enz_notes_

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Uses of Enzymes

1. LEATHER AND WOOL INDUSTRIES

The leather industry consumes a sini!cant "ro"ortion o# the $orld%s en&yme"roduction. Al'aline "roteases are used to remo(e hair #rom hides. This"rocess is #ar sa#er and more "leasant than the traditional methods in(ol(insodium sul!de. Relati(ely lare amounts o# en&yme are re)uired *+.1,1.+ -*$$// and the "rocess must 0e closely controlled to a(oid reducin the)uality o# the leather. A#ter dehairin hides $hich are to 0e used #or"roducin so#t leather clothin and oods are 0ated a "rocess o#tenin(ol(in "ancreatic en&ymes that increases their su""leness and im"ro(esthe so#tness o# their a""earance.

2roteases ha(e 0een used in the "ast to %shrin'"roo#% $ool. Wool !0res areco(ered in o(erla""in scales "ointin to$ards the !0re ti". These i(e the!0res hihly directional #rictional "ro"erties mo(ement in the direction a$ay#rom the ti" 0ein #a(oured relati(e to mo(ement to$ards it. This "ro"ensity#or mo(ement solely in the one direction may lead to shrin'ae and manymethods ha(e 0een used in attem"ts to eliminate the "ro0lem *e.. chemicalo3idation or coatin the !0res in "olymer/. A success#ul method in(ol(ed the

"artial hydrolysis o# the scale ti"s $ith the "rotease "a"ain. This method alsoa(e the $ool a sil'y lustre and added to its (alue. The method $asa0andoned some years ao "rimarily #or economic reasons. It is notunreasona0le to e3"ect its use to 0e re,esta0lished no$ that chea"eren&yme sources are a(aila0le.

2. FOOD INDUSSTRY

2.1 The use of enzymes in starch hydroysis

Starch is the commonest storae car0ohydrate in "lants. It is used 0y the"lants themsel(es 0y micro0es and 0y hiher oranisms so there is a reatdi(ersity o# en&ymes a0le to catalyse its hydrolysis. Starch #rom all "lantsources occurs in the #orm o# ranules $hich di4er mar'edly in si&e and"hysical characteristics #rom s"ecies to s"ecies. 5hemical di4erences areless mar'ed. The ma6or di4erence is the ratio o# amylose to amylo"ectin7 e..corn starch #rom $a3y mai&e contains only 8- amylose 0ut that #romamylomai&e is a0out 9+- amylose. Some starches #or instance #rom "otato


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contain co(alently 0ound "hos"hate in small amounts *+.8- a""ro3imately/$hich has sini!cant e4ects on the "hysical "ro"erties o# the starch 0ut doesnot inter#ere $ith its hydrolysis. Acid hydrolysis o# starch has had $ides"readuse in the "ast. It is no$ larely re"laced 0y en&ymic "rocesses as itre)uired the use o# corrosion resistant materials a(e rise to hih colour and

saltash content *a#ter neutralisation/ needed more enery #or heatin and$as relati(ely di:cult to control.

Fi! 1 The use o# en&ymes in "rocessin starch. Ty"ical conditions are i(en.

O# the t$o com"onents o# starch amylo"ectin "resents the reat challeneto hydrolytic en&yme systems. This is due to the residues in(ol(ed in a,1;,lycosidic 0ranch "oints $hich constitute a0out < , ;- o# the lucose

"resent. =ost hydrolytic en&ymes are s"eci!c #or a,1


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1. "rocesses in $hich the starch hydrolysate is to 0e used 0y micro0es orman and

8. "rocesses in $hich it is necessary to eliminate starch.

In the #ormer "rocesses such as lucose syru" "roduction starch is usually

the ma6or com"onent o# reaction mi3tures $hereas in the latter "rocessessuch as the "rocessin o# suar cane 6uice small amounts o# starch $hichcontaminate non,starchy materials are remo(ed. En&ymes o# (arious ty"esare used in these "rocesses. Althouh starches #rom di(erse "lants may 0eutilised corn is the $orld%s most a0undant source and "ro(ides most o# thesu0strate used in the "re"aration o# starch hydrolysates.

There are three staes in the con(ersion o# starch. ?i 1

1. elatinisation in(ol(in the dissolution o# the nanoram,si&ed starchranules to #orm a (iscous sus"ension7

8. li)ue#action in(ol(in the "artial hydrolysis o# the starch $ith

concomitant loss in (iscosity7 and@. sacchari!cation in(ol(in the "roduction o# lucose and maltose 0y

#urther hydrolysis.

elatinisation is achie(ed 0y heatin starch $ith $ater and occursnecessarily and naturally $hen starchy #oods are coo'ed. elatinised starchis readily li)ue!ed 0y "artial hydrolysis $ith en&ymes or acids andsacchari!ed 0y #urther acidic or en&ymic hydrolysis.

The starch and lucose syru" industry uses the e3"ression de3trosee)ui(alent or DE similar in de!nition to the DH units o# "roteolysis todescri0e its "roducts $here>

*.1/

In "ractice this is usually determined analytically 0y use o# the closelyrelated 0ut not identical e3"ression>

*.8/

Thus DE re"resents the "ercentae hydrolysis o# the lycosidic lin'aes

"resent. 2ure lucose has a DE o# 1++ "ure maltose has a DE o# a0out B+*de"endin u"on the analytical methods used7 see e)uation *


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classi!cation is inade)uate to encom"ass all the en&ymes that are used incommercial starch hydrolysis. One reason #or the con#usion in thenomenclature is the use o# the anomeric #orm o# the released reducin rou"in the "roduct rather than that o# the 0ond 0ein hydrolysed7 the "roducts o# 0acterial and #unal a,amylases are in the a,con!uration and the "roducts

o# 0,amylases are in the 0,con!uration althouh all these en&ymes clea(e0et$een a,1


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?unal a,amylase also !nds use in the 0a'in industry. It o#ten needs to 0eadded to 0read,ma'in ours to "romote ade)uate as "roduction andstarch modi!cation durin #ermentation. This has 0ecome necessary sincethe introduction o# com0ine har(esters. They reduce the time 0et$eencuttin and threshin o# the $heat $hich "re(iously $as su:cient to allo$ a

limited s"routin so increasin the amounts o# endoenous en&ymes. The#unal en&ymes are used rather than those #rom 0acteria as their action iseasier to control due to their relati(e heat la0ility denaturin ra"idly durin0a'in.

#roduction of !ucose syru$

The li)ue!ed starch at 9 ,18 DE is suita0le #or sacchari!cation to "roducesyru"s $ith DE (alues o# #rom


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Fi!ure 2. The - lucose #ormed #rom @+- *$$/ 18 DE maltode3trin at;+F5 and "H


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hydrolysin the 1


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amylase $ould 0e used to "roduce maltose,rich syru"s #rom corn starches"ecially as the com0ined action o# 0,amylase and "ullulanase i(e almost)uantitati(e yields o# maltose. This is not done on a sini!cant scaleno$adays 0ecause "resently a(aila0le 0,amylases are relati(ely e3"ensi(enot su:ciently tem"erature sta0le *althouh some thermosta0le 0,amylases

#rom s"ecies o# Clostridium ha(e recently 0een re"orted/ and are easilyinhi0ited 0y co""er and other hea(y metal ions. Instead #unal a,amylasescharacterised 0y their a0ility to hydrolyse maltotriose *@/ rather thanmaltose *8/ are em"loyed o#ten in com0ination $ith lucoamylase.2resently a(aila0le en&ymes ho$e(er are not totally com"ati0le7 #unal a,amylases re)uirin a "H o# not less than B.+ and a reaction tem"erature note3ceedin BBF5.

Hih,maltose syru"s *see ?iure


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Lactose +.8

Ra:nose +.8

Hydrolysed sucrose 1.1

Hydrolysed lactose +.

lucose syru" 11 DE J+.1

lucose syru"


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(1.2)

Traditionally in(ertase $as "roduced on site 0y autolysin yeast cells. Theautolysate $as added to the syru" *+- sucrose *$$// to 0e in(ertedtoether $ith small amounts o# 3ylene to "re(ent micro0ial ro$th. In(ersion$as com"lete in


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Other "ro0lems in(ol(in de3tran and ra:nose re)uired the de(elo"ment o# ne$ industrial en&ymes. A de3tran is "roduced 0y the action o# de3transucrase *E5 8.


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is readily stirred in slurries containin

a. cellulase *E5 @.8.1.


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re"lace starch as a source o# lucose syru"s #or #ood use. It is ho$e(er)uite "ossi0le that it may 0e used in a "rocess in(ol(in the simultaneoususe o# 0oth en&ymes and #ermentati(e yeasts to "roduce ethanol7 theutilisation o# the lucose 0y the yeast remo(in its inhi0itory e4ect on theen&ymes. It should 0e noted that cello0iose is a non,#ermenta0le suar and

must 0e hydrolysed 0y additional 0,lucosidase *E5 @.8.1.81 also calledcello0iase #or ma3imum "rocess e:ciency.

he use o# lactases in the dairy industry

Lactose is "resent at concentrations o# a0out


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5ommercially it may 0e "re"ared #rom the dairy yeast Kluyveromycesfragilis *K. mar!ianus (ar. mar3ianus/ $ith a "H o"timum *"H ;.B,.+/suita0le #or the treatment o# mil' or #rom the #uni Aspergillus oryzae or A.niger" $ith "H o"tima *"H


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is easily remo(ed 0y !ltration or settlin and decantation once thesta0ilisin e4ect o# the "ectins on the colloidal ha&e has 0een remo(ed.

5ommercial "ectolytic en&yme "re"arations are "roduced #rom Aspergillusniger and consist o# a syneristic mi3ture o# en&ymes>

a. "olyalacturonase *E5 @.8.1.1B/ res"onsi0le #or the random hydrolysiso# 1


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a much loner "eriod *e.. @+ min/ to inacti(ate it "rior to #ermentation.2a"ain is used in the later "ost,#ermentation staes o# 0eer,ma'in to"re(ent the occurrence o# "rotein, and tannin,containin %chill,ha&e%other$ise #ormed on coolin the 0eer.

Recently %liht% 0eers o# lo$er calori!c content ha(e 0ecome more "o"ular.

These re)uire a hiher deree o# sacchari!cation at lo$er starchconcentrations to reduce the alcohol and total solids contents o# the 0eer. This may 0e achie(ed 0y the use o# lucoamylase andor #unal a,amylasedurin the #ermentation.

A reat (ariety o# car0ohydrate sources are used $orld $ide to "roducedistilled alcoholic drin's. =any o# these contain su:cient )uantities o# #ermenta0le suar *e.. rum #rom molasses and 0randy #rom ra"es/ otherscontain mainly starch and must 0e sacchari!ed 0e#ore use *e.. $his'ey #rom0arley malt corn or rye/. In the distillin industry sacchari!cation continuesthrouhout the #ermentation "eriod. In some cases *e.. Scotch malt $his'y

manu#acture uses 0arley malt e3clusi(ely/ the en&ymes are naturally "resent0ut in others *e.. rain s"irits "roduction/ the more heat,sta0le 0acterial a,amylases may 0e used in the sacchari!cation.

lucose o3idase and catalase in the #ood industry

lucose o3idase is a hihly s"eci!c en&yme *#or D,lucose 0ut see 5ha"ter9/ #rom the #uni Aspergillus niger and Penicillium $hich catalyses theo3idation o# 0,lucose to lucono,1B,lactone *$hich s"ontaneouslyhydrolyses non,en&ymically to luconic acid/ usin molecular o3yen and

releasin hydroen "ero3ide *see reaction scheme 1.1P/. It !nds uses in theremo(al o# either lucose or o3yen #rom #oodstu4s in order to im"ro(e theirstorae ca"a0ility. Hydroen "ero3ide is an e4ecti(e 0acteriocide and may0e remo(ed a#ter use 0y treatment $ith catalase *deri(ed #rom the same#unal #ermentations as the lucose o3idase/ $hich con(erts it to $ater andmolecular o3yen>

catalase8H8O8 8H8O G O8 (+.+)

?or most lare,scale a""lications the t$o en&ymic acti(ities are notse"arated. lucose o3idase and catalase may 0e used toether $hen nethydroen "ero3ide "roduction is to 0e a(oided.

A ma6or a""lication o# the lucose o3idasecatalase system is in the remo(alo# lucose #rom e,$hite 0e#ore dryin #or use in the 0a'in industry. Ami3ture o# the en&ymes is used *1;B U ',1/ toether $ith additional

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hydroen "ero3ide *a0out +.1 - *$$// to ensure that su:cient molecularo3yen is a(aila0le 0y catalase action to o3idise the lucose. Other uses arein the remo(al o# o3yen #rom the head,s"ace a0o(e 0ottled and canneddrin's and reducin non,en&ymic 0ro$nin in $ines and mayonnaises.

'. #/0R00EUTI030S 0ND EDIINES

De(elo"ment o# medical a""lications #or en&ymes ha(e 0een at least ase3tensi(e as those #or industrial a""lications reectin the manitude o# the"otential re$ards> #or e3am"le "ancreatic en&ymes ha(e 0een in use sincethe nineteenth century #or the treatment o# diesti(e disorders. The (arietyo# en&ymes and their "otential thera"eutic a""lications are considera0le. Aselection o# those en&ymes $hich ha(e realised this "otential to 0ecomeim"ortant thera"eutic aents is sho$n in Ta0le


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Stre"to'inase c

@.

a. They are too lare to 0e distri0uted sim"ly $ithin the 0ody%s cells. Thisis the ma6or reason $hy en&ymes ha(e not yet 0een success#ul a""liedto the lare num0er o# human enetic diseases. A num0er o# methodsare 0ein de(elo"ed in order to o(ercome this 0y taretin en&ymes7as e3am"les en&ymes $ith co(alently attached e3ternal 0,alactoseresidues are tareted at he"atocytes and en&ymes co(alently cou"ledto taret,s"eci!c monoclonal anti0odies are 0ein used to a(oid non,s"eci!c side,reactions.

0. ein enerally #orein "roteins to the 0ody they are antienic andcan elicit an immune res"onse $hich may cause se(ere and li#e,threatenin alleric reactions "articularly .on continued use. It has"ro(ed "ossi0le to circum(ent this "ro0lem in some cases 0ydisuisin the en&yme as an a""arently non,"roteinaceous molecule0y co(alent modi!cation. As"arainase modi!ed 0y co(alentattachment o# "olyethylene lycol has 0een sho$n to retain its anti,tumour e4ect $hilst "ossessin no immunoenicity. 5learly the

"resence o# to3ins "yroens and other harm#ul materials $ithin athera"eutic en&yme "re"aration is totally #or0idden. E4ecti(ely thisencouraes the use o# animal en&ymes in s"ite o# their hih costrelati(e to those o# micro0ial oriin.

c. Their e4ecti(e li#etime $ithin the circulation may 0e only a matter o# minutes. This has "ro(ed easier than the immunoloical "ro0lem tocom0at 0y disuise usin co(alent modi!cation. Other methods ha(e

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also 0een sho$n to 0e success#ul "articularly those in(ol(inentra"ment o# the en&yme $ithin arti!cial li"osomes syntheticmicros"heres and red 0lood cell hosts. Ho$e(er althouh thesemethods are e:cacious at e3tendin the circulatory li#etime o# theen&ymes they o#ten cause increased immunoloical res"onse and

additionally may cause 0lood clots.In contrast to the industrial use o# en&ymes thera"eutically use#ul en&ymesare re)uired in relati(ely tiny amounts 0ut at a (ery hih deree o# "urityand *enerally/ s"eci!city. The #a(oured 'inetic "ro"erties o# these en&ymesare lo$ m and hih Cma3 in order to 0e ma3imally e:cient e(en at (ery lo$en&yme and su0strate concentrations. Thus the sources o# such en&ymes arechosen $ith care to a(oid any "ossi0ility o# un$anted contamination 0yincom"ati0le material and to ena0le ready "uri!cation. Thera"eutic en&yme"re"arations are enerally o4ered #or sale as lyo"hilised "ure "re"arations$ith only 0iocom"ati0le 0u4erin salts and mannitol diluent added. Thecosts o# such en&ymes may 0e )uite hih 0ut still com"ara0le to those o#

com"etin thera"eutic aents or treatments. As an e3am"le uro'inase *aserine "rotease see Ta0le


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0ioloical system directly. This (ery 0road de!nition is used 0y somescienti!c 6ournals *e.. iosensors Else(ier A""lied Science/ 0ut $ill not 0ea""lied to the co(erae here. The em"hasis o# this 5ha"ter concernsen&ymes as the 0ioloically res"onsi(e material 0ut it should 0e reconisedthat other 0ioloical systems may 0e utilised 0y 0iosensors #or e3am"le

$hole cell meta0olism liand 0indin and the anti0ody,antien reaction.iosensors re"resent a ra"idly e3"andin !eld at the "resent time $ith anestimated ;+- annual ro$th rate7 the ma6or im"etus comin #rom thehealth,care industry *e.. ;- o# the $estern $orld are dia0etic and $ould0ene!t #rom the a(aila0ility o# a ra"id accurate and sim"le 0iosensor #orlucose/ 0ut $ith some "ressure #rom other areas such as #ood )ualitya""raisal and en(ironmental monitorin. The estimated $orld analyticalmar'et is a0out F18+++++++++ year,1 o# $hich @+- is in the health carearea. There is clearly a (ast mar'et e3"ansion "otential as less than +.1- o# this mar'et is currently usin 0iosensors. Research and de(elo"ment in this!eld is $ide and multidisci"linary s"annin 0iochemistry 0ioreactor science

"hysical chemistry electrochemistry electronics and so#t$are enineerin.=ost o# this current endea(our concerns "otentiometric and am"erometric0iosensors and colorimetric "a"er en&yme stri"s. Ho$e(er all the maintransducer ty"es are li'ely to 0e thorouhly e3amined #or use in 0iosensorso(er the ne3t #e$ years.

A success#ul 0iosensor must "ossess at least some o# the #ollo$in 0ene!cial#eatures>

1. The 0iocatalyst must 0e hihly s"eci!c #or the "ur"ose o# the analyses0e sta0le under normal storae conditions and e3ce"t in the case o# colorimetric en&yme stri"s and di"stic's *see later/ sho$ ood

sta0ility o(er a lare num0er o# assays *i.e. much reater than 1++/.8. The reaction should 0e as inde"endent o# such "hysical "arameters as

stirrin "H and tem"erature as is manaea0le. This $ould allo$ theanalysis o# sam"les $ith minimal "re,treatment. I# the reactionin(ol(es co#actors or coen&ymes these should "re#era0ly also 0e co,immo0ilised $ith the en&yme *see 5ha"ter 9/.

@. The res"onse should 0e accurate "recise re"roduci0le and linear o(erthe use#ul analytical rane $ithout dilution or concentration. It shouldalso 0e #ree #rom electrical noise.


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B. The com"lete 0iosensor should 0e chea" small "orta0le and ca"a0leo# 0ein used 0y semi,s'illed o"erators.

;. There should 0e a mar'et #or the 0iosensor. There is clearly little"ur"ose de(elo"in a 0iosensor i# other #actors *e.. o(ernmentsu0sidies the continued em"loyment o# s'illed analysts or "oor

customer "erce"tion/ encourae the use o# traditional methods anddiscourae the decentralisation o# la0oratory testin.

The 0ioloical res"onse o# the 0iosensor is determined 0y the 0iocatalyticmem0rane $hich accom"lishes the con(ersion o# reactant to "roduct.Immo0ilised en&ymes "ossess a num0er o# ad(antaeous #eatures $hichma'es them "articularly a""lica0le #or use in such systems. They may 0e re,used $hich ensures that the same catalytic acti(ity is "resent #or a series o# analyses. This is an im"ortant #actor in securin re"roduci0le results anda(oids the "it#alls associated $ith the re"licate "i"ettin o# #ree en&ymeother$ise necessary in analytical "rotocols. =any en&ymes are intrinsically

sta0ilised 0y the immo0ilisation "rocess *see 5ha"ter @/ 0ut e(en $here thisdoes not occur there is usually considera0le a""arent sta0ilisation. It isnormal to use an e3cess o# the en&yme $ithin the immo0ilised sensorsystem. This i(es a catalytic redundancy *i.e. h JJ 1/ $hich is su:cient toensure an increase in the a""arent sta0ilisation o# the immo0ilised en&yme*see #or e3am"le ?iures @.11 @.1 and B.9/. E(en $here there is someinacti(ation o# the immo0ilised en&yme o(er a "eriod o# time thisinacti(ation is usually steady and "redicta0le. Any acti(ity decay is easilyincor"orated into an analytical scheme 0y reularly inter"olatin standards0et$een the analyses o# un'no$n sam"les. ?or these reasons many suchimmo0ilised en&yme systems are re,usa0le u" to 1++++ times o(er a "eriod

o# se(eral months. 5learly this results in a considera0le sa(in in terms o# the en&ymes% cost relati(e to the analytical usae o# #ree solu0le en&ymes.

When the reaction occurrin at the immo0ilised en&yme mem0rane o# a0iosensor is limited 0y the rate o# e3ternal di4usion the reaction "rocess$ill "ossess a num0er o# (alua0le analytical assets. In "articular it $ill o0eythe relationshi" sho$n in e)uation @.8. It #ollo$s that the 0iocatalyst i(es a"ro"ortional chane in reaction rate in res"onse to the reactant *su0strate/concentration o(er a su0stantial linear rane se(eral times the intrinsic m*see ?iure @.18 line e/. This is (ery use#ul as analyte concentrations areo#ten a""ro3imately e)ual to the ms o# their a""ro"riate en&ymes $hich isrouhly 1+ times more concentrated than can 0e normally determined$ithout dilution 0y use o# the #ree en&yme in solution. Also #ollo$in #rome)uation @.8 is the inde"endence o# the reaction rate $ith res"ect to "Hionic strenth tem"erature and inhi0itors. This sim"ly a(oids the tric'y"ro0lems o#ten encountered due to the (aria0ility o# real analytical sam"les*e. #ermentation 0roth 0lood and urine/ and e3ternal conditions. 5ontrol o# 0iosensor res"onse 0y the e3ternal di4usion o# the analyte can 0eencouraed 0y the use o# "ermea0le mem0ranes 0et$een the en&yme andthe 0ul' solution. The thic'ness o# these can 0e (aried $ith associated

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e4ects on the "ro"ortionality constant 0et$een the su0strate concentrationand the rate o# reaction *i.e. increasin mem0rane thic'ness increases theunstirred layer *d/ $hich in turn decreases the "ro"ortionality constant 'Lin e)uation @.8/. E(en i# total de"endence on the e3ternal di4usional rate isnot achie(ed *or achie(a0le/ any increase in the de"endence o# the reaction

rate on e3ternal or internal di4usion $ill cause a reduction in thede"endence on the "H ionic strenth tem"erature and inhi0itorconcentrations.

Fi!ure 7.1. Schematic diaram sho$in the main com"onents o# a0iosensor. The 0iocatalyst *a/ con(erts the su0strate to "roduct. This reactionis determined 0y the transducer *0/ $hich con(erts it to an electrical sinal. The out"ut #rom the transducer is am"li!ed *c/ "rocessed *d/ and dis"layed*e/.

The 'ey "art o# a 0iosensor is the transducer *sho$n as the %0lac' 0o3% in?iure ;.1/ $hich ma'es use o# a "hysical chane accom"anyin thereaction. This may 0e

1. the heat out"ut *or a0sor0ed/ 0y the reaction *calorimetric 0iosensors/

8. chanes in the distri0ution o# chares causin an electrical "otential to0e "roduced *"otentiometric 0iosensors/

@. mo(ement o# electrons "roduced in a redo3 reaction *am"erometric0iosensors/


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$hich in(ol(e s"eci!c %mediators% 0et$een the reaction and the transducer inorder to enerate im"ro(ed res"onse and third eneration 0iosensors $herethe reaction itsel# causes the res"onse and no "roduct or mediator di4usionis directly in(ol(ed.

The electrical sinal #rom the transducer is o#ten lo$ and su"erim"osed u"on

a relati(ely hih and noisy *i.e. containin a hih #re)uency sinalcom"onent o# an a""arently random nature due to electrical inter#erence orenerated $ithin the electronic com"onents o# the transducer/ 0aseline. Thesinal "rocessin normally in(ol(es su0tractin a %re#erence% 0aseline sinalderi(ed #rom a similar transducer $ithout any 0iocatalytic mem0rane #romthe sam"le sinal am"li#yin the resultant sinal di4erence andelectronically !lterin *smoothin/ out the un$anted sinal noise. Therelati(ely slo$ nature o# the 0iosensor res"onse considera0ly eases the"ro0lem o# electrical noise !ltration. The analoue sinal "roduced at thisstae may 0e out"ut directly 0ut is usually con(erted to a diital sinal and"assed to a micro"rocessor stae $here the data is "rocessed con(erted to

concentration units and out"ut to a dis"lay de(ice or data store.aorimetric "iosensors

=any en&yme catalysed reactions are e3othermic eneratin heat *Ta0le;.1/ $hich may 0e used as a 0asis #or measurin the rate o# reaction andhence the analyte concentration. This re"resents the most enerallya""lica0le ty"e o# 0iosensor. The tem"erature chanes are usuallydetermined 0y means o# thermistors at the entrance and e3it o# small "ac'ed0ed columns containin immo0ilised en&ymes $ithin a constant tem"eratureen(ironment *?iure ;.8/. Under such closely controlled conditions u" to9+- o# the heat enerated in the reaction may 0e reistered as a

tem"erature chane in the sam"le stream. This may 0e sim"ly calculated#rom the enthal"y chane and the amount reacted. I# a 1 m= reactant iscom"letely con(erted to "roduct in a reaction eneratin 1++ 'Q mole ,1 theneach ml o# solution enerates +.1 Q o# heat. At 9+- e:ciency this $ill causea chane in tem"erature o# the solution amountin to a""ro3imately+.+8F5. This is a0out the tem"erature chane commonly encountered andnecessitates a tem"erature resolution o# +.+++1F5 #or the 0iosensor to 0eenerally use#ul.

Ta"e 7.1. Heat out"ut *molar enthal"ies/ o# en&yme catalysed reactions.

Reactant Enzyme/eat out$ut%D/ 89: moe%1;

5holesterol 5holesterol o3idase B@

Esters 5hymotry"sin < , 1;


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lucose lucose o3idase 9+

Hydroen "ero3ide 5atalase 1++

2enicillin 2enicillinase ;

2e"tides Try"sin 1+ , @+

Starch Amylase 9

Sucrose In(ertase 8+

Urea Urease ;1

Uric acid Uricase


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E3ternal electronics *l/ determines the di4erence in the resistance andhence tem"erature 0et$een the thermistors.

The thermistors used to detect the tem"erature chane #unction 0ychanin their electrical resistance $ith the tem"erature o0eyin therelationshi"

*;.8/

there#ore>

*;.80/

$here R1 and R8 are the resistances o# the thermistors at a0solutetem"eratures T1 and T8 res"ecti(ely and is a characteristic tem"eratureconstant #or the thermistor. When the tem"erature chane is (ery small asin the "resent case *1T1/ , *1T8/ is (ery much smaller than one and thisrelationshi" may 0e su0stantially sim"li!ed usin the a""ro3imation $hen3JJ1 that e3F1 G 3 *3 here 0ein *1T1/ , *1T8/

*;.@/

As T1 F T8 they 0oth may 0e re"laced in the denominator 0y T1.

*;.


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inclusion o# the $ell,insulated aluminium 0loc' in the 0iosensor desin *see?iure ;.8/.

The sensiti(ity *1+,


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Reactions in(ol(in the eneration o# hydroen ions can 0e made moresensiti(e 0y the inclusion o# a 0ase ha(in a hih heat o# "rotonation. ?ore3am"le the heat out"ut 0y the "enicillinase reaction may 0e almostdou0led 0y the use o# Tris *tris,*hydro3ymethyl/aminomethane/ as the0u4er.In conclusion the main ad(antaes o# the thermistor 0iosensor are its

eneral a""lica0ility and the "ossi0ility #or its use on tur0id or stronlycoloured solutions. The most im"ortant disad(antae is the di:culty inensurin that the tem"erature o# the sam"le stream remains constant *F+.+1F5/.

#otentiometric "iosensors

2otentiometric 0iosensors ma'e use o# ion,selecti(e electrodes in order totransduce the 0ioloical reaction into an electrical sinal. In the sim"lestterms this consists o# an immo0ilised en&yme mem0rane surroundin the"ro0e #rom a "H,meter *?iure ;.@/ $here the catalysed reaction eneratesor a0sor0s hydroen ions * Ta0le ;.8/. The reaction occurrin ne3t to the thin

sensin lass mem0rane causes a chane in "H $hich may 0e read directly#rom the "H,meter%s dis"lay. Ty"ical o# the use o# such electrodes is that theelectrical "otential is determined at (ery hih im"edance allo$in e4ecti(ely&ero current o$ and causin no inter#erence $ith the reaction.

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Fi!ure 7.'. A sim"le "otentiometric 0iosensor. A semi,"ermea0lemem0rane *a/ surrounds the 0iocatalyst *0/ entra""ed ne3t to the acti(elass mem0rane *c/ o# a "H "ro0e *d/. The electrical "otential *e/ isenerated 0et$een the internal AA5l electrode *#/ 0athed in dilute H5l */and an e3ternal re#erence electrode *h/.

There are three ty"es o# ion,selecti(e electrodes $hich are o# use in0iosensors>

1. lass electrodes #or cations *e.. normal "H electrodes/ in $hich thesensin element is a (ery thin hydrated lass mem0rane $hichenerates a trans(erse electrical "otential due to the concentration,de"endent com"etition 0et$een the cations #or s"eci!c 0indin sites.


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The selecti(ity o# this mem0rane is determined 0y the com"osition o# the lass. The sensiti(ity to HG is reater than that achie(a0le #or NH


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0,lucosidaseamydalin G 8H8O 8lucose G 0en&aldehyde G HG G 5N, (7.12)a 5an also 0e used in NH


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ho$e(er may 0e a4ected 0y the com"osition ionic strenth andconcentrations o# the solutions analysed.

Fi!ure 7.+. Schematic diaram o# the section across the $idth o# an EN?ET. The actual dimensions o# the acti(e area is a0out B++ mm lon 0y B+ mm$ide 0y @++ mm thic'. The main 0ody o# the 0iosensor is a ",ty"e siliconchi" $ith t$o n,ty"e silicon areas7 the neati(e source and the "ositi(e drain. The chi" is insulated 0y a thin layer *+.1 mm thic'/ o# silica *SiO8/ $hich#orms the ate o# the ?ET. A0o(e this ate is an e)ually thin layer o# HG,sensiti(e material *e.. tantalum o3ide/ a "rotecti(e ion selecti(emem0rane the 0iocatalyst and the analyte solution $hich is se"arated #romsensiti(e "arts o# the ?ET 0y an inert enca"sulatin "olyimide "hoto"olymer.When a "otential is a""lied 0et$een the electrodes a current o$s throuhthe ?ET de"endent u"on the "ositi(e "otential detected at the ion,selecti(eate and its conse)uent attraction o# electrons into the de"letion layer. This

current *I/ is com"ared $ith that #rom a similar 0ut non,catalytic IS?ETimmersed in the same solution. *Note that the electric current is 0ycon(ention in the o""osite direction to the o$ o# electrons/.

0m$erometric "iosensors

Am"erometric 0iosensors #unction 0y the "roduction o# a current $hen a"otential is a""lied 0et$een t$o electrodes. They enerally ha(e res"onsetimes dynamic ranes and sensiti(ities similar to the "otentiometric0iosensors. The sim"lest am"erometric 0iosensors in common usae in(ol(e

the 5lar' o3yen electrode *?iure ;.B/. This consists o# a "latinum cathodeat $hich o3yen is reduced and a sil(ersil(er chloride re#erence electrode.When a "otential o# ,+.; C relati(e to the AA5l electrode is a""lied to the"latinum cathode a current "ro"ortional to the o3yen concentration is"roduced. Normally 0oth electrodes are 0athed in a solution o# saturated"otassium chloride and se"arated #rom the 0ul' solution 0y an o3yen,"ermea0le "lastic mem0rane *e.. Teon "olytetrauoroethylene/. The#ollo$in reactions occur>


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A anode


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Fi!ure 7.


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2t anode H8O8 O8 G 8HG G 8e, (7.1


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mediator or oranic conductors re"resent the three enerations in 0iosensorde(elo"ment *?iure ;.9/. The reduction in o3idation "otential #ound $henmediators are used reatly reduces the "ro0lem o# inter#erence 0ye3traneous material.

Fi!ure 7.=. *a/ ?errocene *eB,0is,cyclo"entadienyl iron/ the "arentcom"ound o# a num0er o# mediators. *0/ T=2G the cationic "art o# conductin oranic crystals. *c/ T5N., the anionic "art o# conductin oranic

crystals. It is a resonance,sta0ilised radical #ormed 0y the one,electrono3idation o# T5NH8.

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Fi!ure 7.>. Am"erometric 0iosensors #or a(o,o3idase en&ymes illustratinthe three enerations in the de(elo"ment o# a 0iosensor. The 0iocatalyst is

sho$n schematically 0y the cross,hatchin. *a/ ?irst eneration electrodeutilisin the H8O8 "roduced 0y the reaction. *E+ G+.;9 C/. *0/ Secondeneration electrode utilisin a mediator *#errocene/ to trans#er theelectrons "roduced 0y the reaction to the electrode. *E+ G+.1 C/. *c/ Third eneration electrode directly utilisin the electrons "roduced 0y thereaction. *E+ G+.1+ C/. All electrode "otentials *E+/ are relati(e to the5l,A5lA+ electrode. The #ollo$in reaction occurs at the en&yme in allthree 0iosensors>


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Su0strate*8H/ G ?AD,o3idase 2roduct G ?ADH8,o3idase! (7.1>)

This is #ollo$ed 0y the "rocesses>

*a/0iocatalyst

?ADH8,o3idase G O8 ?AD,o3idase G H8O8 (7.1?)electrode

H8O8 O8 G 8HG G 8e, (7.2@)

*0/0iocatalyst

?ADH8,o3idase G 8 ?erriciniumG ?AD,o3idase G 8 ?errocene G 8HG (7.21)

electrode

8 ?errocene 8 ?erriciniumG

G 8e,

(7.22)*c/0iocatalystelectrode

?ADH8,o3idase ?AD,o3idase G 8HG G 8e, (7.2')

The current *i/ "roduced 0y such am"erometric 0iosensors is related to therate o# reaction *(A/ 0y the e3"ression>

i n?A(A *;.;/

$here n re"resents the num0er o# electrons trans#erred A is the electrode

area and ? is the ?araday. Usually the rate o# reaction is made di4usionallycontrolled *see e)uation @.8/ 0y use o# e3ternal mem0ranes. Under thesecircumstances the electric current "roduced is "ro"ortional to the analyteconcentration and inde"endent 0oth o# the en&yme and electrochemical'inetics.

O"tical 0iosensors

There are t$o main areas o# de(elo"ment in o"tical 0iosensors. These

in(ol(e determinin chanes in liht a0sor"tion 0et$een the reactants and"roducts o# a reaction or measurin the liht out"ut 0y a luminescent"rocess. The #ormer usually in(ol(e the $idely esta0lished i# rather lo$technoloy use o# colorimetric test stri"s. These are dis"osa0le sinle,usecellulose "ads im"renated $ith en&yme and reaents. The most commonuse o# this technoloy is #or $hole,0lood monitorin in dia0etes control. Inthis case the stri"s include lucose o3idase horseradish "ero3idase *E5

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1.11.1./ and a chromoen *e.. o,toluidine or @@%BB%,tetramethyl0en&idine/. The hydroen "ero3ide "roduced 0y the aero0ico3idation o# lucose *see reaction scheme (1.1)/ o3idisin the $ea'lycoloured chromoen to a hihly coloured dye.

"ero3idasechromoen*8H/ G H8O8 dye G 8H8O (7.2+)

The e(aluation o# the dyed stri"s is 0est achie(ed 0y the use o# "orta0lereectance meters althouh direct (isual com"arison $ith a coloured chartis o#ten used. A $ide (ariety o# test stri"s in(ol(in other en&ymes arecommercially a(aila0le at the "resent time.A most "romisin 0iosensorin(ol(in luminescence uses !rey luci#erase *Photinus,luci#erin


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*;./

$here D# is the chane in resonant #re)uency *H&/ Dm is the chane in mass

o# adsor0ed material */ is a constant #or the "articular crystal de"endenton such #actors as its density and cut and A is the adsor0in sur#ace area*cm8/. ?or any "ie&o,electric crystal the chane in #re)uency is "ro"ortionalto the mass o# a0sor0ed material u" to a0out a 8- chane. This #re)uencychane is easily detected 0y relati(ely unso"histicated electronic circuits. Asim"le use o# such a transducer is a #ormaldehyde 0iosensor utilisin a#ormaldehyde dehydroenase coatin immo0ilised to a )uart& crystal andsensiti(e to aseous #ormaldehyde. The ma6or dra$0ac' o# these de(ices isthe inter#erence #rom atmos"heric humidity and the di:culty in usin them#or the determination o# material in solution. They are ho$e(er ine3"ensi(e

small and ro0ust and ca"a0le o# i(in a ra"id res"onse.

Immunosensors

iosensors may 0e used in con6unction $ith en&yme,lin'ed immunosor0entassays *E3IS0/. The "rinci"les 0ehind the ELISA techni)ue is sho$n in ?iure;.. ELISA is used to detect and am"li#y an antien,anti0ody reaction7 theamount o# en&yme,lin'ed antien 0ound to the immo0ilised anti0ody 0eindetermined 0y the relati(e concentration o# the #ree and con6uated antienand )uanti!ed 0y the rate o# en&ymic reaction. En&ymes $ith hih turno(ernum0ers are used in order to achie(e ra"id res"onse. The sensiti(ity o# such

assays may 0e #urther enhanced 0y utilisin en&yme,catalysed reactions$hich i(e intrinsically reater res"onse7 #or instance those i(in rise tohihly coloured uorescent or 0ioluminescent "roducts. Assay 'its usin thistechni)ue are no$ a(aila0le #or a (ast rane o# analyses.

http://www1.lsbu.ac.uk/water/enztech/immuno.html#fig6_9http://www1.lsbu.ac.uk/water/enztech/immuno.html#fig6_9http://www1.lsbu.ac.uk/water/enztech/immuno.html#fig6_9http://www1.lsbu.ac.uk/water/enztech/immuno.html#fig6_9


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Fi!ure 7.?. 2rinci"les o# a direct com"etiti(e ELISA. *i/ Anti0ody s"eci!c #orthe antien o# interest is immo0ilised on the sur#ace o# a tu0e. A mi3ture o# a'no$n amount o# antien,en&yme con6uate "lus un'no$n concentration o# sam"le antien is "laced in the tu0e and allo$ed to e)uili0rate. *ii/ A#ter asuita0le "eriod the antien and antien,en&yme con6uate $ill 0e distri0uted0et$een the 0ound and #ree states de"endent u"on their relati(econcentrations. *iii/ Un0ound material is $ashed o4 and discarded. Theamount o# antien,en&yme con6uate that is 0ound may 0e determined 0ythe rate o# the su0se)uent en&ymic reaction.

Recently ELISA techni)ues ha(e 0een com0ined $ith 0iosensors to #ormimmunosensors in order to increase their rane s"eed and sensiti(ity. Asim"le immunosensor con!uration is sho$n in ?iure ;.1+ *a/ $here the0iosensor merely re"laces the traditional colorimetric detection system.Ho$e(er more ad(anced immunosensors are 0ein de(elo"ed *?iure ;.1+* 0// $hich rely on the direct detection o# antien 0ound to the anti0ody,coated sur#ace o# the 0iosensor. 2ie&oelectric and ?ET,0ased 0iosensors are"articularly suited to such a""lications.

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Fi!ure 7.1@. 2rinci"les o# immunosensors. *a/*i/ A tu0e is coated $ith*immo0ilised/ antien. An e3cess o# s"eci!c anti0ody,en&yme con6uate is"laced in the tu0e and allo$ed to 0ind. *a/*ii/ A#ter a suita0le "eriod anyun0ound material is $ashed o4. *a/*iii/ The analyte antien solution is"assed into the tu0e 0indin and releasin some o# the anti0ody,en&ymecon6uate de"endent u"on the antien%s concentration. The amount o# anti0ody,en&yme con6uate released is determined 0y the res"onse #rom the

0iosensor. *0/*i/ A transducer is coated $ith *immo0ilised/ anti0ody s"eci!c#or the antien o# interest. The transducer is immersed in a solutioncontainin a mi3ture o# a 'no$n amount o# antien,en&yme con6uate "lusun'no$n concentration o# sam"le antien. *0/*ii/ A#ter a suita0le "eriod theantien and antien,en&yme con6uate $ill 0e distri0uted 0et$een the0ound and #ree states de"endent u"on their relati(e concentrations. *0/*iii/Un0ound material is $ashed o4 and discarded. The amount o# antien,en&yme con6uate 0ound is determined directly #rom the transduced sinal.


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