enzyme electrode in 1962 (Clark et al, 1962). Afterwards, Cammann placed the term
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International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN 0976
6480(Print), ISSN 0976 6499(Online), Volume 6, Issue 2, February (2015), pp. 36-62 IAEME
Biosensor into the dictionary of research in 1977 (Arora, 2013). In accordance with International
Union of Pure and Applied Chemistry (IUPAC), biosensor is self-dependent bio-analytical appliance
which has biomolecules layer in intimate contact with the transducer resulting in electrical signals (
Singh & Choi, 2009; Urban, 2009; Sun et al., 2010; Justino et al., 2010; Faridbod et al, 2014). It
consolidates biomolecules within or in intimate contact with a transducer which yield an electrical
signal equivalent to a single analyte (Evtugyn et al., 1998; Newman et al., 2001; Rinken et al., 2001;
Prodromidis et al., 2002; Jin & Brennan et al., 2002; Keane et al., 2002; Radke et al., 2005; Tsai et
al., 2005; Pohanka et al., 2008). Basic components of a general biosensor are shown in Figure 1.
Wherein, biomolecules can be enzyme, DNA, protein, whole cell, antibody etc (Corcuera and
Cavalieri, 2003; Yang et al., 2005; Faridbod et al, 2014). Sensors platform, where chemical reaction
between analyte and biomolecule ocuurs, is surface of a transducer (Ciucu 2014). A transducer
transforms one type of energy into another like chemical energy into an electrical signal. Further,
electronic circuit processes the signal, to get the signal in utilizable form (Evtugyn et al., 1998;
Karube & Nomura 2000).
Figure 1: Fundamental units of a biosensor

Due to the fact that biomolecules have singular selectivity (Velusamy et al., 2010; Ciucu
2014), these biosensors are found to be extremely beneficial in various domains for single analyte
investigation, as if in medical examination (Lee et al. 2000; Pickup et al. 2005; Newman and Turner
2005), water characteristic test (Pogacnik and Franko 2003; Vakurov et al. 2005) and nutrient
analysis (Mello and Kubota 2002). Another reason for attracting intense interest of researchers is that
it creates a way to unite entirely varying fields of biology, material science, electronics, optics,
chemistry and physics. Moreover, in the real world, biosensors replace tedious, costly and complex
conventional analytical techniques (Corcuera and Cavalieri, 2003). For instance, in biomedical and
biotechnology areas, tiresome and complicated processes which need prior clean up of samples, like
biochemical assays, immunoassays and PCR, have been subsituted by biosensors (Arora 2013).
Regarding construction of biosensors, adherence of biomolecules onto transducer is the most
significant and first footstep, called immobilzation. So far, we have four major techniques for
biomolecules immobilization: adsorption, covalent bonding, crosslinking and encapsulation (Lin et
al.,1997; Singhal et al., 2002; Sharma et al., 2003; Rad et al., 2012; Faridbod et al, 2014). Adsorption
and encapsulation belong to physical methods, and crosslinking and covalent bonding are placed
under chemical methods of immobilization (Sharma et al., 2003). For an efficient biosensor,
immobilization technique must have following features: decent and rapid, no percolation of
immobilized biomolecules from the trandsucer, long lifetime and biomolecules must carry its
individuality after immobilization and during sensing, and reproducibility (Lin et al., 1997; Nakamu37
ra & Karube, 2003; Alqasaimeh et al., 2014). But, these features do not come up in a single
technique. Beginning with adsorption technique, figure 2 shows how biomolecules are adsorbed
onto the surface of tranducer. As the interaction between transducer and biomolecule is non-covalent
(Soares et al., 2012), the biomolecules flood out from the floor of transducer. Due to which, response
of biosensor sinks with time and hence, they experience short life span (lin et al., 1997).
Figure 2: Biomolecules adsorbed on a surface

Whereas, covalent bonding and cross-linking techniques utilize the phenomenon of formation
of chemical bonds between the biomolecule and transducer as shown in figure 3 and 4.
Figure 3: Biomolecules attached

covalently with substrate
Figure 4: Crosslinking between

Biomolecules and substrate
Both of these chemical immobilization techniques have long life period, if compared with
adsorption, due to stronger bond formation between the biomolecule and transducer. But, still the
process is regarded to be complex and time consuming, as it requires analysis of complicated
chemical structures. Furthermore, the method utilizes hazardous chemicals which alter identity of
biomolecule. Fortunately, encapsulation process combines the advantages and eliminates the
drawbacks of the chemical method and adsorption. Here, biomolecule is trapped into a porous
polymer matrix on transducer surface as shown in figure 5 (lin et al., 1997; Prodromidis et al., 2002;
Sharma et al., 2003). Matchless feature of polymer matrix is that their arrangement and design can be
easily adjusted. Study of chemical structure is not necessitated in this technique as it was a big
compulsion in chemical bonding and cross-linking (Prodromidis et al., 2002). Moreover, percolation
of biomolecule from the matrix is infrequently seen in the process of encapsulation (Lin et al., 1997;
Zusman et al., 1990; Chernyak et al., 1990; Eguchi et al., 1990). Besides, this method does not harm
integrity of the biomolecule (Dave et al., 1994).
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Figure 5: Encapsulation of biomolecules within the matrix

After picking suitable immobilization technique, other chief component for an efficient
biosensor, is a transducer. Transducer transforms a chemical, optical, mass or temperature change
into an electrical signal. Depending upon various types of transducers or signals transduced,
biosensors are divided majorily into: electrochemical, optical, thermal, piezoelectric, etc
(Kauffman, 2002; Faridbod et al, 2014). Electrochemical biosensors generate an electrical signal
when analyte reaction with biomolecule produces chemical change onto the floor of electrodes (Ho
et al. 1999; Magalhaes et al., 1998). Whereas, optical biosensors analyze alteration in the properties
of light rays when analyte communicates with biomolecule (Chen et al., 2000; Tsai et al., 2003; Tsai
et al., 2005). For example: Fluorescene based ( Lee & Han, 2010; Gervais et al, 2011; Zubair et al,
2011; Buffi et al, 2011), surface plasmon resonance (Fang et al, 2006; Chou et al, 2010; Springer et
al, 2010; Malic et al, 2011) etc. Likewise, thermal biosensors feel changes in temperature and
piezoelectric biosensors sense the modification in mass due to the interaction between analyte and
biomolecule (Faradbod et al, 2014). Table 1 summarizes types of biosensors based on transducers/
signals transduced. From above discussion, it is easily understood that for raising efficiency of a
biosensor, prime components to be focussed are immobilzation and transduction method. Hence,
researchers are introducing new combinations of immobilzation and transduction method to evolve a
better biosensor. And this is how the field of biosenors is growing.
Table 1: Types of biosensors depending on transducers/ signals transduced
SIGNALS TRANSDUCED
NAME OF BIOSENSOR
Chemical signal
electrical signal
Electrochemical biosensor
Optical signal
electrical signal
Optical biosensor
Change in mass
electrical signal
Piezoelectric biosensor
Temperature signal
electrical Signal
Thermal biosensor
In this manuscipt, we review various kinds of electrochemical techniques employed in
biosensors to achieve different goals. In addition, few previous achievements and present trends of
electrochemical biosensos are also briefed. Along with, future prospects are also incorporated to
imagine the world with biosensors.
ELECTROCHEMICAL BIOSENSOR
Numerous diversified fields, such as pharmaceutical, clinical, military, food and
environmental etc show great interest in electrochemical biosensors, due to the fact that they have
following advantages over optical, piezoelectric, thermal biosensors: simple, portable, short response
time, sensitive, low cost, specific and selective. Moreover, it requires less amount of sample under
inspection (Mendez et al., 2012; Faridbod et al, 2014). A biosensor is named electrochemical
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biosensor when interaction between biomolecule and analyte creates chemical change on its surface
which is further converted into an electric signal which changes in accordance with concentration of
a specific analyte. Here, the sensing platform or transducer is an electrode, mostly made up of gold,
silver, carbon, platinum etc (Corcuera and Cavalieri, 2003; Sooducho & Cabaj, 2013). These
biosensors can be classified into voltammetric, potentiometric and impedimetric biosensors as shown
in figure 6.
Figure 6: Types of Electrochemical biosensor

1. VOLTAMMETRIC BIOSENSOR
In analytical chemistry, voltammetry naming technique is, sometimes, used to quantify an
analyte. Under this technique, varying voltage is applied to investigate an analyte and at output,
some informative current flows according to the concentration of the analyte. As the name proposes,
in these biosensors, this ancient technique is used to sense an analyte. A potential is applied onto
electrode surface and change in current is measured by utilization of 2 or 3 electrode systems
(Sooducho & Cabaj, 2013). At least, two electrodes are employed: a working electrode to sense the
chemical changes taking place on its surface and a reference electrode to provide a constant
reference voltage to circuit (Pohanka et al., 2008; Ciucu 2014). Along with, third counter electrode
can be supplemented to eliminate resistance between electrodes and complete the circuit.
Additionally, another chief reason to use a counter electrode is that 2- electrode system has less
control of potential when high current is utilized, which gives rise to reduction in linear range
(Pohanka et al., 2008; Iqbal et al, 2012). 3-electrode system not only offers above advantages, it
allows charge to flow from working to counter electrode, which, keeps reference electrodes voltage
constant. Usually, disposable biosensors prefer 2-electrode system, as long-term stability is not
required (Sooducho & Cabaj, 2013). Sometimes, it is called dynamic process as redox species
movement is involved in voltammetry. Rather, potentiometry, which is described later, is called
static process because it is related to charged species (Ravishankara et al, 2001). Different voltage
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patterns and corresponding name given to the biosensors under voltammetry technique is tabulated in
Table 2.
Table 2: Different voltage patterns used in voltammetry
TYPE OF VOLTAGE PROVIDED
NAME GIVEN TO THE BIOSENSOR
Constant voltage
Amperometric biosensor
One step of voltage
Potential step voltammetric biosensor
Triangular wave voltage
Cyclic voltammetric biosensor
Linearly increasing voltage
Linear sweep voltammetric biosensor
Linearly increasing voltage superimposed by
Differential pulse voltammetric biosensor
small voltage pulses
Linearly increasing voltage superimposed by
Square wave voltammetric biosensor
square waves of constant amplitudes
1.1. Linear Sweep Voltammetric (Lsv) Biosensor
Biosensor in which a linear voltage is applied to investigate an analyte is known as linear
sweep voltammetric biosensor. Here, a linearly increasing voltage running from zero to positive limit
(1), zero to negative limit (2) or negative to positive limit (3) is applied onto the electrode to detect a
redox couple at a particular voltage during the linear voltage scan, shown in figure 7.
Figure 7: Three Different linear sweep voltage pattern
Figure 8: Oxidation of Fe2+ to Fe3+
When the given voltage pattern is provided to the electrode dipped in an electrolyte solution,
then a current-voltage (I-V) curve is obtained in which the variation in current is slow until a redox
couple reduces or oxidizes at a particular potential. Current shoots up when reduction or oxidation is
initialized and increases until whole reduction or oxidation process is over or the present
concentration gradient in the solution gets ruined. And, then the current start decreasing. Hence, a
peak is obtained after which current decreases. As a result, this peak can be used to quantify the
concentration of an analyte (Sooducho & Cabaj, 2013). To exemplify it: Fe2+ gets oxidized to Fe3+
(figure 8) when a potential of 400mV is applied and after this potential, an oxidation peak is
observed by anodic current shown in figure 9. Initially, when no voltage was provided, there was
equilibrium between every electroactive species like Fe2+. But, when the voltage is applied as shown
in figure 7 with voltage pattern (1), the equilibrium gets altered and slow current flows due to the
diffusion of Fe2+ towards electrode because of its concentration gradient.
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Figure 9: Oxidation peak of Fe2+ ions

Figure 9 shows, as the voltage is increased, a voltage is attained after which oxidation starts
(400mV) and more of diffusion of Fe2+ is observed and hence, more rapid increase in current is seen.
This current due to oxidation process is known as anodic current (Ipa). Further, when whole oxidation
process gets over or no concentration gradient is available, i.e., no Fe2+ available for diffusion, then,
current value decays (Zuman et al., 2006). Furthermore, similar kind of peak is obtained in reverse
direction for reduction of Fe3+ to Fe2+ but with varying current (Ipc) and potential (Epc). This current
is called cathodic current (Ipc). Similar reasons of diffusion are applied for the current behaviour
shown in figure 10. A simple conclusion can be made from above discussion that oxidation/
reduction starts and gives a peak according to the concentration of the analyte. As well as, analyte
presence/concentration can shift peaks upwards or downwards/ towards more positive or negative
potential and shift is directly proportional to concentration of analyte as described further.
Figure 10: Reduction peak of Fe3+ ions

Along with previous section, the current response depends upon the scan rate also as shown
in figure 11. Scan rate is explained in terms of slope of linear voltage curve. If we see figure 11 and
12 simultaneously, then we observe that if the scan rate is slow (figure 11), then the current change is
also slow (figure 12) because the diffusion layer goes farther from the electrode. Due to which
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diffusion flow effect is also less. And, therefore, the current also reduces. Higher value of current is
obtained at high scan rates (Pandey et al., 1999; Zuman et al., 2006, Tang et al., 2004; Devadas et al.,
2012).
Figure 11: Effect of Different scan rates on voltage

vs. time plot
Figure 12: Current vs. voltage graph with different

scan rates
Initially, being a new technique in the field of biosensors, LSV was used only for
quantification of an analyte as Mizutani et al., 1997 reported a urea detecting biosensor where urease
enzyme was present in mercaptohydroquinone H2Q modified gold electrodes. Wherein, urease
hydrolyzed urea to generate pH change. That, further, alters the original oxidation and reduction
peaks of H2Q according to the pH change. LSV proved that with increase in urea concentration, the
anodic peak for H2Q moved more towards negative potential. Similarly, hydrogen peroxide was
detected with immobilized horseradish peroxidase attached to modified platinum disk electrode by
Liu et al., 2006 using LSV. These days, LSV technique is applied for oil analysis also as it was used
by Tomaskova et al., 2013 in which they investigated effect of amine containing antioxidants on the
examination of BHT (phenol-type antioxidant butylated hydroxytoluene).
LSV can not only be employed for identification of particles like urea, hydrogen, sodium,
potassium, uric acid, hydrogen peroxide etc, rather, it can be applied for DNA and RNA also. It was
applied by Sun et al., 2005 to quantify concentration of fsDNA in a liquid sample. A liquid sample
was prepared by inserting methyl violet (MV) into fsDNA, to get a supramolecule. The interaction of
fsDNA with MV changed the current values originally obtained for MV in LSV. The current peak
decreased as the fsDNA concentration multiplied. Similar class of work was presented by Sun et al.,
2007 for finding out concentration of yeast RNA (yRNA). Safranine T was intermingled with yRNA
which reduced the peak current, according to the supplementation of yRNA, of standard safranine T
solution. Parallely, other researchers applied LSV technique for investigating chief components of
biosensors, i.e., immobilization technique and electrodes. To choose best out of various electrodes
for a biosensor, LSV was employed by Hu et al., 2001. Various researchers have employed LSV for
understanding behaviour and modeling of ultramicrodisc electrodes for biosensors (Jin et.al., 1996,
Gavaghan, 1998).
With advancement in biosensors, various nanomaterials are used with electrodes for
increasing sensitivity of biosensor like CNTs, graphene, gold nanoparticles etc. LSV was employed
to compare graphite electrode and platinum deposited carbon nanotube (CNT) electrode by Tang et
al., 2004. Peaks, obtained in the case of CNT electrode by LSV technique, showed high current
peaks as compared to ordinary graphite electrodes. A simultaneously detecting adenine and guanine
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biosensor was constructed by Shahrokhian et al., 2012 in which glassy carbon electrodes were
modified by Fe3O4NPs/MWCNT. LSV was applied to compare the oxidation peaks for adenine and
guanine which showed low oxidation peaks and less sensistivity with bare electrodes in comparison
to Fe3O4NPs/MWCNT electrodes. In addition, LSV was employed to find the linear range of the
biosensor in different conditions: increase in guanine concentration with adenine concentration fixed
and vice-versa, and then simultaneous detection of increase in guanine and adenine. Gold
nanoparticles were used in biosensors by Noh et al., 2012 for quantitative analysis of glutathione
disulfide. In addition, each step of immobilization was checked by LSV technique and various
conclusions were made on the basis of obtained peaks. Then, graphene based electrodes were
designed by Devadas et al., 2012. They fabricated electrochemically reduced graphene oxide and
neodymium hexacyanoferrate layered glassy carbon electrodes (ERGO/ NdHCF/ GCE) for the
detection of paracetamol. LSV was employed to study the impact of continuous rise in paracetamol
concentration on its oxidation peak, sensitivity and linear range.
1.2. Cyclic Voltammetric (Cv) Biosensors
In this technique, one sided scan of LSV is also reversed in opposite direction. It can be
called bidirectional LSV technique. Electrodes of biosensors are treated with repetitive triangular
potential to scan the current change shown in figure 13. LSV is one of the extensively used
techniques (Grieshaber et al., 2008).
Figure 13: Potential waveform applied for cyclic

voltammetry
Figure 14: Resultant Current vs. Voltage graph

obtained from cyclic voltammetry
In Figure 14, two separate scans represented for LSV biosensors, are united together which
gives CV current curve. Figure 14 shows a pure reversible process where the oxidized species at the
electrode surface get reduced by reduction and substituted by the reduced species. When the process
is reversed, then reverse process is observed. Hence, CV technique can be used to check reversibility
of a reaction (Gosser 1994). For example: Fe2+ to Fe3+ and Fe3+ to Fe2+ conversion is reversible. The
same theory, shown in figure 15, exploited behind scan rate is applied here also (Pandey et al., 1999;
Wei et al., 2011; Wang et al., 2012).
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Figure 15: Resultant current vs. Voltage graph with different scan rates
Various applications of CV technique in biosensing are discussed briefly in the given below
section. In medical sciences, diabetes is one of the common diseases which is affecting many people
life and cause of other huge diseases. So, to get rid of diabetes, loads of glucose biosensors are made
using CV sensing technique. Like, an unmediated glucose biosensor was developed by Pandey et al.,
1999 using sol-gel as matrix. They made three platinum electrodes with different thickness of sol-gel
layer. CV technique was applied not only to sense glucose but to know which thickness works better
than the other. Together with, they examined the effect of different scan rates on current. In
advanced glucose biosensors, nanomaterials are incorporated to raise their sensitivity for finding
concentration of glucose. Li et al., 2008 fabricated a CNT containing glucose biosensor with
potassium ferricinide mediated glucose dehydrogenase with coenzyme pyrrole quinoline quinone.
They observed that the oxidation peaks, which were not visible in the case of simple carbon
electrodes, were easily observable in CNT modified carbon electrodes. CV results proved CNTs
purpose to increase the conductivity. While fabricating a glucose biosensor, cyclic voltammograms
can be used to know the optimized amount of enzyme, working potential and effect of different pHs
on glucose biosensor as did by Monosik (A) et al., 2012. They worked on a biosensor based on FAD
dependent glucose dehydrogenase enzyme on graphite nanocomposite with multi-walled CNTs
electrode, to detect glucose with N-methylphenazonium methyl sulfate (PMS) mediator.
Identifying concentration of hydrogen peroxide in various market products, like cosmetics,
drugs, antiseptics, bleaching agents etc, is one of vital step in an industry. Its concentration was
detected by Du et al., 2005 using CV technique. They used carbohydrate antigen 19-9 (CA19-9),
attached with horse peroxidase, encapsulated in sol-gel of titania to develop an immunosensor. CV
technique was used to sense current changes in the presence of hydrogen peroxide. To predict
concentration of hydrogen peroxide, another biosensor, made up of nanoparticles, was introduced by
Wei et al., 2011. A unit of Fe3O4 /nano-Au /HRP was attached to the carbon electrode by application
of external magnetic field for finding hydrogen peroxide. From cyclic voltammograms, it was
observed that increase in hydrogen peroxide made current peak to climb. Li (A) et al., 2012
introduced a sensitive biosensor to investigate hydrogen peroxide where polyacrylamide-P123
(PAM-P123) was utilized to entrap haemoglobin. Cyclic voltammograms proved the purpose of Hb
that it made the biosensor more sensitive due to increased electron transfer capability. In addition,
cyclic voltammograms showed rise in cathodic peak for every increase in hydrogen peroxide
concentration due to reduction of haemoglobin.
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In immunosensing also, CV technique plays a crucial role. Like, Wu et al., 2005 informed
about a human immunoglobulin G detecting capacitive biosensor in which the immobilization
technique used was sol-gel along with gold nanoparticles. To check the effect of every step of
immobilization of IgG antibody on insulating property, CV technique was used. Further, concluded
from the cyclic voltammograms that the insulating property of the sol-gel layer ascended with each
step.
A Coprinus cinereus peroxidase (CIP) based biosensor was reported by Savizi et al., 2012 for
determination of sulfide, which is mostly detected during waste water treatment. CV was applied to
view the inhibiting influence of sulfide group on the catalytic property of CIP. Sulfide is not the only
component present in waste water, various ions are also present. For estimating such ions
concentration in water, like As(V), an arsenic As(V) determination biosensor where acid
phosphatase was cross-linked with bovine serum albumine (BSA) and glutaraldehyde (GA) on
screen-printed carbon electrodes with substrate 2-Phospho-L-ascorbic acid was developed by
Mendez et al., 2012. Supplementation of As(V) reduced the activity of enzyme that was shown by
cyclic voltammograms.
1.3. Potential Step Voltammetric (Psv) Biosensors
Another name of this technique is chronoamperometry. Up till now, those techniques were
discussed in which voltage was swept with a constant pace. But here, voltage is increased with a step
instead of sweeping. Initially, a constant low potential Va is applied at which no electroactive species
can reduce/oxidize, then in one step, the potential is increased to get potential of Vb which is
remained constant for a period of time as displayed in figure 16. Here, the change in current is
measured with respect to time. Lets exemplify it:
Fe3+ (s) + e-
Fe2+
Usually, starting voltage Va is insufficient to start reduction reaction. So, when voltage Vb is
provided in one single step, current rises instantly due to the reduction of reactant Fe3+ located near
the electrode. But, as soon as, most of Fe3+ gets over, biosensor needs fresh supply of Fe3+ ions to
continue reduction process further. But, it is not available due to less concentration gradient, hence
the resultant current decays exponentially (Grieshaber et al., 2008). This behaviour of current is
shown in figure 17.
Figure 16: Potential Waveform applied for potential step

Voltammetry
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Figure 17: Resultant Current vs. time graph

by potential step voltammetry
In the beginning, this technique was experimented by few researchers for testing its
significance in the field of biosensors. Jordan and Ciolkosz, 1991, verified electron transfer in their
chronoamperometric biosensor based on enzyme glucose oxidase and alcohol oxidase. After
identifying importance of this method, various biosensors were developed. Like, it was used in
pharmaceutical industries for determination of paracetamol by Filho et al., 2001. As
chronoamperometry is not behind any technique, hence, it can be used for same applications
discussed for CV and LSV. Like, chronoamperometry can also be applied to find concentration of
hydrogen peroxide as done by Liu et al., 2006. Together with, linear range of a horseradish
peroxidase based biosensor was also determined by chronoamperometry. Another horseradish
peroxidase based biosensor to detect hydrogen peroxide was introduced by Wei et al., 2011. A new
complex of Fe3O4 / nano-Au /HRP was reported in this biosensor with hydroquinone mediator that
was magnetically attached to the glassy carbon electrode. Chronoamperometry was applied to the
biosensor to observe the changes in current with rise in hydrogen peroxide concentration that was,
further, utilized to calculate linear range of the biosensor. Glucose concentration was also estimated
using chronoamperometry by Wang et al., 2007. In addition, performance of various electrodes was
also checked. Concentration of arsenic can also be detected in a sample as experimented by Mendez
et al., 2012. They introduced an arsenic As(V) determination biosensor with substrate 2-Phospho-Lascorbic acid where screen-printed carbon electrode was covered with enzyme acid phosphatase
cross-linked with bovine serum albumine (BSA) and glutaraldehyde (GA). Chronoamperometry was
applied to evaluate the linear range of the biosensor with successive addition of As(V) where the
current decrease with each step. Further progress in chronoamperometry, lead to designing of
biosensors like biosensor based on polyphenol oxidase from apple tissue that detects effects of
atrazine, which is a herbicide, on the enzymatic activity of polyphenol oxidase (Majidi et al., 2008).
Diffusion coefficient of the biosensor was also detected by chronoamperometry. Alike, Zare et al.,
2010 introduced a rutin biosensor which catalyzed NADH oxidation. To determine the diffusion
coefficient of NADH, chronoamperometry was utilized.
Alongside, various advancements were seen in the form of introduction of new materials like
CNTs, gold nanoparticles and other nano-structures. Like, Shi et al., 2005 reported a cholesterol
oxidase immobilized in sol-gel layer on platinum deposited with carbon nanotubes intermingled with
graphite electrode paste. Chronoamperometry was employed to watch the change in current with
every addition of cholesterol. Then, Noh et al., 2012 developed a biosensor for glutathione disulfide
using gold nanoparticles (AuNPs). Chronoamperometry was applied to find the linear range of the
biosensor which showed that the current increased with successive increase in the concentration of
glutathione disulfide. Recently, Pohanka et al., 2013, fabricated a chronoamperometry based
biosensor for detection of neurotoxic agents which causes inhibition of enzyme acetylcholinesterase
in a human body. Not only this, chronoamperometry has also entered in the field of metallurgy
where different species of chromium were identified by Perez et al., 2014. Identical work has been
performed by Quiros et al, 2014 to detect Al(III) by investigating inhibition of activity of
acetylcholinesterase. Likewise, vanadium ions concentration can be quantified (Gamez et al., 2014).
1.4. Differential Pulse Voltammetric (Dpv) Biosensor
In this method of sensing, voltage patterns of LSV and PSV are superimposed. In
other words, continuous small voltage pulses are applied over a linear sweep potential as shown in
figure 18. This voltammetric technique is applied to prevent the effects of charging current because
of which the biosensor can not detect current values below the charging current limit. As displayed
in figure 18, current value is sampled before implementation of the pulse as shown by green dot and
then during the last 20% of the pulse duration indicated by black dot (Wang et al., 2012). These two
current values, cathodic and baseline, are considered for distinction. Thereby, the difference in two
47
current values is represented against potential as shown in figure 19. In comparison to cyclic
voltammetry, it has higher current sensitivity (Du et al., 2003; Zare et al., 2010).
Figure 18: Potential waveform applied for

differential pulse voltammetry
Figure 19: Difference between two current values

means the cathodic and baseline that provides
current against voltage curve in differential
pulse voltammetry
The technique is utilized in various ways for different purposes and few of them are
summarized below: during the initial stages of biosensing technology, in the trial stages, DPV was
used to check the behaviour of reactants around electrodes (Brown and Anson, 1977). Then,
response of DPV was studied with various reactants by Wang and Freiha, 1983. And various
measures were found out to improve signal to noise ratio. Further, behaviour of ultra microelectrodes
was studied using DPV by Howard et al, 1998. Mainly, work on detection of analyte started in 21st
century using DPV. Du et al., 2003 utilized DPV to detect the effect of different concentrations of
catechol in his CA19-9 antigen entrapped titania sol-gel based immunosensor. In the field of
genetics, Bang et al., 2005 detailed an aptamer biosensor in which DPV technique was utilized again
to watch the current sensitivity with analyte concentration change. A beacon aptamer was
immobilized with intercalated methylene blue onto the gold electrodes. Aptamers stem and loop
structure got altered when thrombin interacted with it. This process resulted in release of MB which
ended up with decrease in current value. Similarly, to study the interaction of dsDNA with glivec
drug, Diculescu et al., 2006 applied DPV for biosensing. Here, glivec drug links with dsDNA, leads
to oxidation of adenine residues in DNA structure to give rise to electrochemically detectable
changes in oxidation peaks of adenine bases that further gives a product of 2,8-dihydroxyadenine
which has its own peak. To detect various tumors, DPV based biosensors were developed. Wu et al.,
2008 reported a tumor marker detecting biosensor. Immobilized gold nanoparticles joined HRP
labeled tumor antibody (CA 153, CA 125, carbohydrate antigen 199 (CA 199)) was encapsulated in
the biopolymer called chitosan and sol-gel matrix. DPV was applied onto the electrodes whose
current response reduced due to the formation of immunocomplex of antigen and antibody resulting
in blockage of direct electron transport happening between HRP and electrode because of Fe(III) to
Fe(II) transformation. Various proteins like albumin can also be quantified in human body using
DPV biosensors (Lu et al., 2008). For checking presence of flavonoids like rutin, Zare et al., 2010
fabricated a rutin biosensor. They judged the presence of rutin by using a simple principle that
NADH could oxidize at very high potential value without any catalyst like rutin. Usage of rutin runs
the oxidation reaction at lesser value of potential.
48
Various nanoparticles containing biosensors have been made applying DPV as biosensing
technique. Like, Zhang et al., 2012 developed a DNA hydrization detecting biosensor where the
probe DNA was covalently binded to the gold nanoparticles and CuO nanospindles present on glassy
carbon electrode. The current response of biosensor using DPV, reduced with increase in
hybridization due to the fact that methylene blue binds less with dsDNA. To check water and milk
purity, a lead detecting biosensor was introduced by Ion et al., 2012 where amino-funtionalized
exfoliated graphite nanoplatelet modified glassy carbon electrode covered with bismuth films was
used. DPV was applied to observe the increase in the current peak with increase in lead value and
find the linear range of the biosensor. Not only this, DPV has been used to compare different types
of electrodes like Pt-based, carbon-based screen printed electrodes and nafion layered screen printed
electrodes by Falciola et al., 2012. Recently, DPV has been used to detect dopamine and ascorbic
acid (Li et al., 2014).
1.5. Square-Wave Voltammetric Biosensors
This technique is known to be most superior and advanced (Osteryoung et al., 1986; Kahlert
et al., 2001; Lovric et al., 2001). In this method, linear sweep voltage is superimposed by proper
square-waves of constant amplitude as shown in figure 20. Here, current values are sampled before
the implementation of square wave and at the end of the square wave. Wherein, one current value
shows oxidative current and the other shows reductive current. As conventionally, reductive currents
are negative in sign, so, difference of oxidative and reductive current actually gives rise to a higher
peak due to addition of both currents as shown in figure 21.The method and limitations are similar to
differential pulse voltammetry but it has higher sensitivity than DPV and other voltammetry
techniques (Xiao et al., 2012). Moreover, this technique is recommended over DPV when higher
scan rates and high current sensitivity is needed.
Figure 20: Potential waveform applied for square

wave voltammetry
Figure 21: The difference between cathodic and

anodic current values is shown in square wave
voltammetry
Various experiments were done primarily for evolution of square wave voltammetry. But,
majorly it was applied when Ramaley and Matthew, 1969 explained the theory of this technique.
After attracting interests of researchers, this technique was employed for various applications. Like,
Ianniello, 1988, found out presence of various impurities during polymerization reaction of
povidone. This technique can be used to check water purity as well, as concentration of EDTA was
detected in water by Zhao et al., 2003. To check uric acid concentration in various fluids like urine,
Chen et al., 2005 developed a single-use non-enzymatic uric acid detecting biosensor in which SWV
was employed to find out the concentration of uric acid. In the field of farming, concentration of
49
various pesticides, like organophosphate and carbamate, can be detected using SWV (Somerset et al.,
2006). Not only this, SWV can be used in DNA biosensors. Valerio et al., 2008 detailed fabrication
of a DNA biosensor by immobilization and modification of 4-aminothiophenol surface which
detected cylindrospermopsin. In this biosensor, SWV was utilized not only for detection, but, also to
compare the characteristics of various modified gold electrodes. Glucose concentration can also be
quantized using SWV method (Yan et al., 2008). SWV has been used to find out blood vessel
stimulator called angiogenin by Li L. et al., 2011.
Neurotoxic compounds like paraoxon was identified by Pohanka et al., 2012 using SWV.
They used acetylcholinesterase which could split its indoxylacetate acetyl group, but its activity
decreases when any inhibitor is present like paraoxon. From SWV, they resulted out that by raising
concentration of paraoxon, the oxidation of indoxylacetate decreased and it remained intact. Hence,
current response increased. Not limited to above described applications, in microbiology, growth and
presence of microbes can also be checked as done by Xiao et al., 2012. They employed SWV and
cyclic voltammetry to check the presence of E. coli utilizing bare glassy carbon electrodes (GCE)
and MWCNTs modified GCE (MWCNTs/GCE) to compare. They found out that square wave
voltammograms were better than the cyclic voltammograms in higher current peak and improved
peak shape. Nevertheless, concentration of antioxidants like glutathione can be found out using SWV
(Corra-da-Silva et al., 2013). In various fluids like water, blood, urine, milk etc, traces of metal ions
can be identified using SWV technique (Fan et al., 2013; Meng et al., 2014).
1.6. Amperometric Biosensors
First biosensor, mentioned in the introduction of this manuscript, was amperometric
biosensor (Clark et al, 1962). In Amperometric biosensors, a constant voltage is applied to the
electrode system due to which current flows in the system, relative to the amount of a specific
analyte. Upon application of voltage, the redox reaction occurring on the surface of electrode
generates an extra electric current proportional to the concentration of the analyte. These biosensors
show high sensitivity with low detection limits (Pizzariello et al., 2001). Amperometric biosensors
are further branched into three generation based on the evolution of electrochemical biosensors: (1)
First generation biosensors rely on electrochemical recognition of substrate or product (2) Second
generation biosensors make use of redox mediators for enhanced electron transport (3) Third
generation biosensors exclude the use of redox mediators and enzyme-polymer interaction are
responsible for electron transfer.
First generation biosensors are based on Clark model where substrate or product concentration is
under analysis for quantification of analyte. For example: Glucose is oxidized in the existence of
glucose oxidase to give hydrogen peroxide (Prodromidis et al., 2002).
G OX
glucose + O2 -------------------> D-gluconic acid + H2O2
Here, the analyte, substrate and product are glucose, oxygen and hydrogen peroxide. Oxygen
consumption or hydrogen peroxide production directly shows concentration of glucose present in
unknown sample. Figure 22 shows that in first generation biosensors, analyte reacts with enzyme to
give rise to products. Product, whose concentration is in accordance with analyte, gets oxidized at
the electrode surface which actually produces current.
50
Figure 22: Schematic representation of first generation biosensors

First major drawback of first generation biosensors was that high oxidation potential for
hydrogen peroxide or high reduction potential for oxygen was to be applied, at which other species
like dopamine, ascorbic acid etc could also interfere. Another is that oxygen fluctuations are possible
from environment during quantification of oxygen (Rinken et al., 2001). A first generation glucose
biosensor was also presented by Glezer et al., 1993 where sol-gel vanadium pentaoxide was used to
immobilize glucose oxidase enzyme on Pt electrodes. To remove high potential drawback of first
generation biosensors, Second generation biosensors were made to enter into the field of
biosensors, in which, utilization of redox mediators, like potassium ferricyanide, is the main
principle (Li et al., 2005; Li et al., 2008). In the above mentioned glucose biosensors, a modification
with addition of ferrocyanide, as redox couple, was made. Wherein, redox couple enabled the
electron transfer to the electrode floor. Other mediators that are usually used are ferrocene,
hydroquinone and tetrathiafulvalene. Figure 23 shows how a mediator helps in electron transfer.
is the redox mediator
Figure 23: Schematic representation of second generation biosensors

Some of the mediators used with biosensors are discussed here: Tetrathiafulvalene was
consumed by Wang et al., 1998 with glucose oxidase to detect glucose in sol-gel based amperometric
biosensor. Then, Wang et al., 2000 immobilized horseradish peroxidase in composite film of sol-gel
and hydrogel for examination of hydrogen peroxide with potassium hexacyanoferrate as mediator.
Different rhodium compounds, as redox mediators, was utilized by Sockup et al., 2011 for glucose
detection and made a conclusion that RhO2 is the most successful rhodium compound as mediator for
glucose determination. Further, Wei et al., 2011 employed hydroquinone as redox mediator for
51
finding hydrogen peroxide with immobilized horseradish peroxidase on gold nanoparticles that were
further linked to Fe3O4 nanoparticles on glassy carbon electrode. Usage of N-methylphenazonium
methyl sulfate (PMS) mediator was reported by Monosik et al., 2012 in a glucose biosensor using
FAD dependent glucose dehydrogenase enzyme on graphite nanocomposite with multi-walled CNTs
electrode. A Coprinus cinereus peroxidase (CIP) based biosensor with hydroquinone as redox
mediator for the determination of sulfide was detailed by Savizi et al., 2012. Various researchers
used quinones, quinoid-like dyes, etc as mediators for detection of different analytes (Murugaiyan et
al., 2014).
Interest of researchers diverted towards third generation biosensors which includes direct
electron transfer because usage of redox mediators gives birth to complicated and complex reactions.
However, there are huge difficulties for carrying out direct electrochemical reactions like: the redox
centre of the enzyme is deeply placed inside the protein shell that gives rise to long distance due to
which direct electron transfer is not possibble; when the proteins are adsorbed on the surface, then
there are chances of loss of its activity with time. Third generation biosensors are also known as
reagent-less biosensors that do not utilize electron shuttling redox mediators. These biosensors
exploit the phenomena of direct electron transport between enzyme redox centre and electrode as
shown in figure 24.
Figure 24: Schematic representation of third generation biosensors

Electropolymerised conducting films are the polymer films that are used these days for the
development of third generation biosensors (Murugaiyan et al., 2014). New materials are obtained in
the search of direct electron transfer and some of them are inscribed below: Polyanilineperfluorosulfonated ionomer was utilized by Cho et al., 1998 for the entrapment of urease that
reduced polyaniline on the electrode to allow the flow of reduction current. Ferreira et al., 2004
developed glucose oxidase amperometric biosensor where the enzyme was adsorbed in layer-bylayer films of poly (allylamine) hydrochloride (PAH) on Prussian blue (PB) layer modified indiumtin oxide substrate. Not only this, Branzoi et al., 2011 detailed an amperometric urea biosensor with
entrapped urease in polyaniline that got reduced with pH increase. Polypyrrole films were used to
entrap urease enzyme for an unmediated amperometric urea sensors where the film got deprotonated
with rise in ammonium ion concentration by Soares et al., 2012. Vostiar et al., 2002 detailed third
generation amperometric urea biosensor consuming electropolymerized toluidine blue dye as a
polymer for immobilization of urease enzyme. Transition metals like osmium are also utilized to
dope polymer films (Antiochia et al., 2007). Zafar et al., 2012 developed a Corynascus thermophilus
cellobiose dehydrogenase (CtCDH) based biosensor adsorbed into the membrane of Poly
52
(ethyleneglycol) (400) diglycidyl ether (PEGDGE) for glucose detection. Various other third
generation biosensors are reviewed by Murugaiyan et al., 2014.
2. POTENTIOMETRIC BIOSENSOR
This is second type of electrochemical biosensors where a constant electric current is applied
as shown in figure 25, a redox reaction is initiated on the floor of an electrode which generates a
potential difference in electrodes in accordance with the concentration of analyte (Newman and
Setford, 2006; Iqbal et al, 2012; Ciucu 2014).
Figure 25: Current provided for potentiometric technique

But it must be noted that the sensitivity of potentiometric biosensors is less than
amperometric biosensors. Potentiometric biosensors have to suffer from slow responses, interference
of other ions in sample solution (Ling et al., 2012), but on other hand, it shows relatively high
detection limits (Pizzariello et al., 2001). Main fundamental behind the development of
potentiometric biosensor is that an ion sensitive layer is prepared that can easily detect the change in
concentration of that specific ion, shown in figure 26 (Kumar et al., 2007; Ling et al., 2010; Ganjali
et al., 2010).
Figure 26: A typical representation of ion sensitive layer usually used in potentiometric biosensors
53
Some of the applications of potentiometric biosensors are enlisted here in this review. In
agricultural field, concentration of pesticides is a big issue. Mulchandani et al., 1998 fabricated a
potentiometric biosensor to quantify one of the types of pesticides called organophosphates. In milk,
urine, blood etc, urea can be detected by using potentiometric urease based biosensor designed by
Magalhaes et al., 1998. Four urea biosensors were developed with urease immobilized in chitosan
membrane by different methods of adsorption: simple physical adsorption, adsorption followed by
glutaraldehyde reticulation, adsorption followed by activation and activation followed by reduction
of sodium borohydride. Potentiometric study was applied to select the best method of immobilization
among the four methods. They ended up with result that the adsorption with glutaraldehyde
reticulation is the most successful method of adsorption. Another urea identifying potentiometric
biosensor was developed by Eggenstein et al., 1999. They utilized silver paste covered filter paper as
substrate that was covered with PVC-membrane (ammonium ion sensitive membrane) and then
urease comprising poly (carbamoylsulfonate) for working electrode. The interaction between urea
and urease generated ammonium ions which went to the PVC membrane. And a voltage difference
between working and reference electrode was developed. Various other urea detecting
potentiometric biosensors were developed by Lakard et al., 2004 and Chou et al., 2006. In
pharmaceutical industries, to know concentration of drugs, potentiometric biosensors have been used
as by Kumar et al., 2007. They constructed a nimesulide detecting potentiometric biosensor for
which a potentiometric sensing layer was formed of nimesulide-molybdophosphoric acid ion pair
complex in polyvinyl chloride with bis(2-ethyl hexyl) phthalate plasticizer. Potentiometric technique
was applied to find detection limit, response time, pH range and shelf life of biosensor. Similar sort
of study was repeated by Ganjali et al., 2010 also. They developed a potentiometric biosensor for the
quantification of terazosin hydrochloride in pharmaceutical drugs where the potentiometric sensing
membrane was generated by the terazosin-tetraphenyl borate ion pairs present in the polyvinyl
chloride matrix. Membrane composition effect and pH effect on biosensor were studied via
potentiometric technique. In textile industries, paint industries etc, formaldehyde is one of the major
ingredient whose unlimited concentration may effect health of human beings. So, for quantification
of formaldehyde, Ling et al., 2010, fabricated potentiometric biosensor. Oxidation of formaldehyde
by alcohol oxidase AOX gave rise to generation of protons that changed the potential at the
electrode. The potentiometric method was applied to find repeatability, reproducibility, response
time, linear range and detection limit. For glucose monitoring, various potentiometric biosensors are
discussed by Pisoschi 2012 in his review article. Not only in above applications, these potentiometric
biosensors can be used in milk industries to check presence of various ions. Like, concentration of
Pb(II) ions in milk was made known by Kaur et al., 2014 by its urease based potentiometric
biosensor.
3. IMPEDIMETRIC BIOSENSOR
Whenever current flows, hindrance in the form of impedance always exists. Impedance is the
opposition exhibited by a system to the flow of an alternating current upon employment of an
alternating voltage explained below by the equation (Pohanka et al., 2008):
Z=E/I, Z is impedance, E is applied voltage & I is the current.
The real equation of impedance is given below that explains the dependence of impedance on
resistance and capacitance of a system (Pohanka et al., 2008):
Z2 = R2 + 1/(2fC)2
Where Z is impedance, R is resistance, F is the frequency & C is the capacitance.
The impedimetric devices either follow impedance, resistance or capacitance of the system.
This technique is sometimes known as conductometric technique due to the fact that conductance is
inverse of resistance (Pohanka et al., 2008). For low frequencies, only resistance contributes towards
54
impedance and for high frequencies, capacitance takes over resistance. Upon application of
alternating current, resistance and capacitance of the solution alters that is the fundamental for
detection of any analyte. Analyte changes the impedance of the system after falling on the surface of
biosensor.
Behaviour of impedimetry based systems is described with equations:
Excitation signal: E(t)= Eocos(wt) where E(t) is voltage at time t, Eo is amplitude of signal, and w is
radial frequency.
Current equation is I(t)= Iocos(wt-)
Both signals are shown in figure 27 with phase difference.
Figure 27: Phase difference between voltage and current

Impedance is calculated by: Z=Excitation voltage/ current
= Eocos(wt)/ Iocos(wt-)
= Zo cos(wt)/cos(wt-)
The potential is defined: E(t)= Eoexp(jwt)
The current is defined: I(t)= Ioexp(jwt-j)
Impedance can be represented as complex number:
Z= Zoexp(j) = Zo(cos + jsin)
Z= Zreal + Zimaginary = Z' + Z"
When the real part of impedance on X axis is plotted against imaginary part on Y axis, then the plot
is called Nyquist Plot, shown in figure 28.
Figure 28: Nyquist plot between imaginary and real part of impedance
55
A plot obtained between impedance and frequency or phase difference and frequency is
named as bode plot, shown in figure 29 & 30.
Figure 29: Bode plot between impedance and

frequency
Figure 30: Bode plot between phase difference

and frequency
Gathering the information from impedance changes, nyquist and bode plots are obtained
(Guan et al., 2004; Wang et al., 2012).
Day by day, region of application of impedimetry is expanding, For waste water treatment,
cosmetic applications, pharmaceutical industries etc, estimation of hydrogen peroxide is very
necessary. Impedimetric biosensors can be used to measure the concentration of hydrogen peroxide
in various samples (Liu et al., 2006; Sun et al., 2010; Rad et al., 2012). In pharmaceutical industries,
choline is supplemented in drugs to treat various diseases. Its concentration can be measured as it
was did by Pundir et al., 2012 using impedimetric biosensors. Equivalently, paracetamol can also be
quantified (Devadas et al., 2012). Not only this, it has been employed in DNA sensing appliactions
(Li et al., 2011; Zhang et al., 2012). Like in cell cultures, growth of Salmonella typhimurium was
detected by Yang et al., 2004 using impedimetric biosensors. Similarly, E. coli growth can be
identified using impedimetric biosensors (Yang et al., 2005). Application of impedimetric biosensors
is not confined to already discussed topics. In medical application, impedimetric biosensors have
been used for detection of neurotoxic species like Alzheimer's amyloid-beta oligomers (Rushworth et
al., 2014).
APPLICATION
BIOSENSORS
AND
RECENT
ADVANCEMENTS
OF
ELECTROCHEMICAL
So far, we have summarized various electrochemical techniques hired for different purposes
in biosensors: 1. to compare behaviour of electrodes made of different materials, which means, we
can choose the best one to work with. 2. to find out best immobization material. 3. to watch the
changes in the characteristics of biosensor with each step of immobilization. 4. to test various
chemicals to increase the rate of reaction. 5. to check effect of various redox mediator on the reaction
ocuuring on biosensor. 6. to quantify an analyte. 7. to detect an analyte. 8. to find out sensitivity,
shelf life, response time and linearity. There are many more goals which can be achieved with these
electrochemical biosensing techniques (Iqbal et al, 2012). Biosensors were firstly used in medical
field, but now, the era has changed. The above mentioned goals can not only be applied in medical
field, but in pharmaceutical, clinical, environmental, food, agricultural etc industries. To detect a
disease at beginning stages or self identifying purpose by patients, biosensors joined clinical field
(Faridbod et al, 2014). For example: thyriod (Wang et al, 2014) detecting electrochemical
biosensors. As pesticides are nerve poisons for humans, so these biosensors can also be employed to
56
find out traces of pesticides on crops in agricultural industry (Corcuera & Cavalieri, 2003; Ciucu,
2014). Moreover, in pharmaceutical industries, concentration of various ingredients in a drug are
examined by biosensors (Gil et al, 2010). Numerous biosensors have been fabricated to find BOD
and different river water contaminants for environmental check (Arora, 2013). Not confined to above
specified fields, biosensors have entered into food industry as well, to quantify carbohydrates, acids,
amides, amino acids, amines, inorganic ions and alcohol. Alongwith, to check the quality of food
against bacteria, viruses and microbes, biosensors are extensively used (Corcuera & Cavalieri, 2003).
These days, areas of defence and military are also not left of biosensors. They operate biosensors to
detect bioterrorist activities (Arora, 2013). For widening region of applications, use of conductive
polymers (Malhotra et al., 2006), CNTs (He et al, 2006), nanomaterials (Mousty, 2004), biomimetic
ionophore channels (Kohli et al, 2004; Keusgen, 2002) etc has been reported which adds new
features to a biosensor.
MAJOR CHALLENGE IN FRONT OF BIOSENSORS IS ACTUALLY LEADING TO
ADVANCEMENT IN THE FIELD OF BIOSENSORS
With such diversified applications of biosensors, major challenge for biosensors is that out of
hundred biosensors, only one is commercialized. Efforts of researchers can be seen in the form of
advancements in the areas of biosensors. For commercialization, reseachers main focus is on low
cost immobilization techniques (Corcuera & Cavalieri, 2003). Research on these new immobilzation
materials is unstoppable as new materials are experimented daily in laboratories to get new best one.
Secondly, to miniaturize and increase precision and accuracy to sell biosensor in market,
nanostructures like nanowires, nanorods and nanotubes are utilized in electrochemical biosensors
(Das et al, 2006; Yogeswaran & Chen, 2007; Hubalek et al, 2007). Carbon nanotubes (CNTs) not
only increase stability of immobilized biomolecules, rather, in addition, enhances sensitivity of
biosensor. Besides, these CNTs can be used to fabricate electrodes which offers advantage of rise in
electron transfer, reproducibility and stability of biosensor (Basu et al., 2008). To state the matter
differently, CNTs can be used as amplifiers in biosensors (He et al., 2006). Apart from CNTs, other
materials like porous silicon, also are of great importance as substrate/support in biosensors (Stewart
et al, 2000). Other flexible particles which are in focus today are magnetic nanoparticles (Jianrong et
al., 2004). Moreover, due to the fact of high sensitivity and specificity of nanostuctures, they have
been used to sense various analytes like hydrogen peroxide, glucose, cholesterol, DNA, inosine,
bacteria, cancer etc (Yogeswaran & Chen, 2007; Zhang et al., 2009; Faridbod et al, 2014). For
further miniaturization of biosensors, graphene entered into the field of biosensors due to its more
surface area and electrical conductivity (Shao et al, 2009). Various graphene based electrochemical
biosensors, to detect concentration of heavy ions in environment, have been developed (li et al,
2009). In medical and forensic science, graphene based electrochemical DNA biosensors have been
developed to detect genetic disorders and criminals (Zhou et al, 2009). In addition to
commercialization, researchers are concentrating on other aspects of biosensors; it is to make
muliple analyte detecting integrated biosensors in every possible field (Arora, 2013) and implantable
biosensors in medical field (Faridbod et al, 2014).
FUTURE PROSPECTS
A conclusion can be made from above advancements of biosensors that worm of biosensors
has been spreaded to every field. There is not a single field which does not belong to biosensors. In
future, every person would be using biosensors for examination of urine, blood, saliva etc. If the
biosensor would show some indication of problem, then only, the person will go to a doctor. Chances
of heart attack would be identified before emergency. Diseases like cancer would be easily
57
detectable at early stages by biosensors. A traffic police would be checking, whether the driver is
drunk or not, by disposable alcohol detecting biosensors. Farmers would be knowing how much
pesticides are safe for humans. Also, for food and drug analysis, tedious procedures would be totally
replaced by biosensors. A comparison would be possible using biosensors between organic and
inorganic vegetables. A biosensor would be available in future to tell concentration of ingredients
present in tea, coffee or any other solution. So that, a general conclusion can be made regarding
biosensors that they can make our future more hygienic, protected from diseases, less tiresome and
healthier.
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Figure 1: Fundamental units of a biosensor

Figure 2: Biomolecules adsorbed on a surface

Figure 3: Biomolecules attached

Figure 4: Crosslinking between

Figure 5: Encapsulation of biomolecules within the matrix

Figure 6: Types of Electrochemical biosensor

Figure 7: Three Different linear sweep voltage pattern

Figure 8: Oxidation of Fe2+ to Fe3+

Figure 9: Oxidation peak of Fe2+ ions

Figure 10: Reduction peak of Fe3+ ions

Figure 11: Effect of Different scan rates on voltage

Figure 12: Current vs. voltage graph with different

Figure 13: Potential waveform applied for cyclic

Figure 14: Resultant Current vs. Voltage graph

Figure 16: Potential Waveform applied for potential step

Figure 17: Resultant Current vs. time graph

Figure 18: Potential waveform applied for

Figure 19: Difference between two current values

Figure 20: Potential waveform applied for square

Figure 21: The difference between cathodic and

Figure 22: Schematic representation of first generation biosensors

is the redox mediator

Figure 23: Schematic representation of second generation biosensors

Figure 24: Schematic representation of third generation biosensors

Figure 25: Current provided for potentiometric technique

Figure 27: Phase difference between voltage and current

Figure 29: Bode plot between impedance and

Figure 30: Bode plot between phase difference

Lovric, M., 2001, Square-wave voltammetry, in Electroanalytical Methods, Scholz, F. (Ed.),

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