Electrochemical Biosensors for Pollutants in the Environment

Review

Electrochemical Biosensors for Pollutants in the EnvironmentMichal Badihi-Mossberg, Virginia Buchner, Judith Rishpon*

Faculty of Life Sciences, Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat-Aviv 69978, Israel*e-mail: [email protected]

Received: May 14, 2007Accepted: June 27, 2007

AbstractThis article reviews recent advances in electrochemical biosensing and detection of environmental pollutants.Electrochemical biosensors offer precision, sensitivity, rapidity, and ease of operation for on-site environmentalanalysis. An electrochemical biosensor is an analytical device in which a specific biological recognition element(bioreceptor) is integrated within or intimately associated with an electrode (transducer) that converts the recognitionevent to a measurable electrical signal for the purpose of detecting a target compound (analyte) in solution. Thisapproach not only provides the means for on-site analysis but also removes the time delay and sample alteration thatcan occur during transport to a centralized laboratory. We first address the basic principles of merging ofelectrochemistry and biology into a biosensing system, and then we discuss various environmental monitoringstrategies involving this technology.

Keywords: Environmental monitoring, Electrochemical biosensors, Contaminants, Pollutants

DOI: 10.1002/elan.200703946

1. Introduction

Environmental monitoring typically involves several stepssuch as sampling, sample handling, and sample transporta-tion to specialized laboratories. The challenge of environ-mental monitoring in situ requires new and improvedanalytical devices featuring precision, sensitivity, specificity,rapidity, and ease of operation to detect decreasing concen-trations of an ever growing array of pollutants. Such devicesmust be comparable to or better than traditional analyticalsystems, and must be simple to handle, small, cheap, able toprovide reliable information in real-time, and must besensitive and selective for the analyte of interest, andsuitable for in situ monitoring. Biosensors not only fulfill allthese requirements but also have awide range of applicationin the areas of clinical diagnostics, forensic chemistry,pharmaceutical studies, food quality control, biologicalwarfare detection, and environmental monitoring. Biosen-sors are small devices linking biological elements with signalproviding mechanisms for fast and efficient warning ofpollution incidents. Themain advantage of biosensors is thatthey provide real-time, on-site detection and analysis in thefield and often eliminate the need for sample collection,preparation, and laboratory analysis.The challenge of continuous in situ monitoring of

environmental pollution in the field requires instrumentsthat are robust and with sufficient sensitivity and longlifetime. Commonly used conventional methods are time-consuming, expensive, require skilled operators, and lackthe required selectivity. Biosensors have the advantage ofbeing simple, uniform whole structures featuring directtransduction, high bioselectivity, high sensitivity, miniatur-

ization, electrical/ optoelectronic readout, continuous mon-itoring, ease of use, and cost effectiveness. User advantagesinclude low price, reliability, no sample preparation, dis-posability, and clean technology. Hence, biosensors showthe potential to complement both laboratory-based andfield analytical methods for environmental monitoring [1 –4].The biocatalytic recognition element provides a high

degree of selectivity for the analyte to be measured withoutcomplex sample processing. Hence, biosensors can detectbiological or chemical species directly with an accuracyapproaching that of traditional laboratory-based analyzers.This direct approach precludes not only the time delay butalso the possibility of sample alteration that can occurduring transport to a centralized analytical laboratory. Suchdevices are compact, portable, and cost-effective. Mostbiosensors are based on reactions catalyzed by macro-molecules that are present in their original biologicalenvironment, previously isolated, or manufactured [5].The “lock and key” conformation of enzyme-substrate,hormone-receptor, or antigen-antibody brings the ligandsnear to the working electrode. When such an agent isincorporated into the sensor, a successive consumption ofsubstrate(s) can be accomplished. Miniaturized screen-printed electrodes in a multisensor array can be used for theparallel determination of several toxicants in real time. Thehigh level of precision and reproducibility of screen-printingtechnology is ideal for measuring toxicants outdoors in aportable monitoring system. The biosensor can produceeither individual or successive digital electronic signals thatare equivalent to the concentration of a single analyte or agroup of analytes to be monitored. Biosensors have been

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produced for many chemical compounds of environmentalinterest. Promising applications include groundwater mon-itoring, drinking-water analysis, and the rapid analysis ofextracts of soils and sediments at hazardous waste sites [6].Environmental monitoring requires rugged sensors for

the detection of pollution and toxic chemicals, and auto-mated and continuous, remote and in situmonitoring will beincreasingly required. Today, electrochemical biosensorsare at the forefront of a multidisciplinary science combiningthe fields of electrochemistry and biology. Devices combin-ing the selectivity of biological molecules with the process-ing power of microelectronics offer a new approach forenvironmental monitoring that can be carried out in situ oronline.An electrochemical biosensor comprises a biologicalrecognition element immobilized on the surface of anelectrode and a physicochemical detector component. Thebioreceptor translates information from the analyte into achemical or physical output signal with a defined sensitivityfor quantifying the analyte to be monitored, and thetransducer converts the recognition event to a measurableoutput signal. Concentration range, disposability, reusabil-ity, or renewability, accuracy and reproducibility, size ofsensor, and size of analyte sample are important aspects forchoosing electrochemical biosensors for environmentalmonitoring.The advantage of distinguishing oxidation states is highly

important. The electrochemical approach can give a rapidanswer, without digestion, as to the labile fraction of a givenelement in a particular oxidation state, and the experimentcan be performed on-site in the field. The advantage ofelectrochemical biosensors is their high specificity, sensitiv-ity, rapid response time, and ease of operation, thus fulfillingall the requisites for on-site environmental monitoring.Enzymes, antibodies, nucleic acids, hormone receptors,microorganisms, and tissue have been used widely in theconstruction of electrochemical biosensors, which havebecome indispensable for water and food quality controlapplications.Because they can be taken to the sampling area, electro-

chemical biosensing devices have amajor impact upon the insitu monitoring of priority pollutants. The ability of suchdevices to provide rapid and reliable real-time informationabout the chemical composition of the surrounding environ-ment is an important property for monitoring a variety ofpublic health hazards. A group of chemicals known aspersistent organic pollutants (POPs) can travel thousands ofmiles, accumulate in the food chain, and can resist degra-dation in the environment for centuries.Immobilization of the bioreceptor is crucial for electro-

chemical biosensing. The element can be immobilized in athin layer at the transducer surface using such techniques asentrapment behind amembrane, within a polymeric matrix,or within self-assembled monolayers or bilayer lipid mem-branes; covalent bonding of receptors on membranes orsurfaces activated by means of bifunctional groups orspacers; or the bulk modification of entire electrodematerial.

2. Enzyme Biosensors

Enzyme biosensors are based on absorbing enzymes, whoseproducts canbemeasured after degradationof the substrate,to the electrode surface. The electrode amperometrically orpotentiometrically monitors changes by following thebiocatalytic reaction. The current or potential measured isproportional to the rate-limiting step in the overall reaction.Enzyme biosensors are prepared by attaching to theelectrode surface an enzyme whose products can bemeasured after the degradation of a substrate. Such systemsusually involve the catalysis of redox reactions where eitherthe substrate or the product is electrically charged. Manydifferent types of enzyme biosensors have been developedfor environmental monitoring. Environmental pollutantslike parathion, nitrate, and formaldehyde canbedetected bysulfite parathion hydrolase, nitrate reductase, and formal-dehyde dehydrogenase [7 – 10]. Additionally, several bio-sensors for pesticides and toxic metals monitoring are basedon the inhibition of enzymes [11 – 14].

2.1. Pesticides

Pesticides account for the greatest number of reports forenvironmental biosensors. A pesticide is any substance ormixture of substances intended for preventing, destroying,repelling, or lessening the damage of any pest, as defined bytheUSEnvironmental ProtectionAgency (EPA). Of all theenvironmental pollutants, pesticides are themost abundant,present in water, the atmosphere, soil, plants, and food.

2.1.1. Organophosphorus (OP) Compounds

Organophosphorus (OP) compounds are a group of chem-icals that are widely used as insecticides in modernagriculture for controlling a wide variety of insect pests,weeds, and disease-transmitting vectors. The acute toxicproperties ofOPs are due to their ability to inhibit a group ofhydrolytic enzymes called esterases [15]. Acetylcholine(ACh) is one of several chemicals (neurotransmitters) thatare essential for the proper function of the nervous systemsof both humans and insects.Acetylcholine causesmuscles tocontract. To preventmuscle paralysis and death, the enzymeacetylcholine esterase (AChE) immediately cleaves theneurotransmitter to enable muscle relaxation. Because OPsfirmly bind to AChE to form a stable complex that disablesits enzymatic activity, the ability to detect OP in theenvironment is vital.Direct, selective, rapid and simple determination of

organophosphate pesticides has been achieved by integrat-ing organophosphorus hydrolase with electrochemical andoptical transducers. Organophosphorus hydrolase catalyzesthe hydrolysis of a wide range of organophosphate com-pounds, releasing an acid andanalcohol that canbedetecteddirectly. Wang et al. [16] and Mulchandani et al. [17 – 21]developed applications of organophosphorus hydrolase-based potentiometric, amperometric and optical biosensors.

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Several enzyme electrodes have been developed for theenvironmental monitoring of OPs based on pesticide-induced decreases in AChE activity. Neufeld et al. [22]developed a disposable enzymatic biosensor based on theAChE-acetylthiocholine-hexacyanoferrate(III) reaction.The sensor comprises an electrochemical cell consisting ofscreen-printed electrodes covered with an enzymatic mem-brane,which is placed in ahome-made flowcell. ThebindingbetweenAChEand anOPcompoundhinders the enzymaticdegradation of acetylthiocholine chloride to thiocholine andacetic acid. The free thiocholine then reacts with hexacya-noferrate ion in theworking solution. The ensuing reductionof ferricyanide to ferrocyanide and its subsequent reoxida-tion by the electrode generates very sharp, rapid, andreproducible electric signals that are proportional to thethiocholine concentration. The decrease in enzymaticactivity in response to the OP compound dimethyl 2,2-dichlorovinyl phosphate is presented in Figure 1. Thesystem can trace small quantities of a desired analyte. Theadvantage this system offers lies in the ability to work withsmall amounts or volumes of samples, preventing risks thatcan threaten human health.

2.1.2. Parathion

Parathion (O,O-diethyl-O-4-nitro-phenylthiophosphate), isa broad-spectrum OP pesticide having a wide range ofapplications against numerous insect species on severalcrops. Parathion is also used as a preharvest soil fumigantand foliage treatment for awide variety of plants, both in thefield and in the greenhouse [23]. Parathion is highly toxic byall routes of exposure – ingestion, skin adsorption, andinhalation, all of which have resulted in human fatalities.Like allOPpesticides, parathion irreversibly inhibitsAChE.Sacks et al. [24] developed an amperometric organo-

phosphorus hydrolase (OPH)-based biosensor for the directmeasurement of parathion. The enzyme,which catalyzes thehydrolysis of parathion to form p-nitrophenol, was immo-

bilized on a carbon electrode. Enzymatic activity is detect-able by its anodic oxidation. The current signal is linearlyproportional to the parathion concentration, and thedetection limit is lower than 1 ng mL�1. A significantreduction in sample volume was achieved by using screen-printed electrodes and microflow-injection methods, whichenhanced the sensitivity of the system; the use of pulsedtechniques further increased the sensitivity.

2.2. Air Pollutants

Air monitoring presents a special challenge because highselectivity, high sensitivity, real-time monitoring, and inex-pensive analyses are required. Especially during emergen-cies, major incidents (fires and releases to the atmosphere)require real time data that can assist in making decisionsabout safety warnings and/or evacuations. For this purpose,emergency air monitoring mobile laboratories are designedtomonitor a number of substances and to provide the publicwith an immediate snapshot of the air quality of the locationmonitored. Presently used chromatographic methods usu-ally involve the use of a passive air pollutant sampling deviceand analysis in GC/MS systems that are expensive topurchase andmaintain. Electrochemical biosensing systemswould be ideal for use in such situations because they offergreater selectivity than many direct reading systems in useand are inexpensive to purchase and operate.

2.2.1. Formaldehyde

The chemical compound formaldehyde (also known asmethanal) is a gas with an acrid smell. Most formaldehyde isused in the production of toothpaste, polymers, chemicals,and permanent adhesives. The compound is used as the wetresin added to sanitary paper products, such as facial tissue,napkins, and roll towels. Formaldehyde is one of thehazardous air pollutants (HAP) that emerged from theindustrial revolution. The compound, which is present insmoke from forest fires, in automobile exhaust, and intobacco smoke, is toxic, allergenic, and accumulates in theair.In 2000, Herschkovitz et al. [25] presented an electro-

chemical biosensor for formaldehyde based on a flow-injection system using formaldehyde dehydrogenase and aOs(bpy)2-poly(vinylpyridine) (POs-EA) chemically modi-fied, screen-printed electrode. The dehydrogenase enzymesoxidize a substrate by transferring one or more protons anda pair of electrons to an acceptor, usually NAD/NADP. Thecontinuous flux of substrate to the system guaranteed by theflowcell prevents the accumulationor adsorption of productto the electrode. Another advantage of this system is theability to work with low volumes of compounds andreagents, which is important when dealing with hazardouselements. The biosensor is stable over several days, dispos-able, and simple to operate. Flow-injection systems haveproved to be practical applications in water and environ-mental control, agricultural, and pharmaceutical analysis.

Fig. 1. Enzyme activity in response to DDVP at differentexposure times. (Adapted from [22] with permission.)

2017Electrochemical Biosensors for Pollutants



The system is a platform for using different dehydrogenaseenzymeswith their corresponding substrates. Approximate-ly 250 NADH-dependent dehydrogenase enzymes havebeen identified [26].The sensitivity of the formaldehyde dehydrogenase

biosensor depends upon the efficiency of the electrontransfer from theNADHvia the POs-EApolymermediatorattached to the working electrode. The enzyme wasimmobilized onto a membrane on top of this mediator.This type of sensor is designed to measure the pollutant inaqueous solution. The detection limit of formaldehyde is30 ng mL�1 in the solution, which is equivalent to subpartsper billion (ppb) concentrations of formaldehyde in theatmosphere. Figure 2 presents the rapid and sensitiveresponse to successive additions of different concentrationsof formaldehyde, ranging from 30 ng mL�1 to 4.5 mg mL�1.Excluding tails observed in low formaldehyde concentra-tions, the response to the pollutant is reproducible andlinear.Formaldehyde is also an air pollutant; the gas must be

transferred to an aqueous solution from an air samplingdevice. Formaldehyde can be removed from the atmosphereon site by pumping air through a glass coil together with anaqueous solution, which dissolves the formaldehyde andcarries it to the electrode containing the enzyme, asdescribed by Vianello et al. [27].

2.2.2. Sulfur Dioxide

Sulfur dioxide (SO2) is a colorless gas that occurs as acontaminant in the atmosphere. Natural sources includereleases from volcanoes, oceans, biological decay, and forestfires. Anthropogenic SO2 pollutants are products of fossilfuel combustion, smelting, manufacture of sulfuric acid,wood pulp industry oil and coal burning. Natural gasprocessing plants are responsible for close to half of theSO2 emissions in certain areas. Sulfur dioxide, which

produces acidity in rain water and fogs, is a major sourceof corrosion for buildings and metal objects. Healthconcerns associated with exposure to high concentrationsof SO2 include effects on breathing, respiratory sickness, anddeterioration of cardiovascular disease [28]. During coldweather, SO2 in the air is associated with changes in bothsystolic and diastolic blood pressure [29].An amperometric biosensor was developed by Hart et al.

[30] for the measurement of SO2 in flowing gas streams. Thebiosensor is based on the enzyme sulfite oxidase withcytochrome c as the electron acceptor and a screen-printedtransducer. Enzymatic reactions involve the sulfite ion(SO2�

3 ), formed when SO2 gas is dissolved in the supportingelectrolyte. Two types of the biosensorwhere established: ans-type biosensor, in which cytochrome c and sulfite oxidasewere incorporated at the transducer surface; and a b-typebiosensor, in which the components were mixed thoroughlywith the same ink used to produce the screen-printedelectrode. The modified ink was spread over the workingelectrode. The s-type biosensor was found to have theadvantage of providing higher sensitivity and a fasterresponse when compared with the b-type biosensor. Al-though both types showed linear responses in pollutantconcentrations of 4 to 50 ppm, the sensitivity of the s-typewas approximately twice that of the b-type biosensor.Figure 3 describes s-type biosensor response to differentSO2 concentrations.

3. Antibody Biosensors

A biosensor having an antibody as its receptor is called animmunosensor. Antibody-based biosensors (immunosen-sor) are based on the principle that antigen – antibodyinteractions can be transduced directly into a measurablephysical signal. Antibodies are immune system-relatedproteins (or immunoglobulins) that are secreted into the

Fig. 2. Detection of formaldehyde. Amperometric response ofthe sensor to injections of formaldehyde. 0.1 M potassiumphosphate buffer pH 8. Eapp¼�0.35 V. Numbers depict formal-dehyde concentrations. (Adapted from [25] with permission.)

Fig. 3. Amperometric responses for different SO2 concentra-tions, and recovery after air exposure, at an s-type biosensorcoated with 1.2 mg cytochrome c and 0.45 U SOD. (Adapted from[30] with permission.)




blood in response to stimuli by foreign substances (anti-gens). Specificity is the hallmark of the antibody-mediatedimmune response. Namely, the immune system responds togiven antigenic substance by making a structurally uniqueantibody that specifically binds to and neutralizes only onesite on the antigen. If an antigen has many sites, a differentantibody is elicited for each site, collectively called poly-clonal antibodies. An antibody produced against only oneantigenic site is called a monoclonal antibody. Electro-chemical immunosensors have been constructed using bothtypes of antibodies. A hapten is a small molecule containinga single antigenic site that by itself cannot stimulate anantibody response but can do so when coupled to a largeimmunogenic molecule like a protein. The specific bindingpairs employed in immunoassays are either an antigen or ahapten, and the antibody produced in an immune responseto the antigen or hapten.

3.1. Coliforms

Total coliform bacteria, represented byKlebsiella, and fecalcoliforms, represented by Escherichia coli, are used asindicators of fecal pollution in the environment. As water-borne gastroenteric disease caused by high levels of thesebacteria is a major public health problem, the quantitativedetermination of total and fecal coliforms is essential forwater quality control. The presence of E. coli in anenvironmental sample implies the potential presence of avariety of pathogens originating from humans and warm-blooded animals. Conventional microbiological methodsfor determining the number of coliforms in drinking waterare time-consuming, with a long incubation period (from 24to 48 hours) required to detect bacterial colonies. Mittel-mann et al. [31] described a rapid and sensitive electro-analytical technique for the determination of total coliformbacteria and for the specific detection of E. coli. The sensormonitors the activity of b-d-galactosidase originating fromthe bacteria. The enzyme is often used as a general markerfor total coliforms because its interference from nontargetpositive bacteria is low and insignificant. The results aremeasured as colony forming units (cfu), with each unitrepresenting one or more living bacterial cells. The electro-des were coated with polyclonal anti-E. coli antibodiesprepared against the bacterial lysate.ThedetectionofE. coliat the very low concentration of 1.2 cfumL�1 after 5 hours ofincubation indicates that even very small concentrations ofbacteria can be detected within a single working day usingthis sensitive technique. The results are presented in Fig-ure 4.

3.2. Polycyclic Aromatic Hydrocarbons (PAHs)

Polycyclic aromatic hydrocarbons (PAHs) are chemicalcompounds consisting of fused aromatic rings and do notcontain heteroatoms or carry substituents. ThePAHs, one ofthemost widespread organic pollutants, are always found as

amixture of individual compounds, primarily formedduringthe incomplete combustion of carbon-containing fuels.Their origin can be found in oil and coal industrial processes,fires, traffic, or heating. Some PAHs are known or suspectedcarcinogens and are linked to other health problems [32].Polycyclic aromatic hydrocarbons that are present in non-processed foodstuffs are associated with environmentalpollution from both human and industrial activities. [33] Anamperometric biosensor for detecting PAHs was developedby Fahnrich [34]. The coating screen-printed carbon elec-trode antigen is phenanthrene-9-carboxaldehyde coupled tobovine serum albumin (BSA). The enzyme alkaline phos-phatase (AP) was used, with the substrate p-aminophenylphosphate (PAPP). The sensor was tested in tap and riverwater and showed only slightly decreased sensitivity com-pared tomeasurements carried out in buffer. Because manyPAHs are very similar in electron density, molecularstructure and weight, with a lack of side groups, producingantibodies specific for only one compound is impossible.Several antibodies have been raised against various PAHcompounds, such as benzopyrene, pyrene, fluorene, phen-anthrene (PHE), and anthracene, with benzopyrene cer-tainly themost investigated. The biosensor is not specific forPHE, but shows cross-reactivity of varying degrees towardother PAH compounds.

3.3. Food Pathogens

Listeria monocytogenes, a bacterium motile by means offlagella, can be isolated from soil, silage, and other environ-mental sources. It has been associated with foods as rawmilk, cheeses, raw vegetables, rawmeats, and smoked fish.Arelatively low percentage of the human population may be

Fig. 4. Detection of E. coli, at concentrations of 60 and 1.2 cfu/mL, and K. pneumoniae, at concentrations of 60 and 1.3 cfu/mL,in 1 L of water after filtration, incubation in LB medium at 37 8C,and permeabilization. Each point represents the mean of threemeasurementsþ standard deviation. (Adapted from [31] withpermission.)




intestinal carriers of L. monocytogenes. Infection by thebacterium causes listeriosis, a general group of disordersthat may include septicemia, meningitis encephalitis, pneu-monia and cervical infections in pregnant women that couldresult in spontaneous abortion [35]. Once the bacteriumenters the hostMs monocytes, macrophages, or polymorpho-nuclear leukocytes, it becomes blood-borne (septicemic)and can grow. Susmel et al. [36] demonstrated an immuno-sensor for the detection of the pathogenic bacteria; L.monocytogenes and Bacillus cereus, using screen printedgold electrodes (SPGEs). The gold electrode surface wasmodified with a thiol based self assembled monolayer(SAM) to expedite antibody immobilization. The SAMs arebased on different chain lengths of thiols allowing optimumantibody immobilization, electrochemical response, orien-tation, and accessibility of the antigen binding site. Theformation of the complex between the antigen and theantibody introduces a barrier for the electron transferresulting in diffusion coefficient (D) changes of the redoxprobe. This change was measured chronocolometry. Nochange in the diffusion co-efficient was observed when anonspecific antibodywas immobilized and antigen added.Alinear relationship between D and antigen concentrationwas observed.

3.4. Herbicides

Enzyme immunoassays have been used for detectingherbicides contamination. Dzantiev et al. [37] combinedthe essential properties of antibody- and enzyme-basedsystems to construct an electrochemical immunoassaytechnique for chlorsulfuron determination. Chlorsulfuronis an herbicide used worldwide as an agrochemical for theselective control of weeds in wheat and barley [38]. Thecompound is part of a relatively new class of chemicalsinhibiting the action of plant enzymes, stopping plantgrowth, and killing the plant [39]. The enzyme horseradishperoxidase was attached to a screen-printed electrode. Ontop of the enzyme, anti-chlorsulfuron antibodies wereattached through a membrane. The assay is based on acompetition for the available binding sites of themembrane-immobilized antibodies; between the monitored free pollu-tant chlorsulfuron and a chlorsulfuron-glucose oxidaseconjugate. The addition of glucose to a solution containingboth forms of the pollutant induced the generation ofhydrogen peroxide by the glucose oxidase conjugateenzyme. The hydrogen peroxide reduced by the peroxidaseon the electrode results in an electrical current change, dueto the direct electron transfer of the enzyme, which reflectsthe chlorsulfuron content in the sample. The pollutantdetermination time was 15 minutes, and the detection rangewas 0.01 – 1 ng mL�1.Yulaev et al. [40] presented a biosensor for simazine,

Simazine, an herbicide of the triazine class, is used to controlbroad-leaved weeds and annual grasses. The biosensor isbased on the potentiometric detection of the peroxidaseactivity after a competitive immune reaction on the

electrode surface. This biosensor detection limit was 3 ngmL�1 of simazine. The pollutant was detected quantitativelywithout pre-treatment in meat extracts, milk and tomatoes,hence was found efficient for use in food quality control

4. Receptor Biosensors

A receptor is a structure on the surface of a cell (or inside acell) that selectively receives and binds a specific substance.Receptors canbe adsorbedon theworking electrode surfaceusing several methods: capture behind a membrane, apolymeric matrix, or bilayer lipid membranes.

4.1. Endocrine-Disrupting Compounds

The endocrine system consists of glands that secretehormones for the control of growth, maturation, develop-ment, and regulation within the body, usually by binding toreceptors. Endocrine disrupting chemicals are compoundsthat can mimic a hormone, entering the hormoneMs receptorin lieu of the hormone or blocking the normal passage ofhormones into receptors. In the United States, the monitor-ing of water and food for the presence of endocrinedisrupting chemicals is mandated by the Safe DrinkingWater Act and the Food Quality Protection Act ((http://www.epa.gov/safewater/sdwa/index.html; http://www.fda.-gov/opacom/laws/foodqual/fqpatoc.htm).Estrogen is a fundamental hormone produced by the

ovaries in the process of maturation of the female repro-ductive system. The hormone is also secreted by the adrenalglands and the male testes. Estrogen regulates cellularreactions through a specific intracellular receptor thatfunctions as a ligand-inducible transcription factor. Xen-oestrogens are endocrine disrupting chemicals that bind tothe estrogen receptor and mimic estrogen activity. Xenoes-trogens are commonly found as natural or synthetic in theenvironment. The lack of sufficient evidence for a clear-cutrelation between xenoestrogen exposure and major humanhealth concerns has created a need for highly sensitivescreening systems to detect xenoestrogens in human andenvironmental samples for epidemiologic monitoring stud-ies. Granek et al. [41] presented a novel impedance biosen-sor for monitoring such compounds based on a nativeestrogen receptor adsorbed to a synthetic lipid bilayerattached to gold electrodes. Estrogen or xenoestrogenbinding to the receptor-modified electrode causes confor-mational alteration in the lipid layer, leading to electricalcircuit changes detected by fast impedance measurements.Short galvanostatic pulses are applied and the changes in thepotential are followed. The electrodes are left at open circuitpotential for 2 min for equilibration, and then a constantcurrent (1 mA) is applied. The values of the equivalentcircuit components (Rs, Rp1, t1, C1, Rp2, and t2) for themonolayer, bilayer, bilayer-receptor, and bilayer receptor-1 pg mL�1 hormone are calculated. The capacitances,calculated by t1/Rp1, agree with the ac impedance data.




The obtained values of the capacitors and resistors wereused to calculate Z’ and Z’’ in the complex plane plot, andthe results are presented in Figure 5. Two types of xenoes-trogens were monitored; bisphenol A, a synthetic xenoes-trogen, and genistein, a phytoestrogen. The system wasfound to be highly sensitive, providing an efficient tool formonitoring small amounts of endocrine-disrupting chem-icals. The effects of estrogen, bisphenol A, and genistein onthe electrical properties of the bilayer-receptor modifiedelectrode are summarized in Table 1. The bilayer – receptor-modified electrode gave a similar response to either thenatural hormone or the xenoestrogen. The Rs valuesincreased, the Rp1 values decreased, the lifetime t1 valuesdecreased, and accordingly the calculated C1 values de-creased. These alterations can be attributed to the dimeri-zation and conformational changes of the receptor, whichincreases its hydrophobicity and allows it to enter into thelipid layer.Schwartz-Mittelman et al. [42] tested the effects of

various estrogens, xenoestrogens, phytoestrogens, and ster-oidal and nonsteroidal drugs on the estradiol-induceddimerization of the human estrogen receptor alpha(hERa). Most xenoestrogens have the ability to bind theER and interfere with the natural function of the endocrine

system. The method used was a modified yeast two-hybrid(YTH) system with electrochemical detection, in which b-galactosidase activity is under estrogenic control throughER dimerization. [43] The YTH system is based on theobservation that many eukaryotic transcription factors aredivided into two separate functional domains that mediateDNA binding and transcriptional activation. Both domainscontribute to dimerization. The YTH assay includes re-building b-galactosidase activity via protein – protein inter-actions. An ERa monomer is fused to the DNA-bindingdomain, and a second is fused to the activationdomain of thesame transcription factor.When both fused receptors are coexpressed in yeast, ER dimerization leads to the reconsti-tution of a functional transcription factor, measured as b-galactosidase activity. The substrate used in this experimentwas p-aminophenyl b-d-galactopyranoside. The product ofthis enzymatic reaction p-aminophenol (PAP) is oxidized atan electrode. The sensitive modified YTH electrochemicalbioassay was used to characterize hERa dimerizationinduced by natural estrogens, phytoestrogens, xenoestro-gens, EDCs, and the commonly used anticancer drug,tamoxifen, as well as for the antagonist activity of theanalgesic drug, acetaminophen. This drug inhibited the 17-b-estradiol-induced dimerization of human hERa at phys-iological concentrations of estradiol (10�11 to 10�12 M). Theinhibition was determined by a reduction in the activity ofthe reporter enzyme, monitored by electrochemical meas-urements. Table 2 shows a summary of the results for yeastcells exposed overnight to various xenoestrogens and 17-b-estradiol measured with the electrochemical two-hybridsystem. For bisphenol-A, the lowest concentration mea-sured was 10�6 M, whereas that of 17-b-estradiol was 10�11

M. Diethylstilbestrol (DES), a synthetic estrogen, inducedb-galactosidase activity expressed in yeast cell cultures atthe same order of magnitude as that of 17-b-estradiol,implying a similar induction of receptor dimerization byboth compounds. An influence of genistein and naringeninwas also demonstrated.

5. Bacteriophage Biosensors

A bacteriophage (phage) is an intracellular viral-like para-site that infects only one specific bacterial species. Hence,phages are useful for the identification of bacterial contam-inants. Typically, bacteriophages consist of an outer proteinshell enclosing genetic material and multiply by using the

Fig. 5. Nyquist plot for biolayer construction as drawn using thecalculated capacitance and resistance values measured by galva-nostatic pulses: (—) monolayer, (– –) bilayer, (- -) receptor, ( · · · · · )1 pg/mL estrogen. Inset: Equivalent circuit. (Adapted from [41]with permission.)

Table 1. Summary of effects of exposure to different estrogen and xenoestrogen concentrations on electrical properties of bilayer-receptor-modified electrodes. (Adapted from [41] with permission.)

D% Rs (MW) D% t (s) D% Rp (MW) D% C (mF)

0.015 pg/mL estrogen no change no change no change no change44 pg/mL estrogen 74.28 �19.96 �45.74 �28.9830 ng/mL bisphenol A 24.43 �56.63 �16.40 �47.343 mg/mL bisphenol A 33.84 �63.26 �26.54 �48.691 ng/mL genistein 56.28 �21.71 �30.18 �9.78




hostMs biosynthetic machinery. This process begins when thephase attaches to specific receptors on the bacterial cellsurface. The range of bacteria influenced is usually deter-mined by the proteins on the bacterial cell surface. Thephase can attach to proteins, lipopolysaccharides (LPS), pili,and lipoprotein presented on the outer membrane of thecell. Lytic or virulent phages canmultiply in bacteria and killthe cell by lysis at the end of the life cycle, due to theaccumulation of a phage lysis protein, and intracellularphage are released into the medium. The specific selectivityof the phage can be used for constructing a sensitivebiosensor for bacteria, therebyprecluding the need for time-consuming conventional microbiological pretreatments.The linkage of phage-specific identification and the releaseof the inner enzymatic cell markers after the lysis of the cellprovide a powerful tool as a highly specific detectionmethod of a given bacterial strain.

5.1. E. coli

A phage-based biosensor was developed by Neufeld et al.[44] , who combined specific phage detection and the releaseof intracellular enzymes to produce a highly specific markerfor the bacterial strain E. coli K-12, MG1655. The virulentphage l vir serves not only as the specific recognitionelement for E. coli but also as the releasing agent of theenzyme b-d-galactosidase (which is widely used for identi-fying E. coli in water and food samples). The product of itsenzymatic activity is measured amperometrically by mon-itoring its oxidation at the carbon anode. The amperometricdetection enables the use of a wide range of bacteriaconcentrations, reaching as lowas 1 cfu 100 mL�1within 6 – 8hours, as shown in Figure 6. The electrochemical methodcan be applied to any type of bacterium–phage combina-tion by measuring the enzymatic marker released by thelytic cycle of a specific phage.The detection of E. coli was also demonstrated using a

phage based biosensor and the enzyme alkaline phospha-tase. Plasmids are DNA molecules found in bacteria cells,separate from the bacterial chromosome, that are capable ofautonomous replication. Neufeld et al. [45] constructed a

bacteriophage containing a bacterial plasmid encoding forthe enzyme alkaline phosphatase, known as a phagemid. Inthe bacteria, this enzyme reacts with the substrate, p-aminophenyl phosphate (PAPP), in the periplamic spaceseparating the outer plasma membrane from the cell wall.Thus, the activity of the reporter enzyme can be measureddirectly without further treatment. The product of theenzymatic activity, p-aminophenol, diffuses out and isoxidized at the working electrode. Using a phagemidcombines the advantages of the specific recognition con-tributed by bacteriophage and the effortless genetic manip-ulation of a plasmid, a concentration of 1 cfu mL�1 E. colifrom 50 mL of a contaminated water sample was detectedrapidly within 2 – 3 hours. This method is specific and can beexercised for water or food borne bacterial contaminationdetection.

Table 2. Summary of results displayed as Dcurrent/Dtime (nA/s), for yeast cells exposed overnight to various xenoestrogens and 17-b-estradiol measured with the electrochemical two-hybrid system. Each result represents the mean of three measurements. (Adapted from[42] with permission.)

Concentration (M) Naringenin BPA Genistein 2,4-Dihydroxy BP DES 17-b-Estradiol

10�3 0.008� 0.1110�4 0.005� 0.05310�5 0.0014� 0.03 0.031� 0.33 0.67� 0.085 0.0095� 0.2510�6 0.002� 0.05 0.009� 0.045 0.015� 0.17 0.034� 0.5810�7 0.0035� 0.035 0.0099� 0.051 0.078� 0.5410�8 0.0011� 0.018 0.025� 0.4210�9 0.015� 0.1710�10 0.0004� 0.037 0.29� 0.002710�11 0.11� 0.027

Fig. 6. Detection of low concentration of E. coli. A) 1 – 3, 10 cfumL�1; 4 – 6, 1 colony forming unit mL�1; 7 – 8, without phage. B)1 cfu 100 mL�1. (Adapted from [44] with permission.)




6. Liposome Biosensors

A liposome is an artificial spherical vesicle composed of aphospholipid bilayer surrounding an aqueous cavity, orig-inally developed to study cell membranes. The ability tobear different molecules inside the cavity offered a greatpotential for using liposomes for diagnostics, drug delivery,and environmental monitoring. Different molecules can beassociated with liposomes in several ways; encapsulationwithin the aqueous inner cavity, partitioning within the lipidtails of the bilayer, or covalent and electrostatic interactionswith the polar head-groups of the lipids [46]. For surveyingenvironmental contamination, liposomes enclosing a mark-er can be tagged on their surfacewith haptens, antibodies, orDNA [47]. The marker is released by lysing the liposomes.

6.1. Triazine Pesticides

As a chemical family, the triazines are a group of pesticideswith a wide range of uses. Their chemical structures areheterocyclic, composedof carbonandnitrogen in their rings.Herbicide members of this family include atrazine, hexazi-none, metribuzin, prometon, prometryn, and simazine.Atrazine is one of the most used herbicides in Europe andthe USA [48]. These compounds are known to have amoderate toxicity; yet can undergo transformation to moretoxic, mutagenic, and carcinogenic forms. Such pollutantscan be found in the human food chain or directly in drinkingwater, as a result of their presence in ground water [49].Baumner et al. [50] developed a disposable amperometric

sensor for the detection of triazine pesticides in watersamples. The biosensor is based on the competition betweenthe free pollutant and tagged liposomes. Thick film electro-des printed on PVCwere used as strip-type transducers, andmonoclonal antibodies against atrazine and terbutylazineattached on top served as the biorecognition element.Hapten-tagged liposomes entrapping ascorbic acid as amarker molecule were used to generate and amplify thesignal. For signal detection on a graphite electrode, theliposomes were lysed by Triton X-100 and the releasedascorbic acid was quantified at a potential of þ300 mV vs.printed Ag/AgCl. The biosensor response time was 1 –3 min, and the sensitivity of measurements in tap waterwas below 1 mg L�1 of atrazine which correlates well withstandard detection procedures.

6.2. Cholera Toxin

The cholera toxin (CT) secreted by the bacterium Vibriocholerae is a known causative agent of diarrhea, vomiting,and cramps, often leading to death in humans. Whenreleased from bacteria in the infected intestine, the choleratoxin binds to intestinal cells, triggering endocytosis of thetoxin into the cell. Once inside, the toxin causing a dramaticefflux of ions and water from the infected cell, leading towatery diarrhoea. A sensitive biosensor for the detection of

CT, described by Viswanathan et al., [51] is based onliposomes containing potassium ferrocyanide and labeledwith highly specific recognition molecules for the analyte.The monitoring platform consists of a monoclonal antibodyagainst the B subunit of the CT polymer coated on nafion-supported multi walled carbon nanotube on a glassy carbonelectrode. The CT is first bound to the anti-CTantibody andthen to the specific molecule attached to the liposome. Thepotassium ferrocyanide is released from the bounded lip-osomes by lysis with a methanolic solution of Triton X-100and measured by adsorptive square-wave stripping voltam-metry. The detection limit of this biosensor is 10�16 g ofcholera toxin. This device offers an effective tool for clinicaldiagnostics, food andwater safetymonitoring, and epidemiccontrol. Todetermine the sensitivity of the immunosensor tothe CT; analytical calibration at different concentrations ofthe target analyte was conducted. The calibration curve forthe voltammetric detection of CTat optimum experimentalconditions is presented in Figure 7.

7. Whole-Cell Biosensors

In recent times, the use of whole cell biosensors inmonitoring technologies has become more common. Suchbiosensors can monitor general toxicity or specific toxicitycaused by one or more pollutants using whole cells as thebiorecognition agent. A variety whole cell biosensors havebeen developed to enable the monitoring of pollutants byquantifying light, fluorescence, color, or electric current.Such biosensors can be used in a wide range of applicationsin the fields of pharmacology, medicine, cell biology,toxicology, and environmental monitoring. The biosensoris based on whole cell sensing systems carrying a geneticallyengineered reporter gene that is inserted into the cell understudy and is expressed only upon exposure to a monitoredtoxicant, which can be quantitatively measured.

Fig. 7. Calibration plot for cholera toxin. The inset shows alinear part of the main curve. (Adapted from [51] with permis-sion.)




Microorganisms, bacteria in particular, provide a goodtool for monitoring because of their rapid growth, fastresponse, and ease of genetic adjustment. Two approachesto using whole bacterial cells as biosensors are used today;turn off or turn on mechanisms [52]. In the turn onmechanism, a signal appears following exposure to themonitored toxic compound, while in the turn off mecha-nism, a measurable signal decreases by toxicity, The secondmechanism is more common, assembled by fusion ofreporter genes to stress-response promoters, a regulatoryregion of DNA that provides a control point for genetranscription into RNA. [53, 54]. Such promoters, which canbe activated by toxic or hazardous chemicals, can be fused toreporter genes to monitor the presence of those chemicals.The use of promoters sensitive to DNA damage, proteindamage, and membrane-damage have been demonstratedin the past [55] .Promoters are sensitive elements located upstream of the

translated gene; they control activation or repression andare sensitive to temperature, ionic strength, or compoundslike metabolites or environmental stress agents. Hence,promoters can be useful for monitoring environmentalpollution. Whole cell biosensors based on bacteria can beengineered by placing a reporter gene encoding reporterproteins like b-galactosidase (lacZ), green fluorescent(GFP), or alkaline phosphatase (AP, phoA), under atranscriptional control mediated by the monitored analyte.The thus engineered cell will then produce the reporterprotein in the presence of the monitored analyte, which canbe electrochemically detected and quantified [53, 54].As a whole cell biosensor does not require pretreatment

of the bacterial cells, the assay can be conducted usingportable and simple equipment and disposable electro-chemical electrodes. The detection can be carried out in situ

to monitor a wide range of toxicants and to determinepromoter activities in the environment, as well as tounderstand complex microbial interactions [56].

7.1. Genotoxic Agents

Genotoxic chemicals are capable of causing damage toDNA. General toxicity is often caused by genotoxic agentslike mutagens and carcinogens that induce the cell regu-latory systems, like stress response or the SOS DNA repairsystem, which allows bacteria to survive sudden increases inDNAdamage.Thedrug4-nitroquinoline 1-oxide (4NQO) isa genotoxic agent and model carcinogen that damagesDNA, thereby inducing the SOS response in cells. Using E.coli as a model system, Paitan et al. [57] fused a lacZ geneencoding for the reporter enzyme b-galactosidase to theSOS promoter. The genetically engineeredE. coli producedthe protein b-galactosidase in response to the DNAdamaged elicited by 4NQO. The amperometric monitoringof various concentration of the genotoxin 4NQO is shown inFigure 8.

7.2. Aromatic Hydrocarbons

Pollution of water resources is an increasing problemworldwide. A biosensor based on genetically engineeredcells can provide valuable information on the level oftoxicity of wastewater and the quality of drinking water.Phenol is frequently used in oil refinery wastes and is alsoproduced in the conversion of coal into gaseous or liquidfuels and in the production of metallurgical coke from coal.Phenol discharges can enter the environment from oil

Fig. 8. Monitoring genotoxin 4NQO. The assay is based on the response of E. coli to DNA-damaging agents using a strain which carriesa lacZ gene fused to a gene promoter that responds to DNA damage (sfiA) [58] . b-Galactosidase activity was measured on-line withincreasing concentrations of the genotoxin 4NQO. (Adapted from [57] with permission.)




refineries, coal conversion plants, municipal waste treat-ment plants, or spills. Recombinant bacteria are micro-organisms whose genetic makeup has been altered by thedeliberate introduction of new genetic elements. Neufeldet al. [59] used a recombinant E. coli containing twoplasmids – pfabA encoding the protein b-galactosidaseunder the control of the fabA promoter; and pfabR, whichencodes the repressor of this promoter. The recombinantmicroorganisms were exposed to the test chemicals in anelectrochemical cell and the induced b-galactosidase activ-ity was determined amperometrically. The product of theenzymatic reaction, PAP, is oxidized at the working elec-trode, resulting in signal that is proportional to themonitored analyte concentration. This biosensor is verysensitive to low concentrations of phenol (1.6 ppm), andresults are obtained within a very short time � 20 min. Thesensor responds to phenol derivatives like nonylphenol, 4,4’-biphenol (DHBP), toluene, hydrazine, and ethanol, whileremaining insensitive to bisphenol A and the organophos-phate DDVP. Characterization of the toxicity detectionpotential of the recombinant bacteria containing the twoplasmids pfabA and pfabR, in the presence of phenol ispresented in Figure 9.Other aromatic hydrocarbons of great concern to the

environment are toluene and xylene. Paitan et al. [60]described a bacterial whole cell electrochemical biosensorthat can be used for monitoring those aromatic hydro-carbons. The sensor is based on an aromatic compounds-sensitive promoter that induces the production of analytethat can be monitored electrochemically at real-time andon-line. The promoter xylS was fused inE. coli, upstream totwo DNA sequences containing two reporter genes, lacZand phoA. The result is a whole cell electrochemicalbiosensor with b-galactosidase or AP as reporter genes,using their respective substrates p-aminophenyl-b-d-galac-topyranoside (PAPG) and PAPP. The product of bothenzymatic reactions, PAP, is oxidized at the electrode

resulting in a signal proportional to the aromatic hydro-carbons concentration. This system was found to besensitive enough to detect vapors of toluene and benzenein 35 and 25 min, respectively. Monitoring all three xylenestereoisomers – meta-, ortho-, and para-xylene in a shorttime (20 – 40 min) was achieved.

7.3. Heavy Metals

Cadmium (Cd) is a chemical element ubiquitous in theenvironment. The concentrations of Cd in soils, plants, andother environmental media have escalated due to a growinguse of the chemical in industrial processes. Cadmium canaccumulate in specific organs of the human body; hence it isconsidered a cumulative poison. Cadmium is classified as ahuman carcinogen, and exposure to Cd has also beenassociated with renal dysfunction and bone diseases [61].Biran et al. [62] constructed a biosensor for cadmiumcontent in E. coli consisting of a lacZ gene that is expressedunder the control of a cadmium- responsive promoter ofzntA, which has been shown to be involved in the efflux ofheavy metals [63]. Awide range of cadmium concentrationswas monitored using an electrochemical assay of b-galacto-sidase activity, the reporter protein of the lacZ gene. Thewhole-cell biosensor could detect, within minutes, nano-molar concentrations of cadmium in water. Cadmiummonitoring was also demonstrated using various types ofgrowth media. Cadmium was detected at concentration aslow as 25 nM in less than 1 hour. This biosensor was alsoused for the detection of Cd in soil samples. The signalsobtained by the biosensor were proportional to the Cdconcentration in the soil, as demonstrate in Figure 10 below.600 ppb. (5.34 mM) of the metal could be detected withoutany pretreatment of the soil.

8. Biochip Based on Whole-Cell Biosensor

Popovtzer et al. [64] presented a nano-biochip, whichcontains an array of nano volume electrochemical cells,based on siliconmicrosystem technology (MST). The wholecell bacteria used is a genetically engineered E. coli. Themicroorganisms integrated into the chip will express elec-trochemically detectable signals in the presence of toxicants,as described before by Biran et al. [65]. The devicearchitecture includes an array of eight nano volumechambers functioning as electrochemical cells containingthe bacteria. Using this method one can concurrentlymonitor eight different toxicants with the general stressresponsive promoter. The E. coli strain used contains adeletion in the lacZ gene and carries recombinant plasmidsthat include fusions of the lacZ gene to promoters of heatshock genes coding for the GrpE and DnaK heat shockproteins. The promoter of those proteins responds to avariety of stresses, such as elevation in temperature andexposure to a variety of chemicals like ethanol or heavymetals [66]. In the presence of a toxin, the lacZ promoter is

Fig. 9. E.coli sensor, pfabA pfabR response and induction by thephenol derivatives bisphenol A, DHBP, and nonylphenol atdifferent toxicant concentrations. Reporter b-galactosidase activ-ity is presented by DA/Dt. (Adapted from [59] with permission.)




activated and induces the production of the reporter enzymeb-galactosidase. This enzyme reacts with the substratePAPG to generate the product PAP; the oxidation of PAPcreates a current which is monitored. To exemplify thenanobiochip ability to detect water toxicity, the authorstested various chemicals. The toxicants ethanol and phenol,inducers of the heat shock proteins, were introduced togenetically engineered E. coli in the presence of the PAPGsubstrate. The induced b-galactosidase activity was moni-tored electrochemically as presented in Figure 11.These results using this biosensor showed a direct

correlation between the currents signals and the toxicantconcentrations. Concentrations as low as 0.5% of ethanoland 1.6 ppm of phenol could be detected in less than 10 min.The novel technology described is a combination of biologyand engineering, enabling multi analyte detection, highscreening, and miniaturization in real time detection.

9. Conclusions

The vast potentialmarket for biosensors is only beginning tobe exploited. Awide variety of laboratory-based biosensortechniques that could be applied to environmental mea-surement have been reported; and some have been com-mercialized. Electrochemical biosensors provide precise,rapid, sensitive, and easy to use tools for on-site environ-mental monitoring and analysis. The devices are ideal forenvironmental monitoring because only a small amount ofthe sample is needed for the analysis, and usually, pre

treatment of the sample is not required. Progress andbreakthroughs in biotechnology, biochemistry, genetic en-gineering, and immunochemistry offer a wide range ofplatforms for recognition elements to be used in advancedbiosensors. Using low cost materials as screen printedelectrodes, enzymes and genetically engineered microor-ganisms provide an essential tool for monitoring pollutantsin the environment. An electrochemical biosensor is apowerful tool for real time, on-site environmental analysis.One limitation of this approach is that often only a limited

amount of information about the nature of pollutants isavailable for contaminated sites, thus monitoring methodsmust be capable of identifying expected as well as unex-pected pollutants at low levels. At present, most biosensorsare typically designed for specific applications that involve anarrow range of compounds. Nevertheless, as part of anintegrated site study plan, the commensurate increase in thesampling frequency allowed by the lower cost of fieldscreening analyses can reduce the overall uncertaintyinvolved in characterizing the contaminated site. Once thekey contaminating compounds have been identified, how-ever, biosensor field screening methods could be used tomap their spatial distribution. Analytical tasks associatedwith remediation and post-closure monitoring may requirefrequent and repetitive analysis at specific locations forparticular compounds of interest. Biosensors are particu-larly well suited for this purpose because are optimized torapidly measure a single compound or class of compounds.

10. Future Directions

Biosensors and biosensor-related techniques that showpotential for environmental applications must overcome anumber of obstacles to become commercially viable in the

Fig. 11. Amperometric response curves for real-time monitoringof ethanol using the nano-biochip. The recombinant E. colicontaining a promoterless lacZ gene fused to promoter grpEexposed to 0.5 – 2% concentration of ethanol. The bacteriacultures with the substrate PAPG and the ethanol were placedinto the 100 nL volume electrochemical cells on the chipimmediately after the ethanol addition (ca. 1 min) and weremeasured at 220 mV. (Adapted from [64] with permission.).

Fig. 10. Current signals obtained from the induction of the zntA-lacZ fusion in cadmium-contaminated soil samples. The soilsamples (30 mg) were added directly, without any pretreatment, tothe bacterial culture in the electrochemical cells. The signalsobtained 1 h after the additions of the soil samples are shown.Each point is the mean of four replicates from two separateexperiments. The cadmium concentrations were also determinedby ICP (Spectro), as shown by the numbers in the abscissa. 1 ppm8.9 mM. (Adapted from [62] with permission.)




highly competitive area of field analytical methods. Some ofthe obstacles common to all field analytical methodsinclude: the diversity of compounds and the complexity ofmatrices in environmental samples, the variability in dataquality requirements among environmental programs, andthe broad range of possible environmental monitoringapplications. More specific to biosensor technology, thesehurdles include: relatively high development costs for singleanalyte systems, limited shelf and operational lifetimes forpremanufactured biorecognition components and relativeassay format complexity for many potentially portable (butcurrently laboratory-based) biosensor systems.Nevertheless, there are a number of areas where the

unique capabilities of electrochemical biosensors might beexploited to meet the requirements of environmentalmonitoring. Advances in areas such as toxicity, bioavaila-bility, and multipollutant-screening, could widen the poten-tial market and allow these techniques to be more com-petitive. Miniaturization, reversibility and continuous oper-ation may allow biosensor techniques to be incorporated asdetectors in chromatographic systems.Due to unique characteristics and flexibility in opera-

tional design, biosensors continue to show significantpromise for use in environmental monitoring applications.Nevertheless, because of a variety of obstacles (manyunique to the environmental monitoring area), the intro-duction (and early successes) of these devices into thiscommercial market will likely involve narrowly focusedapplications. Successful biosensors will likely incorporatesome of the following features: sensor platforms that areversatile enough to support interchangeable recognitionelements (to measure a number of analytes), miniaturiza-tion to allow automation and convenience at a competitivecost, and other capabilities not currently available such asautomated, continuous and remote detection of multiple,complex organic analytes.

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