bio monitoring of aquatic environment
TRANSCRIPT
Bio-monitoring of Aquatic
Environment
Jitendra Kumar
DFK-1303
Department of Fisheries Resources and Management
College of Fisheries, [email protected]
Objectives
• Definition and Principle of biosensors and Biochips
• History and types of biosensors
• Outline - the current and potential application/s ofbiosensors and in detection of environmentalpollution
• Biochips and their use
• Conclusion
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 functionaland/or structural aspects of biological communities.
• Toxicity bioassays are a laboratory-based methodology forinvestigating and predicting the effect of compounds on testorganisms.
• Behavioral bioassays explore sub-lethal effects of fish or otherspecies when exposed to contaminated water; usually as on-site,early warning systems.
• Bioaccumulation studies monitor the uptake and retention ofchemicals in the body of an organism and the consequent effectshigher up the food chain.
• Fish health studies deal with causes, processes and effects ofdiseases; and can form a complementary indication of overallecosystem 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 amphotericenzyme 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. Thisrequires five days of incubation but a BOD biosensor using theyeast Trichosporon cutaneum with oxygen probe takes only 15 minutesto detect organic pollution.
CONTND……
• Miscellaneous biosensors- A graphite electrode with Cynobacterium andSynechococcus - electron transport inhibition during the photosynthesisdue to certain pollutants e.g. herbicides.
• Biosensors - polychlorinated biphenyls (PCBs) and chlorinatedhydrocarbons and certain other organic compounds.
• Biosensors employing acetylcholine esterase which can be obtained frombovine RBC can be used for the detection of organophosphoruscompounds in water.
Use of Biosensors In Detection of Envt. Pollution
• Toxicity assays - Microtox or Tox Alert - are based on the use ofluminescent bacteria, Vibrio fischeri. Bacterial bioluminescence hasproved to be a convenient measure of cellular metabolism - a reliablesensor.
• Cellsense, which is an amperometric sensor that incorporates Escherichiacoli bacterial cells for rapid ecotoxicity analysis.
• Cell sense - investigate the toxicity of 3,5-dichlorophenol and otherphenols in wastewater for the determination of nonionic surfactants andbenzene 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 becauseof their high toxicity, their increasing environmental levels (due to their use inindustrial 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, andchromium in soil.
• The luminescence-based bacterial sensor strains - Pseudomonas fluo- rescens OS8- mercury and arsenite detection in soil extracts.
Polychlorinated biphenyls (PCBs)
• Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutantswidely used as industrial chemicals, particularly as dielectric fluids inelectrical 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 DNAbiosensor with chronopotentiometric detection and variousimmunosensors with fluorescence, SPR , and electrochemical detectionprinciples.
Dioxins
• Released as by-products in a number of chemical processes involving chlorine
• Processes - production of some pesticides, the manufacture of PVC plastics, thechlorine bleaching of pulp and paper and waste incineration - included in lists ofpotential EDCs.
• Conventional dioxins analysis requires laborious multistep clean-up proceduresthat increase the cost of each analysis. A significant number of immunoassays fordioxins have been developed in an effort to provide simplified and routine analysis.
• The SPR biosensor developed by Shimomura et al. for the determination of PCBwas 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 wascharacterized 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 thesecompounds.
• 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 theenvironment. About 30 % of herbicides, including phenyl urea, triazine, andphenolic herbicides, inhibit photosynthetic electron flow by blocking the PSIIquinone-binding site and thus modify chlorophyll fluorescence
• Recently, a label-free direct piezoelectric immunosensor built on a flow-throughcell was used for the determination of 2,4-D in water with a limit of detectionaround 0.2 µg/l
Antibiotics
• The increasing use of antibiotics for therapeutic purposes or as growth promotersin dairy cattle and as feed additives in fish farms or in livestock - caused a geneticselection of more harmful bacteria, which is a matter of great concern.
• A commercial biosensor BIACORE 3000 - the cross-reactivity between twosulfonamides, sulfa- methazine and furosemide. Sulfonamides sometimes causeallergic reactions, whereas their effect in the human inmunosystem is of highinterest 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 induciblepromoter 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 theproduction of drugs, dyes, and antioxidants.
• Phenolic compounds, and especially chlorophenols, - imp. - because of their hightoxicity and possible accumulation in the environment . They are also considered asprecursors of the dioxins.
• Parellada et al. developed an amperometric biosensor, with tyrosinase (apolyphenol oxidase with a relatively wide selectivity for phenolic compounds)immobilized in a higrogel on a graphite electrode, which correlated satisfactorilywith the official method for the determination of the phenol index inenvironmental samples.
• Chlorophenols - with a flow-injection chemiluminescence fiber optic biosensorexploiting the ability of certain substituted phenols to enhance thechemiluminescence reaction of luminol, catalyzed by horseradish peroxidases.Finally, a Lux-based biosensor was used to assess the toxicity of a paper mill sludgebeing some metals (Cd and Cu) and pentachloro-phenol (PCP).
Toxins
• Toxins are a very heterogeneous group capable of affecting different biochemicalprocesses including membrane function, ion transport, transmitter release, andDNA 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 platinumfilm with an immobilized layer of antibodies against the staphylococcalenterotoxin B. Various evanescent wave immunosensors have also been reportedto be capable of detecting botulin with very low limits of detection . A rapid andsensitive immunosensor for the detection of the Clostridium botulinum toxin Ahas also been developed.
• Tetanus toxin and detection of the cholera toxin - A portable fiber optic biosensorfor quantification of the staphylococcal enterotoxin.
Inorganic phosphate
• Inorganic phosphate found in surface waters is used as a measure of the degree ofeutrophism.
• Traditional methods for its determination are chromatography, volumetric titration,or spectrophotometry. Therefore, the development of simple and fast biosensorsrepresents an interesting alternative to them. Enzymatic phosphate biosensors forphosphate determination.
• Parellada et al. described a configuration based on the sequential action of threeenzymes that opens up a way to the construction of reagentless enzymaticphosphate sensors.
BOD
• BOD sensors have been developed and marketed by various manufacturesin both biofilm and bioreactor-type configurations.
• Most commercially available BOD sensors are flow-type systems that canbe more easily automated, but generally require high maintenance toprevent fouling and clogging.
• Many BOD biosensors - determination of high BOD values in indus- trialwastewater 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 concernbecause they can harm the water environment.
• A biosensor containing immobilized denitrifying - determination of NO3 in tapwater. Through the reduction of NO3– in a reaction chamber, N2O was formed anddetermined by a N2O microelectrode, which was the sensing element of thebiosensor.
• A microscale biosensor for nitrate/nitrite determination was used for in-sitemonitoring in an activated sludge plant. The biosensor was based on the diffusionof nitrate/nitrite through a tip membrane into a dense mass of bacteria convertingthe ions into nitrous oxide with subsequent electrochemical detection
Hormones
• Endogenous hormones of human or animal origin have been reaching theenvironment for thousands of years, Besides endogenous hormones, exogenoussteroids used as growth promoters in several countries have become a matter ofconcern.
• These residues may have endocrine-disrupting activity in aquatic fauna or eventerrestrial. Although very low concentrations (ng/l range) of hormones such asestradiol, estrone, and ethynil- estradiol have been found in water their widespreaduse and their capability to induce responses in fish at concentrations as low as ng/lor even pg/l level, have alerted scientists to the potential dangerous consequencesof 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 andreceptor-based sensors for the rapid detection of androgens such as testosteroneand metabolites.
Endocrine disrupting chemicals
• Nowadays - increasing concern regarding many environmentalcontaminants that produce adverse effects by interfering withendogenous hormone systems, the so-called EDCs.
• Many endocrine disruptors are also believed to bind to the estrogenreceptor (ER). Thus, the binding ability of the chemicals toward the ERwould be a crucial factor for screening or testing their potentialenvironmental toxicity.
• The SPR biosensor BIAcore has been applied in the determination ofestrogens and xenoestrogens.
Surfactants
• Detergent products use surfactants as the basic “active” component. The anionicsurfactants are the most widely used, while the cationic surfactants represent only5 % of the total.
• An amperometric biosensor for detection of anionic surfactants was constructedwith Pseudomonas rathonis T (bearing a plasmid for surfactant degradation) as abiological 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 petroleumproducts is a serious environmental problem. Of particular concern for drinkingwater quality are water-soluble aromatic components (e.g., benzene, toluene,ethylbenzene, and xylenes) of petroleum products. Although many of thesecontaminants 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 aqueoussolutions.
• Another microbial whole-cell biosensor, using E. coli with the promoter luciferaselux AB gene- determination of water-dissolved linear alkanes by luminescence. Thebiosensor was used to detect the bioavailable concentration of alkanes in heatingoil-contaminated groundwater samples.
• PAHs are carcinogenic compounds generally formed during incomplete combustionor pyrolysis of organic matter containing carbon and hydrogen. They are veryabundant, ubiquitous, and recognized carcinogenic compounds. Amperometricbiosensors for naphthalene found in contaminated soils, were constructed usingSphingomonas yanoikuyae B1. For benzo(a)pyrene (BaP) and related adducts, afiber optic fluoro-immunosensor - high sensitivity is achieved by laser excitationand optical detection.
Bioremediation
• Bioremediation is an application of the microbial capacity to transformcomplex organic molecules into simpler inorganic constituents.
• Biosensors that can monitor these parameters will help to better controlthe bioremediation process. Different molecular biosensors implementedto monitor these parameters were reviewed by Purohit. These biosensorsuse the luciferase expression system.
• The biological component in this molecular biosensor is a recombinantplasmid. It has a specific promoter, whose expression is sensitive to atarget 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 usingan SPR sensor based on antibodies immobilized on the gold sensor surface.
• Salmonella and Listeria - sensor at concentrations down to 106 cell/ml. Recently, anumber of piezoelectric biosensors are used.
• Salmonella typhimurium detection in liquid samples by an immunosensor based onthe 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 geneof A. hydrophila and of distinguishing between samples that contained the geneand samples that did not.
CONTND…
• Ercole et al. described a biosensor for the determination of E. coli inwater samples by an immunochemical potentiometric alternatingbiosensor.
• Hasebe et al. described an amperometric tyrosinase-based biosensor forthe detection of E. coli in wastewater. The detection was based ontyrosinase-catalyzed oxidation of polyphenolic compounds, which areproduced microbiologically from salicylic acid, and the subsequent signalamplification. The sensor was capable of detecting 103–104 cells/ml afteran 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 IMMNUNOBIOSENSOR 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 changesunderpins the formulation of effective management strategies forsustainable use and protection of the marine environment.
• Sensors are traditionally used in marine studies to determine physicalparameters, but there is increasing demand for real-time informationabout chemical and biological parameters.
• These parameters are currently measured in samples collected at sea andsubsequently 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 freshwaterand wastewater.
BIOCHIPS
• BIOCHIPS carry a high-density array of test sites that can analyze a singledroplet of a sample for hundreds to thousands of biological agentssimultaneously.
• BIOCHIPS contain hundreds to thousands of test sites, each chip being amatrix 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 glassor plastic substrate. • The array of sites can be tailored for the specificneeds of users, whether to detect bioterrorism agents, specific strains ofinfectious diseases or other biological organisms.
• Each site can detect the presence of trace quantities of the agents forwhich 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 aspecific biological agent or biochemical signature. These pads are in knownpositions 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 onlyhindrance towards development lies on currentpolicies and financial restrictions. World HealthOrganization as well as Environmental ProtectionAgencies of various countries should take fulladvantage of these biosensors in environmentalmonitoring, detection of various kinds of pollutants,bioremediation.
CONTND…
• However, the use of such devices may besubjected to abuse, therefore appropriatelaws, policies, rules and regulations should bein place prior to full launching of researchesassociated with clinical trials and ecosystemelement participation. In the end, disposal ofused or consumed biosensors may also posesignificant ecological and health risk if notproperly done.