Chapter-2 Review of Literatureshodhganga.inflibnet.ac.in/bitstream/10603/13742/10/10...Ilangovan et...

Chapter-2

Review of Literature

11

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To determine heavy metals at very low concentrations in biological material has

become a matter of high priority in many analytical laboratories all over the world (Su

et al., 2011). Trace metal analysis at the low µg/kg-level, which often is the normal

level of heavy metals in foodstuffs, was formerly a rather difficult task before the

development of techniques like flameless atomic absorption spectrophotometry

(FAAS) and differential-pulse anodic-stripping voltammetry (DPASV) etc. With the

developed techniques, possibilities for accurate determinations of metals e.g. cadmium

and lead, in milk have increased (Jonsson, 1976).

However these methods of analytical chemistry measure the total metal content of the

sample, therefore to access bio-available concentration in a more convenient way the

scientific community has been attracted towards “Biosensors”.

2.1 General Characteristics & Classification of Biosensors (Hu et al., 2011)

General characteristics of biosensors are following:

Immobilized bioactive materials are used which is of low cost or occasionally

can be utilized repeatedly, which overcome the shortcomings of the analysis

method that have a higher expenditure and excessive complexity in the past.

Biosensors generally have high accuracy and specificity.

Biosensors comparatively have a lower cost and a higher pace in the monitoring

analysis; sometimes you get the results only in minutes.


12

Easy to handle, experimental analysis is simple; usually don’t require a tiresome

& time consuming sample pre-treatment and easy to implement automatic

analysis.

Classification: There are many kinds of classification in biosensors many ways

basically:

On the basis of bio-recognition clement or sensing components, biosensors

can be divided into microbial biosensor, antibody biosensor/immuno

biosensor, enzymatic biosensor, DNA biosensor etc.

On the basis of transducer biosensors can be divided into thermal biosensor,

field-effect tube biosensor, piezoelectric biosensor, optical biosensor,

acoustic biosensor, enzyme electrode biosensor, electrochemical biosensor,

etc.

On the basis of type of interaction between the biological sensitive

materials, biosensors can be divided into two kinds: affinity biosensor and

metabolic biosensor.

In addition to above sporadically the biosensors are named after the analyte

also such as cadmium biosensor, uranium biosensor (Hillson et al., 2007)

etc.

Researchers have tried different approaches to develop biosensors for the desired

analytes e.g. different combinations of bio-components/bio-reporters, transducers,

detector systems.


13

2.2 Bio-recognition element

Bio-recognition element or bio-component is responsible for detection and interaction

with the analyte specifically and therefore is a very important part of any kind of

biosensor. For the construction of heavy metal biosensors various types of bio-

recognition elements as represented in Fig. 2.1 have been used including whole cells,

enzymes, non-enzymatic purified proteins, recombinant microbes and antibodies etc.

(Verma & Singh, 2005). Probably the major challenge and the most important part in

designing of a heavy metal biosensor is the selection of bio-receptors with strong

metal-binding capabilities with specificity. The interaction of heavy-metal ions with

biological molecules such as proteins, antibodies, or nucleic acids offers remarkable

advantages in this field in terms of selectivity and limits of detection. Some examples

of different class of biological receptors are depicted in Table 2.1.

Fig. 2.1: Classification of heavy metal biosensors on the basis of bio-component

applied.


14

Table 2.1: Classification of Bio-receptors or Bio-recognition molecules used for

development of heavy metal biosensors

Type of Bio-receptor Metal analyzed Reference

Antibody 2A81G5 Cd Khosraviani et al., 1998

ISB4 Cd Blake et al., 2001

12F6 U Melton et al., 2009

Enzyme Alkaline

Phosphatase Zn Satoh, 1991

Pyruvate Oxidase Hg Gayet et al., 1993

Urease Cd May & Russell, 2003

Glucose Oxidase Hg Malitesta & Guascito, 2005

Trienzymatic

(invertase,

mutarotase and

glucose oxidase)

Hg, Ag Soldatkin et al., 2012

Proteins Glutathione S

Transferase Cd and Zn

Corbisier et al., 1999 Saatci

et al., 2007

Mer R Hg, Cu, Cd, Zn, Pb Bontidean, 2003

Cue R Cu Changela et al., 2003

Metallothionein Cd, Zn and Ni Wu & Lin, 2004;

Varriale et al., 2007

Phytochelatin Cd and Zn Adam et al., 2005; Lin &

Chung, 2009

DNA T-T mismatch based Hg

Miyake et al., 2006; Long

et al., 2011; Wang et al.,

2012

DNAzyme based Pb

Breaker & Joyce, 1994; Lu,

2002; Shen et al., 2008; Li

et al., 2010

Metal-DNA

interaction based Pb, Cd and Ni Oliveira et al., 2008

Natural Whole cells P. phosphoreum Cr Lee et al., 1992

Chlorela vulgaris Cd and Zn Chouteaua et al., 2005.

Cardiac cells Hg, Pb, Cd, Fe, Cu, Zn Liu et al., 2007

Genetically

Engineered

E. coli DH5α

(pVLCD 1) Pb, Cd Liao et al., 2006

Saccharomyces

cerevisiae Y2805 Cd, As, Hg Park et al., 2007

E. coli DH5α

(pMOL30) Pb Chakraborty et al., (2008)

D. radiodurans

(KDH081) Cd Joe et al., 2012


15

2.2.1 Enzymes as Bio-recognition element

Enzyme based biosensors for the analysis of metal ions use either enzyme inhibition or

activation as its bio-assay principle. Normally metal ion combine with thiol groups

present in the enzyme structures which results in conformational changes and thereby

affect catalytic activity. For the construction of inhibition based biosensor many

different enzymes have been used such as glucose oxidase (Malitesta et al., 2005),

urease (Ilangovan et al., 2006; Gani et al., 2010), glutathione-S-transferase (Saatci et

al., 2007), alkaline phosphatase (Berezhetskyy et al., 2008), lactate dehydrogenase

(Tan et al., 2011), acid phosphatase (Kulkarni et al., 2011) and invertase (Soldatkin et

al., 2012) utilized for the detection of various metals like cadmium, lead, copper,

mercury and zinc etc. However, inhibition based biosensor suffer from a major

drawback of lack of selectivity as some of the enzymes are inhibited by several metals,

pesticides etc. Attempts have been made by different researchers to lighten this

complication.

Malitesta et al., (2005) immobilized glucose oxidase in poly-o-phenylenediamine

detected Hg2+

based on enzyme inhibition. Saatci et al., (2007) compared the

performance of pure bovine liver glutathione S-transferase Theta 2-2 (GST-Theta 2-2)

and recombinant 6His-Tag glutathione S-transferase (GST-(His) 6) based heavy metal

biosensors. GST-Theta 2-2 biosensor was able to detect Zn2+

from 1fM to 1mM and

Cd2+

from 10pM to 1mM while GST-(His)6 biosensor could detect Zn2+

and Cd2+

in the

range of 1fM to 10mM, and Hg2+

in the range of 1fM to 10mM. A thermostable

bacterial lactate dehydrogenase (LDH) was used to construct an inhibition based

electrochemical biosensor for mercury. Enzyme was purified and immobilized onto a

gold sheet coated by PGA-pyrrole polymeric material (Tan et al., 2011).


16

Kulkarni et al., (2011) constructed a biosensor using acid phosphatase for the first time

for heavy metal analysis, in acidic medium acid phosphatase catalyzes the conversion

of 1-naphthyl phosphate to 1-naphthol a highly fluorescent product having λex 346 nm

and λem 463 nm. For the construction of a fluorescent biosensor enzyme was entrapped

in A-J biocomposite, increased metal ion concentration resulted in increased inhibition

of enzyme and thereby decreased fluorescence. The inhibition effect was found in order

of Hg2+

>Cu2+

> Cr2+

. Enzyme was stable for more than two months at 4ºC and

biosensor could be regenerated successfully.

Recently Soldatkin et al., (2012) constructed a biosensor through immobilizing a

battery of enzymes containing invertase, mutarotase and glucose oxidase on the

transducer surface. Three enzyme system was used as bioselective element, bioassay

principle was based on inhibition of invertase by the metal ions; developed biosensor

showed the best sensitivity towards Hg2+

and Ag2+

.

2.2.2 Urease enzyme as Bio-recognition element

Urease enzyme has been exploited by different researchers either single or in

combination with other enzymes. Zhylyak et al., (1995) applied a conductometric

biosensor based on urease to detect heavy metals. Rodriguez et al., (2004) developed

bienzymatic bioassay principle with urease and glutamic dehydrogenase (GLDH) to

develop an electrochemical biosensor for the detection of heavy metal in polluted

samples with a detection limit of 7.2µg/l, 8.5µg/l, 0.3mg/l and 0.3mg/l respectively.

Screen printed disposable urease based Potentiometric biosensor was developed by

Ogonczyk et al., (2005) which could detect silver and copper to sub-ppm levels.

Ilangovan et al., (2006) immobilized urease through sol-gel to develop a

conductometric biosensor for heavy metal ions determination in liquid samples in the


17

range of 0.1mM to 10mM. Among the three metals used, the amount of inhibition is

found in order of Cd > Cu > Pb. Haron & Ray (2006) developed an optical biosensor

for Pb and Cd exploiting urease and acetyl choline esterase (AchE) as biocomponent,

biosensor based on urease performed better, could detect upto 1ppb of Cd and Pb in

water sample. Nepomuscene et al., (2007) immobilized crude urease extracted from

Dolichos uniflorus on non woven cellulose swab to construct a biosensor for

chromium.

2.2.3 Whole cell Biosensors for heavy metal ions and other analytes

Microorganisms (eukaryotic and prokaryotic cells), animal or plant tissues, or cell

receptors are being used as biological recognition elements in whole-cell biosensors.

The use of microorganisms as the sensing elements of a biosensor has several

advantages over the use of other sensing elements, such as enzymes, antibodies, or sub-

cellular components. There are typically a variety of microorganisms suitable for a

given purpose, and they may be easily prepared through simple cultivation in relatively

inexpensive media. In addition, microorganisms are capable of detecting a wide range

of chemicals, they are amenable to genetic modification, and they can often adapt to a

broad range of reaction conditions (Lei et al., 2006; Yagi 2007).

The use of whole cells as biological recognition elements has many attractive

advantages:

(i) Whole-cell biosensors are usually cheaper than enzyme based biosensor,

because whole cells culturing and harvesting is easier than isolation and

purification of enzymes.


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(ii) Whole cells are more tolerant to a significant change in pH, temperature or ionic

concentration than purified enzymes.

(iii)A multi-step reaction is possible because a single cell can contain all the

enzymes and co-factors needed for detection of the analyte.

(iv) Biosensors can easily be regenerated or maintained by letting the cells re-grow

while operating in situ.

(v) Extensive sample preparation is usually not required.

However, there are some drawbacks that limit the possible applications of whole-cell

biosensors, for example:

i. They are susceptible to interference by contaminants other than target

analyte as compared with enzyme-based biosensors.

ii. They have a relatively slow response compared to other types of biosensors.

A bi-enzymatic conductometric whole cell conductometric biosensor for pesticides and

heavy metal ions detection in water samples has been developed by Chouteaua et al.,

(2005). Whole cells of Chlorella vulgaris microalgae were immobilized inside the BSA

membranes which were reticulated with glutaraldehyde vapours deposited on

interdigitated conductometric electrodes. A local conductivity variation is created by

algal enzymes; alkaline phosphatase and acetylcholinesterase which could be detected

electrochemically. Bioassay principle has been based upon the inhibition of these

enzymes by distinct families of toxic compounds. Heavy metal ions are known to

inhibit alkaline phosphatase while acetylcholinesterase is inhibited by carbamates and

organophosphorous (OP) pesticides. Sensitivity of developed biosensor towards Cd2+

and Zn2+

was found to be 10ppb (10µg/l) for both. There was no significant inhibition

by Pb ions. As far as pesticides are concerned paraoxon-methyl was found to inhibit C.


19

vulgaris AChE contrary to parathion-methyl and carbofuran. Interference of heavy

metal ions with pesticide and vice versa was also analyzed, it was found that there is no

synergetic or antagonist effect of metal ions on pesticide or vice versa.

Liu et al., (2007) developed a cardiac cell based biosensor for heavy metals by light

addressable potentiometric sensors (LAPS). Distinct advantage of using mammalian

cell for the development of biosensor is that it offers insight into the physiological

effect of the analyte. Distinctive changes in terms of beating frequency, amplitude and

duration was noted under the exposure of different metal ions (Hg2+

, Pb2+

, Cd2+

, Fe3+

,

Cu2+

, Zn2+

in concentration of 10µM) in less than 15 min.

Many researchers exploited whole cells as a choice for the development of biosensor

for different analytes. Tsybulskii & Sazykina (2010) could isolate sixteen luminescent

strains of bacteria including Vibrio fisheri V_9579 and Vibrio fisheri V_9580 from

water of the Azov and Black seas. The isolated luminescent strains showed high

individual sensitivity to heavy metal salts, oil derived products, phenol and sodium

dodecyl sulfate (SDS).

Yucea et al., (2010) developed an algal cell based electrochemical biosensor for

detection of lead, selectivity of biosensor towards lead has also been studied.

Techniques used in the study include cyclic voltammetry and differential pulse

stripping voltammetry. Linear range of detection for Pb (II) was from 5.0×10−8

M to

2.0×10−5

M (10µg/l –4000µg/l).

Gammoudi et al., (2011) developed a whole cell (Escherichia coli) based biosensor for

the detection of heavy metals in liquid medium. Escherichia coli were used as

bioreceptors, fixed onto the acoustic path of the sensor which was coated with a


20

polyelectrolyte multilayer (PEM). A small platform was created for fast analysis by

inserting acoustic delay-line in an oscillation loop and associating it with a

Polydimethylsiloxane (PDMS) microfluidic network. Variation in frequency was noted

when a sample solution of Cd2+

and Hg2+

ions was pumped through the microchannels.

The system could response up to 10−12

mol·l−1

concentration.

2.3 Immobilization of microorganisms

As described earlier biosensor has got three components i.e. bio-receptor, transducer

and read out device; bio-receptor is made in close contact of transducer so that signal

produced by interaction of analyte with bio-component could be transferred to

transducer with a minimum or no loss of signal intensity. Thus, for the development of

a whole cell biosensor, cells are either immobilized directly immobilized on transducers

itself (Mikkelsen and Corton, 2004) or on some platform and then brought in close

proximity of the transducer (Verma et al., 2011a). Immobilization strategy plays a very

important role in the response of biosensor, operational stability and long term use

therefore choice of immobilization strategy is critical.

Theoretically, design of an interface must satisfy five conditions (Eltzov &. Marks,

2011):

1. Maintenance of biological activity after immobilization

2. Proximity of the bio-component to the transducer

3. Stability and durability of the bio-component

4. Sensing specificity of the biological component for its analyte

5. Possible reuse of some biomaterial

Immobilization of micro-organism on to a support matrices or transducers can be


21

carried out by chemical or physical methods (Blum, 1991; Turner et al., 1992; Tran,

1993; Mulchandani and Rogers, 1998; Nikolelis et al., 1998; Mikkelsen and Corton,

2004).

2.3.1 Chemical methods

Covalent bonding and cross linking are the two chemical methods used for the

immobilization of microbial cells (Blum, 1991; Turner et al., 1992; Tran, 1993;

D’Souza, 2001; Mikkelsen and Corton, 2004).

In covalent binding a stable covalent is formed between the functional groups of

transducer e.g. carboxyl, amine, epoxy or tosyl with functional groups of the

microorganisms’ cell wall components such as carboxylic, amine or sulphydryl.

Covalent bonding has got a drawback of exposing the whole cells to harmful chemicals

and harsh reaction condition, which may decrease the biological activity and damage

the cell membrane. Another chemical method for immobilization is cross-linking that

involve formation of a network by making bridge between functional groups present on

the outer membrane of the cells using multifunctional reagents e.g. glutaraldehyde and

cyanuric chloride. Cross-linking is found to be fast and simple method and therefore

has got wide acceptance for immobilization of microorganisms. Cross-linking of cells

can be carried out directly onto the transducer surface or on a support membrane, which

can then be placed on the transducer (Karube et al., 1977; Margineanu et al., 1985;

Karube, 1990; Blum, 1991; Turner et al., 1992; Tran, 1993; Korpan & Elskaya, 1995;

Nomura & Karube, 1996; Mulchandani & Rogers, 1998; Nikolelis et al., 1998;

Ramsay, 1998; Riedel, 1998; Arikawa et al., 1998; Simonian et al., 1998; Matrubutham

& Sayler, 1998; Souza, 2001; Mikkelsen & Corton, 2004).


22

Replacement of the membrane with the immobilized cells is an advantage of the cross-

linking approach; however cross-linking agents can affect the cell viability and/or the

cell membrane biomolecules (Souza, 2001).

2.3.2 Physical methods

Physical methods of immobilization include adsorption and entrapment, Physical

methods do not involve covalent bond formation with microbes therefore do not

interfere with microorganism native structure and function, physical methods are

preferred when viable cells are required for development of biosensor (Riedel, 1998;

Arikawa, et al., 1998; Simonian, et al., 1998; Matrubutham & Sayler, 1998; Souza,

2001). For the physical adsorption a microbial suspension is incubated with an

immobilization matrix such as alumina and glass bead (Souza, 2001; Mikkelsen &

Corton, 2004) subsequently rinsing with buffer to remove unadsorbed cells. Adsorptive

interactions include hydrogen bonding, ionic, polar and hydrophobic interaction.

Immobilization using adsorption alone suffers from the drawbacks of poor long-term

stability due to desorption of microbes.

Physical entrapment of microbial cells is carried out by either using filter or dialysis

membrane or in biological/chemical polymers/gels such as polyacrylamide,

polyvinylachohol, carrageenan, alginate, agarose, collagen, chitosan, polyethylene

glycol, polyurethane, etc. A major drawback of immobilization through physical

entrapment is the additional diffusion resistance by the entrapment material, which

usually affect the detection limit and sensitivity of developed biosensor. (Arikawa et

al., 1998; Mikkelsen & Corton, 2004).


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2.3.2.1 Sol –gel- immobilization

Sol-gel immobilization is a kind of physical entrapment of biological material; sol-gel

immobilizes the bio-component, at the same time allows the analyte to diffuse freely

and allow interaction with bio-component thus do away with a major drawback of

physical entrapment method of immobilization. Different bio-components such as

proteins, enzymes, antibodies, etc. can be immobilized through sol-gel method, sol-gel

matrix provide structural integrity, usually a full biological function and stability and

resistance against chemical and thermal deactivation to the immobilized bio-molecule.

2.3.2.2 Sol–gel Process and Matrix Characteristics

Sol–gel is formed with hydrolysis of alkoxide precursors under acidic or basic

conditions, subsequent condensation and polycondensation of the hydroxylated units,

which result in the formation of a porous gel. Concentration and strength of the catalyst

have an effect on rate and extent of the hydrolysis (Aelion et al., 1950). The

immobilization of biomolecules by the sol–gel method has been used for various

purposes (Avnir et al., 1994; Gill & Ballesteros,1998)

Normally a low-molecular weight metal alkoxide precursor molecule for example tetra

ethoxysilane (TEOS) or tetramethoxy silane or (TMOS) is hydrolyzed first in the

presence of water, acid catalyst and a common solvent (Avnir et al., 1994; Gill and

Ballesteros, 2000).

As a result of hydrolysis of metal alkoxide (e.g., TEOS or TMOS) silanol groups (Si-

OH) are formed; these silanol moieties react further to form siloxanes (Si-O-Si);

through condensation and finally SiO2 matrices are formed through polycondensation

of silanol and siloxanes after aging and drying processes (equations 1–3).


24

Sol-gel a network of polymeric chain of an average length of greater than a micrometer

with pores of sub-micrometer.

Among the catalyst used for the hydrolysis the most commonly used catalyst are HCl

and ammonia; however, other catalysts such as KOH, NaOH, amines, acetic acid, KF,

and HF are also used (Brinker &Scherer 1990).

(RO)3SiOR + H2O (RO)3SiOH + ROH [1]

2(RO)3SiOH (RO)3Si-O-Si(OR)3 + H2O [2]

(RO)3SiOH+ROSi(OR)3 (RO)3Si-O-Si(RO)3 + ROH [3]

2.3.2.3 Application of Sol–gel for immobilization of Biomolecules

Researchers have exploited sol-gel method enormously for immobilization of

biomolecule for various purposes (Kandimalla et al., 2006) including development of

biosensor; among biosensors a few are being quoted here.

Sol-gel method was used to immobilize urease by Lee & Lee (2002). A fluorescently-

labelled dextran co-entrapped with a hydrolytic enzyme (urease and lipase) in sol–gel

derived nanocomposite films has been prepared by Gulcev et al., (2003) and used for

biosensing urea and glyceryl tributyrate (GTB). Tsai et al., (2003) also used sol-gel

method to immobilize urease and fluorescent dye, FITC-dextran to develop an optical

biosensor for determination of heavy metal ions. Ilangovan et al., (2006) also used sol-

gel method to immobilize urease for developing biosensors for heavy metals. Recently

Kato et al., (2012) immobilized urease in a composite sol-gel silica matrix.


25

Researchers have used some non-silica sol–gel materials also to immobilize bioactive

molecules for the construction of biosensors (Liu et al., 2000) and synthesized new

catalysts for the functional devices (Bach et al., 1998; Jiang & Zuo, 2001).

Liu et al., (2000) immobilized tyrosinase in alumina sol–gel for detection of trace

phenols. Yu & Ju (2002) prepared a composite film of titania sol–gel to immobilize

horseradish peroxidase (HRP), the enzyme was retaining its activity. Latter on Yu & Ju

(2003), Dai et al., (2003) and Du et al., (2003) used the same for immobilization of

haemoglobin carcinoma antigen and carbohydrate antigen respectively for the

development of biosensors.

2.4 Transducers

Transducer is another important component of biosensor; transforms the biological

signal produced from the interaction of analyte with bio-component in some kind of

readable. Table 2.2 illustrates the merits and demerits of different kinds of transducers.

Recently developed monitoring technologies use whole cell biosensors because such

biosensors are essential for analyzing the environmental stress e.g. specific toxicity,

general toxicity caused by the pollutants (Su et al., 2011).

A number of microbial biosensors have been developed using electrochemical (CV,

Potential, current etc.) and optical transducers (light, fluorescence, color, luminescence

etc.)

2.4.1 Electrochemical Transducer

The electrochemical transducer is of special interest for in situ measurements as it can

be carried out with use of compact, simple and mobile equipment and is easily


26

adaptable for online measurements. The following techniques are commonly used

among electrochemical transducers.

2.4.1.1 Amperometric

A large proportion of electrochemical sensors are based on the amperometric principle.

In the amperometric type, the biomolecular recognition of the analyte is coupled to an

oxidation or reduction process that gives rise to an electrical current. Within this sensor

type, proteins can fulfil two different functional roles, namely the specific recognition

of the analyte molecule and the transduction of the recognition event into an

electrochemical signal (Heller, 2004; Willner & Katz, 2005).

Whole cell based amperometric sensing system for Cu has been developed based on

recombinant Saccharomyces cerevisae strains transformed with plasmid carrying Cu

inducible promoter with lacZ gene (Tag et al., 2007). In electrochemical biosensors

biomolecular recognition signal is transformed into an electrical signal.

2.4.1.2 Potentiometric

In potentiometric biosensors the interaction of biological molecule with analyte results

in a measurable change in potential. Low cost small size all-solid state pH-urease

electrodes useful for determination of heavy metals ions have been developed by means

of screen printing, Cadmium was inhibiting at the conc. of 1 mM (Ogonczyk et al.,

2005). Cardiac cells based heavy metal biosensor with a transducer system based on

light addressable potentiometer has been developed by Liu et al., (2007).


27

2.4.1.3 Conductometric

Conductometric biosensors involve the measurement of difference in conductivity of

the system due to the interaction of biomolecule with the analyte. A conductometric

biosensor using immobilized Chlorella vulgaris microalgae was used as a bi-enzymatic

biosensor (Chouteau et al., 2005) lower limit of detection achieved was 10 ppb for Cd

after 30 min long exposure based on alkaline phosophatase, acetyl choline transferase

inhibition.

Chong et al., (2008) developed a whole cell biosensor on a diamond electrode.

Unicellular microalgae Chlorella vulgaris was entrapped in the BSA membrane and

immobilized directly onto the surface of a diamond electrode for heavy metal detection.

The cell based diamond biosensor could attain a detection limit of 0.1ppb for cadmium.

2.4.2 Optical Transducer

Optical transducers have their own properties/advantage over electrochemical

transducers such as being free from electromagnetic interference, wide dynamic range

etc. Therefore making them a choice to be used for the development of biosensor,

following are some techniques used for the development of optical biosensors.

2.4.2.1 Surface Plasmon response

May & Russell (2003) developed a biosensor based on changes in structure of urease

enzyme after binding with cadmium being the basis of surface plasmon resonance

biosensing system. The enzyme was modified with N-succinimidyl 3-(2-pyridylthiol)

propionate (SPDP) to facilitate the formation of a self assembled monolayer of urease

on the gold coated glass SPR sensor disk.


28

A biosensor based on mammalian metallothionein for the detection of metal ions was

developed. MT was immobilized onto a carboxymethylated dextran matrix as a

biocomponent for the detection of metal ions by surface Plasmon response (SPR).

Cadmium was detected up to the range 2 to 10 micro mole (Wu & Lin, 2004).

A novel transmission-based localized surface plasmon response (LSPR) fiber-optic

probe has been developed to determine Cd ion concentration. The LSPR sensor was

constructed by immobilizing phytochelatins (PCs), (gammaGlc-Cys)(8)-Gly, onto gold

nanoparticle-modified optical fiber (NM(Au)OF); got a detection limit of 0.16ppb. The

sensor retained 85% of its original activity after nine cycles of deactivation &

reactivations; in addition sensor retains its activity upto 35 days at 40C in 5% d-(+)-

trehalose (Lin & Chung; 2008).

2.4.2.2 Luminescence Based Biosensors

Tauriainen et al., (1998) constructed a recombinant plasmid by inserting the regulation

unit from cadA determinant of plasmid pI258 to control the expression of firefly

luciferase. The resultant plasmid was expressed in two different strains Staphylococcus

aureus strain RN4220 and Bacillus subtilis strain BR 151, thus produced luminescent

bacterial sensor for cadmium and lead. Strain BR 151 responded to cadmium at 3.3 nM

while Strain RN4220 responded at 10nM; the results were obtained with 2-3 hrs

incubation.

Different genetically modified lux-based biosensor have been cited in literature and

used for different purposes e.g. Staphylococcus aureus strain RN4220 (pTOO24) and

Bacillus subtilis strain BR 151 (pTOO24) (Ivask et al., 2004), E. coli HB101 pUCD607


29

(Rattray et al., 1990); Pseudomonas fluorescence 10586r pUCCD607 (Dawson et al.,

2006) etc.

2.4.2.3 Fluorescence Based Biosensors

Liao et al., (2006) developed a GFP based biosensor E coli DH5α (pVLCDI) carrying

GFP under the control of cad promoter and the cadC gene of Staphylococus aureus

plasmid pI258. DH5α (pVLCDI) responded to Cadmium 0.1nM being the lowest

detectable concentration with 2 hr exposure. Haron and Ray (2006) developed an

optical biosensor for cadmium and lead by employing the technique of total reflection

at the interface between Si3N4 core and composite polyelectrolyte self-assembled

(PESA) membrane containing cycloptetrachromotropylene (CTCT) as an indicator;

achieved a detection limit as low as 1ppb for both the metals. A protein based

biosensor for Cd detection using a revered-displacement format was developed by

Varriale et al., (2007); a column containing chelex resin saturated with Zn2+

and a

rodhamine-labeled metallothionein (MT) comprised the assay reactive chamber. Lower

detection limit achieved was 0.5 µM for Cd.


30

Table2.2: Merits and demerits of different transducers (Eltzov &. Marks, 2011)

Type of

Transducer

Method of

Transduction Merits Demerits

Electro-

chemical

Amperometry

Surface conductivity

Potentiometry

Voltammetry

Conductivity

Sensitive

Compatible with modern

microfabrication

technologies

Portable

Disposable Require minimal power

Their uses are confined to a liquid, generally

aqueous, environment

Interferences (related to electrochemical

reaction of species

other than the one or

ones of interest)

Fouling

Optical

Surface plasmon

resonance (SPR)

Absorption

Reflection

Fluorescence

Luminescence

Simple Flexible

Multichannel sensing

Remote sensing

Free from electromagnetic

interference

Electrically passive

Wide dynamic range

Light interference (need a dark

environment)

Irreversibility of immobilized

bioreporter molecules

A need exists for more selective indicators and

more immobilization

steps

Mass-

responsive

Acoustic wave

Piezoelectric

Quartz crystal

microbalance

(adsorption)

Real-time output

Simplicity of use

Wider working pH range

Cost-effectiveness

Lack of specificity or sensitivity or

selectivity

Interferences from the liquid medium where

the analysis takes place

Sensitive to environmental

temperature changes

Thermal

Heat changes during

bioreporter activity

Calorimetry

Multichannel sensing

Stability for continuously

monitoring

Flexible shape and size

Losses of heat during signal measurement

due to the irradiation

Influence of the system from external

temperatures

Lack of specificity


31

2.5 Recombinant Biosensors for Heavy metals

Basic approach for the development of recombinant biosensor for heavy metals

involves the incorporation of a metal specific promoter upstream to a reporter gene.

The most commonly used reporter genes have been summarized in Table- 2.3 (Kohler

et al., 2000)

Table 2.3 Reporter genes and proteins used for development of heavy metal

biosensors

Reporter

protein

Reporter

gene Potential substrate Detection method References

Insect

luciferase luc Luciferin Luminescence Bronstein et al., 1994a

b-

Galactosidase lacZ Galactopyranosides

Colorimetric,

electrochemical,

fluorescence,

chemiluminescence

Cartwright et al., 1994;

Bronstein et al., 1996

b-

Glucuronidase

uidA

(gusA,

gurA)

b-Glucuronides

Colorimetric,

fluorescence,

luminescence

Bronstein et al., 1994b

Bacterial

luciferase Lux

Long chain aldehydes

(C9–C14) Luminescence Bronstein et al., 1994a

Alkaline

phosphatase phoA

Phosporylated

organics

Colorimetric,

chemiluminescence

Bronstein et al., 1994a;

Beck et al., 1994

b-Lactamase bla Lactamides Colorimetric

Yoon et al., 1991;

Cartwright et al., 1994;

Moore et al., 1997

Green

fluorescent

protein

gfp ---------- Fluorescence Kain and Kitts, 1997

SOE1:gfp ---------- Fluorescence Park et al., 2007

Recent examples of recombinant biosensors developed for analysis of heavy metals are

being mentioned here. Hillson et al., (2007) engineered a strain of the bacterium

Caulobacter crescentus to fluoresce in the presence of uranium at micromolar levels


32

when exposed to UV. urcA promoter was fused with gfp to produce a UV-excitable

green fluorescent protein in the presence of the uranyl cations. Tag et al., (2007) fused

CUP1 promoter from S. cerevisiae to the lacZ gene from E. coli developed an

amperometric biosensor for the detection of copper. The two selected recombinant

strains from the study were applied to real samples and could measure a concentration

of copper between 1.6 to 6.4 mg/l and 0.05 to 0.35 mg/l.

Fu et al., (2008) used bacterial luciferase as a reporter gene for the construction of

mercury specific biosensor. Pseudomonas putida X4 and Enterobacter aerogenes

NTG- 01were used as host strains. Recombinant Pseudomonas putida X4

(pmerR:luxCDABE-Kan) showed maximum bioluminescence during mid exponential

phase. The developed biosensors could detect Hg up to a concentration of 100pM at

28ºC and pH 7.0.

Wei et al., (2010) developed a luminescent biosensor assay for detection of mercury, a

mercury-inducible promoter, PmerT, and its regulatory gene was fused with

promoterless reporter gene EGFP. The developed biosensor was applied to detect bio

available Hg in soil samples. The limit of detection for Hg was found to be 200nM.

Ba2+

, Mg2+

, Fe2+

, Mn2+

, Sn2+

, Zn2+

, Co2+

, Cu2+

, Ag+ and Pb

2+ ions did not interfere with

the measurement system at nanomolar level.

Raja & Selvam (2011) constructed a green fluorescent protein based bacterial biosensor

for heavy metals. They developed a recombinant strain Escherichia coli cadR30 in

which green fluorescent protein was expressed under the control of cadR gene which

was isolated from Pseudomonas aeruginosa BC15. The developed biosensor was

applied for determining the availability of heavy metals in soil and wastewater.

Whole-cell biosensor for analysis of heavy metal ions in environmental samples based

on metallothionein promoters of a ciliated protozoan Tetrahymena thermophila has


33

been developed by Amaro et al., (2011) prepared two gene constructs by linking

eukaryotic luciferase gene as a reporter with Tetrahymena thermophila MTT1 and

MTT5 metallothionein promoters.

Recombinant Escherichia coli and Shigella sonnei, transformed with the pLUX

plasmid, harbouring the Lux-CDABE operon have been developed by Ademola et al.,

(2011) for rapid and effective monitoring of heavy metals in wastewater with a

detection limit of 53µg/l, l2.7µg/l, 61µg/l and 42µg/l for As, Cd, Hg and Pb

respectively.

Recently a colorimetric whole cell biosensor has been developed by Joe et al., (2012)

for cadmium. Promoter regions of Cd inducible genes (DR_0070, DR_0659, DR_0745,

and DR_2626) from Deinococcus radiodurans were screened. DR_0659 was found

highest specific among the screened promoters and used for the lacZ reporter gene

cassette. The study resulted in development of a genetically engineered biosensor D.

radiodurans (KDH081) with a range of detection was from 50 nM to 1 mM of Cd.

2.6 Biosensors developed and applied on Milk samples

Researchers have developed biosensors and applied on milk for detection and

determination of different analytes such as urea, melamine, Carbamate insecticides,

organo-phosphorus pesticides, antibiotics, lactose and mycotoxins etc.

Amarita et al., (1997) developed a hybrid biosensor to estimate lactose in milk.

Bioassay principle was based on series of biochemical reactions involving Lactase

activity which hydrolyze the lactose in glucose and fructose which were then fermented

by Saccharomyces cerevisiae to produce CO2. CO2 produced in the system could be

measured by a CO2 electrode.


34

Guidi et al., (2003) applied recombinant human IGF-1 and goat polyclonal antibodies

against human for the development of Low-cost Biosensors.

A potentiometric whole cell biosensor for analysis of urea in milk has been developed

by Verma & Singh (2003). Whole cells of urease producing Bacillus sp. were

immobilized onto a membrane and brought in close proximity to ammonium ISE; urea

present in sample is hydrolyzed to produce ammonia which results in change in

potential. Change in potential was correlated with concentration urea.

Biosensor for Organophosphate and Carbamate insecticides detection in milk was

developed by Zhang et al., (2005) using three engineered variants of Nippostrongylus

brasiliensis acetylcholinesterase (NbAChE) combined with wild type enzyme as

bioreceptor. Developed biosensor assay could detect paraxon and carbaryl down to the

contraction of 1 μg/l and 20 μg/l respectively.

Akerstedt et al., (2006) developed biosensor assay for haptoglobin (marker for

inflammatory reaction in case of mastitis). They developed a biosensor based on

affinity between haemoglobin and haptoglobin (Hp) using surface plasmon response

(SPR). The limit of detection was determined as 1.1 mg/l.

Carboxypeptidase activity of a bacterial penicllin binding protein was used by Sternesjo

& Gustavsson (2006) for developing a surface plasmon resonance (SPR) biosensor for

determination of beta-lactams in milk. Carboxypeptidase activity converts tri-peptides

into di-peptides. Bioassay principle is based on inhibition of carboxypeptidase activity

by beta-lactams. Polyclonal antibodies developed against the 2 peptides were used to

measure the amount of enzymatic product formed (di-peptide assay) or the amount of

remaining enzymatic substrate (tri-peptide assay), respectively. The detection limits of

developed biosensors were 1.2 and 1.5 µg/kg, respectively.

http://www.ncbi.nlm.nih.gov/pubmed?term=Sternesj%C3%B6%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16792082http://www.ncbi.nlm.nih.gov/pubmed?term=Sternesj%C3%B6%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16792082http://www.ncbi.nlm.nih.gov/pubmed?term=Sternesj%C3%B6%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16792082


35

A potentiometric biosensor for urea determination in milk has been developed by

Trivedi et al., (2009) fabricated a basal conducting track of the potentiometric electrode

using thick film screen printing technique and used the same for developing biosensor

for determination of urea by immobilizing urease. The detection limit of developed

biosensor was reported to be 2.5×10−5

mol/l.

Keegan et al., (2009) developed surface plasmon resonance (SPR) biosensor for

detection of Benzimidazole carbamate residues in milk using polyclonal antibodies

raised against methyl 5(6)-[(carboxypentyl)-thio]-2-benzimidazole carbamate protein

conjugate with limit of detection as low as 2.7µg/kg.

Zhang et al., 2010 developed an aptamer based electrochemical biosensor for analysis

of tetracycline in milk. Aptamer was immobilized on glassy carbon electrode surface

and used for development of biosensor. Aptamer specifically bind with tetracycline in

the samples without any pre-treatment subsequently the electrochemical signal

produced was correlated with concentration of tetracycline. The system was sensitive

up to a concentration of 1ng/ml with a response time of 5min.

Valimaa et al., (2010) developed biosensor for detection of zearalenone family

mycotoxins in milk. Saccharomyces cerevisiae strain was genetically modified to

produce firefly luciferase-enzyme in presence of mycotoxins. D-luciferin was used as a

substrate. An amperometric biosensor for the analysis of lactose in milk and dairy

products has been developed by Conzuelo et al., (2010). A bioelectrode was designed

by immobilizing enzymes beta-galactosidase (beta-Gal), glucose oxidase (GOD),

peroxidase (HRP) and the mediator tetrathiafulvalene (TTF) on a dialysis membrane

attaching that to 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-

modified gold electrode. Presence of lactose initiates a series of reaction which

ultimately resulted in reduction of TTF which is measured amperometrically and


36

correlated with concentration of lactose. The limit of detection 4.6 x 10-7

M could be

achieved.

An optical biosensor has been developed by Fodey et al., (2011) based on

immunoassay for the detection of melamine in infant formula milk. Polyclonal

antibodies were developed using a chemical molecule similar to melamine. A detection

limit of 0.5µg/ml could be achieved.

Recently a uric acid amperometric biosensor has been developed by Ivekovic et al.,

(2012). To prepare an alkaline stable H2O2 transducer electrode was modified with

Prussian blue containing structurally incorporated Ni2+

ions. Urate oxidase was used as

bio-component; linear range of detection of biosensor is found from 2.5 to 200μM, and

detection limit of 0.65 μM.

Mishra et al., (2012) developed an automated flow based biosensor for the

determination of organophosphorus pesticide in milk. Genetically modified

acetylcholinesterase (AChE) enzymes B394, B4 and wild type B131 were immobilized

on cobalt (II) phthalocyanine (CoPC) modified electrodes by entrapment in a

photocrosslinkable polymer (PVA-AWP). The limit of detection in milk achieved for

chlorpyriphos-oxon , ethyl paraoxon and malaoxon were 5×10-12

M, 5×10-9

M and 5×10-

10M respectively.

Yakovleva et al., (2012) developed a novel combined thermometric and amperometric

biosensor for lactose determination in milk based on immobilised cellobiose

dehydrogenase. Results of developed biosensor were highly reproducible in the range

of 0.05 mM and 30 mM.

Conclusively various biosensors have been developed for determination of different

analytes in milk by researchers at different times. No biosensor has been found in

literature especially for determination of heavy metal in milk.

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