Gheorghe Asachi Technical University Iasi Faculty of Chemical Engineering and Environmental Protection Domain Environmental Engineering !

eciali"ation Environmental #anagement and ustaina$le Energy

Biosensors for environmental monitoring

Professor% Prof& Dr& Eng& Igor Cretescu

tudents% 'ulai Alice Diana (anganu Andrei

Introduction A $iosensor is a self)contained integrated device* consisting of a $iological recognition element in direct contact +ith a transduction element* +hich converts the $iological recognition event into a usea$le out!ut signal& 'iosensors should thus $e distinguished from $ioassays +here the transducer is not an integral !art of the analytical system& 'iosensors are usually classified into various $asic grou!s according either to the method of signal transduction or to the $iorecognition !rinci!le& Accordingly* $iosensors can $e categori"ed as electrochemical* o!tical* !ie"oelectric and thermal sensors on the $asis of the transducing element* and as immunochemical* en"ymatic* non)en"ymatic rece!tor* +hole)cell and D,A $iosensors on the $asis of the $iorecognition !rinci!le& -ne .ey ste! in the develo!ment of $iosensors is the immo$ili"ation of the $iological com!onent at the transducer surface& The immo$ili"ation !rocures $oth the sta$ili"ation of the $iomaterial and the !ro/imity $et+een the $iomaterial and the transducer& The immo$ili"ation methods most generally em!loyed are% !hysical adsor!tion at a solid surface* cross)lin.ing $et+een molecules* covalent $inding to a surface* and entra!ment +ithin a mem$rane* surfactant matri/* !olymer or microca!sule In addition to these conventional methods* sol0gel entra!ment* 1angmuir0'lodgett 21'3 de!osition* electro!olymeri"ation* self)assem$led $iomem$ranes and $ul. modification have $een recently used& The main advantages of the $iosensors over other .inds of sensors are their s!ecificity of res!onse and* in some cases* their a$ility to +or. in very dirty environments& (o+ever* much research is needed in order to im!rove their !erformance in a variety of +ays & 'iosensor technology has reached maturity for determination of the $iological o/ygen demand 2'-D3* !henols* heavy metals* and many !esticides

'iosensors offer the !ossi$ility of determining not only s!ecific chemicals $ut also $iological effects* such as to/icity or endocrine disru!ting effects* +hich are $ecoming more demanded information a$out the sam!le&

2. BIOSENSORS CLASSIFICATION 'iosensors can $e grou!ed according to their $iological element or their transduction element& 'iological elements include en"ymes* anti$odies* micro)organisms* $iological tissue* and organelles& Anti$ody)$ased $iosensors are also called immunosensors& 4hen the $inding of the sensing element and the analyte is the detected event* the instrument is descri$ed as an af5nity sensor& 4hen the interaction $et+een the $iological element and the analyte is accom!anied or follo+ed $y a chemical change in +hich the concentration of one of the su$strates or !roducts is measured the instrument is descri$ed as a meta$olism sensor& Finally* +hen the signal is !roduced after $inding the analyte +ithout chemically changing it $ut $y converting an au/iliary su$strate* the $iosensor is called a catalytic sensor& The method of transduction de!ends on the ty!e of !hysicochemical change resulting from the sensing event& -ften* an im!ortant ancillary !art of a $iosensor is a mem$rane that covers the $iological sensing element and has the main functions of selective !ermeation and diffusion control of analyte* !rotection against mechanical stresses* and su!!ort for the $iological element& The most commonly used sensing elements and transducers are descri$ed $elo+& 2.1Sensing Elements Enzymes

En"ymes are !roteins +ith high catalytic activity and selectivity to+ards su$strates & They have $een used for decades to assay the concentration of diverse analytes& Their commercial availa$ility at high !urity levels ma.es them very attractive for mass !roduction of en"yme sensors& Their main limitations are that !(* ionic strength* chemical inhi$itors* and tem!erature affect their activity& #ost en"ymes lose their activity +hen e/!osed to tem!eratures a$ove 678C& #ost of the en"ymes used in $iosensor fa$rication are o/idases that consume dissolved o/ygen and !roduce hydrogen !ero/ide 2 Fig& 93& En"ymes have $een immo$ili"ed at the surface of the transducer $y adsor!tion* covalent attachment* entra!ment in a gel or an electrochemically generated !olymer* in $ili!id mem$ranes or in solution $ehind a selective mem$rane& to electrochemical and 5$er o!tic transducers& everal revie+s of en"yme immo$ili"ation have $een !u$lished& En"ymes are commonly cou!led

Fig1 Schematic representation of amperometric enzyme membrane electrode Anti odies Anti$odies are !roteins that sho+ outstanding selectivity& They are !roduced $y $) lym!hocytes in res!onse to antigenic structures* that is* su$stances foreign to the organism& #olecules larger than a$out 97 .Da can stimulate an immune res!onse& maller molecules li.e vitamins or steroids can $e antigenic 2also called ha!tens3 $ut
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they do not cause an immune res!onse unless they are con:ugated to larger ones li.e $ovine serum al$umin& #any anti$odies are commercially availa$le and commonly used in immunoassays& Anti$odies are usually immo$ili"ed on the surface of the transducer $y covalent attachment $y con:ugation of amino* car$o/yl* aldehyde* or sulfhydryl grou!s& The surface of the transducer must $e !reviously functionali"ed +ith an amino* car$o/yl* hydro/yl* or other grou!& A revie+ of con:ugation techni;ues can $e found else+here& Anti$odies share similar limitations +ith en"ymes& Furthermore* $inding may not $e reversi$le and regeneration of the surface may re;uire drastic changes in conditions li.e lo+ !(* high ionic strength* detergents* etc& Therefore* efforts are $eing made to !roduce lo+ cost* single use sensors& Pro$a$ly the main !otential advantage of immunosensors over traditional immunoassays is that they could allo+ faster and in)5eld measurements& Immunosensors usually em!loy o!tical or acoustic transducers & !icro es The use micro)organisms as $iological elements in $iosensors is $ased on the measurement of their meta$olism* in many cases accom!anied $y the consum!tion of o/ygen or car$on dio/ide* and is* in most cases* measured electrochemically& #icro$ial cells have the advantage of $eing chea!er than en"ymes or anti$odies*can $e more sta$le* and can carry out several com!le/ reactions involving en"ymes and cofactors& Conversely* they are less selective than en"ymes* they have longer res!onse and recovery times* and may re;uire more fre;uent cali$ration& #icro)organisms have $een immo$ili"ed* for e/am!le* in nylon nets* cellulose nitrate mem$ranes* or acetyl cellulose& 2.2Tr"nsducer elements Electroc#emic"l. Am!erometric and !otentiometric transducers are the most commonly used electrochemical transducers& In am!erometric transducers* the !otential $et+een the t+o

electrodes is set and the current !roduced $y the o/idation or reduction of electroactive s!ecies is measured and correlated to the concentration of the analyte of interest& #ost electrodes are made of metals li.e !latinum* gold* sliver* and stainless steel* or car$on) $ased materials that are inert at the !otentials at +hich the electrochemical reaction ta.es !lace& (o+ever* $ecause some s!ecies react at !otentials +here other s!ecies are !resent* either a selective mem$rane is used or an electron mediator that reacts at lo+er !otential is incor!orated into the immo$ili"ation matri/ or to the sam!le containing the analyte& Potentiometric transducers measure the !otential of electrochemical cells +ith very lo+ current& Field effect transistors 2FET3 are !otentiometric devices $ased on the measurement of !otential at an insulator0 electrolyte interface& The metal gate of a FET can $e su$stituted $y an ion selective mem$rane to ma.e a !( transducer 2!( I FET3& En"ymes have $een immo$ili"ed on the surface of such !( I FET to !roduce en"ymesensiti"ed 5eld effect transistors 2E,FET3& A com!lete descri!tion of such sensors can $e found else+here& O$tic"l.

Fig 2. Schematic representation of ber optic enzyme sensor Fi$er o!tic !ro$es on the ti! of +hich en"ymes and dyes 2often <uorescent3 have $een co)immo$ili"ed are used& These !ro$es consist of at least t+o 5$ers& -ne is connected to a light source of a given +ave length range that !roduces the e/citation +ave& The other* connected to a !hotodiode* detects the change in o!tical density at the
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a!!ro!riate +avelength 2fig =3 urface !lasmon resonance transducers* +hich measure minute changes in refractive inde/ at and near the surface of the sensing element* have $een !ro!osed& urface !lasmon resonance2 P>3 transducers have $een !ro!osed& P> measurement is $ased on the detection of the attenuated total re<ection of light in a !rism +ith one side coated +ith a metal& 4hen a !)!olari"ed incident light !asses through the !rism and stri.es the metal at an ade;uate angle* it induces a resonant charge +ave at the metal?dielectric interface that !ro!agates a fe+ microns& The total re<ection is measured +ith a !hotodetector* as a function of the incident angle& For e/am!le* +hen an antigen $inds to an anti$ody that is immo$ili"ed on the e/!osed surface of the metal the measured re<ectivity increases& This increase in re<ectivity can then $e correlated to the concentration of antigen& The $asic theory of A fe+ P> e/citation and some e/am!les of its a!!lication to $iosensors are !resented else+here& P> $iosensors have $een commerciali"ed $ut no com!act ine/!ensive !orta$le device is availa$le yet& Acoustic.

Fig 3. Schematic representation of surface acoustic wave propagation sensor Electroacoustic devices used in $iosensors are $ased on the detection of a change of mass density* elastic* viscoelastic* electric* or dielectric !ro!erties of a mem$rane made of chemically interactive materials in contact +ith a !ie"oelectric material& 'ul. acoustic +ave 2'A43 and surface acoustic +ave 2 A43 !ro!agation transducers are commonly used& In the 5rst* a crystal resonator* usually ;uart"* is connected to an

am!li5er to form an oscillator +hose resonant fre;uency is a function of the !ro!erties of t+o mem$ranes attached to it& The latter is $ased on the !ro!agation of A4s along a layer of a su$strate covered $y the mem$rane +hose !ro!erties affect the !ro!agation loss and !hase velocity of the +ave& A4s are !roduced and measured $y metal interdigital transducers de!osited on the !ie"oelectric su$strate as sho+n in Fig& @ C"lorimetric.

Fig 4. Schematic representation of enzyme thermistor

Calorimetric transducers measure the heat of a $iochemical reaction at the sensing element& These de%ices c"n e cl"ssi&ed "ccording to t#e '"y #e"t is tr"ns(erred. Isot#erm"l c"lorimeters m"int"in t#e re"ction cell "t const"nt tem$er"ture using )oul #e"ting or *eltier cooling "nd t#e "mount o( energy re+uired is me"sured. ,e"t conduction c"lorimeters me"sure t#e tem$er"ture di((erence et'een t#e re"ction %essel "nd "n isot#erm"l #e"t sin- surrounding it. .sing #ig#ly conducting m"teri"ls ensure +uic- #e"t tr"ns(erred et'een t#e re"ction cell "nd t#e #e"t sin-. Fin"lly/ t#e most commonly used is t#e iso$eri ol c"lorimeter t#"t "lso me"sures t#e

tem$er"ture di((erence

et'een t#e re"ction cell "nd "n isot#erm"l 0"c-et eing e"sily cou$led to 3o'

surrounding it. ,o'e%er/ in t#is c"se t#e re"ction cell is t#erm"lly insul"ted 1"di" "tic2. T#is c"lorimeter #"s t#e "d%"nt"ge o( in0ection "n"lysis systems 1 (ig 42 5.EN6IRON!ENTAL A**LICATIONS To7icity In environmental !ollution monitoring* it is $ecoming a general o!inion that chemical analysis $y itself does not !rovide sufficient information to assess the ecological ris. of !olluted +aters and +aste+aters* thus* much effort has $een made during the last years to develo! and use different $ioassays and $iosensors for to/icity evaluation of +ater sam!les 4hole organisms are used to measure the !otential $iological im!act 2to/icity3 of a +ater or soil sam!le& These systems are $ased on the use of luminescent $acteria* ibrio fischeri* to easure to/icity from environmental sam!les& 'acterial $ioluminescence has !roved to $e a convenient measure of cellular meta$olism and* conse;uently* a relia$le sensor for measuring the !resence of to/ic chemicals in a;uatic sam!les& ome $ioassay methods are integrated no+ in $iosensors such as the CellsenseA* +hich is an am!erometric sensor that incor!orates !scherichia coli $acterial cells for ra!id ecoto/icity analysis& It uses ferricyanine* a solu$le electron mediator* to divert electrons from the res!iratory system of the immo$ili"ed $acteria of a suita$le car$on electrode& The resulting current is* thus* a measure of $acterial res!iratory activity* and the !ertur$ation $y !ollutants can $e detected as a change in the magnitude of the current& Cellsense has $een a!!lied to investigate the to/icity of @*B)dichloro!henol and other !henols in +aste+ater * for the determination of nonionic surfactants and $en"ene sulfonate com!ounds * for the analysis of +aste+ater treatment +or.s 244T43 influent and effluent * and for the to/icity testing of +aste+aters and se+age sludge & #oreover* Cellsense has $een !ro!osed as one of the ne+er ra!id to/icity assessment methods +ithin the direct to/icity assessment 2DTA3 demonstration !rogram of the UC Environmental Agency &
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Biocides The e/tensive use of !esticides for agricultural !ur!oses is the cause of their +ides!read !resence in natural +aters& Concerns a$out their to/icity and !ersistence in the environment has led the Euro!ean Although conventional techni;ues such gives satisfactory analytical results for !esticide determination* ne+ assays and sensors for chea!er and faster on)site analysis are $eing develo!ed& En"ymatic sensors* $ased on the inhi$ition of a selected en"yme* are the most e/tended $iosensors used for the determination of these com!ounds& 'ased on the inhi$ition of acetyl cholinesterase 2AChE3 and colin o/idase* various $iosensors have $een develo!ed for the detection of organo!hos!horous and car$amate !esticides &Although sensitive* $iosensors $ased on AchE inhi$ition are not selective 2since the AchE is inhi$ited $y neuroto/ins* +hich include organo!hos!horous !esticides 2-Ps3* car$amate !esticides* and many other com!ounds3 and cannot* therefore* $e used for ;uantitation of either an individual or a class of !esticides& -ne a!!roach to solve the lac. of s!ecificity of AchE involves the genetic engineering of cholinesterase en"yme to o$tain ne+ s!ecific en"ymes for desired analytes or families& Different e/!ression systems for the !roduction of recom$inant AChEs for $iosensor a!!lications +ere revie+ed The organo!hos!horous hydrolase 2-P(3* on the other hand* is a$le to hydroly"e a num$er of -P !esticides such as !arao/on and !arathion* and chemical +arfare agents such as sarin and soman& (ydrolysis of these -P !esticides generates p) nitro!henol* +hich is an electroactive and chromo!horic !roduct& Thus* -P( could $e com$ined +ith an o!tical transducer to measure the a$sor$ance of p)nitro!henol or +ith an am!erometric transducer to monitor the o/idation or reduction current of this !roduct & & Photosynthesis inhi$ition is an interesting indicator that ra!idly reflects the to/ic effect of certain !ollutants& Ta.ing advantage of this feature* some $iosensors $ased on Photosystem II 2P II3 have $een re!orted to $e a$le to detect her$icides in the environment A$out @7 D of her$icides* including henylurea* tria"ine* and !henolic

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her$icides* inhi$it !hotosynthetic electron flo+ $y $loc.ing the P II ;uinone)$inding site and thus modify chloro!hyll fluorescence Another P II)$ased $iosensor allo+ed the detection of her$icides such as atra"ine* sima"ine* iso!roturon* and diuron at su$)Eg?l concentration levels& ,ormones Endogenous hormones of human or animal origin have $een reaching the environment for thousands of years* even though to an increasing e/tent they are due to gro+ing !o!ulation and more intensive farming& 'esides endogenous hormones* e/ogenous se/ steroids used as gro+th !romoters in several countries have $ecome a matter of concern not only $ecause of the residues !otentially found in meat* $ut also $ecause environmental soil and +ater sam!les may $e contaminated as result of the animal e/creta& These residues may have endocrine)disru!ting activity in a;uatic fauna or even terrestrial Although very lo+ concentrations 2ng?l range3 of hormones such as estradiol* estrone* and ethynilestradiol have $een found in +ater their +ides!read use and their ca!a$ility to induce res!onses in fish at concentrations as lo+ as ng?l or even !g?l level* have alerted scientists to the !otential dangerous conse;uences of their !resence in the a;uatic environment & 4ith an o!tical immunosensor in real +ater sam!les& The Euro!ean Union 2EU3 is funding the develo!ment of ne+ $iosensing strategies for the control of hormone residues in an effort to im!rove food ;uality controls and to !rotect !u$lic health against the adverse effect of these su$stances& As a model of e/am!le* in the frame+or. of the EU !ro:ect Develo!ment of single and multianalyte affinity sensors for ra!id detection of androgen residues in live an !ost) mortem animalsa grou! of researchers are develo!ing single and multi)analyte affinity sensors and rece!tor)$ased sensors for the ra!id detection of androgens such as testosterone and meta$olites *CBs Polychlorinated $i!henyls 2PC's3 are u$i;uitous environmental !ollutants +idely used as industrial chemicals* !articularly as dielectric fluids in electrical transformers and ca!acitors& The high to/icity of some PC' congeners re!resents a ris. for !u$lic health

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as these com!ounds are still !resent in the environment* even though the !roduction of PC's has $een $anned in several countries many years ago& Different $iosensor configurations have $een designed to determine PC's in the environment* and these include the D,A $iosensor +ith chrono!otentiometric detection and various immunosensors +ith fluorescence P> &and electrochemical F6=G detection !rinci!les& 8io7ins A!art from PC's* other !olychlorinated com!ounds of environmental concern are the dio/ins* +hich are released as $y)!roducts in a num$er of chemical !rocesses involving chlorine& Thus* !rocesses such as the !roduction of some !esticides* the manufacture of PHC !lastics* the chlorine $leaching of !ul! and !a!er and +aste incineration generate dio/ins& They are considered carcinogenic and are a !otential threat to human health * and more recently* they have $een included in lists of !otential EDCs Conventional dio/ins analysis re;uires la$orious multiste! clean)u! !rocedures that increase the cost of each analysis& A significant num$er of immunoassays for dio/ins have $een develo!ed in an effort to !rovide sim!lified and routine analysis The P> $iosensor develo!ed $y himomura et for the determination of PC' 2mentioned a$ove3 +as also em!loyed in the determination of the dio/in =*@*I*J)TCDD& imilarly* another $iosensor for detection of dio/in)li.e chemicals 2!olyhalogenated dio/ins* furans* and $i!henyls3 $ased on a recom$inant mouse he!atoma cell line +as characteri"ed and o!timi"ed $y Pasini et al& *#enols Phenolic com!ounds that a!!ear in the environment originate from the !a!er and !ul! industry and from the !roduction of drugs* dyes* and antio/idants& Phenolic com!ounds* and es!ecially chloro!henols* are im!ortant environmental !ollutants $ecause of their high to/icity and !ossi$le accumulation in the environment They are also considered as !recursors of the dio/ins FParellada et al& F=G develo!ed an am!erometric $iosensor* +ith tyrosinase 2a !oly!henol o/idase +ith a relatively +ide selectivity for !henolic com!ounds3 immo$ili"ed in a higrogel on a gra!hite electrode*

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+hich correlated satisfactorily +ith the official method for the determination of the !henol inde/ in environmental sam!les& Chloro!henols have $een also detected +ith a flo+)in:ection chemiluminescence fi$er o!tic $iosensor * e/!loiting the a$ility of certain su$stituted !henols to enhance the chemiluminescence reaction of luminol* cataly"ed $y horseradish !ero/idases& Sur("ct"nts Detergent !roducts use surfactants as the $asic active com!onent& The anionic surfactants are the most +idely used* +hile the cationic surfactants re!resent only B D of the total & An am!erometric $iosensor for detection of anionic surfactants +as constructed +ith "seudomonas rathonis # 2$earing a !lasmid for surfactant degradation3 as a $iological element& -/ygen consum!tion acted as an indicator of cell meta$olism and thus of the surfactant content of the sam!le 'iosensors $ased on $acterial strain "seudomonas and $chromobacter are used $ecause of their a$ility to degrade the anionic surfactants& The degradation of surfactants $y the $acteria caused a decrease in dissolved o/ygen and a change in the o/ygen electrode current& The micro$ial $iosensor en) a$led detection of surfactants +ith high selectivity* sensitivity* and re!roduci$ility& Al-"nes/ "rom"tic com$ounds/ "nd $olycyclic "rom"tic #ydroc"r ons 1*A,s2 Contamination of soils and surface and ground+ater su!!lies +ith !etroleum !roducts is a serious environmental !ro$lem& -f !articular concern for drin.ing +ater ;uality are +ater)solu$le aromatic com!onents 2e&g&* $en"ene* toluene* ethyl$en"ene* and /ylenes3 of !etroleum !roducts& Although many of these contaminants are readily $iodegrada$le* they often !ersist in the environment & A green fluorescent !rotein)$ased "seudomonas fluorescens strain $iosensor +as constructed and characteri"ed for its !otential to measure $en"ene* toluene* ethyl$en"ene* and related com!ounds in a;ueous solutions& The $iosensor is $ased on a !lasmid carrying the toluene)$en"ene transcri!tional activator & Another micro$ial +hole)cell $iosensor* using !. coli +ith the !romoter luciferase lu/A' gene* +as develo!ed for the determination of +ater)dissolved linear al.anes $y luminescence & The $iosensor +as

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used to detect the $ioavaila$le concentration of al.anes in heating oil)contaminated ground+ater sam!les& PA(s are carcinogenic com!ounds generally formed during incom!lete com$ustion or !yrolysis of organic matter containing car$on and hydrogen& They are very a$undant* u$i;uitous* and recogni"ed arcinogenic com!ounds& Am!erometric $iosensors for na!hthalene found in contaminated soils* +ere constructed using Sphingomonas yanoi%uyae & Anti iotics #edical su$stances have $een released into the environment +ith very little attention until recently& The !resence of anti$iotics in the environment is +orrying since they !romote anti$iotic resistance& The increasing use of anti$iotics for thera!eutic !ur!oses or as gro+th !romoters in dairy cattle and as feed additives in fish farms or in livestoc. during the last five decades has caused a genetic selection of more harmful $acteria* +hich is a matter of great concern & The +ides!read administration of anti$iotics raises significant food safety issues since anti$iotic resistance can $e transferred to humans on ingestion of affected meat and mil. !roducts & Therefore* most of the $iosensors develo!ed are aimed at determining them in $iological or food sam!les& For e/am!le* a commercial $iosensor 'IAC->E @777 +as used to study the cross)reactivity $et+een t+o sulfonamides 2a grou! of anti$iotics3% sulfametha"ine and furosemide & ulfonamides sometimes cause allergic reactions* +hereas their effect in the human inmunosystem is of high interest for their thera!eutical a!!lication& To7ins To/ins are a very heterogeneous grou! ca!a$le of affecting different $iochemical !rocesses including mem$rane function* ion trans!ort* transmitter release* and D,A and !rotein synthesis& In many cases* s!ecific details of the site and mode of action of a to/in at the molecular level are not .no+n& A num$er of attem!ts have $een made to detect to/ins in environmental and clinical sam!les using rece!tor sensors & The induction of re!orter genes $y a !romoter* +hich res!onds to a +ide variety of to/ic com!ounds* can $e used to !roduce $iosensors useful as first indicators of the !resence of !ollutants in the

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environment& 4hen the cells are e/!osed to to/ic su$stances* there can $e either a reduction 2negative signal3 or an activation 2!ositive3 in re!orter)!rotein !roduction & To/in)sensitive cells that e/!ress a re!orter gene can $e used* therefore* as a $iosensor for the nons!ecific detection of to/ics* $ut not for a s!ecific to/in& A great num$er of s!ecific sensors for $acterial to/ins and mycoto/ins have $een develo!ed for food and environmental control & !icroorg"nisms 'acteria* viruses* and other microorganisms are found +idely in !olluted* untreated* and treated +aters* +hich im!lies a +orld+ide !u$lic health !ro$lem& In the case of untreated +ater systems* fecal coliforms may enter rivers and streams through agricultural runoff and non!oint source !ollution and via domestic +aste+ater& Pathogenic com!ounds may reach humans $y various routes* such as the use of these +aters for recreation or s!orts* for the irrigation of fruit and vegeta$les and as drin.ing +ater& Therefore* surface +aters may !lay an im!ortant role in the transmission of !athogens& uita$le monitoring of the +ater su!!ly for the !resence of !athogens can assist in !reventing disease from these sources &Current methods for detection of !athogenic viruses* $acteria* !roto"oa* and helminthes tend to $e inaccurate* time) consuming* and e/!ensive& As a result* indicator $acteria such as Salmonella* total fecal coliforms* and total fecal stre!tococci are commonly used to determine the ris. of fecal contamination and the !ossi$le !resence of !athogens in +ater and +aste+aters & -ther emerging !athogenic microorganisms* &ampylobacter* $eromonas* and 'ersinia genera and in recent years* also $acterio!hages* have $een !ro!osed $y several grou!s as indicators for the micro$ial ;uality of +ater since they are more suita$le to signal viral contamination of +ater sources -n the other hand* monitoring of microorganisms is not only of environmental concernK from a military !oint of vie+* there are a num$er of !athogenic $acteria that can $e considered !ossi$le $iological +arfare agents Conventional analytical methods for microorganisms are $ased on colony)forming unit 2CFU3 count and re;uire selective culture* $iochemical* and serological

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characteri"ation & In most cases* they re;uire also some enrichment ste!s& These methods are sensitive and selective* $ut time)consuming% days to +ee.s are needed to get a result& The increasing !u$lic concern over environment safety has led to a search for technologies ca!a$le of ra!idly identifying contamination !ro$lems at source& 'iosensors ca!a$le of detecting an organism ;uic.ly +ill $e im!ortant in the environmental monitoring of !athogens in real time& The micro$ial content of a sam!le can $e determined $y monitoring micro$ial meta$olism& The transducer can either detect consum!tion of o/ygen or the a!!earance?disa!!earance of an electrochemically active meta$olite & (o+ever* !roliferation of nucleic acid and immuno)$ased detection technologies has !rovided sensitive and s!ecific detection systems for !athogenic $acteria and viruses& D,A detection may $e more s!ecific than immunologically $ased detection* and the sensitivity can im!rove than.s to its com$ination +ith !olymerase chain reaction 2PC>3 methods& Gene !ro$es are already finding a!!lication in detection of disease)causing microorganisms in +ater su!!lies* food* or in !lants* animal or human tissues &Immunological detection* on the other hand* is faster and more ro$ust than D,A detection& #oreover* it has the a$ility to detect not only contaminating organisms* $ut also their $ioto/ins * it has $een re!orted the detection of Salmonella enteriditis and (isteria monocytogenes in real time using an P> sensor $ased on anti$odies immo$ili"ed on the gold sensor surface& Salmonella and (isteria +ere detected $y the sensor at concentrations do+n to 976 cell?ml& >ecently* a num$er of !ie"oelectric $iosensors formats have $een develo!ed for the detection of several micro$ial contaminants Salmonella typhimurium detection in li;uid sam!les $y an immunosensor $ased on the acoustic +ave !rinci!le +as re!orted $y Pathirana Ercole et al& descri$ed a $iosensor for the determination of !. coli in +ater sam!les $y an immunochemical !otentiometric alternating $iosensor& The monitored change in the redo/ !otential +as due to the !roduction of ,(@ $y a urease) !. coli anti$ody con:ugate lin.ed +ith the !. coli cells !resent in the +ater& !et"ls The determination of traces of heavy metals such as Cu* Cd* (g* and Ln in the environment is very im!ortant $ecause of their high to/icity* their increasing

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environmental levels 2due to their use in industrial !rocesses3* and $ecause metals can $ioaccumulate in living organisms* es!ecially in marine organisms #etals are usually determined after digestion +ith strong acids& Common analytical techni;ues used are ion chromatogra!hy* inductively cou!led !lasma* and !olarogra!hy & (eavy metals can also $e determined +ith ion)selective electrodes& (o+ever* these methods are not a$le to distinguish $et+een availa$le 2!otentially ha"ardous3 and nonavaila$le 2!otentially nonha"ardous3 fractions of metals to $iological systems F99BG& -ne advantage of the +hole cell)sensors is their a$ility to react only to the availa$le fraction of metal ions& >ecent !rogress has $een made in the develo!ment of $iosensors relying on intact $acterial cells to monitor to/ic metals& 'oth nons!ecific and s!ecific $iosensors that utili"e intact cells have $een develo!ed for this !ur!ose & ,ons!ecific micro$ial $iosensors* +hich have $een used for several years* measure only general to/icity* such as the already mentioned Cellsense& -n the other hand* heavy metals are +ell .no+n to inhi$it the activity of en"ymes* and a!!lication of this !henomenon to the determination of these ha"ardous to/ic elements offers several advantages such as sim!licity and sensitivity& 'esides* in many cases* the inhi$ition effect is related to its $iological to/icity &Durrieu and Tran)#inh descri$ed a $iosensor for the determination of heavy metals $ased on inhi$ition of the AP !resent on the e/ternal mem$rane of &hlorella vulgaris microalgae& For the determination of a s!ecific metal* recom$inant $acterial sensors have $een constructed and used& !ecific $iosensors* $ased on induci$le !romoters fused to re!orter genes 2e&g&* those that code for $ioluminescence !roteins* such as luciferase3* are more sensitive than $oth chemical analysis methods and nons!ecific to/icity $iosensors & The light emitted can $e detected $y !hotometers* illuminometers* and charge)cou!led devices & >ecom$inant luminescent $acterial sensors +ere used $y Ivas. et al& for the determination of the $ioavaila$le fraction of cadmium* "inc* mercury* and chromium in soil& In this +or.* t+o $acterial recom$inant heavy metal sensors +ere constructed $ased on t+o different rece!tor)re!orter systems% one +as induci$le $y Ln=M* Cd=M* and (g=M* and the other $y Cr2HI3 and Cr2III3& The $acterial sensors used +ere not !erfectly s!ecific to one heavy metal* $ut res!onded to some nontarget metals as +ell& In another e/am!le* the mer)lu/ gene fusion in !. coli +as used to estimate $ioavaila$le mercury in

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soil& The $ioavaila$le fraction +as defined here as $eing !art of the +ater)leacha$le fraction& The mer)!romoter +as activated +hen (g2II3* !resent in the cyto!lasm of the $iosensor $acterium* $inds to #er>* resulting in transcri!tion of the lu/ genes and su$se;uent light emission&The luminescence)$ased $acterial sensor strains "seudomonas fluorescens - J 2!TPT993 and "seudomonas fluorescens - J have also $een used for mercury and arsenite detection* res!ectively* in soil e/tracts F99JG& oil sam!les +ere e/tracted +ith +ater* ammonium acetate* hydrogen !ero/ide* and nitric acid* and the results o$tained +ere com!ared +ith those attained +ith traditional methods& -ther $iosensors have $een designed* $ased on $ioengineered !roteins& In these cases* the $iosensor monitors conformational changes caused $y the $inding of the metal ion to the engineered !rotein& Nitr"te The increasing nitrate levels found in ground and surface +aters are of concern $ecause they can harm the +ater environment& In line +ith this* ur$an +aste+ater treatment regulations aim to reduce !ollution* including nitrate !ollution* from se+age treatment +or.s and industry& A $iosensor containing immo$ili"ed denitrifying $acteria +as a!!lied for the determination of ,-@ 0 in ta! +ater& Through the reduction of ,-@0 in a reaction cham$er* ,=- +as formed and determined $y a ,=- microelectrode* +hich +as the sensing element of the $iosensor & A microscale $iosensor for nitrate?nitrite determination +as used for in)site monitoring in an activated sludge !lant& The $iosensor +as $ased on the diffusion of nitrate?nitrite through a ti! mem$rane into a dense mass of $acteria converting the ions into nitrous o/ide +ith su$se;uent electrochemical detection&

Bioc#emic"l o7ygen dem"nd 'iochemical o/ygen demand 2'-D or '-DB3 is defined as the o/ygen re;uired to neutrali"e organic +astes over B days at =7 8C* and is a !arameter +idely used to indicate the amount of $iodegrada$le organic material in +ater & The conventional '-D test has certain $enefits such as $eing a universal method of measuring most +aste+ater sam!les* and* furthermore* no e/!ensive e;ui!ment is needed&

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It has* ho+ever* the limitation of $eing time)consuming* and conse;uently it is not suita$le for online !rocess monitoring& Thus* it is necessary to develo! an alternative method that could circumvent the +ea.ness of the conventional '-D test & Fast determination of '-D could $e achieved $y $iosensor) $ased methods& A common feature of these sensors is that they consist of a micro$ial film that can $ioo/idi"e the organic su$strate to $e ;uantified* sand+iched $et+een a !orous cellulose mem$rane and a gas)!ermea$le mem$rane as the $iological recognition element& The res!onse is usually a change in concentration of dissolved o/ygen or other !henomena such as light emission & #ost '-D sensors rely on measuring the $acterial res!iration rate in close !ro/imity to a transducer* commonly Clar.ty!e 2an am!erometric sensor for measuring dissolved o/ygen develo!ed $y Clar. in 9NB6 3& ome '-D sensors have $een develo!ed and mar.eted $y various manufactures in $oth $iofilm and $ioreactor)ty!e configurations& #ost commercially availa$le '-D sensors are flo+)ty!e systems that can $e more easily automated* $ut generally re;uire high maintenance to !revent fouling and clogging Instrument information a$out many '-D commercial $iosensors +as !rovided $y 1iu and #attiason & Des!ite the good agreement $et+een $iosensor results and conventional '-D analysis* and des!ite the short res!onse time of $iosensors* current '-D $iosensor systems still !resent a series of limitations that restrict their industrial a!!licatios% the lac. of standardi"ation and legislation in most countries* com!licated maintenance re;uirements* and insufficient resistance to various to/ic com!ounds such as heavy metal ions* C,0 and !henol in the +aste+ater& It is !ossi$le to eliminate theto/ic effects of heavy metal ions $y using a chelating agent that com!le/ the ions& #any '-D $iosensors have $een develo!ed for the determination of high '-D values in industrial +aste+ater and not ada!ted to the measurement of lo+ '-D values An o!tical fi$er $iosensor +as develo!ed for the evaluation of lo+ '-D values in river +aters $y Chee et al&The immo$ili"ed "seudomonas putida $acterium mem$rane +as !laced on the to! of an o!tode* +hich +as lin.ed to a !hotodiode that detected fluorescence signal& The res!onse time +as 9B min for chloride u! to 9777 mg?l&

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CONCL.SIONS Des!ite the huge !otential of $iosensors* and the ever)increasing num$er of $iosensors develo!ed* commercially availa$le $iosensors are $eing a!!lied to a restricted area of the !otential mar.et& In general* $iosensors for environmental analysis have several limitations% sensitivity* res!onse time* and lifetime* +hich should $e im!roved for them to $ecome a com!etitive analytical tool& The areas of develo!ment that are e/!ected to have an im!act in $iosensor technology are% immo$ili"ation techni;ues* nanotechnology* miniaturi"ation* and multisensor array determinations& (o+ever* a crucial as!ect may $e the !roduction of ne+ sensing elements easy to synthesi"e and +ith the ca!a$ility to $roaden the s!ectra of selectivities that can $e reached $y a $iosensor& At !resent* the !re!aration and !roduction in large scales of $iomolecules such as en"ymes or anti$odies need an investment of time and .no+ledge&

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Re(erences 9& ara >odrigue")#o"a" et all 'iosensors for environmental a!!lications% Future develo!ment trends& Chem&* Hol& I6* ,o& O* !!& I=@0IB=* =77O* IUPAC =& PoseQ I& >eyes De Corcuera >al!h P& Cavalieri 0 'iosensors 4ashington tate University* Pullman* 4ashington* U& &A& =77@ @& ara >odrigue")#o"a"a* #aria P& 1Qo!e" de Aldaa* #aria)Pilar #arco$* DamiRa 'arcelQ 'iosensors for environmental monitoring% A glo$al !ers!ective* De!artment of Environmental Chemistry* IISA')C IC* C?Pordi Girona 9J0=6* 7J7@O 'arcelona* !ain =77B O& Frieder 9NN= chelfer and Florian chu$ert*Techni;ues and instrumentation in

analytical chemistry H-1U#E 99/ Amsterdam ) 1ondon ) ,e+ Tor. )To.yo

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