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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

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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, Immobilization, Electrochemical, Voltammetric, Potentiometric,

Impedimetric

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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“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 biomolecule’s 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). Sensor’s 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; Nakamu-



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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 Figure 4: Crosslinking between

covalently with substrate 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; Sołoducho & 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

(Sołoducho & 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 electrode’s voltage

constant. Usually, disposable biosensors prefer 2-electrode system, as long-term stability is not

required (Sołoducho & 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

small voltage pulses Differential pulse voltammetric biosensor

Linearly increasing voltage superimposed by

square waves of constant amplitudes Square wave voltammetric biosensor

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 (Sołoducho & 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 Figure 12: Current vs. voltage graph with different

vs. time plot 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 Figure 14: Resultant Current vs. Voltage graph

voltammetry 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. Let’s exemplify it:

Fe3+

(s) + e- Fe

2+

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 Figure 17: Resultant Current vs. time graph

Voltammetry by potential step voltammetry



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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-L-

ascorbic 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



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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 Figure 19: Difference between two current values

differential pulse voltammetry 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. Aptamer’s 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.



49

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 Figure 21: The difference between cathodic and

wave voltammetry 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



50

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

(Corrêa-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).

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.

G OX



51

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



52

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: Polyaniline-

perfluorosulfonated 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-by-

layer films of poly (allylamine) hydrochloride (PAH) on Prussian blue (PB) layer modified indium-

tin 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



53

(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



54

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



55

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-θ)

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-θ)

= Zo cos(wt)/cos(wt-θ)

The potential is defined: E(t)= Eoexp(jwt)

The current is defined: I(t)= Ioexp(jwt-jθ)

Impedance can be represented as complex number:

Z= Zoexp(jθ) = Zo(cosθ + jsinθ)

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



56

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 Figure 30: Bode plot between phase difference

frequency 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 AND RECENT ADVANCEMENTS OF ELECTROCHEMICAL

BIOSENSORS

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



57

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



58

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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