CA2104711A1

CA2104711A1 - Electrobiochemical analytical method and electrodes

Info

Publication number: CA2104711A1
Application number: CA002104711A
Authority: CA
Inventors: Itamar Willner; Evgeny Katz; Azalia Riklin; Ron Kasher; Benjamin Shoham
Original assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 1992-08-25
Filing date: 1993-08-24
Publication date: 1994-02-26
Also published as: DE69331257T2; IL102930A0; EP0584794A2; IL102930A; EP0584794A3; DE69331257D1; JP2633459B2; JPH06281613A; EP0584794B1; US5443701A

Abstract

A B S T R A C T

An analytical method for determining the presence or concentration of an analyte in a liquid medium is provided in which electrons are transferred from an electrode material of an electrode to the redox center of an enzyme, by the mediation of an electron mediator, whereby in the presence of an analyte the enzyme calalyzed a redox reaction in which the analyte is converted into a product. By measuring either the concentration or the product or the charge which flows in this process, the presence and/or concentration of the analyte in the medium is determined.
Either or both of the enzyme and the electron mediator are immobilized on the electrode.

Description

`` 21 0~711 ~UG ~4 '93 10:03 R.COHN ~ PTNS. 972 3 5606405 P.4 ELECI~OBIOC~iEMlCAL ANAI.ltTICAL MET~OD
AND ELECT~ODES

~ELD OF THE TNVE;NTIOI~
Thc present ulvontlon concerns an analytical method for the dctc~mination Oe the prcscnce and concenlration of an analytei in a liquid med~um ~ho m~ithod of thc prcsent invenllon is an clectr~biochcmical 5 mcthod in which the conccntratioD. of the analyte in a medium is determined by means of an electrically induccd enzymatic redox ~action iu which lhe analyte is convcrted in~o a product. The detcrmination of the concentratiorls of the product or the dctermination of thc char~e flow servcs as a meaisurc of the allalytc s concc~tratio~.
rhe method of the prcsent i~vonition makcs use of a novel kind of electrodes having immobil~ed thereo~ cither thc enzyme rnolccules al~
clectro mediator which transfers elcctrons from thc electrode malerial to thc reto~c center of the enziyme or both.

15 B~CK.G~O~JN~ OF TI~I~ INVEN rlON AND PRX03R ART
The spccificity of enzymes to a specific analy~c rcllders thcm uscful as p~obcs for thc dctcction of the ptcscncc of analytcs in a liquid medium. ~ spccific class of cnzymes which has been proposed for the usei . , :' .

2 ~ O ~
RUG 24 '93 1~:03 R.CO~N ~ PTNS. 97Z 3 5606405 P.5 .

in analytical biochemical methods ue ~edox enzymes i~ which case tho dctoction of tho prosoncc and concentration af an analyte in a medium may eithcr bc b~scd on thc mcasuremcnt of ~hc flo~v of charge resulting &o~ lhc enzymatlc redox reaction or on lho accumulatlorl of products obtained in thc S enzymatic redo~ rcaction. In such mcthods an clectrode is used which comprises an elcctrodc malerial made ~or examplc of gold or platinum on which therc are immobilized tedox enz~mcs. ~ redox reaction involves lhe Iransfer of electrons from lhe enzyme to the anal~te (in a teduction reaction~
or vice versa (in an oxidation reaction) and if there is an clec~ical 10 communication betwcen the rcdox ccn~er ot the enzyme molecules and ~hc clcctrodc material, therc is ~n clcctrical ctlarge flo~ which can scrve as an indication o~ the prcsence of ~he analyte and lhe extent of charge flow may sorvo ~s arl ;ndic~ion of tho analytc's conccnttat;on. Altcrna~ively, the dctcnnination may be based on thc rneasu~cmenL oE a product of the lS reacthn.
Thc ba6ic requi~cment in such clec~roblochcmical syslcms is lo devclop an electrical ~ommunication between thc cnzyrnes' rcdox ccnter and thc clectrode matcrial (~ellcr, 1990; Wilson e~ al., 1987). Such olectrial communication may be cstablishcct by irnmobilizadon of thc redox 2~ cnzymcs j~ ~unctionalized rcdo~ polymers (Degani et al., 1989; Gorton et al., 1990; Foulds et al., 1988) or by chemical modification of protcins with elcctron transfcr mcdiators (~cgani et ~l., 19B8; Heller, 1992). Most of tbe prcscn~ly dcvelopod electrobiochcmical electrodes were uti~iæed in an o~idative palhway. Howcvor, it was rccently shown that immobiliz~tion of 25 enzymes in bipyridinium functionalized polymcrs (Willncr er al., 1990) or funclionalization of protcirls by bipyridinium componen~s (Willner et al., 1991) givcs risc to an clcclrical communlcation il~ pholochemical s~stcms whicb, m~y bc uscd in rcduclive routes.

- .

.

' ~

21 ~7~i ~UG Z4 '~3 10:~34 R.COHN & PT~S. 972 3 5613641~5 P.6 ., .
It is the object ol thc present invenlion to provide an analylical eloc~robiochemical mcthod for thc determination of the prescnco of an analytc In both reductive and oxidativc pathways.
It Is furthcrrnore the object of thc present invention to pravide 5 an elcctrodc for usc in Ihe above mcthod.
It is still a further objccl o~ thc p~csent in cntion lo providc a proccss for the preparation of such elcclrodes.

GE~IER~L D13:SCRlPIION O~ THE INVENTION
In accordancc witb the pres6nt invcntion it has been, found that very cfficicnt clectron transfer between the surfacc of thc clcctrode mstlcrial of an elcctrade to rcdox en2ymes by mcans of an electron mediator group can ~e achioved if cither or both of lhe enzymes or the clectron meciiator ~oups arc ~mmobilizcd on thc surfacc of thc clcctron material by mc8ns of lS groups havin~ 9ulphut cont~uling moicties which are chcmisorbcd on said surfacc, or by linking thc clcctron mcdiator to the en2ymc bcing immobi-Uzed on tho electrode matcrial by means of sulphur containin~ moic~ics.
Ihc prcsent ~nvontion thu~ pro~idcs, in accotdance with one of its aspects, an analytical method for delermining the presence or 20 conccntr~tion of an ~nalyte in a liguid mcdium by an electrobiochemical enzymatic rcdox rcactiorl in which olectrons are transferred betwcen thc surfacc of an elcctrode matcrial and a redo~ enzymc by the mediat~on of molecular electron mediators whe~cby the onzyme is capablc of catalyzing a redo~c rcactio~ i~ which the ~alytc is convertcd into a product; said 2S mcthod Compris~ng mcasuring tbe conccntration of ~he product obtained in thc rcdo~ rcaction or measuring lhe flow of charge; tho method bcing characlor~zed ~ that said clcctrode material is of a Icind which is capable of chemisorption of sulpbue cont- nin~ moiclies aDd tha~ al leu~ o~ f the componcnts, ule ~ , . .

21~71 ~
RUG 24 ' ~13 10: 04 R . COHN 8~ PTNS. 972 3 5606405 P. 7 erLzymc or the clcctro~ medialor, is immobilized o~ thc surf~cc of thc c1ectrode matcrial by mcans o~ a ffrsl lir~ing group covalently bound th~teto having a sulphur cootaining moi¢ly chomisorbed to s~id surface, and t~e other of sa~d components bcing eithct (i) tumbling in thc liquid medium S surrounding Ihc electrodc material, (ii) irnmobilizcd on said surfacc by means of a second li~king uoup having a sulphur containing moiety chemisorbcd onlo said surface wbich onay bc Ihc samc or diffcrent lhan said first llnking group, or (iii) immobili~cd on said surface by bcing covalen~ly bound to said first linking group or to said one of the components.
' T116 ptesent invent;on also provides in another o~ its aspccts, electrodcs for use in Ihe above melhod, comprising an el~ctrode malerial of Ihe l~ind capablc of chcmisorption of sulphur containing maieties, having immobilized thcreon a plurality of complcxes cach comprising a linking ~roup havu~g a sulphur containing moicty and at least one of a redo.x 15 enzyme or an clectron medialor, all componenls of lhc compkx bein~
covalently bound to onc utolher~
By a still further aspect of the prescnt invc~tion thcre is provlded ar~ electrobiochcmical systom for carryirlg out the above method~
By a yel still f~hcr aspect of the present in~cntion thele is 20 provided a ~rocess for prcparing lhc above clectrodcs, which process will be outlined further bclow.
The ele~trode material may be selected from a large numbcr of conducting or semi-condllcting substances havin~ the capa~ility to chemisorb a sulphur coatai~ g moiet~. Examplcs of such clectrodc 25 matcrials ma~ bc gold, silvcr, plalinum or copper and semicorlductors such as gallium arsenide.
Tho 1inking group may ha~c ~hc following gencral fonnula (l):

Z-RI-Q (1) ~, , .
: .

21Bll711 QUG Z4 '93 1E~:05 R.COHl & PTNS. 972 3 5606435 P.3 whercin Z rcpresents a sulphur contair~ing moiety;
Q ls a group X~ or P; ~C' is a functio~al group which is capableof forming a ca~alent bond with a moiety of said at least one component, i.e. eithcr tho cDzymc or the electron mcdia~or, as the case may be; ~ is a protcin, a polypcptidc or a polymer having a plurality of functional ~oups ~ having thc meaning o~ X~ abovc;
Ri represonts a connec~ing group.
, Z may for examplc be a sulphur atom, obtained from a thiol group or ~ disulphidc gro~lp, a solfonate or st-lfatc groups.
X~ or x2 may for example be a functional group capable of bindiDg to a carboxyl rcsiduc of a protcin such as an amine g~O~lp, a carboxyl group capable of binding to,amine rcsi~ucs o~ thc protein; Bll 15 isocyanato or is~thiocyanate groups or an acyl group capable of bh1ding to an smine residuc of a protoin; a halidc group capable Oe bulding to hydroxy reslducs of a protein or a pol$peplido, or the polymer P as del5ncd abovc.
Particular examplcs of Xl are thc groups ~ CO~H;
-N~S; N=C_O; or an acyl group having thc fonnula -R'~CO-G
20 whcrcin (3 may bc a group such as OH, halogen, OR~, or a C~ o-o group; R' and R~ bcl~g, indcpcndently, a C~ 2 aLt~yl, alkenyl, alkynyl or a phenyl containing chaia, which is optionally substitutcd, e.~. by halogen.
The functional groups ;n P may be an amis~c or a carboxyl group carricd on Ihe polypcptide chain, o~ may bc groups carricd OD a sidc 25 chain, P may be a polymor or a polypeptidc ca~ying a plurality of functional groups ~ which may bc all the ~ame or different and ha-c the same meaning as given above fo~ X~.
.

: ` :
~UG 24 ' 93 113: 05 R. COHN ~ PTN5. 972 3 5606405 P . 9 .

E~amples o~ P are polypcplidcs havillg free carboxyl or amine groups capablc of ~indhg to aminc and carboxyl groups in the enzymc moleculc, rcspcctivc1y. Particular examples o~ P are polyamincs such as polycthylcllcimine and polypeptides nch in glutamate or lysinc. P may als~
S cornprisc funct;onalized side groups.
R' may bc a covalent bond or may bc selectcd ~om a vcry wide varicl~ of suilablc groups such as alkylene, alkenylenc, alkyn~lcne phenyl contain~n~ chains, and many others;
Particular cxamples o~ Rt are a chemical bond or a group 10 having the following fonnulae (Ua), (llb) or (III): ~

R2 ll R2 . N~-- (II) 15(a) (b) A B
--R3 - NH - C ~ I~IH - Ph CH - CH - Ph - NH a (m) .

wherein R~ or R3 may bc 1hc sanlc or difforcnt and represcnt snaight or bra~ch alkylene, alkenylene, alkynylonc having 1-16 carbon ~lon-Y or tepreserlt a covalc~t bond, A and B may bo the samc or diKcrent and reprcSenl O or S, Ph is a phenyl group which is optio~ally substituted, c~g.
by onc or morc mcmbers selected &om ~hc group consisting of SO,~ ol alkyl.
. ~he elcctron mediator is a compound having an clect~on mcdiator mo;ety (rcprescnted hercinbelow at timc~ by the Ictte~ "U") which 2~ 7~ ~

~UG Z4 '93 10:05 R.COHN ~ PTNS 972 3 56a64135 P.10 is capablc of car~ing an extra electron and transferring same from the clectrode material to th~ redox center of lhc cnz~me molecule. U should havc a redox pot~ntiii suitablo for such an electron tra~sfcr.
The electron m~diator may eithct be saluble or may be 5 immobilizecl od thc surfacc of thc èleclrode ma~erial, or may bc covalently linkc~;l lo the enzyme.
Wherc thc cnzymc is a reductng enzyme, U may be an optionally wbstitu~cd viologon such as alkyl violog6t~ - e ~. carboxalkyl bipyridinium ha~ring thc following f~rmula (l~
H3C -~3N~ ~NG3 ( CH2 )n COOH (IV) wherein n~ 1-I6.
an optionally substi~ulcd pyridillium such as carboxyl substitutcd pyridinium, 8 that sho~n In thc following formula (V):
CGOH

an optionally substituted acridinc tbe subs~ituen~ being f~r example ca~boxyl, 6.g. thc compound having the following formuta (Vl):

25 ~ COO~
~ (~) 210~711 FlUG 24 '93 113:06 R.COHN & PTNS. 972 3 5606405 P.ll Whcrc thc enzymc is an oxidizing enzyme, U may for cxample bc an optionallg substitutcd fcrrocene, thc substilucnt being for e.~cample alkyl, earboxyl, alko~y-carbonyl, alk~rlamlde or ~Ikylcarboxyl fenoceno such as thc compound shown in the following fonnula (Vll):

~3-R Y

10, ~ ~VJI) wherein ~ is an C02H or NE~2 grt~P
Anothcr cxample for U bein~ an optionally substitutcd phenolhiazinc, such as the one shown i~ the follo~ng fonnula ~5~

~ 111) Electron mcdiators capable - of being immobiIized on the eicclrodc matcrial have thc general formula (I~):

U-R4-X' (IX) 25 wherein U has tho meanings given above, R~ acd X3 havc the meanings of ~nd ~' in ~omlula tl), ~ pectively.
In accordanc0 wilh one cmbodimcrlt vf the prescnt inven~ion the or zymc mo2ec~l1es are immobilized on the clectrodo material by means of the linking g~oup and lhe eléctron mediato~ is tumbling in thc surrounding ' '- '. . : ' , ' ~

21 ~ 711 , ~
~UC 24 '53 10:06 R.COHN æ PTNS. 972 3 5606405 P.12 _ 9 _ ., , solution. In accordance with this embodiment, the electrode malerial is Goated by a plurality of complexes, cach comprising a linking ~roup and one or more enzyme moleculcs.
bl accordance with anolher cnlbodimenl of the prcsent S invcntion, thc electron mediatot groups are immobiliz~:d on the electrodc matenal al d thc cnzyme molccules are tumbling in the surrounding solution.
In accordance with this cmbodiment, the eleclrodc matcrial is coaled by a plurali~y ot complexcs cach compris~ng B linlciug ~roup and onc or morc elcctron mediator graups.
In accardance wi~h a third embadiment of tho prescnt invcrltion bolh thc electron mediator ~roups alld thc cnzymcs are immo~ilized on thc electrode makrial. The electron medialor groups and the cnzyme mokcules may be in a single ~olccular complex comprising both thc linkin~ group, tho el6ctron mediator ~roups and Ihe enzymc molecule, or may be in 15 scparate ~omplcxes, one comprising a first linking group and the clcctron mediator g~oup and another comprising a second linking ~oup which may be tho same or d~ffcrent than tho ~ust linking ~roup and Ihe enzytne.
Whoro one of Ihe compollenls, namely cither Ihe en~yule o~ c electron medialor, is frcel~ tumbling in thc solution surrounding thc 20 elecl~odc and thc other comporlent is immobilizcd, Ihe eloctrode may c~mprisc a 5e~i-permeable membrane permeable ~o the lested ana1y~e but impenncablc to tlle tumbling component, enclosing a small volume of 50htlo~ wlth the tumbling componenl bctwcen it alld Ihc clcctrode matcrial.
Thc cîectrodc may comprise a slnglc layer of enzymes all 2S ~ound dueclly to a linlcing group. It may at times bc prcfcrred to include se eral layers of onzymc molecules, in which e~zymc molecules o~ one laycr connccted to tho cnzyme molecules of a previous layer by means of bndging ~oups. Such bridgiIIg groups may for cxample bc a group having the following formula (X):

. .~ . . . ~ . .
.

' . "-' '' . :';'' ' ~ ' 2~ 0'~7tl QUG Z4 '93 10:06 R.COHN & PTNS. 972 3 5i~06405 P.l~l . .

, Wl-R6-W~ (X) Whercin, Wl and W~ may bc thc samc or diffetent &om one another and have thc same meanings of Xl in forrnula (1), and R~ has the S meanlngs of Rl In forrnula (J)- ~
The brid~ing gtoup may alsu be a polymcr or a po1ypeplide havitl~ thc same rneaninKs as P in formula (1).
Wherc the electrode compriscs a pluralily of layers of enzymo molecu1cs, suitably some of thc cnzyme molecules in Ihc extemal layer ue sccondary eozymes intended to dccompose interfcring agcnls which may be oxitized or reducui in ~ non-specific manner, i.e. not by the enzysnc, when coming closc so îhc electrodc matcria1. Such agents if not decornposcd, may scriously affect th6 results and rcndcr thctn inaccurale. Thus for cxample, whore Ihc cnzyme is ~lucose oxidasc, such secondar~ enzymes may for e%amplc be peroxidasc to avoid a rcdox teaction which may be caused by agents such as ascorbate utate or scetaminophen.
1~ orter lo propare electtodes ot lhc prcscnt invcntioo, it is possible e;thor fir~t to ct~emisorb the linlcing E~roup and thcn. bind thc component to be immobilized thcreon it, i.c. the erlzym~ or the electron 20 mediator group. Alternativcly, it is possible fir6t to perfonn the binding betwccn thc linking gsoup and said componenl and then chemisorb thc comp1ex on to thc elcctrode matcrlal.
Wherc the elcctron mediators aro immobilized onto the enzymc, it is prcferrcd to immobilizc at Icast some electron mcdiator groups 25 cl,oso to the redo~ sito of Ihc enzyme and for that purposo the enz~/me moleculc is flrst unfolded, e.g. ~y the usc of hlgh concclltrations o~ urca, andthe ctcctrol- mcdlator group is then bound to the ullfoldcd enzyme mokcul6 whlch is subsequentlr Pfoldcd by decreasing the urca conce~tratio~.

. . ..
-- :
. .
.
... . .

. ~ , , , ,, , ~
- . .
. - ~ : .

21~7~1 RUI~ 24 '93 10:07 R.COHN & PTNS. 97Z 3 5606405 - P.14~

Suitably, the cnzymc moleculc is Ixtodi~led by binding to a plurality of elcctron mediator groups, c g 4-12 DErAlLED DESCRlPTlON OF TlIE PRESENT INVENTION
S I~ Ihe followin~ Ihe invenlion wilî be described at times with refercnce to the annoxed drawings In Ihe drawings Fi~. 1 is a schematic rcp~esenta~ion of an oloctrode's surface carrying complexei each comprising a single rcdox cslzymc molecu1e, an electron mediator group and a linking group ln (a) X' and ~3 ~re such with bind to an amine group of the enzyme molecule; in (b) Xl and X' are such with bind to a carbo~yl group of the enzym6 molcculc;
Fig. 2 show~ a schcma~ic repreSentation of an electrode's surface in accordance with ~nothcr embodimenl in which each cornplcx on thc clcctrode surfacc comp-ises an en2yme molecule and ~ linlcing group and Iho electton modiatots are tumbling in tho surroundit g liquid medium;
Fig 3 is a schematic reptesentation of tbc surface of all elec~rode in acaordanco with anothcr embodiment of the present invention in vJhich each complcx chcmisorbed on the surfaco of lhc clcctrode material compriscs a linking group and an clectron mediator ~roup and thc enzymc moleculcs arc ~eely tumbl~ng In Ihe solution and optionally have bourld ~hcreto n plurality Oe eloctron medialor groups;
Fi~. 4 i~ a schematic representation of the surface of an clectrode in accordance with ano~het embodiment of Ihc prcser~ ven~ion in w~ch t~e electrode carries two types of complcxcs, one comprising a linking ~roup ~5 and atl electton mediator group and anothcr comprising a linkirlg group andan enzyme molecule, thc enzyme molecule optionally has electron medialor groups bound therelo;
Flg. S is a schematic reprcsentation Oe IhC sureace of an clectrode in accordance with another embodiment of the present inven~ion carryi~g , ' - . : ' ~ . :. . . : : ~ .
.. ~ . . ...................... . .
- . .. . . . . . . . . .

21~711 .
f~UG 2'1 '93 10:07 R.COHN 8~ PTNS. 97Z 3 5606405 P. 15 complcxes cach compris~ng B plurality of layers o~ redox enzyme molccules, a plurality of electron-mediator groups covslently bound to the enzymes, a lir~ing group cl~emisorbed on Ihe surface of the eleclrode material and covalently bound to onc of thc rcdox cnzyme molccules in thc first layer and S a plurality of bridging groups linkin~ thè redox enzyme molecules of ouc laycr to those of ~olhcr;
~1~5. 6 is a schematic representation of the surtace of an elect ode in accordancc witb another embodi~nent of thc prescnl invcntion canying complcxes each comprising a plurality of rcdox enzymè molecules in diffcrcn~ laycts, ~hc ellzyme molcculcs of one laycr bcing linlccd to thosc of another by bridg1ng g~oups comp~ising a polymeric or a polypeptide chain;
~g. 7 is a schcn~atic rcpresentation of Ihe surface of an clcctrode in accordance ~ith another embodiment of Ihe pr~sent invention carrying complexes each comprising a plurality of rcdox el~zyme molecules all bound lo a linlcing group which comprises a polymenc or a polypeplide chain;
FiK. 8 is a schematic representation of onc manner of prepatation of a~ olectrode of Ihc kind shown in Fig l(a) whe~ein thc cnzymc is glutathi-one ~eductasc, and of the elcctron-transfer pathways in the obtained complcx;
Flg. 9 shows a cyclic vollammo~am of a gold clcctrodc modified with DSP and uscd subsequently for immobilkation of amino terivatives of naphthoquinonc tpolential scan rate 200 mV/s; background 01 M
phosphate buffer, p~ 7.51;
Fl~. 10 shows ano~hcr proccdure o~ immobilization Oe an enzymc on 2S ~he surface of a gold electrode;
Fi~. 11 shows a cyclic ~roltammogram of a gold electrod~ modified with cystamine a~ld 2,3-dichloro-1,4-naphtboquinone quinone immobili-2ation was carricd out before (solid Imc) and after (dashed linc) clectrode '', "' '' . ' ' '' " ' ~ '. ~ ' :
. -- ' '' ' ~ :- ' ' ' .
': , ~ '' ' ~ ' " ' :

- 21~71~ .
~UG ~4 '~3 10:E~8 R.COHN ~ PTNS. 972 3 56i364OE; P 16 .

trealmcnt with DIDS for 1 hr ipotontial scan ratc: 200 m~/s; background:
0,1 M phosphatc buffcr, pH 7.5];
Fi~. 12 shows surface conccntration of q~inone immobilizcd ~ia amino grou~s of cystamine versus time of electrode pretreatment with DlDS;
S Ihe concentrations worc calcula~ed by integration of peaks on cyclic vo1tammograms sunilar to lhc one shown in Fig. 11;
Fi~. 13 shows cyclic vol~ammograms of a ~old elcctrode modified by cystaminc, thcn by DIDS ànd subscqucntl~ by aminoquinonc (solid linc) and of a control cxperuncnt in which prior lo reaction with aminoquinonc, thc DlDS-modified elccltode was trca~ed with n-butylamine ~o dcactivate isolhiocyano groups on Ihe e1ectrode surface (dashed line);
~ig. 14 is a schcmatic reprcscntation of a surfare of an electrode motifiet wllh DSP and glulalhione reductase and the electron transfcr fiom Ihe ctectrode to the aclive centc~ of Ihe enzyme being ~via diffusionally mobile ele~tron ~rans~er mediator (MV2~ which rcsults in reduction o~ thc substrate;
Fi~ shows a cyclic voltammogram with a gold elc~trode modificd with glutathJonc rcductas¢ al~ached to lhc elcctrode surfacc via DSP id Ihe presencc of lx10-~ M me~hylviologen in the solu~ion, shown schematically in Fig. 14 (solid line) [potcntial scan rate: 200 mVlscc; back~round~ 0.1 M pho,sphate buffcr, pH 7.5 (dashed line)];
~ig. 16 shows tho glutathionc reduced forrm (GS~I) accumulation during clectrolysis o~ Iho elcctrodo modi~cd with DSP and wi~h glutathionc rcduct~sc and in thc presencc of lx1~-3 M methylviologen in thc solution as a.dlffusionally immobile electron trar~fer mediator and lx1~-2 M af GSSG
as initial substratc [cle~rodc potcntial: -0.7 V (~g. SCE)];
~g. 17 is ~ schematic representation of the preparation of a con~plex on thc surfacc of an clcclrode consisting of DSP, glula~hione reductase and ., , . . : : : . .: -. ....... :: , . ... : . , ~ ~
.: ..

:
- .- . .

-`` 21047i ~
~UG 24 ' 93 1~: 0e R. COHN & PTNS. 972 3 56064el5 P. 17 ~- 14 --an clectton mcdiator group bound to the e~zyme via groups of differcat chain lengtl~;
Fi~. 18 sbows GS~ accumulalion during eloctrolysis on an clccttode ot thc Idnd show~l schcmatlcally in Fig 17 with thc viologen attachcd to Ihc S enzyme ~ria a~ alk~lcnc ~roup of dif~etc~t lengths: (a)-C2, (b)-C5 and (c)-Cl~ lelectr~de p~tential; -0.7 V ~s SCE); GSSG concentration 1xlO-~ M, pH 7 3 (phosphate buffer)l;
Fl~. 19 shows tbc glutathione reduccd fonn (GSH) accunnulation during olcctrolysis of GSSH using tho clectrodc shown in Fig 10, w~h a viologen attached to the c~ymc via Cs - spacel [ckctrodc potential ~ 7V
(vs. SC~); inttid concentration of GSSG - 1x10-~ Ml;
F1g. 20 Is a schematic rcprcselltslion o~ a~ elccode cartying glucose oxidase cnzymcs ~E) immobilizcd on Ihe surface of thc elcct~odc by means of a 1-thioheptanoic acid linking group;
Fig. 21 shows cyclic voltammo~rams of the clcctsode of Fig 20, with 1x10-~ M fcrroceno in the s~lutio~, either without (a) or wilh 10 mM of g1uc~se irl tho solution (b) ~clect~odo potential +0 5 V (vs Ag/AgCI)];
Fi~. 22 shows an elcctrode having fertocene compr~sing complexes immobllizcd thereon;
~0 Fl~. 23 shows thc cu~ent obtaincd ~t differcnl ~lucose concentrations with a g1ucosc oxid~sc cnzymo (2mg/ml) in thc solutlon (clectrode potet~tial +0,5 V (vs. ~g/AgCl)l;
Flg. 24 is a schcmatlc represcntation of thc manner of prcparing clcct odes having bound theteto complcxes of the kind shown in Fig. S;
2S ~ig. 25 shows GSH accumulalion during clectrol~sis using au clcctrodc o~ tho Icind shown schemlltically in Fig. 24 (with soluble mcthyl vjologe~ rathcr than an immobilized viologell ~roup) wilh difforcnt layers (2 or 9) of onzymc moleculcs ~clectsode poten~ial 1 0 5 V (vs SCE); initlal :, ' ' .. ..
. -.
.
.
.

210471 ~
~UG 24 '93 10:1a9 R.COHN & PTNS. 9~2 3 5606405 P.18 : -- 15 --"
conccntration of GSSG: 1X10~2 M; concentralion of solubili~ed mctbyl viologen (electron tral~sfer mediator): lx10 2Ml;
Fig. 26 sho~s cyclic voltammo~ams of electrodes of the kind which arc prcparcd ill accordancc with the procedure shown schcmatically in ~ig S 24 wlth n ~ 10. Electrode~ with onc 1aycr four Iayers and cight layers of e~zymes were tested [potential scan rate: 200 m~/s; background 01M
phosphate buffcr pH 7 31;
F;g. 27 shows the radioactive lab~ling of an clectrodc versus thc number of laycrs of radioactivc labcled erlzymes on its surface;
10Fig. 28 is a schcmatic reprcsentation of th~ manner of pfeparing e1cctrodes of Fig 7;
Flg. 29 shows thc configuralion of Ihe bilirubin oxidase electrode;
Fi~. 30 shows the ampcrometric responsc of an electrode compnsing of 3 6 or 12 laycrs towàrd5 a concentralion o~ bi1irubin corrcsporlding to 15 1.7x10~ M In lhis cxpelimenl SxlO ~ M ~errocenecarboxylic acid is used as an clcctron t~ansfcr mcdiatot; and Fi~. 31 shows thc almperometric response o~ thc electrodc toward ~he concentration of bilirubin In thi8 experiment an electrode comprisirlg o~ 8 layols is applied as a sensing clcctrodc OEIAlL~D DESCRIPI'101~1 OF T~l~ INVENT101~
nle Invenlion will now bc illustrated b~ sevcral speci~1c cmbodimenls it being underslood that tllc present invcntion is ~ot limited thcrcto The altisan will no doubt apprecia~e tha~ the invcntion can als~ b~
25 ca~ried out by varjous modifications of its discloscd cmbodimcnts as well as by other embodimcrlts and thc artisan will havo no difficulties of calTying out such other embodiments on the basis of thc disclosure ir thls specifica-tion.

.

~. . . . .
, .. . : ::
: . : , , . ~ - .: :

'.. ': . ' ' , ~ ': ' ,. . . :
.

21~7~1 ~UG 24 ' 9~ 09 R . ~OHN & PTNS. !372 3 56064;35 P. l9 Refc~cnce is fust boing madc to Fig. 1 which is a schemalic represcntation of lhc surface of an elcc~ode in accordancc with on¢ embodi-ment of thc inwnllon. Thc surface of a gold electrodc 1 is covered by a plurality of complexes 2, cach of which consists of a linl~ing group 3, a S redox enzymc 4 and a~ clect-o me~iator group 5. The meaning of R, X', X', R~ and U are as defincd abovo for formula (l).
The nalure of Ihe elcctro medialor moiety U will vary dcpcnding on lho nature of the redox enzjmc. Wherc the enzyme is an reducing enzyme, sui~able clcct~o mcdiator moictics aN ~or examplc the 10 groups having thé ~o~mulae (IV), (V) and (Vl) as defined above and where Ihe enzymc i8 an oxidiziDg cnzyme, ~ suitable clectro med;ator moiety is for example a g~oup having the formula0 (~11) or (~II~) as d~fincd abo~e.
Exampks of cnzymcs are oxidizipg en~yme~ such as bilirubin oxidasc, glucosc o~tidase, alanine oxidase, xanthenc oxid~se and lactate lS oxldase and choleshrol oxidasei reducing enzymes such as gl~l~athionc reduct~se, nitrate reductàse, nilrile rcduclasc, and isocytratc dchydrogenase.
In Fig. la Xl of the linkin~ group 3 is of a kind which can bind lo an amino uoup i~ the enzymc. Suitable such X' groups arc for c~tunplc -N. C=S, -Nsc~o or an acyl group ha~ing Ihe gcneral ~onnula 20 It-CO-O whcrcin ~ is as dcfined above. X' in ~ig. lb is of a kind capablc of bindlng lo a carboxyl group of the enzyme such as - ~.
Thc clcctron tran~fcr path bctwccn ~he olectrode material 1 and thc rcdo~ centcr of thc cnzyme 6 is shown schcmaticatly by bidttectional ~rows in ~ig. la, the dircctioll of the electron transfer depending on whclher 2S lhe enzyme is a reductase or an oxldasc.
Fig. 2 shows another embodiment of th~ p;esent invention in whjcl~ only thc cnzymc 10 is lmmobilizcd by ~cans of the linlcing g~up 11 an the surface af thc clcctrode matcrisl 12 and the elcct~on medialors U arc .

...... . . . . . . . .

: .. .. .: . . . : ~ .
.
... . , ,. ~ .

-' .

210l~7 11 ~liG 24 '93 10:11a R.COHN & PTNS. ~72 3 56064E~!~; P.20 freely tumbllng in 1hc swTounding solutioll. Trans~er of elec;trons to the rcdox ccntcr of thc enzyme is cnsurcd by diffusion of thc elcctron mediators.
In the embodiment shown in Fig 3, only an clcctron mediator group 13 Is immobilizcd by rncans of a linking group 14 on to thc surface S of thc electrode material 15 and thc cnzyme molecules 16 are tumbling &cely in thc solu~lon. The c~zyme molecules 16 may also, if desired, carry elcctron media10t g~oups 13. Electron ~ransfer is ensured by the diffusion of Ihe enzymcs whlch thereby como into contact with thc imm~bilized clectron mediator groups.
10 ' ln ~hc embodimenl shown ;n Fig. 4, both thc elcctton mediator group 17 and Ihe enzymc molecules 18 are lmmobilizcd by separate linking groups 19.
Rcference is now being made lo Fig. S which is a schemalic b roprosontat~on of the sur~ace of an clcctrode in accordance with anothcr 15 cmbodiment of lhe invontion. Each complex 20 in accordarlce with lhis embodimenl carricd on the surfacc of ~he electrodc material 21 compAscs a pluralily of redox onzymc molcculcs, two o~ which 2~ and 23 arc shown.
Thc complex is bound to the olectrodc material ~1 by mcans of a liuking ~roup 24 bou~d to one ot the e~ZymeS 22. Thc enzyrncs cany electron 20 mctiatol groups 25. (As rcgard thc mcanings o~ Z, R~ , Xl and R~, sce above).
Thc cn~ymc molecules arc arranged in laycrs, the cn~yme molccules of the flrst laye- being linked by means of linking ~roup 24 to Ihe clectrodc matenal a~d the enzyme molccules of each subsequerlt layer, being 25 bound îo enz~rrne molecules of a prcvious layer, by means of bAdginR
groups 26. l~rid~ng groups 26 havc thc scneral ~ormu1a W-R~-W, wherein W and 1R' havc Ihc same meanings as X' and R' in formula (1).
Somc of the electroll transfcr pathways are shown by mea~s ot bldlroctlon~l ~ows, and also hore the direction of thc clectron Iransfer - .

-' ' , -. , ,' ~ ' .'' ' .~
,, ~ , . - . ' . . . ' .:
.

" 2104711 fWG 24 '93 10:10 R.COHN & PTNS. 972 3 56E~6405 P.21 depcr~ds <~n thc nature of the et~zyme, i.e. whether Ihe pathway is rcductive or oxidative.
Rcfercnce is now being made to Fig. 6 showing a schcmatic rcpresentathn of the surface of an elcctrodc in accortance with a furthel 5 embodiment of thc presen~ inVenliOn. In this figure, symbols havc the samc meaAings ss thosc which appear alrcady in Figs. 1 and 2 and the readcr is ~e~erred lo thc description in these figures for theit exp1anation. Similarly as in thc cmbotimcnt showil in Fig. 2, also in this embadiment cach complex 30 compt~scs a plurality of ~edox cnzymc molecula, of which two, 10 32 ~d ~3 arc shown. The complcxcs are bound to the eleclrode substratc 31 by means of a linking ~oup 34 covalcnlly bound ~o c~zymc molecule 32. Each of thc er~zyme molecules has associatcd thercwith electron mediator groups 35, covalcntly bound thereto.
The enzymes in the complex aro lillJccd to one ano~hcr by 15 bridging groups 36 comprising a polymcr ot a polypeptide P catrying a .. . plurality of functional g~oup~ capablo of binding to groups in the proleia or to the elcctr~ medlat~r Rroup. For example, whcre P is a polypeptitc rich in lydnc, c.g. a polylysine, lbC amine graups of 1ysirle can readily bW ~o the carbo~ylic groups ;n the onzyme. Where, fot exunple, P is a polypep-20 ~ido ri~h i~ glutimate, c.g. a polyglutamate, thc carboxyl sroups ~f glutamalc can readily bind t~ amine groups in the ctlzyme molecule. Where P is a polypeptide, it carries bi-functional groups 37 having the same nature as the bridging groups 26 in Ihc cmbadimcnt shown in Fig. 5.
Bi-directioltal a~ows show somc of the comple~ eleclros 2S traD,sfcr pathway~ bctwoon the elcctrode material 31 and the redox centers 38of tho enzyme moleculcs. Here as well, thc di~ection of thc electron traAsfcr depcndls o~ whether thc catalytic pathway is oxidative or rcducdve.
l~efcrcncc is now being madc to Fig. 7 showing yct another embodiment of Iho prcsent inventian. in this figure all liko symbals have ~ .

210~7l3L
RU~ 24 '9~ 1~ 11 R.COH~ 8~ PTNS. 972 3 5006405 P.22 .

~ 19- ' lhc same meAni~gs as ill p~ovious t;gurcs. Complcxcs 41 of this embodi-ment comprise a plurality of enzyme molealles 42 linked to electrode m~terial 40 by mea~s of linkln~ group 43. Liruking ~,roup 43 has a polymer or a polypcptlde P which Is substanlially the sarnc as P in the embodiment S shown in Fig. 6.
Some of thc complex electron trans~er pathways in this clectr~de a~e shown also here by means of bi-di~eclional ar~ows, and again thc actual direction bcing dcpcndcnt on the nalurc of their redox ptthway.
Also in the cmbodimenl of Fi~. 7, additional layers o~ cnzymcs can be crea~ed.
', ~LS pointcd out already abovc, in cmbodiments in accordance wi~h Ihe present invention where the electrodc comprises several layers of cnzyme molecules, such'as thc embodimentg shown in Figs. 5 to 7, tbe extcrnal layers may consist of non rcdox enzymcs inlended to disintcgrate agents othcr than 1he analyte in order lo avoit non specific oxidation or r reduction, as the ca~c may be, jf thesc agents will corne inlo contact wlth Ihe electrodc material or with the electron mediators. Such agents, if not dis~ntcg~atod may give r,ise to non-spocific currcn~ and thus a dccrcasc in the ~ccuracy of the results.
' ln accord~nce wit~ a modiftcation of the embodiments of IFigs.
5 to 7, instesd l~f havin~ an 016ctron mcdiator group within the complexes or as an addilion thereto, it is possible to provide for elcctron mediation by having elcctron mediators dissolvcd in the solution sur,ro~nding the e~ectrode. In such a ca~e electron transfer will be ensured by Ihe free 25 diffusion of these compounds ill Ihe solulion.
E~lectrodes of the inYen~ion are usef,ul for lesting for Ihe presence and coqcontratioll of specific ana'iytes in a tested sample. 1~ the presencc of an analyte in a solu~ion,surrounding the electrode, (which solution should also is~clude an electron mediator compound if an elcctron . ; ., :
.
.
. , 21~711 RUG 74 '93 10:11 R.COHN ~ PTNS. 972 3 '606~a5 P.Z3 mediator group i~ nol illcluded as part of the complexes on the electrode) which anal~te can bo reduced or oxidiz~d by tho redox cnzyme as thc case may be and approprlate potential on lhe electrode is applicd a chargc will be IraDsferred to or ~om the enzyme respeciively which will provide an 5 indicatioll of thc presencc of thc analyte in the solution. The magnitude of the charge fl~w will bc proportional lo its concenlratlon in the solution.
Whcrc the enzyme is a reductase enzyme a negali~e potential lo Ihe electrodo shauld bc applied and where tho cnzyme is an o~cidasc enzyme the potcnlial should be po~itivc (in the former caso the transfer o~ elect~ons is 10 frdm the electrode substrale to tho redox conler and vlce versa in thc latter casc).
At tlmes rathcr than deterrnining the concentr~tlon o~ lhe anal~tc by mcasuring thc charge flow Ihc concenlra~ion will bc dc~crmincd by ~ea~uring Ihe concenlra~ion of ~he product of ~he redox rcaction ~ er a lS cortain period of ~ime.
The invention will now be further illustraled by the followin~
ex~mplcs.
I

amplo 1 Co~alentl~ biodin~ eoz~nne to an electrode by the use of blfullctlonal r~ents conbining sulphur ul~chor groups in the molecule lllc proccdure descnbod bclow is depicted schematically in Figs. ~.
~ barc ~old (Au) electrode (foil geome~rical area 0.2 cm2) was soaked ~n conccrllnled uitnc acid for about 10 min. following which thc eloctrode was rinsed ~horoughly with water and d~mc~hyl-sulphoxide (~fSO). After such prctrcatmcrlt thc clcctrodc was so~ked In DMSO
conlalnlng lxlO-~M di~hio-bis-(succ~nimidylpropionato) (DSP Fluka) for 30 2 h~ rinsed with DMSO thrce times and one time with water.

:~ :' , ' ............... .
' 210~71~
,~IJG 2~ ' 93 10:12 R. COHN 8. PTNS . 972 3 5606405 P. 24 'rhc elcctrode with bo~nd succilumidyl activc cster groups was then incub~tcd overnight at 4~C in 0.1 M phosphate buffec, pH 7.2, containing 1~10 U/ml glutathione ~eductase (from bakers yeast, E~:: 1.6.4.2, Sigma) and rinscd threc timcs with thc samc buffcr to r~move non-attached S en~yme from thc clectrode surfacc.
1~16 same ptocedure was followed, using however anoth~r bifunctional leag~nt: dimelhyl-3,3-dithiopropionimidatc hydrochloride ~Dl~P, E~luka~, hav~n~, thc following formula:
HN~ NH
/C - ( CH2 )2 S - S - ( CH2 )2 C 2 HCI

The electrode modification can also be carried out in a vater solution rather than DMSO, using watcr soluble sulphonated bifuncliQal reagents such as 3,3'-dithio-bis-(sulfosuccinimidylpropionate) (lDTSSP, Picrcc), having thc following folmula:
-O3S~O ~ ,SO3-~N - (:~ - C - ( CH2 )2 - S - S - ( CH2 )2 - C - O - N~
O O
In addition to glutathione rcductase, the following ~thcr enzymcs have also bcen bound to electrode in the same marmer: lipoamide dehydrogena5e (&om bovine intestinal mucosa, EC 1.8.1.4) a~d ferrcdoxin NADP reductase (from sp~nach leavcs, EC 1.18.1.2., Sigma) and glucosc 2j oxidase.
In ordcr to examine tho ability to immobilize amino corn-pounds on electrodes modified by the abovc mcntioncd bi~unctional rj~ge~ts, the teda~ ~ctive aminoquin~nc, 2-chloro-3-(4-aminobuty1)-1,4-naphthoquinonc, was bourld to thc succinimidyl activc ester groups. A

: . : ,. . .. ~ . , .

. . : .

.

-- 21~4711 ~UG Z~ '93 10:12 R.COHN & PTNS. 972 3 5606405 P.25 --2~ --cyclic voltammogram was obtained a~ainst a water background (0.1 ~1 phosphate buffer, pH 7.2) and surface concenkalion of Ihe active group was validated to bc about 8~10-" mol/cm2 by integration of cathodic or anodic poak (thc cyclic ~oltamrnogram is shown in Fig. 9).
S In ordcr to determine the surfacc concentration of immobili~ederlzyme on Ihe elcctrode surface, glulathione reductase molecuies were labcllcd with H3-iodoacctic acid, and were thcn immobilizcd on Ihe eloctrodc. Surface conccntration was dctormined to bc about ~x10-~
moVcm2 by mcasurin~ Ihe radioactivily Oe the enzyme modified electrode.
Example 2 Prepartttion of electrode bg lhe use o~ c~rstamine or c~steaminc ~OI
~ncttorlng the camplexes Thc manncr of prepatation of the clectrode as descr~bcd belo~r, is depicted in Fig. 10.
Aftcr pretreatmellt with nitric acid as described in Examplc 1, Iho Au clectrodo w~6 soaked in 0.1 M phosphate buffcr, pH 7.2, con~aining 0.02 M cystamlnc (2,2'-diaminodicthyldisulfidc, Fluka) for 2 hr. 'nle oloctrode was then rinscd repca~edly wtth distillct water and immcrscd for 10 min. at O-C in the same phosphatc buffcr containiDg lx10-2 M 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (I)IPS, Piercc).
Thc modified eloctrode was rinsed again with watel and was incubate~l fot 1 ht at 0C in thc phosphatc buffcr containing glutathione 2S reductase, 100 U/ml. The enzyme-modificd electrodc was then rinsed with the phosphate buffer thtce timcs to tcmove non~immobilized er~yme By a similar proccdurc electrodcs were prepared using howevcr cysteaminc containing lhiol anchor group [H~N-(CH2)2-SH] rather than cystamine, bu~ in thi~ casc thc soaking of the Au elect~ode could bc docreascd to up to about 1 min.

2~7~
RUGZ4 '93 10:13 R.COHN 8 PTNS. 97Z ~ 56064~25 P.26 lnstead of DlDS, other bifunctional reagellts can be used such as bis(succinim;dyl)suberate (BS, Piesce), following the same procedurc. BS
ha~ thc following ~ormula:

5~ O3s~J(o 0 0 ,~ "SO3-~N - O ~ C - ( CH2 )~ - C - O - N~
O , O :' ' In order to de~emune su~face concentration of amino groups 10just after clcclsode modification with cystamine (or cysteamine) 2,3-dichloro-1,4-naphthoquinone was bound th~teto. By coMparisOn of ~he su~face concentration of amino g~oups prior to DIDS modification and afle~
"such modification, the ~inding of the quinone i~ both cascs may bc compsred. Fig. 11 shows a cyclic voltammogram of a an cleetrodc modified 15with a cystami~c and then modified with t~c quinone, the second modiaca-tion bcing eithcr imnlcdiately aftcr the cystamine ~odification (solid linc) or followhng treatmenl with :~IDS for 1 hr (dashed line). Fig. 12 shows surfacc conccntra~ion of the quinone as a function of the timc of the DIDS
treatment. ~LS c~n be seen, afte~ abo~t ten rninutes' reaction with ~lDS
20~approximatcly hatf of thc surface arnino groups are blockcd by lhe blfunctional rcgcnt DIDS.
A~other way to es~imate suffacc cancentration o~ DIDS on th~
elcctrod~ suRaee is covalcnt imrnobilization of a quinone wjth an a~nino glOUp on thc end of a 8ide radical b~ ~eac~ion of the ami~e group with the ~5active isothioc~Ano group of DI~S. Cathodic (or anodic) peak integratiou tO~ cyclic ~roltammograms of lhe kind dcpicted in Fig. 13, givcs a mc1sure of the surface conccntration of the quinone and this value, whilc gcne~ally . .
in agrccment with thc value obtained aba~re, shows a slightl~ smallcr conccnlration, which is belicved to ori~inate froln de3ctivalion o~ part of thc ' ~

.
.: . . . - . .
. . ~ .

, 21~7:~1 RlJG 24 '93 10:13 R.COHN & PTNS. 972 3 5~;36405 P.2, , --~4--isot~iocyano groups because o~ their hydrolysis turins electrode modifica-lion.

I:~ample 3 5 ~ctivit~ of the clectrode of IExample I
Ille immobili2cd enzymc was elcctrically coupled with the elecl~odc materi~l by dissolving a mobile elcctron lrsnsfer mediator, melhyl-viologen (MV2~), MV2t having Ihc following formula:
H ~C 0N~N~ Cl 13 .2CI'~

'rhc clcotron kansfer palhway of this system is shown in lS Fig. 14. Fig, lS shows a cyclic volt~mmogram obtained in a soiution containin~ 1~10-~ M~2~ and 0.01 ~I glutathione (oxidized f~)rm, GSSG) which is a substratc for thc c~zyme (salid li~c) and that obtlined withoul MV2~ (dashcd lino). The c~lrvc reveals an electrochcmicallg rcYersiblc rcdox procoss for vio10gen which indicates thal this proccss is not blockcd by Ihc ~0 monolaycr of complcxcs whicb was formcd on the surface o~ thc elcctrode body. Electrolysis in the solution using the enzymc-modified cleclrodc at a constant potcntial of -0. î V (vs. SCE) resu1ts in formation of thc reduccd fonn o~ glutathionc, GS~I. 'rhc accumulation of GSH, which was dete~-mincd by a spectral analy~is, is shown in Fig. 16.
~j , ' ,, ~

` 210~711 RUG 24 ' 9~ 10:13 R. COHN & PTNS . 97Z 3 5606405 P. ZE~
: ` .

--25-- .

Example 4 ~urth~r~ modlflcat~o~ ~ electrodes b)~ co~alently ~ttachiug electrou medhtor ~roups to the complexcs S ~lécttodes prcpared according'to Ex~mples 1 or 2 wcre further modifict by c~rbo~cy dcrivalives of viologen with different spacer length and having thc following fonnula:

~3C ~ ~N~- ( CH2 )n - COS)H

whercjn n - 1-16. Thc modificalion was by rnearls of carbodiimidc coupling of thcir carbox~ ~oup with ~he amino group of a lysine residuc in Ihe enzymo mol~cule. In order to ~chieve such couplin~, thc clccttodcs lS preparcd irl accordance wilh Examples 1 or 2 wcsc immerscd ovcrnigh~ at 4C in a 0.1 M HEPES buffcr solution, pH 7.3, conta~ing O.OI M
carbo~ylic acid derivative of viologen, st~uclurc IV, 0,01 M l-cthyl-3-(3-dimethylaminopropyl) carbodiimid~ (EDC) as a coupling rcagcnt and 1,0 M
urea to ope~ tho irmcr shell Iysinc residucs. Aftcr incubation the electrod~
20 wa~ nnscd wilh phosphatc buffcr, pH 7.3, ~o remove all non-attachcd component~ from thc cleclrodc surfacc. l~c procedure, a~ld the final clcctron lraw~er pathway is showlt schematically in Fig. 1~. ...
Thc clectrodes which werc obtained ~rere st~ldi~d by cyclic volt~lmmetr~ to check for rcdox transformations of immobilized viologens.
25 131cctrochenr~ically rc~/crsiblc teduction was seell having a redox polenlial E
~ -O.S8 V. Similar ~urves wcrc oblain~d for al! c~-a-tboxy dcr.i~ati~res of vhlogen c~c~epl lhosc havirlg short spacers o~ 2 methylene groups.
The surfaco collccntration ~or imn~obiIizcd viologen was c~lcul~ted by in~egration of cathodic (or'an~dic) pcak on thc assumption that 30 onc clcctron reduction is charactcristic for tho first stcp of ~iologe~ redox :

210~7~1 .
~UG 24 '93 10:14 R.COHN & PTNS. 97Z ~ 56~64E!5 P.Z9 --2~--process and a ~aluc o~ about 1.6x10-' mol/cm2 was obtaincd. Sincc surfacc concentr~ltion of ia~mobitized protein was es1im~tcd to bc about 2xlO~
moVcm~ (scc l~amplc I) ~vcragc number of such groups purporting arc about 8, narnely oul of the 39 Iysine residucs in tho lutathione reductasc, 5 8 are modified by the~e redox groups.

Exunple 5 FuDctlon of olectrode prepared according to Example 1 and modlfied b~
electroo n~ediator groups according to E~cnmple 4 Enzyme-modificd clectrodcs prepared according to Example 1 and modified by electron mediator groups according to Example 4 contain redox active components attached directly to Ihe protcin ~lobula. An elcctrodc having mcdiator groups with diffcrcnt spacer Icnglhs (C-2, S, I I) '~ 15 WClO used. The elcctrodes were immers,ed in a solution containing 0.01 1~1 GSSG and 0.1 M phosphatc buffcr, pH7.2, and a negative potential of -0.7 V (vs. SCE) was applicd. GSH accumulation was observed by spectral analysis (as in ~amplc 3). T1~6 accumulation of GSH as a function of tlmc by using bipyridinium rclays having diffcrcnt alkylenc chain length linked 20 to the p~otein, is shown in Fig. 18. ~As can bc scen, therc is an improved elcct~ical communicadon upon lengthening of thc spacers which is likely ;Kributablc to thc cnhanccd intra protcin e1cdron transf~r rates as a re~ult of electron-donar distance ~hortcning. Namcl~, the flcxible alkyl "a~ms"
gencratc a shorter iJltra protcirl clectron transfer distances betwecn the 25 electron mediator molety and thc active sile of thc cnzyrnc resulling i n cnhallced electrical communication.
It was found that thc electroc~zymatic activity of the clectrodc strongls~ deper~dcd on thc presencc of una dunng thc la8t 6tcp of the dcctrodo prcparation: in the ab~cncc of urea tho electrodos which were 30 obtained had tbc sunc clcctrochcmical propertics for the immobilized '' ' ; '' ~ . - " ~

~:
.

210~711 ~UG 24 ' 9~ 14 R. COHN ~ PTNS. 972 3 560O405 P. 30 '' ~27' viologeos but lackcd clcctrical communication between the immobilizcd viologens and Ihe ~CtiYC centet o the enzymes. ~or stlch electrodcs electrocnzymatic scliv~ty was achieved only in thc presence of diffusionslly mobile clectron transfcr mcdiators.
S
~i;xample C
Function of tlectrodes prepared accordlng to l:xample 2 aud modifled br electron trans~cr mediator accordi~g to Example 4 l~lectron modi~ed electrodes preparcd sccording to Example 2 and modified by electron transfer mediator according to Example 4 con~ain redox active comporlents attached not only to thc protein g lobula bul also to ammo groups on the electrode surfacc many of which are not blockcd by DIDS. A~ was shown in Examplc 2 elcctrodes modified by cyslamitle and t eated theresftcr by Plr)S for 10 min. had approximately ~0% of non-blocked amino groups on ~hcir surface (see Fig. 12). A~ter modi~lcation wlth catboxy dcrivativc of viologcn according to Examplc 4 this elcctrodc mediator groups were attached bo~b to the olcctrodo sutface and to the p~otcin globula. Sur~acc conccntration Oe ~ho imtnobilized viologcn was calcubted to bo Sx10-' mollcm2 which i8 sllghlly highet thsn thc va3ue .
obtaincd in Ex~mple 4 Rale constant for elcc~ron transfer bctwccn the elcctrodc and immobilized viologen was calculated as described in Example 4 and very sirnihr valucs about 100 s ' was obtaincd. Electroenzymatic aalv;ty o~ thc electrode was studied as describcd in Example S and very similar ralc of GSH accumulalion was observed as shown in Fi~. 19.

Ex~mple 7 Immobilization of glucose oxidase on an electrode The gold elcctrode was incubated ~n 1-lhioheptanoic acid (0.02 M) in cthanol overnight at room tempcraturc. Thcn the electrod~ was .

. - :. . . .. .
, : . ' ~

, .
.

21~7~1 RUG 24 '9~ lJ:15 R.COHN & PTI`IS. Y7Z 3 5606405 P.31 .

rinsed five tlmes with clhanol and then a~ain wit~ 7.3 phosphatc bu~fer (0.1 M~ and was il~cubalcd with a solution comprising glucose oxid~;c 2 ms/ml and with 0.02 M of El:~C. Thc surface of thc obtained clcctrode is shown schcmat;cally irr ~ig. 20 (E represenls gluc~se oxidase enzymc).
Thc cyclic voltammograms (at a scan rate of 2 mV/iec) in thc presence of 0.1 mM carboxy derivative of ferrocene (Xl) in the --CH2 - Nl I ~ ( Ci-12 )s - COOH
Fe (~) ~

solution, with and without 10 mM glucose arc ShOWII in Fig. 21 in which (a) is Ihe cycllc vo!tammograms witho~ll glucosc ~nd (b) is thc ono in Ihe prcscnco of 10 mh~ glucose.
1~ As can be secn, in the prese~ce of glucosc, thcre is a dramatic increase in the charge flow rcxul~ing from tllC catalytic 07tidation Oe glucose.
Example 8 Use of eloctrode csrr~log Immobilized ferrocene Gold clectrodes wcse incubatcd in a 0.02 M cyslamine dihydrochloridc salution for 2 hr at room temperahlre. Thc eleclrode was rinscd fivc timcs ~rith triply distilled water aud then r~vo timcs with 0.1 M
~P~S buffer ~ 7.~. Thc eleclrodes were lhen incubated wilh 0.02 ~f carboxy dc~iva~ive of ferrocene (Xl) in HEPES buffcr in thc p~esence of 0.02 M EDC at room ~ompcra~ure o~vernight.
The surace of lhe obtained olcctrode is shown schematically in Fig. æ (Fc ferroccne).
~, Aftcr lhis modificalion the electr~de was rinsed five times with lhe H~P~S buffcr and chcckcd for the presencc oE Irnmobilized ferrocene ~ . " . . , ' , ~UG 2~1 ' 93 1~:15 R. COHN & PTNS . 97Z 3 56064i~5 P. 32 2~711.

by cyclic vollammelry 250 units of glucose oxidase were then added to the el~ctrochemical cell and cyclic voltammctry was ~arried at a scan rate of 2 mVlsoc with different concent-ations of glucose The resul~ing current with the electrode fixed at ~0 5 V ~t diffctent glucosc conccntrations is shown in S Fig 23 and as can be seen there is a lincu relationship bctween thc glucose col~contration and the me~sured current ~ample ~
Prep~ tion of electrodes modif~ied with 8 few larers of the enzyme Enzyme-modi~ed electrodcs prepared accord;ng to Exa~ple 1 or 2 can bc trcated again with a bifunctional teagcnt Thc sequcnc~ of modification of such an clectrode is shown schematically in ~ig 24 A~fter modification of the elcctrode in accordance with Examplc 1 or 2 and thc 15 binding of thc clcctron mcdiator ~roup Pg detailed in 12xample 4, thc electrode ~as treated for a second time with Dl~)S for 10 min at O~C
~ter this second nodification, a second 1aycr of enzymes was immobllkcd on thc clcctrodc by following the same ptocedute o~ E~amplc 4 At times, carboxy dcrivativc8 of viologen wete attached to the en~yme molecules i~
20 the ~nc mannor as that described in E~ample 4 In various eXperimenlS, tl~ese carboxy dcrivativcs Oe viologen had various chain lengsh, wilh Ihe numbcr of ca bons in thc alkylene varyin~ bct veen 6-lI
After the above-descnbcd process, the resulting clec1rode hact two layers of enzymes, and in order to obtain a multi-laycr enzymc 25 elcctrode, this process was repeated for a llumber or cyclcs as desired Fig 2S shows Iho accumulation of CSEI as a function of lime us~ng clectrodes which did not co~nprisc immobilizcd carbo%y deAvatives of viologen but rather the methyl viologen dissolved in the solutions surround-ing the electrodo Elcctrodes comprising two layels of cnzymes (El 2) or 30 ninc la~ers of en~mes (El 9) werc used and the concentration of methyl , , . ~
.

.. . . . ..

.

RUG 24 '93 11a:16 R.COHI`~ & PTNS. 972 3 5~,06405 P.33 viologcn i~ 601ution was 10 mM. As can be sccn, the ratc GSH accumula-tion in the El 9 ele~trode is considelably highcr than in thc El 2 electfode.
Electrodes with scveral laycls of.lho et~zyme ~lulathionc reduct~se were prepared in accordancc wi~h the procedu-e shown in Fig. 24 S including the a~lachmcnt o~ carboxy dcriva~ives of viologcn (ha~in~ 10 carbon atoms in thc all~ylenc chain). A cyclic vollammelly e~peliment was conductod and lhe rosults depic~ed in Fig. 26 show a clear dcpcndence of lhe electrodc electsorespo~se on the number of cnzyme laycrs.
In another sct of experiments thc en~ymes wcro radioactively 10 1abcled and thé labcling ~ctsus the numbet of layers was tested. As can bc scen in Fig. 26, a linear relationship betwcen lhe numbcr of layers and thc radioactivity can be seen, indicativc of linear incrcasc in thc numbe~ of cnzyme moleculcs wilh the increaso in the numbcr of layers.

lS Examplo 10 Co-immobillzathll of an enzyme and pol~mcrs cont~ining tall redox-~ct~re g~oups The procedurc for prcparing îhe electrode o~ this e~ample is 20 shown schcmatically in Fig. 28.
An electrodc was n~dified with cystamine and activated wilh DIDS as described in ~xample ~ and ~hen a polyiysinc chain (PL in Fig. 2~) was imtnobilized on thc clectrode following a simila~ procedure to that of the immo~ilization of tho etlzymes described in Example 2. Thc 2S polylysine chains carried bipyridin~um groups which wcrc covalently attached to amino ~toups'of the polylysine chain by carbodiimidc coupling of the carboxy g~oup of th~ bipyridinlum derivatlvcs and amino g~oups of tho polymer. 'Thc loading rate of the bipylidinium g~oups was madc nol to bc vcry high so as to Icavc non-n~odificd amino groups on the polymer.

~IJG 24 '93 10:16 i~.COHN ol PTNS. 97Z 3 5606:105 P.~4 210~711 Following the immob;lizatio~ of Ihe polymer, lhe electrodcwas trcated agaln with ~IDS and ahcr rinsin~, enzymes were immobilized on Ihe electrodes by covalent binding to Ihe Dll:~S groups. The procedure of reactioP. with DIDS and Ihen immobilization o~ a hnhet layer of poly1ysine, S trcalmen~ with DIDS and a further layer of enzyme can bc repeated for a numbcr of times to obtain an clcctrodc with a ,olurality of cnzyme layors.

Example 11 Immobillzalioo of biUtubln oxidasc on electrode and amperometr~
10 d~tennin~lHon of bil~rubln l~c procedure of preparing the e!cctrodo of this Exanlple is shown schcmaticall~ in Fig. 29.
A bare gold elcctrode w~ succcssively treatcd with conccn-trated HNO~, rinscd wilh distillcd wlller, rinsed with DMSO, and then motificd with 2x10-~ M dithio-bis-(succlnimidylpropionate) in D~SO, ir thc m~nncr describcd in Ex~mple 1. Ihe modificd electrodc was removed from the solution, washed twice with DMSO and once with cold (0'C) phospha~e buf~cr (01 M, pH 7.3). Thc flrst bilirubin oxidasc layer was covalcnlly Immobilizcd to thc modi~lod c1ectrode by soakin~ thc electtodc overni~hl at 4C in a solution of 100 U of Bi1irubin Oxidase in 2.5 ml of thc phosphate bu~fer, and then washing it with cold phosphatc buffcr A
successi~,rc laycr of bilirubin oxidasc was linkcd to ~he basc layer by OII:)S.
Thc enzS~me electrode was dipped in 2.5 ml o~ colt (0~C), 0.02 M solutio~
of DIDS in phosphate buffer (0.1 M~t pH 7.3~ for 10 minutes. The electrode was then rcmoved from Ihc DIDS solution, washcd twicc with cold phosphatc buf~cr, and thon soakcd in thc cnzymc solution at 0C for 30 minutes, This proce~;s was tepea~cd until the desired number of layers o~
bilirubi~ oxidaso was linlced to Ihe electtodc.

RUG 2~ '93 10:17 R.COHN ~ PTNS. 972 ~ 561~16405 P.35 210~711 ., .

A catalytic wave with EP _ 0.~8 (vs. Ag/AgnO~) was oblalncd by c~clic voltammctry wilh Ihe multiple layer bilirubin oxidasc electrode in a solution of ~oilimbin ln TRIS bu~fcr (0.~5 M pH 8.0) with ferraccne carbo~cylic acid (Sx10-' M) as an electron transfer mediator frccly tumbling 5 in 1hc solution. The cyclic voltammelry was perfonned in a ~lass cell, with tho multiple laycr bilirubin oxidasc clcctrode as a working electrodc, a graphite rod as a counter clcctrodc, and AglAgNO3 as a teference eleclrode.
The electrodet remained slable and gave the same calalytic wavc for o-~er five weel;s of dry storagc at 4C. Thc anodic pcak currcnt increascd as 10 more onzyme laycrs were addcd as shown ;n Fi~. 30. The amperomclric mu1tiple layer bilirubin oxidase electrode had a lincar calibration curve, and its anodic pcak currcnt was lincarly depcndent on thc bilirubin concenttalion (Fig. 31).
;

~,

Claims

1. An analytical method for determining the presence or concentration of an analyte in a liquid medium by an electrobiochemical enzymatic redox reaction in which electrons are transferred between the surface of an electrode material and a redox enzyme by the mediation of molecular electron mediators whereby the enzyme is capable of catalyzing a redox reaction in which the analyte is converted into a product; said method comprising measuring the concentration of the product obtained in the redox reaction or measuring the flow of charge; the method being characterized in that said electrode material is of a kind which is capable of chemisorption of sulphur containing moieties and that at least one of the components, the enzyme or the electron mediator, is immobilized on the surface of the electrode material by means of a first linking group convalently bound thereto having a sulphur containing moiety chemisorbed to said surface, and the other of said components being either (i) tumbling in the liquid medium surrounding the electrode material, (ii) immobilized on said surface by means of a second linking group having a sulphur containing moiety chemisorbed onto said surface which may be the same or different than said first linking group, or (iii) immobilized on said surface by being convalently bound to said first linking group or to said one of the components.

2. A method according to Claim 1, wherein the enzyme molecules are immobilized on the electrode material by means of a linking group and the electron mediator is tumbling in the surrounding solution.

3. A method according to Claim 1, wherein the electron mediator groups are immobilized on the electrode material and the enzyme molecules are tumbling in the surrounding solution, the electrode material being coated by a plurality of complexes each comprising a linking group and one or more electron mediator groups.

4. A method according to Claim 1, wherein both the electron mediator groups and the enzymes are immobilized on the electrode material.

5. A method according to Claim 1, wherein at least one of said first or said second linking group has the following general formula (I):

Z-R1-Q (I) wherein Z represents a sulphur containing moiety;
Q is a group X1 or P; X1 is a functional group which is capable of forming a covalent bond with a moiety of said at least one component; P is a protein, polypeptide or polymer having a plurality of functional groups X2 capable of forming a covalent bond with a moiety of said at least one component:
R1 represents a connecting group.

6. A method according to Claim 5, wherein X1 or X2 is an amine group, a carboxyl group, -N=C-S, -N=C=O, an acyl group or

7. A method according to Claim 5, comprising linking group of the general formula (I), wherein Q is P, and P has amine or carboxyl groups.

8. A method according to Claim 5, wherein R1 represents a covalent bond or represents alkylene, alkenylene, alkynlene and phenyl containing chains.

9. A method according to Claim 8, wherein R1 represents a chemical bond or a group having the following formulae (IIa), (IIb) or (III):

, (II) (III) wherein R2 or R3 may be the same or different and represent straight or branch alkylene, alkenylene, alkynylene having 1-16 carbon atoms or represent a covalent bond, A and B may be the same or different and represent O or S, Ph is a phenyl group which is optionally substituted, e.g.
by one or more SO3- or alkyl groups.

10. A method according to Claim 5, wherein the electron mediators have a mediator moiety U being a member selected from the group consisting of viologen, pyridinium, acridine, ferrocene and phenothi-azine, all which are optionally substituted.

11. A method according to Claim 10, wherein U is a member selected from the group consisting of the following formulae (IV) to (IX):

(IV) wherein n represents an integer between 1 and 16.

(V) (VI) (VII) wherein Y is CO2H or NH2 (VIII)

12. A method according to Claim 5, wherein the electron mediator group forms part of said complex and has the general formula (IX):

U-R4-X3 (IX) wherein U is a member selected from the group consisting of viologen, pyridinium, acridine, ferrocene and phenothiazine, all which are optionally substituted, R4 is a connecting group, and X3 is a functional group, which is capable of forming a covalent bond with a moiety of said at least one component.

13. A method according to Claim 5, wherein said electrode comprises several layers of enzymes, enzymes of one layer linked to enzymes of an adjacent layer by means of bridging groups.

14. A method according to Claim 12, wherein the bridging groups have the following general formula (X):

W1-R6-W2 (X) wherein W1 and W2 may be the same or different from one another and represent a functional group capable of forming a covalent both with the enzyme or with a molecule bound to the enzyme, and R6 represents a connecting group.

15. A method according to Claim 13, wherein R6 is a protein, a polypeptide or a polymer optionally carrying a plurality of functional groups capable of forming a covalent bond with a moiety on said at least one component, or R6 is an optionally substituted alkylene, alkenylene, alkynylene and phenyl containing chains.

16. A method according to Claim 12, wherein the enzyme molecules in the external enzyme layer are non-redox enzymes capable of decomposing agents other than the analyte.

17. An electrode comprising an electrode material of the kind capable of chemisorption of sulphur containing moieties, the electrode carrying a plurality of complexes immobilized on said electrode material each of which complexes comprises a linking group having a sulphur containing moiety and at least one of a redox enzyme or a molecular electron mediator, all components of the complex being covalently bound to one another, the electron mediator being capable of transferring electrons from the electrode material to the redox center of the redox enzyme, whereby in the presence of an analyte the enzyme catalyzed a reaction in which the analyte is converted into a product.

18. An electrode according to Claim 16, comprising linking groups of the following general formula (I):

Z-R1-Q (I) wherein Z represents a sulphur containing moiety;
Q is a group X1 or P; X1 is a functional group which is capable of forming a covalent bond with a moiety of said at least one component; P is a protein, polypeptide or polymer having a plurality of functional groups X2 capable of forming a covalent bond with a moiety of said at least one component;
R1 represents a connecting group.

19. An electrode according to Claim 17, comprising linking groups of the general formula (I), wherein X1 or X2 is an amine group, a carboxyl group -N=C=S, -N=C=O, an acyl group or

20. An electrode according to Claim 18, wherein said acyl group has the formula -Ra-CO-G wherein G is OH, halogen, ORb; Ra and Rb being independently an optionally substituted C1-C12 alkyl, alkenyl, alkynyl or a phenyl containing chain.

21. An electrode according to Claim 17, comprising linking groups of the general formula (I), wherein P is polyamine or a polypeptide with free amine or carboxyl groups.

22. An electrode according to Claim 20, wherein P is polylysine, polyglutamate or polyethyleneimine.

23. An electrode according to Claim 17, wherein R1 represents a covalent bond or represents alkylene, alkenylene, alkynylene and phenyl containing chains.

24. An electrode according to Claim 22, wherein R1 represents a chemical bond or a group having the following formulae (IIa), (IIb) or (III):
(II) (a) (b) (III) wherein R2 or R3 may be the same or different and represent straight or branch alkylene, alkenylene, alkynylene having 1-12 carbon atoms or represent a covalent bond, A and B may be the same or different and represent O or S, Ph is a phenyl which is optionally substituted, e.g. by one or more SO3- or alkyl groups.

25. An electrode according to Claim 17, wherein the electron mediators have a mediator moiety U being a member selected from the group consisting of viologen, pyridinium, acridine, ferrocene and phenothi-azine, all which are optionally substituted.

26. An electrode according to Claim 25, wherein U is a member selected from the group consisting of the following formulae (IV) to (IX):

(IV) wherein n is an integer between 1 and 16.

(V) (VI) _ 40 _ (VII) wherein Y is CO3H or NH2 (VIII)

27. An electrode according to Claim 17, wherein the electron mediator group forms part of said complex and has the general formula (IX):

U-R4-X3 (IX) wherein U is a member selected from the group consisting of viologen, pyridinium, acridine, ferrocene and phenothiazine, all which are optionally substituted, R4 is a connecting group, and X3 is a functional group, which is capable of forming a covalent bond with a moiety of said at least one component.

28. An electrode according to Claim 17, comprising several layers of enzymes, enzymes of one layer being linked to enzymes of an adjacent layer by means of bridging groups.

29. An electrode according to Claim 28, wherein the bridging groups have the following formula (X):

W1-R6-W2 (X) wherein W1 and W2 may be the same or different from one another and represent a functional group capable of forming a covalent both with the enzyme or with a molecule a priori bound to the enzyme, and R6 represents a connecting group.

30. An electrode according to Claim 29, wherein R6 is a protein, a polypeptide or a polymer optionally carrying a plurality of functional groups capable of forming a covalent bond with a moiety on said at least one component, or is an optionally substituted C1-C12 alkylene, alkenylene, alkynylene and phenyl containing chain.

31. An electrode according to Claim 28, wherein enzyme molecules in external layer ate non-redox enzymes capable of decomposing agents other than the analyte.

32. A process for preparing an electrode comprising an electrode material of the kind capable of chemisorption of sulphur containing moieties, having immobilized thereon a plurality of complexes each comprising a linking group having a sulphur containing moiety and at least one of a redox enzyme or an electron mediator, all components of the complex being covalently bound to one another; said process comprising:
(a) immobilizing the linking group into the electrode material by chemisorption of the sulphur containing moiety;
(b) binding the other components of the complex to the chemisorbed linking group.

33. A process according to Claim 32, further comprising:
(a) unfolding the enzyme molecule;
(b) binding electron mediator to the unfolded enzyme molecule;
(c) refolding the enzyme.

34. A process for preparing an electrode comprising an electrode material of the kind capable of chemisorption of sulphur containing moieties, having immobilized thereon a plurality of complexes each comprising a linking group having a sulphur containing moiety and at least one of a redox enzyme or an electron mediator, all components of the complex being covalently bound to one another; said process comprising:
(a) binding the linking group with other components of the complex;
(b) immobilizing the complex onto the electrode material by chemisorption of the sulphur containing moiety of the linking group.

35. A process according to Claim 34, further comprising:
(a) unfolding the enzyme molecule;
(b) binding electron mediator groups to the unfolded enzyme molecule;
(c) refolding the enzyme.

36. An electrobiochemical system for carrying out the method of Claim 1.

37. An electrobiochemical system, comprising an electrode according to Claim 16.