Chapter-2
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11
CCCHHHAAAPPPTTTEEERRR 2 RRREEEVVVIIIEEEWWW OOOFFF LLLIIITTTEEERRRAAATTTUUURRREEE
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.
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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.
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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.
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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
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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).
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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
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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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18
(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.
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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
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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
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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).
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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).
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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.
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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
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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).
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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.
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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
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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.
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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
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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
Review of Literature
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
Review of Literature
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.
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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
Review of Literature
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
Review of Literature
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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