Bio monitoring of aquatic environment

Bio-monitoring of Aquatic
Environment
Jitendra Kumar
DFK-1303
Department of Fisheries Resources and Management
College of Fisheries, Mangalorejitenderanduat@gmail.com

Objectives
• Definition and Principle of biosensors and Biochips
• History and types of biosensors
• Outline - the current and potential application/s of
biosensors and in detection of environmental
pollution
• Biochips and their use
• Conclusion
jitenderanduat@gmail.com

Biosensors
Biosensor” is defined as-
– A biosensor is an analytical device, used for the
detection of an analyte, that combines a biological
component with a physicochemical detector.
– Biosensors are analytical devices which are
capable of providing either qualitative or
quantitative results.

General principle of biosensors
• A biosensor can be considered as a
combination of a bio-receptor, the biological
component, and a transducer, the detection
method.
• The first link of a biosensor is the bio-receptor,
which has a particularly selective site that
identifies the analyte.

• This localized modification is generally made via
an immobilized enzyme, which transforms the
analyte into a product that is detectable by the
transducer.
• This is the case for enzyme sensors.
• Sometimes, however, the enzyme is only stable in
its natural environment, which cannot be
modified, and the whole cell or microorganism is
immobilized on the biosensor

Principle
Biological detector – enzyme, cell
Physical component - transducer
Processing unit
Biosensor has three parts:

– Principle of detection - The specific binding of the analyte of
interest to the complementary bio-recognition element
immobilized on a suitable support matrix .
– The specific interaction results in a change in one or more physico-
chemical properties (viz. pH change, electron transfer, mass
changes, heat transfer, uptake or release of gases or specific ions)
which can be detected and measured by the transducer
– The usual aim is to produce an electronic signal, which is
proportional to the concentration of a specific analyte or group of
analytes, to which the biosensing element binds.

Biosensors applications
• The most important applications are in
medicine (in hospitals or in the home) and in
the food produce industry for the control of
manufacturing processes.
• More recently, many biosensors have been
applied to environmental control:
• the bio-receptor being purified enzymes or
whole cells directly immobilized on a
transducer.

categories,
• Bioassessments are based on ecological surveys of the functional
and/or structural aspects of biological communities.
• Toxicity bioassays are a laboratory-based methodology for
investigating and predicting the effect of compounds on test
organisms.
• Behavioral bioassays explore sub-lethal effects of fish or other
species when exposed to contaminated water; usually as on-site,
early warning systems.
• Bioaccumulation studies monitor the uptake and retention of
chemicals in the body of an organism and the consequent effects
higher up the food chain.
• Fish health studies deal with causes, processes and effects of
diseases; and can form a complementary indication of overall
ecosystem health.
(Roux et al, 1993):

Brief History of Biosensors
Discovered by Leland C. Clark in 1962
1962 – Leland C. Clark first described a biosensor as an amphoteric
enzyme electrode for glucose
1969 – First potentiometric biosensor
1970s– Ion Selective Field Effect Transistor, Fiber Optic Sensor
1980s–First Surface Plasmon Resonance Immunosensor (SPR)
1990s – SPR based and handheld biosensors
Current – Quantum dots, nanoparticles

Types of Biosensors
 Biosensors can be classified - according to bio- receptor used:
 Enzymes,
 Antibody/ antigens
 Nucleic acids/complementary sequences.
 Microorganisms, animal or plant whole cells and tissue slices
 Depending on the method of signal transduction:
 Electrochemical (Amperometric, Potentiometric or Conductometric),
 Optical,
 Magnetic
 Micromechanical
 Thermometric and
 Piezoelectric.

Some Important Biosensors used in Environmental Pollution monitoring
• Gas biosensors- Sulphur dioxide, Methane, Carbon dioxide
• Microbial biosensors – Thiobacillus - SO2, Methane - Methalomonas.
Pseudomonas - Carbon dioxide
• Immunoassay biosensors- Triazines, Malathion and Carbamates
• BOD biosensor- (BOD) - detect the levels of organic pollution. This
requires five days of incubation but a BOD biosensor using the
yeast Trichosporon cutaneum with oxygen probe takes only 15 minutes
to detect organic pollution.

CONTND……
• Miscellaneous biosensors- A graphite electrode with Cynobacterium and
Synechococcus - electron transport inhibition during the photosynthesis
due to certain pollutants e.g. herbicides.
• Biosensors - polychlorinated biphenyls (PCBs) and chlorinated
hydrocarbons and certain other organic compounds.
• Biosensors employing acetylcholine esterase which can be obtained from
bovine RBC can be used for the detection of organophosphorus
compounds in water.

Use of Biosensors In Detection of Envt. Pollution
• Toxicity assays - Microtox or Tox Alert - are based on the use of
luminescent bacteria, Vibrio fischeri. Bacterial bioluminescence has
proved to be a convenient measure of cellular metabolism - a reliable
sensor.
• Cellsense, which is an amperometric sensor that incorporates Escherichia
coli bacterial cells for rapid ecotoxicity analysis.
• Cell sense - 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.
• Cell sense - one of the newer rapid toxicity assessment

Heavy metals
• Determination - Cu, Cd, Hg, and Zn in the environment is very important because
of their high toxicity, their increasing environmental levels (due to their use in
industrial processes) - because metals can bioaccumulate in living organisms,
especially in marine organisms.
• Recombinant luminescent bacterial sensors were used by Ivask et al -
determination of the bioavailable fraction of cadmium, zinc, mercury, and
chromium in soil.
• The luminescence-based bacterial sensor strains - Pseudomonas fluorescens OS8
- mercury and arsenite detection in soil extracts.

Polychlorinated biphenyls (PCBs)
• Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants
widely used as industrial chemicals, particularly as dielectric fluids in
electrical transformers and capacitors.
• The high toxicity of some PCB congeners represents a risk for public health
• Different biosensor - PCBs in the environment, and these include the DNA
biosensor with chronopotentiometric detection and various
immunosensors with fluorescence, SPR , and electrochemical detection
principles.

Dioxins
• Released as by-products in a number of chemical processes involving chlorine
• Processes - production of some pesticides, the manufacture of PVC plastics, the
chlorine bleaching of pulp and paper and waste incineration - included in lists of
potential EDCs.
• 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 et al. for the determination of PCB
was also employed in the determination of the dioxin 2,3,7,8-TCDD. Similarly,
another biosensor for detection of dioxin-like chemicals (poly- halogenated dioxins,
furans, and biphenyls) based on a recombinant mouse hepatoma cell line was
characterized and optimized by Pasini et al.

Biocides
• 0.1 µg/l - individual pesticides and of 0.5 µg/l - total pesticides.
• Pesticide determination - 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 -
developed for the detection of organophosphorous and carbamate pesticides.
• some biosensors based on Photosystem II (PSII) - able to detect herbicides in the
environment. About 30 % of herbicides, including phenyl urea, triazine, and
phenolic herbicides, inhibit photosynthetic electron flow by blocking the PSII
quinone-binding site and thus modify chlorophyll fluorescence
• Recently, a label-free direct piezoelectric immunosensor built on a flow-through
cell was used for the determination of 2,4-D in water with a limit of detection
around 0.2 µg/l

Antibiotics
• 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 - caused a genetic
selection of more harmful bacteria, which is a matter of great concern.
• A commercial biosensor BIACORE 3000 - the cross-reactivity between two
sulfonamides, sulfa- methazine and furosemide. Sulfonamides sometimes cause
allergic reactions, whereas their effect in the human inmunosystem is of high
interest for their therapeutical application.
• Hansen and Sorensen presented three different reporter gene systems from V.
fischeri, E. coli, and Aequorea victoria all combined with a tetracycline inducible
promoter in the development of three corresponding whole-cell biosensors.
Biosensors were able to determine penicillin G or tetracyclines , both in milk.

Phenol
• Phenolic compounds - originate from the paper and pulp industry and from the
production of drugs, dyes, and antioxidants.
• Phenolic compounds, and especially chlorophenols, - imp. - because of their high
toxicity and possible accumulation in the environment . They are also considered as
precursors of the dioxins.
• Parellada et al. developed an amperometric biosensor, with tyrosinase (a
polyphenol oxidase with a relatively wide selectivity for phenolic compounds)
immobilized in a higrogel on a graphite electrode, which correlated satisfactorily
with the official method for the determination of the phenol index in
environmental samples.
• Chlorophenols - with a flow-injection chemiluminescence fiber optic biosensor
exploiting the ability of certain substituted phenols to enhance the
chemiluminescence reaction of luminol, catalyzed by horseradish peroxidases.
Finally, a Lux-based biosensor was used to assess the toxicity of a paper mill sludge
being some metals (Cd and Cu) and pentachloro-phenol (PCP).

Toxins
• Toxins are a very heterogeneous group capable of affecting different biochemical
processes including membrane function, ion transport, transmitter release, and
DNA and protein synthesis.
• An integrated optical sensor - analysis of aflatoxin B in corn. A potentiometric -
analysis of saxitoxin and ricin has also been described.
• An impedance-based immunosensor - prepared by using an ultrathin platinum
film with an immobilized layer of antibodies against the staphylococcal
enterotoxin B. Various evanescent wave immunosensors have also been reported
to be capable of detecting botulin with very low limits of detection . A rapid and
sensitive immunosensor for the detection of the Clostridium botulinum toxin A
has also been developed.
• Tetanus toxin and detection of the cholera toxin - A portable fiber optic biosensor
for quantification of the staphylococcal enterotoxin.

Inorganic phosphate
• Inorganic phosphate found in surface waters is used as a measure of the degree of
eutrophism.
• Traditional methods for its determination are chromatography, volumetric titration,
or spectrophotometry. Therefore, the development of simple and fast biosensors
represents an interesting alternative to them. Enzymatic phosphate biosensors for
phosphate determination.
• Parellada et al. described a configuration based on the sequential action of three
enzymes that opens up a way to the construction of reagentless enzymatic
phosphate sensors.

BOD
• BOD sensors have been developed and marketed by various manufactures
in both biofilm and bioreactor-type configurations.
• Most commercially available BOD sensors are flow-type systems that can
be more easily automated, but generally require high maintenance to
prevent fouling and clogging.
• Many BOD biosensors - determination of high BOD values in industrial
wastewater and not adapted to the measurement of low BOD values.
• An optical fiber biosensor - evaluation of low BOD values in river waters

Nitrate
• The increasing nitrate levels found in ground and surface waters are of concern
because they can harm the water environment.
• A biosensor containing immobilized denitrifying - determination of NO3 in tap
water. Through the reduction of NO3– in a reaction chamber, N2O was formed and
determined by a N2O microelectrode, which was the sensing element of the
biosensor.
• A microscale biosensor for nitrate/nitrite determination was used for in-site
monitoring in an activated sludge plant. The biosensor was based on the diffusion
of nitrate/nitrite through a tip membrane into a dense mass of bacteria converting
the ions into nitrous oxide with subsequent electrochemical detection

Hormones
• Endogenous hormones of human or animal origin have been reaching the
environment for thousands of years, Besides endogenous hormones, exogenous
steroids used as growth promoters in several countries have become a matter of
concern.
• These residues may have endocrine-disrupting activity in aquatic fauna or even
terrestrial. Although very low concentrations (ng/l range) of hormones such as
estradiol, estrone, and ethynil- estradiol have been found in water their widespread
use and their capability to induce responses in fish at concentrations as low as ng/l
or even pg/l level, have alerted scientists to the potential dangerous consequences
of their presence in the aquatic environment.
• Estrogen, along with other organic pollutants (atrazine and isoproturon) -
determined with an optical immunosensor in real water samples.
• A group of researchers are developing single and multi-analyte affinity sensors and
receptor-based sensors for the rapid detection of androgens such as testosterone
and metabolites.

Endocrine disrupting chemicals
• Nowadays - increasing concern regarding many environmental
contaminants that produce adverse effects by interfering with
endogenous hormone systems, the so-called EDCs.
• Many endocrine disruptors are also believed to bind to the estrogen
receptor (ER). Thus, the binding ability of the chemicals toward the ER
would be a crucial factor for screening or testing their potential
environmental toxicity.
• The SPR biosensor BIAcore has been applied in the determination of
estrogens and xenoestrogens.

Surfactants
• Detergent products use surfactants as the basic “active” component. The anionic
surfactants are the most widely used, while the cationic surfactants represent only
5 % of the total.
• An amperometric biosensor for detection of anionic surfactants was constructed
with Pseudomonas rathonis T (bearing a plasmid for surfactant degradation) as a
biological element.
• The microbial biosensor enabled detection of surfactants with high selectivity,
sensitivity, and reproducibility.
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 .

CONTND…
• A green fluorescent protein-based Pseudomonas fluorescens strain biosensor -
measure benzene, toluene, ethylbenzene, and related compounds in aqueous
solutions.
• Another microbial whole-cell biosensor, using E. coli with the promoter luciferase
lux AB gene- determination of water-dissolved linear alkanes by luminescence. The
biosensor was used to detect the bioavailable concentration of alkanes in heating
oil-contaminated groundwater samples.
• PAHs are carcinogenic compounds generally formed during incomplete combustion
or pyrolysis of organic matter containing carbon and hydrogen. They are very
abundant, ubiquitous, and recognized carcinogenic compounds. Amperometric
biosensors for naphthalene found in contaminated soils, were constructed using
Sphingomonas yanoikuyae B1. For benzo(a)pyrene (BaP) and related adducts, a
fiber optic fluoro-immunosensor - high sensitivity is achieved by laser excitation
and optical detection.

Bioremediation
• Bioremediation is an application of the microbial capacity to transform
complex organic molecules into simpler inorganic constituents.
• Biosensors that can monitor these parameters will help to better control
the bioremediation process. Different molecular biosensors implemented
to monitor these parameters were reviewed by Purohit. These biosensors
use the luciferase expression system.
• The biological component in this molecular biosensor is a recombinant
plasmid. It has a specific promoter, whose expression is sensitive to a
target molecule.

Microorganisms
• Bacteria, viruses, and other microorganisms are found widely in polluted,
untreated, and treated waters, which implies a worldwide public health problem.
• 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 - sensor at concentrations down to 106 cell/ml. Recently, a
number of piezoelectric biosensors are used.
• Salmonella typhimurium detection in liquid samples by an immunosensor based on
the acoustic wave principle was reported by Pathirana et al Piezoelectric biosensors
- Aeromonas hydrophila, were immobilized onto the surface of a streptavidin-
coated gold surface of a quartz crystal.
• This sensor was capable of detecting a PCR product amplified from a specific gene
of A. hydrophila and of distinguishing between samples that contained the gene
and samples that did not.

CONTND…
• Ercole et al. described a biosensor for the determination of E. coli in
water samples by an immunochemical potentiometric alternating
biosensor.
• Hasebe et al. described an amperometric tyrosinase-based biosensor for
the detection of E. coli in wastewater. The detection was based on
tyrosinase-catalyzed oxidation of polyphenolic compounds, which are
produced microbiologically from salicylic acid, and the subsequent signal
amplification. The sensor was capable of detecting 103–104 cells/ml after
an enrichment step.

COMMERCIALLY AVAILABLE BIOSENSORS FOR THE DETECTION OF ENVT.
POLLUTANTS
INSTRUMENT BIOLOGICAL
ELEMENT
TRANSDUCTION ANALYTE
BIOACORE BIOMOLECULAR
INTERACTIONS
OPTICAL (SPR) SULFONAMIDE
PATHOGENS
IBIS BIOMOLECULAR
INTERACTIONS
OPTICAL (SPR)
SPR-CELLIA WHOLE CELLS OR
MACROMOLECULES
OPTICAL (SPR)
REMEDIOS WHOLE CELLS OPTICAL
BIOLUMINESCENCE
TOXICITY
PZ
IMMNUNOBIOSENSO
R SYSTEM
ANTIBODIES PIZOELECTRIC PATHOGENS
ARAS BOD BOD
NECi`s Nitrate
Biosensor
Nitrate reductase Amperometric Nitrate

Biosensors for marine pollution
• Measurement of ecological, climatic and anthropogenic changes
underpins the formulation of effective management strategies for
sustainable use and protection of the marine environment.
• Sensors are traditionally used in marine studies to determine physical
parameters, but there is increasing demand for real-time information
about chemical and biological parameters.
• These parameters are currently measured in samples collected at sea and
subsequently analysed in the laboratory.
• While their potential for use in the marine environment is enormous,
much published work to date has focused on applications in freshwater
and wastewater.

BIOCHIPS
• BIOCHIPS carry a high-density array of test sites that can analyze a single
droplet of a sample for hundreds to thousands of biological agents
simultaneously.
• BIOCHIPS contain hundreds to thousands of test sites, each chip being a
matrix of three- dimensional gel pads about 100x100x20 microns in size
(100 microns is about the width of a human hair).
• Thousands of individual test sites can be immobilized on a 1x3 inch glass
or plastic substrate. • The array of sites can be tailored for the specific
needs of users, whether to detect bioterrorism agents, specific strains of
infectious diseases or other biological organisms.
• Each site can detect the presence of trace quantities of the agents for
which they are set up.

CONTND…..
• BIOCHIP has hundreds to thousands of gel pads. A segment of a DNA strand,
protein, peptide or antibody is inserted into each pad, tailoring it to recognize a
specific biological agent or biochemical signature. These pads are in known
positions so, when a sample reacts, the reaction position can be detected,
identifying the sample.
• Syringe mini-column (20 min). Purified, fragmented and labeled DNA or RNA
• 1 hr hybridization
• Image acquisition (2 min)
• Automated analysis (30 sec)
• Complete analysis in two hours or less
• Biochip Technology - improving energy efficiency, reducing wastes and pollution,
lowering production costs, and improving productivity.
• The system makes use of the polymerase chain reaction (PCR), a universal method
for converting one piece of genetic material into billions of copies. PCR is, in effect,
a biological amplifier that enables low-abundance bacteria and threat agents to be
detected with relative ease, within hours instead of days.

CONCLUSION
• With these technological advancements, the only
hindrance towards development lies on current
policies and financial restrictions. World Health
Organization as well as Environmental Protection
Agencies of various countries should take full
advantage of these biosensors in environmental
monitoring, detection of various kinds of pollutants,
bioremediation.

CONTND…
• However, the use of such devices may be
subjected to abuse, therefore appropriate
laws, policies, rules and regulations should be
in place prior to full launching of researches
associated with clinical trials and ecosystem
element participation. In the end, disposal of
used or consumed biosensors may also pose
significant ecological and health risk if not
properly done.

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