Biosensors for the

Environment

1
 WHY BIOSENSOR?
 Numerous pollutants from industrial or agricultural origins are
found in the environment, and especially in water.
 Compounds such as herbicides, pesticides, chlorinated solvents as
well as compounds linked to petroleum utilization are frequently found as
pollutants of aquifers. These chemicals are generally toxic for humans and
animals and their presence in water must be monitored to avoid the
contamination of drinking water sources.
 Classical analytical tools (GC, GC/MS, etc.) provide accurate, reproducible and
sensitive determination of contaminant concentrations. Nevertheless,
their use requires to take samples on the contaminated sites and to
transport the samples to laboratory for analysis. Such handling of samples
is time-consuming and expensive.
 Biosensors are new analytical tools, whose conception has benefited from
advances in different scientific areas, and particularly in biology; they now
allow the development of highly specific tools. They provide real-time
determination and detection of very low concentrations of 2
contaminant and they can be used directly on-site.
Enzyme biosensors
Biocatalysis-based biosensors depend on the use of enzymes (i.e. proteins) to
moderate a chemical reaction.

An enzyme can be defined as a protein that catalyzes or modifies the rate at

which chemical reactions proceeds; hence it is not consumed during the
reaction. The initial molecules used in the catalysis are called substrates and
the molecules produced during this process are called products.

They can be of two types.

I. Catalytic transformation of a pollutant (typically from a nondetectable form

to a detectable form).

II. Tthe detection of pollutants that inhibit or mediate the enzyme’s activity.

Catalytic transformation mechanisms can be simple to design and operate but

cover a limited number of pollutants and have relatively high detection limits.

Enzyme inhibition approaches tend to cater for a larger number of

environmental pollutants and low concentrations. However, they suffer from
reusability and selectivitysuch formats require the use of substrates and in
Some cases the biosensor may need to be reactivated.
Process realized by an enzyme during the catalysis procedure

The International Union of Biochemistry and Molecular Biology has classified to enzymes by the
mechanism used in the catalysis:
 EC 1 Oxidoreductases: Enzymes that catalyze oxidation/reduction reactions to generate by means of
the substrate a new product.
 EC 2 Transferases: Enzymes that transfer a functional group to the substrate to form a new product.
 EC 3 Hydrolases: Enzymes that catalyze the products using the cleavage of the
substrate and the addition of water.
 EC 4 Lyases: Enzymes that catalyze the products by means of cleaving several bonds using hydrolysis
and oxidation.
 EC 5 Isomerases: Enzymes that catalyze geometrical or structural changes within a substrate
molecule with the aim of forming a single product. 4
 EC 6 Ligases: Enzymes that can catalyze the joining of two substrate molecules by forming a new
chemical bond by hydrolysis.
Affinity biosensors

Bioaffinity-based biosensors rely on the use of proteins or DNA to recognise and

bind a particular target (one receptor, one binding event).

For affinity-based biosensors competitive formats based on immunosensors

are generally used to detect the stoichiometric binding of the analyte to antibodies,
since small molecular weight organic pollutants generally have few distinguishing
optical or electrochemical characteristics. These immunosensors rely on the use
of an antigen-tracer which competes with the analyte for a limited number of
antibody binding sites. The enzyme and the substrate have specific complementary
geometric shapes that fit exactly into one another.
is
Immunosensors present the advantages of sensitivity and selectivity inherent
to the use of immunochemical interactions. Limitations are the troubles derived
from the regeneration of the immunosurface, and cross-reactivity, although a
certain degree of cross-reactivity is often desirable in order to determine different
congeners of the same family.

Nucleic acid-based affinity biosensors for potential environmental applications

include the detection of chemically induced DNA damage and the detection of
microorganisms through the hybridization of species-specific sequences of DNA.
Microbial biosensors
The third category of biosensors relies on micro-organisms as the biological
recognition element. These generally involve the measurement of microbial
respiration, or its inhibition, by the analyte of interest. Such responses have
been measured both optically and electrochemically. Compared to enzyme
Based approaches, microorganism-based biosensors are relatively inexpensive to
construct and can operate over a wide range of pH and temperature. However,
they involve long response time including the initial and return to baseline. These
aspects are primarily determined by the cellular diffusion characteristics and can be
modified by using genetically engineered microorganisms (GMOs). The broad
specificity of microbial biosensors to environmental toxins make them
particularly applicable for general toxicity screening or in situations where
the toxic compounds are well defined, or where there is a desire to measure total
toxicity through a common mode of action.

Biosensors have also been developed using GMOs that recognise and report
the presence of specific environmental pollutants. To date only a limited
number of GMOs have been constructed to respond to specific contaminants.
Systems capable of reporting both the metabolic condition of the relevant
microorganisms as well as the rates of pollutant breakdown could also be
Conceived.
Type of Toxicants
Type of Toxicants Source Nature of toxicity

Phenol and Industry waste, Catechol and

derivative of Microbial degradation of chlorocatechols have been
Phenol. E.g poly chlorophenyl (PCBs) characterised as strong
irritants to eyes, skin, and
catachol
respiratory tract and have
been proven to cause
DNA damage.
Organo phosphorous and Insecticides, pesticidies and Neurotoxin
carbamates bioware agents

Pathogenes Bio Waste Disease

Alkanes, aromatic carcinogenic compounds Cancer

compounds, and generally formed during
polycyclic aromatic incomplete combustion
hydrocarbons (PAHs) or pyrolysis of organic
matter containing carbon
and hydrogen
Dioxins by-products in a carcinogenic and are
number of chemical a potential
processes involving threat to human
chlorine. health

Surfactants Industry

Toxins affecting different

biochemical
processes including
membrane function,
ion transport,
transmitter release,
and DNA and protein
synthesis.
Antibiotics Drugs caused a genetic
selection of more
harmful bacteria,
which is a matter of
great concern

Heavy metal,inorganic Industry waste

phosphate
Schematic
representation of biosensor
components

10
WHAT IS A BIOSENSOR?
• Definition:
Biosensor transforms a
specific molecular signal
into an electric signal, and
comprises several
modules:
– a recognition module,
which can be biological or
biomimetic.
– a transduction module,
which transforms the
recognition event into a
measurable signal.
– and a module of data 11
evaluation.
Prerequisites for a biosensor
• A.Selectivity: The biosensor device should be highly selective for the target analyte
and show minimum or no cross reactivity with moieties having similar chemical
structure.
• b. Sensitivity: The biosensor device should be able to measure in the range of
interest for a given target analyte with minimum additional steps such as pre
cleaning and pre concentration of the samples.

c. Linearity of response: The linear response range of the system should cover the
concentration range over which the target analyte is to be measured.

d. Reproducibility of signal response: When samples having same concentrations are

analyzed several times, they should give same response.

e. Quick response time and recovery time: The biosensor. device response should be
quick enough so that real time monitoring of the target analyte can be done efficiently.
The recovery time should be small for reusability of the biosensor system.

f. Stability and operating life: As such most of the biological compounds are unstable
n different biochemica
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• Although conventional techniques such HPLC/MS and
GC/MS gives satisfactory analytical results for pesticide
determination, new assays and sensors for cheaper and faster
on-site analysis are being developed.
• Enzymatic sensors, based on the inhibition of a selected
enzyme, are the most extended biosensors used for the
determination of these compounds. Based on the inhibition of
acetyl cholinesterase (AChE) and colin oxidase, various
biosensors have been developed for the detection of
organophosphorous and carbamate pesticides.
• Although sensitive, biosensors based on AchE inhibition are
not selective (since the AchE is inhibited by neurotoxins,
which include organophosphorous pesticides (OPs), carbamate
pesticides, and many other compounds) and cannot, therefore,
be used for quantitation of either an individual or a class of
pesticides.
13
 Basic concepts about pesticides and enzymes
• A pesticide is any substance or mixture of substances used to
prevent, destroy, mitigate any pest.
• Pesticides are categorized in accordance with their chemical
substituents: herbicides, fungicides, insecticides and bactericides..
• A herbicide is a pesticide used to kill unwanted plants or to
reduce the growth of the weed, which leaves unperceived
secondary effects.
• A fungicide is a pesticide used to kill or inhibit fungi and fungal
spores, which damages the quantity, quality and profit of yield.

14
Cholinesterase is a family of enzyme
• Cholinesterase is a family of enzymes used to catalyze the hydrolysis
of the neurotransmitter acetylcholine (ACh) into choline and acetic
acid. AChE is found in the blood and neural synapses in multiple
molecular forms.
• AChE or true ChE is found in the central nervous system, bound to the
cellular membranes of excitable tissues and associated with nerve
transmission processes. Among the pesticides, organophosphates
and carbamates form an important class of toxic compounds; their
toxicity is based mainly on the inhibition of AChE during synapsis.
However, small amounts of these classes of chemicals can also
disrupt hormones and reduce their ability to successfully reproduce.
• AChE catalyzes the hydrolysis of ACh into choline and acetic acid, an
essential process for removing ACh from the nerve junction.

15
Acetylcholinesterase(AChE) enzyme based
biosensor
• The esteratic site is made up of the so-called catalytic triad a part of
which is the serine residuum which reacts with the substrate but
also with organophosphates. The mechanism of acetylcholine
hydrolysis catalyzed by the acetylcholinesterase enzyme is shown in
the reaction

Nerve agents, even at very low concentrations, inhibit their hydrolytic

capacity through a bond in this active center. The inhibition takes
place in several steps. In the first one the inhibitor is bonded to a
hydroxyl serine group in the esteratic center of AChE. Thus the
reversible enzyme-inhibitor complex arises. Further serin is
covalently phosphorylated. An electronegative leaving group is
released from the molecule of organophosphorus toxic agent. This
bond is still reversible. A competing process is the ageing of AChE,
the phosphorylated AChE switches to its dealkylated form. It is a 16
process where the alcohol is split off by means of water and the
alkoxyl group bonded to phosphor is replaced by the hydroxyl
group.
This state is irreversible. The result of enzyme inactivation is accumulation of
acetylcholine in the nerve ending and development of toxic exposures
demonstrated in the form of nicotine, muscarinic and central effects.

Inhibition-based biosensors are disposable biosensors and are

designed with this orientation for a cheaper cost. The main drawbacks of the
inhibition based biosensors are slow and tedious, due to they require multiple
steps of reaction such as measuring initial enzyme activity, incubation with 17
inhibitor, measurement of residual activity (after being used), and
regeneration and washing (i.e., add more substrate and remove biochemical
residues found).
 Biosensors for heavy metals

Enzyme-based biosensors

Alkaline phosphatase apoenzyme reactivation by the metal cofactor, a reaction

that is exothermic. The enzyme was covalently immobilized and Zn (II) was
detected over the range of 10 µM–1.0 mM with a response time of 3 min. The
biosensor had a long-term operational stability of 2 months.

The inhibition of urease by mercury using a potentiometric urea biosensor was

also studied. The sensor had a detection range of 0.05–1.0 lM and was
regenerable in EDTA and thioacetamide; however, it showed poor long-term
Stability.
Protein-based biosensors

An engineered green fluorescent protein (GFP), mutant BFPms1, can be used

to monitor metal binding based on alteration in fluorescence properties. Zn(II)
and Cu(II) bind to this protein with different coordination geometries with
Zn(II) causing enhanced fluorescence intensity and Cu(II) leading to
quenching of fluorescence. 18
Naturally occurring whole-cell based biosensors
The area of whole-cell microbial biosensors looks very promising for a multitude
of uses. The microbial strains are cheaper than isolated enzymes and the
enzyme activity is often enhanced in microbial cells owing to optimal
environment provided by the cells . Specific metabolic pathways in
microorganisms are used, resulting in the development of microbial sensors for
more selective analysis of compounds or pollutants, which cannot be measured
by simple enzyme reactions, e.g. the determination of aromatic
compounds and heavy metals a strain of luminous-bacterium.

Photobacterium phosphoreum MT 10204 was immobilized on a cellulose nitrate

membrane filter and used to develop a microbial sensor for the determination of
chromium based on a luminescence- based inhibition bioassay, achieving I50 at
0.85 nM Cr(VI). The use of luminescent bacteria, Vibrio fischeri, is to measure
toxicity from environmental samples. Bacterial bioluminescence has proved to
be a convenient measure of cellular metabolism and, consequently, a reliable
sensor for measuring the presence of toxic chemicals in aquatic samples.

Inhibition of bacterial growth in the presence of copper ions was monitored

based on the change in frequency of an uncoated piezoelectric quartz crystal of
19
a surface acoustic wave transducer, upon contact with biomass. The inhibition
effect of Cu(II) on the biomass was in the range of 18.0–25.0 ppm.
Biosensors for Heavy Metal detection

20
DNA based biosensor
Deoxyribozymes, also called DNA enzymes, DNAzymes, or catalytic DNA,
are DNA oligonucleotides that are capable of performing a specific 
chemical reaction.

A typical trans-cleaving DNAzyme system consists of an enzyme strand and a

substrate strand . In the presence of an analyte, the enzyme carries out catalytic
reactions, such as hydrolytic cleavage of the substrate strand at the scissile
ribonucleic acid adenosine (rA). The substrate can be extended on both the 3’
and 5’ ends as long as the enzyme recognition portion is maintained. It consists
of 5’-thio-modified 12-mer DNA attached to 13-nm-diameter gold nanoparticles
(called DNAAu), a DNAzyme (called 17E), and its substrate (called SubAu). The
sequence of the SubAu is designed so that it can hybridize specifically to a
DNAAu on each end, while maintaining the 17E recognition portion. These
hybridizations cause aggregation of gold nanoparticles and result
in a blue color. However, in the presence of Pb(II), the 17E catalyzes hydrolytic
cleavage of SubAu and prevents the formation of nanoparticle aggregates. A red
color appears as a result. Typical UV-vis spectra of the DNAzyme-gold
nanoparticle sensor in the absence and in the presence of 5 íM Pb(II). The color
change is attributable to the cleavage of SubAu by 17E in the presence of
Pb(II), which prevents the DNAAu from aggregating effectively. Two control 21
experiments supported this conclusion. First, in the absence of Pb(II), only blue
precipitates were observed.
(a) Secondary structure of the “8-17” DNAzyme system that consists of an
enzyme strand (17E) and a substrate strand (17DS). The cleavage site is
indicated by a black arrow. Except for a ribonucleoside adenosine at the
cleavage site (rA), all other nucleosides are deoxyribonucleosides.
(b) Cleavage of 17DS by 17E in the presence of Pb(II). (c) Schematics of
DNAzyme-directed assembly of gold nanoparticles and their application as 22
biosensors for metal ions such as Pb(II). In this system, the 17DS has been
extended on both the 3¢ and 5¢ ends for 12 bases, which are complementary to
the 12-mer DNA attached to the 13-nm gold nanoparticles
Second,the G*T wobble pair in the DNAzyme has been shown to be essential for
the DNAzyme activity. When the G*T wobble pair was changed to a GC
canonical base pair (by changing the 10th nucleotide in 17E from a thymine to a
cytosine and keeping other bases the same), the resulting mutant DNAzyme
(called 17Ec) lost its activity completely.
Although 17Ec was as capable of forming blue nanoparticle aggregates as 17E,
no color change was observed in the presence of Pb(II) . These results indicate
that Pb-(II)-assisted DNAzyme cleavage of SubAu is responsible for the color
change and thus for the metal ion detection..

UV-vis extinction spectra of an active 17E DNAzymenanoparticle sensor (a) and an

inactive 17Ec DNAzyme-nanoparticle sensor (b) in the absence (blue curve) or in the
presence (red curve) of 5 íM Pb(II). (c) Pb(II) detection level of the sensor. When the 23
enzyme strand is the active 17E only, the Pb(II) detection range is 0.1-4 íM (solid green
squares). When the ratio of 17E and 17Ec is 1:20, the Pb(II) detection range
is 10-200 íM (open green squares).
For quantitative analysis of Pb(II), the ratio of extinction at 522 and 700 nm was
used. These two wavelengths are chosen to represent the relative amount of free
and aggregated gold nanoparticles, respectively. The ratiometric method allows
the determination of metal ion concentration independent of sampling
conditions.

A plot of this extinction ratio versus Pb(II) concentration, which shows that this
unoptimized sensor is capable of detecting Pb(II) between 100 nM and 4 µM, and
thus it is well suited for household and environmental monitoring since 480 nM is
considered the toxic level for human .A calibration curve such is necessary for
quantitative analysis.

The color change of the sensor induced by Pb(II) can be monitored by UV-vis
spectroscopy. Typical UV-vis spectra of the DNAzyme-gold nanoparticle sensor in
the absence and in the presence of 5 íM Pb(II) are shown in Figure . The color
change is attributable to the cleavage of SubAu by 17E in the presence of Pb(II),
which prevents the DNA Au from aggregating effectively. Two control
experiments supported this conclusion. First, in the absence of Pb(II), only blue
precipitates were observed. Second,
24
the GâT wobble pair in the DNAzyme has been shown to be essential for the
DNAzyme activity.22,28,29 When the GâT wobble pair was changed to a GC
canonical base pair (by changing the 10th nucleotide in 17E from a thymine to a
cytosine and keeping other bases the same), the resulting mutant DNAzyme
(called 17Ec) lost its activity completely.
Although 17Ec was as capable of forming blue nanoparticle aggregates as 17E,
no color change was observed in the presence of Pb(II). These results indicate
that Pb-(II)-assisted DNAzyme cleavage of SubAu is responsible for the color
change and thus for the metal ion detection.

The color of the sensor developed on an alumina TLC plate with different Pb(II)
concentrations (a) and with 5 íM concentrations of eight other divalent metal ions (b). The
reactions in both (a) and (b) are carried out in 25 mM Tris-acetate buffer, pH 7.2, containing 25
300 mM NaCl. Colorimetric detection and quantification of lead in leaded paint. The color
developed on a TLC plate by the sensor after reacting with 360 (c) and 15 000-fold (d)
dilution of the soaking solution for the leaded paint.
For quantitative analysis of Pb(II), the ratio of extinction at 522 and 700 nm is
used. These two wavelengths are chosen to represent the relative amount of
free and aggregated gold nanoparticles, respectively. A plot of this extinction
ratio versus Pb(II) concentration shows that this sensor is capable of detecting
Pb(II) between 100 nM and 4 íM, and thus it is well suited for household and
environmental monitoring since 480 nM is considered the toxic level for human
beings. Therefore, a calibration curve such as that shown in Figure c is
necessary for quantitative analysis.

An engineered green fluorescent protein (GFP), mutant BFPms1, can be used to

monitor metal binding based on alteration in fluorescence properties. Zn(II) and
Cu(II) bind to this protein with different coordination geometries with Zn(II)
causing enhanced fluorescence intensity and Cu(II) leading to quenching of
fluorescence.

26
Biosensors in food and agriculture

27
Biosensors have potential application in the food sector as in monitoring food
processing, food quality assessment, food packaging, food storage,
monitoring of shelf-life and viability, indicator of food safety and microbial
contamination, toxin and residual contamination in food.

The major implication in the area of agriculture is physical monitoring

of temperature, humidity, soil quality and fertility, sensing microbiological
microenvironment of the soil, indicator for seed viability and shelf-life,
response sensors for irrigation and safety in agronomy, precision agriculture,
detection of residual pesticides, fertilizers and toxins, and plant pathological
monitoring.

28
Schematics depicting the major nanostructures used in the
area of food and agriculture sector. MNPs magnetic nanoparticles,
AuNPs gold nanoparticles, upconversion nanoparticles, QDs quantum
dots, SWNTs single-wall carbon nanotubes, MWNTs multiwall carbon
29
nanotubes, nanobarcode technology and electronic nose are the major
nanotechnological integrations utilized in the development of
the suitable nanosensors or nanobiosensors.
Quality control is the essential part of a food industry and efficient quality
assurance is becoming increasingly important.

Fruit Maturity, Ripening and Quality Relationships

fruits harvested either too early or too late in their season are more susceptible
to post harvest physiological disorders than fruits harvested at proper
maturity. Fruits can be divided into two groups: 1) Fruit that are incapable of
enduring their ripening process once picked from the plant like berries,
cheery, citrus fruits, grapes, lychee, pineapple, pomegranate, and tamarillo. 2)
Fruits that can be harvested mature and ripped off the plant like apple, apricot,
avocado, banana, cherimoya, guava, kiwifruit, mango, nectarine, papaya, passion
fruit, pear, peach, persimmon, plum, quince, sapodilla, Sapote .

Volatile compounds are responsible for the characteristic aroma of fruits and
are present in extremely small quantities (<100< g/g fresh wt.). The major
volatile formed is ethylene.

30
Organic Acid as an Indicator of Fruit Maturity
Organic acids directly play an important role in the growth, maturation and
acidity of the fruit, and also affect the shelf life of the fruit by influencing the
growth of microorganisms. The citric, malic, oxalic, and tartaric acids ranging
from 0.1 to 30 g/L are found in orange, grape, and apple juices.

There is a considerable difference in the organic acid content found in various

types and brands of fruit juice. For example, Minute Maid contains higher levels
of oxalic and citric acids when compared to all other orange juices tested. Grape
concentrate is found to have lower amount of malic acid than other grape juice,
while freshly squeezed grape juice contains higher amount of tartaric acid.

Brae burn apples contains the highest amount of citric acid in apples; however
Granny Smith apples are the overall most acidic apples tested.
Effect of Shelf Life on the Changes in Organic Acids of Fruits
The content and composition of organic acids in apples (Malus domestica Borkh,
cultivars Gloster, Mutsu, Starting), and apricots (Prunus armeniaca L.,
cvs. Ceglédi bíborkajszi, Csongrádi kajszi, Gönci Magyar kajszi, Nagykõrösi óriás)
are studied.
Malic, ascorbic, citric and succinic acids were found in the different extracts,
depending on the type of fruits . The concentration of malic and ascorbic acids 31
decreases with the increase in storage time and with the increasing radiation
doses whereas the concentration of succinic and citric acids in general increased
during storage.
Pyruvic Acid
Onion flavour is principally directed by the perception of pungency. A
disposable prototype electrochemical screen-printed (carbon-based) biosensor.,
Gwent, UK) was constructed using pyruvate dehydrogenase immobilized on
mediated Meldolas Blue electrodes and a combined Ag/AgCl reference/counter
electrode, both screen-printed onto a PVC substrate to determine pungency in
onions.

The biosensor developed was able to differentiate between mild and pungent
bulbs with pyruvate concentrations ranging between ≈4 and 8 mM in
freshly extracted juices. Electrochemical measurements were carried out at +50
mV at 21˚C.

32
e‑NOSE and e‑TONGUE
The electronic nose and electronic tongue are functionally analogous to the
human sensory perception of odor and taste.
The odor of volatile component and taste of nonvolatile component keep crucial
information about the quality and quantity of the material in food, beverages,
agriculture pharmacology, personal care product manufacturing and processing .
The e-nose and e-tongue are becoming the alternatives and substitute of the
human sensory expert panel and consumer panel established for the quality
assessment and quality control during the manufacturing processes to fulfill
maximum consumers satisfaction.
e-nose comprises four components such as sampling headspace system, a sensor
array, electronic data acquisition control system, and a pattern recognition
software.
The sensor array composite of the chemical sensors which upon contact with the
volatile analyte changes the conductance and gives a detection signal to the
acquisition system.

Metal oxide sensors, conductive polymer sensors, quartz crystal microbalance

sensors, optical sensors, surface acoustic wave sensors, gas-sensitive field effect
transistors are the major kind of sensors used as a component of an electronic
nose. The concentration of dopant material determines the sensitivity and 33
response time of the sensor to the analyte.
The data from the sensor array are being analyzed and classified by the
subsequent electronic component by multivariate signal processing, which in turn
processed by the pattern recognition software based on parametric and
nonparametric algorithm such as principal component analysis (PCA),, partial
least squares (PLS), cluster analysis (CA), fuzzy logic or artificial neural network
(ANN).

Typical electronic nose with its component; sample

head space, nanosensor array, unit for algorithmic
processing, and classified data after the detection in
form of a map. In the functional aspects, volatile
organic compound from the specific sample source of 34
food, fruits and
vegetables, microbes, pesticides, plant components,and waste reach to sensory array from sample head
space. After sensory multivariate algorithmic processing, the signal is forwarded to pattern recognition
software for mapping and result output
 The Biosensor Market
• The United States and Europe captured 68.73% of the
biosensor market in 2008
• Due to large development and manufacturing costs, devices
tend to be specialized into areas the will receive the most
response from the market
• Miniaturization has reduced the price of the fabrication of the
sensors
• Makes products more marketable
 The Biosensor Market
• The biosensor market is dominated by only a few products
• For medical diagnostics, approximately 90% of biosensors are
glucose monitors, blood gas monitors, and electrolyte or
metabolite analyzers
• Half of all biosensors produced worldwide are glucose
monitors
• Sales are projected at $1.28 billion in the US in 2012
• The majority of the remaining market includes biosensors
directed at environmental control, fermentation monitoring,
alcohol testing, and food control
 Use in the Food Industry
• There is an increasing demand for biosensors in the food
industry
• In the past little attention was given to using biosensors to
examine food for pathogens
• However, with a rise of incidents involving contaminated food
there is now a need for a sensor that can accurately and
quickly determine if food is contaminated
• There are few sensors designed to do this now but this is a
major field of new research
 Techniques for
Commercialization
• Home blood glucose monitors
• Have shown several keys to making competitive biosensors in the
market
• Limiting cost both to the manufacturer and consumer
• Need for very high quality and accurate sensors
• Especially in the medical industry where potentially life threatening
illnesses are diagnosed
• Understanding the end users needs
• Sight impaired
• Transparency in users life
• Interface with a physicians work regime
 Techniques for
Commercialization
• R&D of Commercial Sensors
• R&D of commercial biosensors tends to focus on the creation of
new sensors and the miniaturization of new sensors
• Research takes place at both universities and private business
• Because of the high cost to manufacture biosensors,
miniaturization allows more sensors to be made with less
material, energy, and effort
• New research keeps companies and universities at the head of
this quickly changing field
 Techniques for
Commercialization
• Miniaturization
• Need for analysis of a large number of assays
• Cost efficient if small amounts of reagents are used
• Allows for multi-analyte assays
• Academic research
 Commercialization Issues
• The commercialization of biosensors has lagged behind their
research and development
• There are significant costs and technical barriers that can slow
down or block the commercialization of new systems
• The amount of initial capital and technical knowledge that is
required to start developing biosensors is so great that many
new companies simply can not handle them
 Commercialization Issues
• Changes in manufacturing processes, automation, and
miniaturization techniques mean that many biosensors are
already obsolete when they are released
• Customers are not willing to pay high prices of a product that
is not the most advanced of its kind
• As a result companies need to sink a large percentage of their
budget into developing new technologies to stay competitive
• If a company does not have enough capital to develop these
technologies quickly enough, even if their product would
normally be in high demand, they will not be successful
 Commercialization Issues
• Changes in manufacturing processes, automation, and
miniaturization techniques mean that many biosensors are
already obsolete when they are released
• Customers are not willing to pay high prices of a product that
is not the most advanced of its kind
• As a result companies need to sink a large percentage of their
budget into developing new technologies to stay competitive
• If a company does not have enough capital to develop these
technologies quickly enough, even if their product would
normally be in high demand, they will not be successful
 Market Development
• The biosensor market is driven by market demand and by the
companies that produce sensors
• This demand can come from the consumer (market pull) or it
can come from the developer (technology push)
• Push and pull have very different market strategies and they
must be treated differently
• Biosensors that are “pulled” directly by the consumer are
generally more profitable and successful
 Technology Push of
Biosensors
• Technology push deals with the development of biosensors
that may not address a true user need
• These products are developed by a company with the desire
to create a market demand
• Many commercial biosensors are designed with the idea that
if they are available people will develop a need for them
• Generally less successful and profitable until the product
develops a need for its own distinct market
 Market Pull of Biosensors
• Market pull is generated by a true need for a product
• Products that are necessary for the health and well-being of
groups and individuals
• These sensors tend to be related to medicine, safety, and
biological sensing
• Glucose sensors, pathogen detection, EKG sensors
• This is currently the largest and most profitable area for the
development and commercialization of biosensors
 Trends in the Medical
Industry
• The medical industry demands biosensors that are fast,
accurate, and noninvasive
• Sensing time needs to be reduced while maintaining accuracy
of the measurements
• There is a growing demand for sensors that are internal
instead of external to the body
• Glucose sensors that are implantable so users are not required to
pick their fingers several times every day
Monoclonal antibodies

Monoclonal antibody has been generated that recognize metal–EDTA

complexes of cadmium, mercury, copper, nickel, lead, cobalt and silver, besides
many other metal ions. The developed antibodies had maximum binding affinity
for Cd(II) and were studied at a concentration of 100 ppm Cd–EDTA–bovine
serum albumin (BSA) complex. an amperometric sensor that incorporates
Escherichia coli bacterial cells for rapid cyotoxicity analysis. It uses ferricyanine,
a soluble electron mediator, to divert electrons from the respiratory system of
the immobilized bacteria of a suitable carbon electrode. The resulting current is,
thus, a measure of bacterial respiratory activity, and the perturbation by
pollutants can be detected as a change in the magnitude of the current.

49
• Cellsense has been applied to investigate the toxicity of 3,5-
dichlorophenol and other phenols in wastewater , for the
determination of nonionic surfactants and benzene sulfonate
compounds, for the analysis of wastewater treatment works
(WWTW) influent and effluent and for the toxicity testing
of wastewaters and sewage sludge Moreover, Cellsense has
been proposed as one of the newer rapid toxicity assessment
methods within the direct toxicity assessment (DTA)
demonstration program of the UK Environmental Agency.

• Most environmental biosensors have focused on bacterial

systems while eukariotic biosensors are rare, even more rare
is the use of mammalian cells. The mammalian cell, which
is more complex than bacteria, can give a more sensitive
response when compared to bacteria while also responding 50
to the estrogenic effects of chemicals.
 ENVIRONMENTAL
APPLICATIONS
• Biosensors developed for environmental monitoring, first, biosensors that measure
an effect such as toxicity and second, biosensors that detect a compound or a group
of compounds based on the specific recognition of a biomolecule.

• Toxicity: In environmental pollution monitoring, it is becoming a general opinion

that chemical analysis by itself does not provide sufficient information to assess the
ecological risk of polluted waters and wastewaters.

• Bacterial bioluminescence has proved to be a convenient measure of cellular

metabolism and, consequently, a reliable sensor for measuring the presence of toxic
chemicals in aquatic samples. These systems are based on the use of luminescent
bacteria, Vibrio fischeri, to measure toxicity from environmental samples.

• Cellsense®, which is an amperometric sensor that incorporates Escherichia coli

bacterial cells for rapid ecotoxicity analysis. It uses ferricyanine, a soluble electron
mediator, to divert electrons from the respiratory system of the immobilized
bacteria of a suitable carbon electrode. The resulting current is, thus, a measure of
bacterial respiratory activity, and the perturbation by pollutants can be detected as51a
change in the magnitude of the current.
• One approach to solve the lack of specificity of AchE involves the
genetic engineering of cholinesterase enzyme to obtain new specific
enzymes for desired analytes or families.
• The organophosphorous hydrolase (OPH), on the other hand, is able
to hydrolyze a number of OP pesticides such as paraoxon and
parathion, and chemical warfare agents such as sarin and soman.
Hydrolysis of these OP pesticides generates p-nitrophenol, which is
an electroactive and chromophoric product. Thus, OPH could be
combined with an optical transducer to measure the absorbance of p-
nitrophenol or with an amperometric transducer to monitor the
oxidation or reduction current of this product.
• Photosynthesis inhibition is an interesting indicator that rapidly
reflects the toxic effect of certain pollutants. Taking advantage of this
feature, some biosensors based on Photosystem II (PSII) have been
reported to be able to detect herbicides in the environment.
• About 30 % of herbicides, including phenylurea, triazine, and
phenolic herbicides, inhibit photosynthetic electron flow by blocking 52
the PSII quinone-binding site and thus modify chlorophyll
fluorescence.
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• PCBs
• Polychlorinated biphenyls (PCBs) are environmental
pollutants widely used as industrial chemicals,
particularly as dielectric fluids in electrical transformers
and capacitors.
• The high toxicity of some PCB represents a risk for
public health as these compounds are still present in the
environment, even though the production of PCBs has
been banned in several countries many years ago.
• Different biosensor configurations have been designed to
determine PCBs in the environment, and these include
the DNA biosensor with chronopotentiometric detection
and various immunosensors with fluorescence, SPR ,
and electrochemical detection principles. 57
• Dioxins
• Apart from PCBs, other polychlorinated compounds of
environmental concern are the dioxins, which are released as by-
products in a number of chemical processes involving chlorine.
Thus, processes such as the production of some pesticides, the
manufacture of PVC plastics, the chlorine bleaching of pulp and
paper and waste incineration generate dioxins. They are
considered carcinogenic and are a potential threat to human
health.
• Conventional dioxins analysis requires laborious multistep clean-
up procedures that increase the cost of each analysis.
• A significant number of immunoassays for dioxins have been
developed in an effort to provide simplified and routine analysis.
• The SPR biosensor developed by Shimomura for the
determination of PCB (mentioned above) was also employed in
the determination of the dioxin 2,3,7,8-TCDD. Similarly, another
biosensor for detection of dioxin-like chemicals (polyhalogenated58
dioxins, furans, and biphenyls) have been developed.
• Alkanes, aromatic compounds, and polycyclic aromatic
hydrocarbons (PAHs)
• Contamination of soils and surface and groundwater supplies with
petroleum products is a serious environmental problem. Of particular
concern for drinking water quality are water-soluble aromatic
components (e.g., benzene, toluene, ethylbenzene, and xylenes) of
petroleum products. Although many of these contaminants are readily
biodegradable, they often persist in the environment
• A green fluorescent protein-based Pseudomonas fluorescens strain
biosensor was constructed and characterized for its potential to
measure benzene, toluene, ethylbenzene, and related compounds in
aqueous solutions.
• PAHs are carcinogenic compounds generally formed during
incomplete combustion or pyrolysis of organic matter containing
carbon and hydrogen. They are very abundant and recognized
carcinogenic compounds. Amperometric biosensors for naphthalene
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found in contaminated soils, were constructed using Sphingomonas
yanoikuyae B1.
• Antibiotics
• Medical substances have been released into the environment with
very little attention until recently. The presence of antibiotics in
the environment is worrying since they promote antibiotic
resistance. The increasing use of antibiotics for therapeutic
purposes or as growth promoters in dairy cattle and as feed
additives in fish farms or in livestock during the last five decades
has caused a genetic selection of more harmful bacteria, which is a
matter of great concern.
• The widespread administration of antibiotics raises significant
food safety issues since antibiotic resistance can be transferred to
humans on ingestion of affected meat and milk products.
Therefore, most of the biosensors developed are aimed at
determining them in biological or food samples.
• For example, a commercial biosensor BIACORE 3000 was used
to study the cross-reactivity between two sulfonamides (a group of
antibiotics): sulfamethazine and furosemide. Sulfonamides
sometimes cause allergic reactions, whereas their effect in the
human inmunosystem is of high interest for their therapeutical 60
application.
• Microorganisms
• Bacteria, viruses, and other microorganisms are found widely in polluted, untreated,
and treated waters, which implies a worldwide public health problem. Pathogenic
compounds may reach humans by various routes, such as the use of these waters for
recreation or sports, for the irrigation of fruit and vegetables and as drinking water.
Therefore, surface waters may play an important role in the transmission of
pathogens.

• Conventional analytical methods for microorganisms are based on colony-forming

unit (CFU) count and require selective culture, biochemical characterization .These
methods are sensitive and selective, but time-consuming: days to weeks are needed
to get a result.

• The increasing public concern over environment safety has led to a search for
technologies capable of rapidly identifying contamination problems at source.
Biosensors capable of detecting an organism quickly will be important in the
environmental monitoring of pathogens in real time.

• The microbial content of a sample can be determined by monitoring microbial

metabolism. The transducer can either detect consumption of oxygen or the
appearance/disappearance of an electrochemically active metabolite. DNA detection
may be more specific than immunologically based detection. Immunological 61
detection, on the other hand, is faster and more robust than DNA detection.
Moreover, it has the ability to detect not only contaminating organisms, but also
their biotoxins.
• Detection of Salmonella enteriditis and Listeria monocytogenes in real time
using an SPR sensor based on antibodies immobilized on the gold sensor
surface. Salmonella and Listeria were detected by the sensor at
concentrations down to 106 cell/ml.
• Recently, a number of piezoelectric biosensors formats have been developed
for the detection of several microbial contaminants.

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Commercially available
biosensors

63
• Despite the high number of biosensors under development and also the
amount of research literature on this area, few practical systems are currently
enjoying market acceptance. The first successful commercial biosensor was
the “glucose pen”, launched by Exactech in 1987. Nowadays, 90 % of sales
come from glucose-detecting biosensors for medical applications

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 FUTURE PERSPECTIVES
• In spite of the past and current large amount of research in
biosensor development, there is still a challenge to create improved
and more reliable devices. This section presents foreseeable future
trends in biosensor research activities.
• Nanotechnology: Nature is full of intricate nanosystems, far more
advanced than all of the man-made systems. A significant effort is
being made nowadays to either mimic or use such systems in the
development of new applications and devices such as sensors and
biosensors. In order to improve biosensing interfaces, design and
analysis on the molecular level have been proposed. All are
addressing biointerfaces in molecular dimensions and thus can be
summarized by the term “molecular nanotechnology”.

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Gap between Academia and industry for commercialization of
biosensor
• The fast pace of technological change and the market demands for
novel and better products requires continuous innovation and fast
market introduction. The key issues emerging from the present work
refer to early information transfer from university to industry for
achieving the product innovation. This implies primarily a market-
targeted research strategy in the universities, focusing on industrial
needs and not on conventional technology substitution, thus
preventing misjustifications of potential and cost-effectiveness.
• Such an approach could minimize the gap between industry (and
• market) and university output, providing that this output is
• suitably handled and managed by industry during its incorporation
• into in-house R&D.

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THANK YOU

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