CA2138856A1 - Electrochemical sensors - Google Patents

Abstract

A solid state, multi-use electrochemical sensor having an electrically nonconductive substrate, a working electrode, and a semi-permeable membrane covering the working electrode. The working electrode includes an electrically conductive material adhered to a portion of the substrate. A first portion of the conductive material is covered with an electrically insulating dielectric coating, and a second portion of the conductive material is covered with an active layer. The active layer includes acatalytically active quantity of an enzyme carried by platinized carbon powder particles, which are distributed throughout the active layer. A sensor package for incorporating a sensor is provided.

Description

2 1 3 8 ~
-ELECTROCHEMICAL SENSORS
The present invention relates generally to electrocht mi~1 sensors and, more particularly, to enzyme catalyzed el~i( ~hemical sensors including glucose and lactate sensors. Novel packages incolyol~ling enzyme electrodes having an enzyme contained in an electrically conductive ~ d~e, responding to the catalytic activity of the enzyme in the presence of the substrate are described.

TECHNICAL REVIEW
The c~ncenllalion of glucose and lactate in the blood is extremely important for... ,i~ inil.g hom~ostq~ For e~ yle, a conc~ntration of g1IJ~Ste below the normal range, or hyyoglyc~.llia, can cause u~c~nY O~ f-Q~ and lowered blood yr~ ule~ and may even result in death. A co~ lion of glucose at levels higher than normal, or hyyerglycemia, can result in synthesis of fatty acids and cholesterol, and in ~ qhet coma. The me~s.lr~ment of the conc~ntration of glucose in a person's blood, therefore, has beo~",e a nec~-Qi;ly for ~liq~eties who control the level of blood glucose by insulin therapy.
In a clinical setting, ac~uldte and relatively fast de~l.-.in~;ons of glucose and/or lactate levels can be dct~."lined from blood samples uti1i7ing electroch~mi~1 sensors.
Conv~n~ionq1 sensors are f^l-ric-qtsd to be large, co,-lyl;sing many serviceable parts, or small, planar-type sensors which may be more convenient in many circlJm~t-q-nc~s. The term ~planar" as used herein refers to the well-known p~ ~ of fabricating a ~s~ 11y planar structure compri~ing layers of relatively thin ma~riq1s, for example, using the well-known thick or thin-film techniques. See, for example, Liu et al., U.S.
Patent No. 4,571,292, and P~rud~ki~ et al., U.S. Patent No. 4,536,274, both of which are inco,~l~ted herein by reference.
In the clinical setting, it is a goal to m~ximi7~ the data obtainable from relatively 213~rj6 -small test sample volumes (microliters) during chPmic~l blood analysis. Fabrication of a sensor sample chamber for holding a blood sample in contact with a sensor is desirable in this regard so that many de~....ina~;olls may be sim~ Po~l~ly ~.Ço~ ed on a test sample, for example, using a series of interconne~ted sensors, each constructed to detect 5 a dirr~enl analyte, from a small test sample volume. However, as a sample chamber is made smaller, the conc~n~.dtion of cQrlt~ in~ in a sqmple, as those released from sensor COIn~ tS themselves, especially col-lponellls d~r.,-ing the sample chamber, and/or certain reaction products of the sensor itself is incleased. Such cont~min~tion may result in p,e~llalule sensor failure.
There are two major types of glucose or lactate electrode sensors. The first is an elecLI~cdtalytic device which utilizes direct oxidation of glucose or lactate for obtaining a measurable rP~ron~P The second is . n enzyme electrode which utilizes an enzyme to convert glucose or lactate to an elec~.oac~i~/e product which is then analyzed ele,lr~xhf.mi.~lly.
With respect to glucose sensors, the latter type of electrode sensors, inclutling an enzyme electrode, converts g1lJCOSe in the presence of ~.~y",es, such as glucose oxidase, and results in the formation of reaction products incl~ ing hydrogen peroxide according to the following reactions:

C6HI2O6 + 2 + H2O [oxidase] C6HI2O7 + H2O2 >

H22 > 2 + 2H+ + 2e~

In these rca^tiQns glllcose reacts with oxygen to form gl~)~QIlol~^tone and hyd~og~n peroxide. A suitable electrode can then measure the formation of hydrogen peroxide, as an el~tlic~l signal. The signal is produced following the transfer of electrons from the peroxide to the electrode, and under suitable conditions the enzyme 30 catalyzed flow of current is plopollional to the glucose conc~ntr~tion~ Lactate electrode sensors including an enzyme electrode, similarly convert lactate in the presence of enzymes, such as lactate oxidase.
Numerous devices for dt;lel nlination of glucose and lactate have been described, ~1388~

however most of them have some limi~tion with respect to reproducibility, speed o r~onse, test same volume, number of effective uses, and the range of detection. Some ting comn~ rcial m~tho~ls rely on utili7~tiQn of hydrogen peroxide measurement as outlined above.
S With respect to glucose sensors, in known enzyme electrodes, glucose and oxygen from blood, as well as some int~.re~ , such as ascorbic acid and uric acid diffuse through a primary membrane of the sensor. As the glucose, oxygen and inlcl r~d,.ts reach a second membrane, an enzyme, such as glucose oxidase, catalyzes the conversion of glucose to hydrogen peroxide and glllconol~ ~t~ne The hydrogen peroxide may diffuse back through the primary membrane, or it may further diffuse through the s~4nd~ry l..e.n~.~nc to an electrode where it can be reacted to form oxygen and a proton to produce a current l~lupollional to the glucose con~ntration. The electrode's .,.e..,bl~ne asselnbly serves several fi)nction~ including selectively allowing the passage of glucose th~ldhlo~lgh, providing a loc~tion bGlweell the primary and ~4n-1~ry 1S IllGlnbldrlCS for an enzyme to catalyæ the reaction b~;n the glucose and oxygen passing llll~l~gh the pli~ Illelllblane, and allowing only hydr~gen peroxide ll-r~u~h the s~4n-~q~y -,G",b,~ne to the electrode.
A single-layered Glectrode mc.-,b,~ne was describe~l by Jones in EP Patent No.
207 370 Bl. This reference is directed to an electrochemi~l sensor inclu~1ing three 20 primary co ~l~nf.-lc a metal electrode, a reactive layer of immobilized enzyme directly on an anode, and a single-layered --e..,b,~ne. The -,e"-b,~dne ~1iselosed in EP 207,370 Bl, is glucose p~.-,le~ble and whole blood colll~ ible, thereby e~ n~t;ng the need for the s~4n~1~ y ...e...l"~ne typical in prior art sensors. The ,-,e",b~ e is formed from a n of a poiy- .~ '^ silicon~4n~;~;ning co..1~und applied in an inco...~letely cured form, having a liquid carrier which is ess~ lly insoluble in the dispersed phase and removable from the dispersion during curing. The membrane cures as a continuous layer, film or Illelllbldne, having high glucose permeability.
It has been found, however, that the single membrane layer ~ ose~ in EP
207,370 Bl prevents only anionic in~.î~ling subst~n~s, such as ascorbic acid and uric acid, from passing thercthloLlgh. Neutral species, such as ~ t~minophen, can diffuse through the membrane and influence the sensor's sensitivity and accuracy.

~1388S~

As noted above, enzyme electrodes convert glucose into hydrogen peroxide, which can be reacted to produce a current pl.)~llional to the glucose concentration 1~LY~IIC electrodes adapted to ",ea~ule other analytes have also been described in the art.
An enzyme electrode having an electrically co~ductive support member which consists 5 of, or comprises, a porous layer of resin-bonded carbon or g.~l hil~ particles is .l~ s~d by Renne~to et al., in U.S. Patent No. 4,970,145. The carbon or gla~ le particles have a finely divided plalil,ulll group metal intim~tçly mixed therewith, to form a porous, sub~ t;~lly holl,ogeneous, substrate layer into which the enzyme is adsorbed or immobilized. The pl~ f~ d s~ o.te materials are resin bonded, plqtini7Pd carbon paper electrodes, comprising p!~t;l-i7~d carbon powder particles bonded onto a carbon paper s~sll~te using a synthetic resin, pl~;fe~bly polytetrafluoroethylene, as the binder. These electrode mqtPriql~ are manufactured by depositing colloidal size particles of platinum, palladium, or other plat m~ln group metal, onto finely dhided particles of carbon or gl~phile, blending the p1~1;ni~ or ~llq~i7~ carbon or graphite particles with a 15 fluorocarbon resin, pl~f~ ~bly polyl~ Qroethylene, and applying the mixture onto an ectrir-q-lly co~luc~ e support, such as carbon paper, or a filq.~Pnto~c carbon fiber web.
The above-le~el~nced enzyme electrodes require premolAing of the graphite or carbon base often under conditions l~uiling sint~ing of the molded compact to fuse the binder, which, as noted, is a high melting point hydrophobic synthetic resin. These high 20 t~lllpel~lulcs would destroy enzymes, such as glucose oxidase or lactate o~ q~e.
Enzyme electrodes comprising an en_yme or Illixlule of enzymes immobili_ed or adsc,ll~d onto a porous layer of resin bonded ~ ;n;,~ or pqll~ ~i7~ carbon or gl~l~ite particles without a high t~ tll~ binder have been ~i~los~d by M--llçn, in U.S.
Patent No. 5,160,418. Mullen disclosed that the high t~!llpe.~ binders can either be 25 di~ns~d with entirely or replaced by a low t~m~dl~lre, p-ere dbly water soluble or water dispersible binder, such as gelatin (a binder which can be activated at room peldlu~ which does not require high tem~dtule sintering).
Despite the above improvements in the art, however, a need remains for accurate,multi-use glucose and lactate sensors, incol~ldting a glucose or lactate and 30 oxygen-permeable membrane and an en_yme electrode. In addition, there is a need for an electrochemical sensor package which can be used with a small blood sample and for 21388S~

extended sampling or uses in a clinical setting. An electrochemical sensor of this type that does not require .-.~i .t~ nce, i.e. reme,-,t"~ning, electrode cle~nin~ etc. would also be desired.
It is therefore an object of the present invention to provide an improved S ele~ ~h~m~ sensor, and method of making the same, generally incorporating en_yme ele~llodes having a m~t~lli7~d carbon base and an overlying silicon-co-l~;ning plot~~ e glucose and/or lactate permeable membrane.
It is a further object of the invention to provide an elecllocllemical sensor inco~ ting an inl~lr~lence correcting electrode onto the sensor to provide efficiency over eYtPn~ed samp!ing periods.
It is a further object of the present invention to provide an improved sensor package, which can be used with a series of int~r~o~ne~tçd sensors, including a small sample çh~mber.
It is a still further object of this invention to provide an improvement in a planar ~l~t,ode comprising using a met~lli7~d carbon active layer over a metal contact, and having an outer me,l,l),~nc which enables rapid testing of ~mplçsJ including blood, to del~ ,ine glucose and/or lactate concentrations.
It is a still further object of this invention to provide a planar sensor having a small sample chamber which incol~l~s a velocity CG"~nsator to allow fluid flow without incurring problems of incuffirient wash-out, i.e. sample carTyover, or velocity mo lifi~tiQn in the ch~mber, thus enabling fast filling and emptying of the cha,.,b~r and inc~ing sample throughput.
It is a still further object of this invention to provide means and method for ~ching small resilient el~ct i~l leads to a plurality of cont~ctc in an el~tric~l sensor with positive predete~",ined positioning rapidly and efficiently and with high precision and accuracy.
It is still a further object of the invention to provide a method for post-treating sensors to prolong the storage life or wet-up of the sensor.
It is still a further object of the invention to provide multi-use glucose and lactate sensors having a long life.
It is still a further object of the invention to provide a method of formulating an Z1~88~6 enzyme into a paste for use in an electrode.
It is still a further object of the invention to provide a method of for nlllqting cellulose acetate into a paste for use in an electrode.
Accordingly, the present invention provides a solid state, planar ele~;l,~he...ic~ql S sensor inclu~ling an elPctrir-q-l1y nonconductive substrate, a working electrode, and a semi-perrneable ,.,c;".bndne covering the working electrode, which permits glucose and oxygen or lactate and oxygen to pass through to the electrode. The working electrode includes an electrically conductive material adhered to a portion of the SllbSlldle. A first portion of the cor-ductive material is covered with an elPctric-q-lly in~ulqtin~ ~iPl~pctric 10 coating, and a second portion of the con~uctive material is covered with an active layer.
The active layer includes a catalytically active quantity of an enzyme, such as glucose oxidase or lactate o~ q~e~ carried by phq~ini7ed carbon powder particles, which are distributed throughout the active layer.
The sensor may further include a counter electrode having a second elPctri~qlly 15 conductive mqteriql adhered to a second portion of the nonconductive substrate. A
portion of the second conductive material is covered with the el~ctric-qlly in~l~lqting dielectric coating, and at least one portion of the second conductive mqteriql r~"~aills uncovered.
In one embodiment of the present invention, the nonc~onductive substrate is made20 from alumina ^~mised with a glass binder, and the conductive mqtPriql~ are thick-film pastes of either silver, gold, or pl-qtinum. The ~iPlpctric coating is made from either ceramics, glasses, polymers, or co",bin~ionc thereof. The semi-permeable membrane can be formed from ~p~ lose qv~ Pt-q~, polyur~ ane, ~ilicQne co",pounds, and other mqt~riql~ known in the art such as Nafion~ mq~teriql available from E.I. DuPont de 25 Nemours & Co., Wilmington, DE. The l.ref~.led "~e",bl~ne is a dispersion of apolymPri7^ble silicon-co-;t;~ining compound applied in an incompletely cured form of a silicone co"~pound dispersed phase in a liquid carrier. The semi-permeable membrane includes a silicone compound having at least about 10.0 percent colloidal silica, by weight. The ~,efel,ed membrane includes at least about 14.0 percent colloidal silica, by 30 weight.
In another embodiment of the present invention, the electrochemical sensor may ~13~

further include a reference electrode including a third elPctric~lly condl~ctive silver ~ l adhered to a third portion of the ~ub~t,dte. A first portion of the third conductive material is covered with the electricaUy in~ ting ~lipl~pctric coating, and a second portion of the third conductive material r~;.l.ains uncovered by the electrically 5 in~ul~ting di~ ctric coating. The third electricaUy conductive m~tPri~l typically includes a silver/silver chloride thick-film paste. The second portion of the c4nd~1ctive m~ri~l is covered by celll)lose acetate.
In still another embodiment of the present invention, the electrochemical sensormay further include an int~lre~nce c~ ing electrode including a fourth ~lectric~lly conductive m~tPri~l adhered to a fourth portion of the substrate. A first portion of the c4nd~lctive mqtPri~l is covered with the electrically in~ul~ting dielectric coating, and a second portion of the c4n-1uctive material is covered with an inactive layer. The inactive layer inc~ d~ps an inactive protein immobili7~d onto p!~;ni~d carbon powder particles, which are distributed ~ fi~;~lly unirol-,-ly throughout the inactive layer.
In another embo~limpnt of the present invention, the semi-permeable membrane is post-treated with a high boiling point, water soluable, hydrophilic liquid anti-drying agent.
In a further aspect of the present invention, an electrochP-mic~l sensor packageis provided. The package includ~Ps a housing having a recess with a pprim~otp-r and at least one passageway col nP~l~d to the recess. A gasket c4rlt~cts the recess perim-pter and a solid state, planar electroch~Pmil~l sensor, as described above, and forms a seal the~t~. The housin~ and electrochp-mi~l sensor define a sample ch~mber.
In yet another embodiment of the sensor pa~ge of the present invention, the package further inrludes a contact lead frame. The lead frarne includes ieads secured to the frame at a first end, and a recess for the sensor at the op~sile end. The contact lead frame may further include a stabilizer bar for ~ligning the leads with contact pads on the surface of the sensor. A groove may also be provided in the package for receipt of the stabili_er bar as the leads are wlapped over the top portion of the lead frarne to be aligned with the sensor contact pads. A pad, or the like, may also be provided in the package of the present invention for ~uppol~ing the sensor in the recess of the contact lead frame.

- ~13~8~i~

In another en.bodi,.,ent of the present invention, the sensor package includes avelocity co-np~Q~Ior or bump within the sample chamber. The velocity comren~tor can be a molded part of the housing and ",er~ldbly faces the sensor.
The method of fo~,lling a solid state, planar electroc-hP-mi~l sensor inC1udps 5 sPlPcting a suitable ~s( dle m~tPri~l made from el~ctric~lly noncollductive m~Pri~l, and forming it into a desired shape and size. An elPctric~l1y conductive m~tPri~l is then de~sil~d onto a portion of the s~s~ldte. Next, a portion of the conduGtive m~t~ri~l is covered with an electrically incul~ting diPlpGtric coating, and a portion of the c~ductive material is uncovered so as to define an electrode area. A working electrode is then 10 formed on the electrode area which includes an active layer comprising a catalytically active ~ tily of an enzyme, such as glucose oxidase or lactate oxidase, immobiliæd onto pl~ini,~ carbon powder particles, which are distributed ~ul~s~ lly uniformly throughout the active layer. L;astly, a semi-permeable membrane covers the working electrode, which permits glucose and oxygen or lactate and oxygen to pass through to the 15 electrode.
A p.~f~.lod solid state, planar ele~t~l.G.ni(~l sensor of the present invention is formed by s~Pctinp a suitably sized and shaped sub~ .te made of an elPctri~lly nonc~rlductive m~tP~i~l, such as a ceramic m~teri~l compricin~ alumina and a glass binder. Four conductive strips are deposited on top of the substrate so as to extend from 20 a first end to a second end thereof. At the first end, the conductive strips define contact pads for electrical c~nll~o~;on, and at the oppo~ile end of the sul,~ e the strips define an electrode area for test sample eA~ lle. The conductive strips may be depositPd using thin or thick-film silk-sc~ g techniques using conductive metal pastes of either silver, gold, and/or r~-;n~..n An e~tri~ ~lly insul~ting tliP1~tric coating is ~imil~rly de~c;~Æ~
on top of portions of the conductive strips, while leaving portions of the strips uncovered to define the reference electrode, cc,unler electrode, working electrode, in~.Ç~c,ence collectii g electrode, and contact pads. The reference electrode is formed by depositing a layer of silver/silver chloride onto the exposed electrode region. A cellulose acetate layer is then applied over the silver/silver chloride reference electrode to protect the silver chloride from cont~ in~nts that would shift the reference potential.
A working electrode is formed by depositing an active layer, comprising a catalytically active quantity of an enzyme immobili~d onto to pl~tini7~d carbon powder particles, upon a co~dlJctive strip using similar screen printing techniques. Anintclr~lcnce collc;ling electrode is formed in a .,.anne, similar to the working electrode.
The intclrer~nce collccling electrode, however, includes an inactive layer, compri~ing S an inactive protein in place of the catalytically active quantit,v of an enzyme immobili~d onto pl~tini7~d carbon particles. The inte,r~,ence correcting electrode serves to adjust for ele~ rmi~lly active neutral species which may diffuse through a semi-permeable cover me..-bl~ne, which is preferably spun-cast over the electrodes.
In an improvement provided by the present invention, a planar electrode for use in glucose and/or lactate del~.. hlations, in vitro, has an in~ul~ting base layer, a conductive layer, an overlying active layer and an outer plolccli~le membrane permeable to glucose and/or lactate. The improvement of the invention incllldes the active layer having an enzyme reactive with one of glucose or lactate, and a pl~t;.-;,Pd carbon powder particle portion,- so that the active layer is capable of c~using formation of hydrogen peroxide in arnounts ~)ro~ollional to the amount of the glucose or lactate being tested when they are e ,~l)osed to the active layer, and the outer ~lu~;li~/e membrane which is a silicone co---~-md havi~g an additive incol~ ted therein for enabling transport of glucose or lactate thclcth~ gh to enable rapid and accurate de~l---inations of glucose or lactate.
In another improvement of this invention, a multi-use electrochemical sensor is provided having a long life of effective use without m~inten~nce In an~ . r impro~e...e.lt of this invention, in a planar sensor having a plurality of electrodes positionPd in a sample ch~mber with the sample chamber having a flow-ll.lougll path, an inlet and an outlet, each having a cross~ tion~l arca less than the 25 cross-scctional area of a portion of the chamber, a velocity co~ cnc~or is provided. The velocity co-,l~nsa~or is a structural barrier mounted in the flow path between the inlet and outlet to reduce the cross-sectional area of the chamber in the flow path so as to prevent co,-~ llt!i from collecting, and to ~ubsti~lially ~ in stability in fluid velocity when flowing through the chamber. The velocity co-npensator is prcrcldbly 30 integral with the sample chamber and extends towards the electrodes without obstructing fluid flow over the electrodes.

In still another improvement in an electrochemical sensor mounted in a housing and having a plurality of electrical contacts spaced close to each other and a plurality of e1ol-g~d axially e~t~nding e1ec~n~1 leads conn~l~ to the cont~t~, the leads are spaced apart by a stabilizer bar. The stabilizer bar is ~tt~h~d to the leads and positively positions the leads to establish electrical ç~nt~ct The leads are resilient and urged into contact by the stabilizer bar which is p,efe,dbly mounted on a lead frame base.
Other reatul- s of the present invention will become appar~nl from the followed detailed descli~lion when taken in conne~tion with the acco,l~panying drawings. It is to be understood that the drawings are desi~ned for the purposes of illustration only and are not intended as a definition of the limits of the invention.

BRIEF DESCRIPIION OF THE DRAWINGS
The above and other features, objects and advantages of the present invention will be better unde~lood from the following spe~ifi~tion when read in conjunction with the lS acco",~nying drawings, in which:
FIG. 1 is a ~ls~ /e view of an ele~t oche ~ 1 sensor package of the present invention;
FIG. 2 is an exploded view of the co"~ponents of the sensor package shown in FIG. l;
FIG. 3 is a cross-sectional side view of a contact lead frarne shown in FIG. 2, with its leads partially open, taken along section line 3-3;
FIG. 4 is a cross-s~tion~l side view of the contact lead frame shown in FIG. 3, with its leads wide open;
FIG. S is a rn~gnified partial view of a sample ch~l~b~, of the sensor package shown in FIG. 2;
FIG SA is a graphical illustration of the cross-sectional area of the sample chamber shown in FIG. S versus the position along the ch~ml~e flow path, with and without a velocity compensator (bump);
FIG. SB is a graphical illustration of the ratio of sensing area to flow path cross-sectional area of the sample rh~mb~r shown in FIG. S versus the position along the chamber flow path, with and without the velocity compensator;

-FIG. 6 is a cross-s~tion~l side view of the sample chamber shown in FIG.S, taken along section line 6-6;
FIG. 6A is a cross-sectional side view of the sample chamber shown in FIG.S
taken along section line 6-6, showing the velocity of fluid flow with the velocity 5 col æ-fi~ tnr (velocity: white > black);
FIG. 6B is a cross-s~ti~n~l side view of the sample ch~mber shown in FIG.S
taken along section line 6-6, showing the velocity of fluid flow without the velocity comren~tQr (velocity: white > black).
FIG.7is a cross-sectional side view of the sample chamber shown in FIG. 6, 10taken along section line 7-7;
FIG.8is a cross-section~l side view of the sample chamber shown in FIG.S, taken along section line 8-8;
FIG.9A is a m~nifi~ top plan view of a sensor used in the sensor pac~ge shown in PIG.l;
15FIG.9B is a m~gnifi~ top plan view of another embodiment of a sensor used in the sensor package shown in FIG.l;
FIG.lOis a cross-s~tion~l side view of a working electrode used in the sensor shown in FIG.9A, taken along section line 10-10;
FIG.llis a cross-se~tion~l side view of a reference electrode used in the sensor20shown in FIG.9A, taken along section line 11-11;
FIG. 12 is a gr~rhic~ tr~ion of a g1ucose sensor l~ponsc to glucose co~ .alion in whole blood ~mpl~s acc~lding to one embo~im~nt of the present invention;
FIG. 13 is a graphical illustration of a lactate sensor les~onse to lactate 25conc~-l.~ion in whole blood s~mrl~s according to one embodiment of the presentinvention;
FIG. 14 is a g~rhi~l illustration of the effect of an in~lrelence coll~;ting electrode acco~ing to one embo~iment of the present invention, as glucose conc~ntrations are measured with and without the correcting electrode applied;
30FIG.lSis a graphical illustration of a glucose sensor output over an extended period of time and sample use;

, FIG. 16 is a graphical illustration of gllJcose sensors response to glucose concf..t.~on, with and without a surfactant post-tre~ment FIG. 17 is a graphical illustration of glucose sensors response to glucose conn~ntration, with a variety of surfactant post-tre~tm-ont~;
S FIG. 18 is a graphical illustration of ~1ucose sensors response to glucose conc~n.l.~;on, after one week in storage at room le",~l~tulc, with and without asurfactant post-tre~tm~nt;
FIG. 19 is a graphical illustration of glucosc sensors response to glucose conc~ntration, and the effect of membrane thic~n~-~s on the line~ily of the response;
FIG. 20 is a graphical illustration of glucose sensors l~onse to glucose concf nl-ation, and a comparison between 2-layer and 4-layer spin~ast membranes on the linearity of the response;
FIG. 21 is a graphical illustration of gll~cos~q sensors response to glucose con~ ;on, and a cG~I~ison between 2-layer spin~ast and s~n~il~ membranes on the linearity of the response;
FIG. æ is a graphical illustration of glucose sensors r~ponse to glucose con~..l.alion, and the effect of storage over an ~Ytçnde~ period of time on the respon~e when no surfactant is added to the pl~tini7~d activated carbon of the active and inactive layers of the sensor electrodes;
FIG. 23 is a graphical illustration of glucQse sensors response to glucose conc~ntration, and the effect of storage over an e~t~nd~ period of time on the r~ponse when surfactant is added to the pl~tini7~d activated carbon of the active and inactive layers of the sensor cle~ odes;
FIG. 24 is a gr~phi~l illu~tr~tion of glucose sensors l~ on~ to glucose conc~ ation~ and the effect of adding a surfactant m~ri~l to the membrane m~te~
covering the sensor electrodes; and FIG. 25 is a view of the steps of formation of a glucose sensor.

DETAILED DESCRIPTION OF THE INVENTION
Referring now to the drawings, in which like reference numerals de~ign~te like or colles~nding parts lhloughoul the several views, an assembled electrochemical sensor package 10 in accor~ance with a ~)r~Çe,.~d embo~1iment of the present invention is sll~d~ed in FIG. 1. Package 10 has a generally J-shaped body, inclu~in~ a handleportion 12, a main body 14, contact portion 16, and fluid or liquid passageway 18.
The internal co",;~onen~s of sensor package 10 are shown in an exploded view in FIG. 2. Package 10 includes a J-shaped housing 20 having a recess 22 formed therein.
The recess 22 which forms a part of a sample ch-qmbe 54 (FIG. 5) incllldes an outer peli",~ t~r 24 and at least one pq~qgeway 18. Housing 20 has a sul~st;~ lly flat upper portion 120, sidewalls 122, 124, a frontal opening 126, and a rear wall 128 which is contiguous with sensor package handle portion 12. Housing 20 further includes a d~l~ ;,s~d inner rim 130 and projections 132 which contact the lead frame 32 when package 10 is assembled. A gasket 26 is provided to contact, and form a seal between, the housing perimeter 24 and a sensor 28. Gasket 26 is substqntiqlly rectangular-shaped and includes a s~lbs~ lly oval-shaped opening 134, and two raised surfaces 136, 138 which run along the length of gasket opening 134. Gasket raised portions 136, 138 allow gasket 26 to fit around the housing recess penmeter 24, while also allowing recess 22 to be exposed to sensor 28. A sensor pad 30 is provided to s.l~l)oll sensor 28. Sensor pad 30 incl-ldes a series of transverse prullus;ons 140 on rear side 142 which provide sensor 28 with added support when l,ac~ge 10 is assembled. Lastly, a contact lead frame 32 is provided to el~tri~qlly colmæ~ sensor 28 to an instrument (not shown) which can 20 .leasur~ and convert a current to d~ ,ine analyte cone~ntrqtiQn, ~, glucose or lactate.
Contact lead frame 32 inr,l~ 5 four leads 34 secured to a base 36 at a first end portion 38, and a sensor recess 40 at a second end portion 42. The lead frame 32 can also include a stabilizer bar 44 for hol~ing the leads in a predelelmined position with respect to each other and q~ nin~ the leads 34 with the sensor 28. An ~-litiotlql recess 46 can be included for receipt of stqbili7Pr bar 44. It is also noted that an electrode O-ring 48, colllnle.cially available from Ciba Cornin~ Di~nostics Corp. or the like, can be provided ta -,aintain a seal b~n ;v~jq~nt sensor packages, or a fluid conduit (not shown), each dirÇel~ sensor used to ~imul~neously detect dirrelent analytes from the same fluid sample.
Referring now to FIGS. 3 and 4, a cross-sectional side view of contact lead frame 32 taken along section line 3-3, is shown with its leads partially and wide open. As noted above, to provide an interface system between an instrument and contacts on the sensor 28 (~sr . ;hed below), contact lead frame 32, including plural leads 34 secured to base 36 at end portion 38, is provided. Leads 34 are typically made from a highly c~llductive, malleable metal, such as copper. Preferably, leads 34 are made of a highly conductive, malleable material, which is beryllium, silver, gold, or platinum plated, due to the lower cost of plated material. Most preferably, photo-etched, gold plated leads 34 are used due to their high conductivity. The leads 34 are molded into one end 38 of the contact lead frame 32. Lead frame 32 can be formed of any material that is co"lpatible with, and can be secured to, the sensor package housing 20. Typically, lead frame 32 is made from a rigid, durable m~tPn~l such as glass, ceramic, st~inl~cc steel, or a plastic material such as acrylic, polyester, polycarbonate, polyvinyl chloride, and the like.
~efelably, an acrylic plastic material, such as V825 acrylic, available from Rohm &
Haas Corp., Phil~delphia, PA, is used to mold lead frame 32 due to its strength,durability, relatively low cost and ease of pl~c~c~ g.
During assembly of the sensor package 10, sensor 28 is placed into recess 40 andleads 34 are bent around the lead frame 32 until they make contact with the sensor.
Leads 34 contact the sensor with rounded spring tips 50 which apply co~t~nt pr~
on the sensor contacts. Stabilizer bar 44, which aligns the leads 34 with the sensor cont~tc, is secured in recess 46 after the leads are bent around the frame.
~felably, the stabilizer bar, if present, is solvent c~-nlent~ in place in recess 46. In;,llu-l-enl contact surfaces 52, which are exposed after the sensor package 10 is assembled, are formed as the leads are bent over the frame (as shown in FIG. 3). Once the leads are in contact with the sensor, hollcitl~ 20 is placed over the lead frame 32.
The housing and lead frame are then secured together by being snap-fit, ult~con~ y welded, adhesive bonded, or by other me~ho~ls known to those skilled in the art.Rerelling now to FIGS. 5-8, a m~gnifi~d view of a sample chamber 54 of the sensor package 10 is shown. As noted above, sample charnber 54 is defined by thehousing 20, the outer perimeter 24 around recess æ, gasket 26, and the sensor 28. At least one passageway 18, having an inlet 56 and an outlet 58, is provided to allow passage of a fluid sample, such as blood, into and out of sample chamber 54. Although, in the embodiment illustrated, inlet 56 and outlet 58 pass through housing 20, these openings can be formed in any manner to provide a passageway through which a fluid sarnple could reach sample chamber 54. For exarnple, opçning~, or c-h~nn~ could be formed in the gasket 26, or other part(s) of the sensor package 10.
The sample chamber 54 of the present sensor package 10 also includes a velocity S co...l~r.~-tor 60 (Bump), which reduces the intern~l volume of the ch~mhPr and creates a cross s~l;o~-~l area close to that of the inlet 56 and outlet 58. FIG. SA graphically ilîustrates the sample rl-~...her 54 cross-sectional area along the ch~mber flow path, with and without velocity co.--l~n~tor 60. As shown in the graph, the cross-sectional area of the sample çh~mher at the velocity cG.~l)en~lor approaches that of the inlet and outlet.
10 The velocity co",i~nsator or bump acts as a structural director of fluid flow.
Conventional sample delivery systems experience problems such as carryover of previous sample m~trri~l~, and trapped air bubbles which are present or within the leading edge of the sample fluid to address a problem common to convention~l sample delivery system. Typically, as a sample enters the ch~mhor 54 its flow velocity abruptly slows 15 until the c~ ber is full. The sample velocity then increases to its initial level, leaving the solution at the cl~-s"~bel walls ~:~n~llt Although sample rh~mh~ers are washed belween lll~ulr-~llr-nt~ air bubbles and fluid can become trapped in the ch~mher in st~n~nt areas. These air bubbles and residual fluid effect the ~rCur~cy of the sample meas..r~."~ nt. Thevelocity co...pen~tor 60 of the present invention, therefore, keeps the flow velocity stable within the ch~mhPr, and reduces or el~ n~tes the sta~nt areas where bubbles and fluid can collect. Referring to FIGS. 6A and 6B, the velocity of fluid flow lhlougl~ the sample Cl~5~bÇr iS ;~b.s~''''t;~lly unifollll in the presence of the velocity co",p~r,~lor. In ^~lflition, velocity co",~ or 60 allows the use of a large sensing area with relatively small inlet 56 and outlet 58 cross-sections. FIG. 5B graphically ill~ es the ratio of sensing area to flow path cross-s~c~ior~l area along the ch~mher flow path, with and without velocity co.--i~-~tor 60. Rec~use the cross-sectional area of the chamber 54 is reduced (as shown in FIG. SA) with the velocity cG",pensator 60 in place, the ratio of sensing area to flow path is inc~ased. This aspect of the present invention allows fluid samples to more efficiently contact sensor 28 as they are passed through package 10. Moreover, by positioning the velocity compensator 60 facing the sensor 28, samples are directed toward the sensor 28 while bubbles are substantially çlimin~t~.

213885~

FIG. 6 illustrates a cross-sectionql side view of the sample ch-q-mher 54, takenalong section line 6-6. The velocity co...l~tor 60 is shown as a molded part of }h~u;~ing 20. Although shown having a rounded shape, a variety of smooth, slopedshapes, without sta~rl-q-nt areas, can be used. Purthermore"qlthough shown as a molded 5 part of the holJ~ing, velocity co...~ or 60 can be a se~ dte component, added to sample chq-mher 54.
Inlet 56 and outlet 58 portions are shown leading in and out of chqmh~r 54.
These sample paths typically have 11is~ t~l~ bcl~n about 0.02 inch and about 0.04 inch; ~,lcÇ~dbly, the ~ qm~t~rs are about 0.03 inch. The sample chqmher 54 has asample cli-q-met~r, with the velocity co.. ~n~or 60, of at least the size of the sample paths to about 0.06 inch. FIG. 8 shows a side view of sensor package 10, taken along section line 8-8, through passageway 18. This view illustrates the relative sizes of the velocity com~nQqtor 60, sample ch-q-mber 54, and passageway 18.
Housing 20, as well as inlet 56 and outlet 58, and velocity co-,-pensator 60, can 15 be fabricated from any mqtoriql that is ~ e with a sample which passes into sample chqmber 54 during analysis. For eYqmpl~ m-q-tPriqls such as glass, ceramics, stqinl steel, or plastic materials such as acrylic, polyester, polycarbonate, polyvinyl rhl~nlle, and the like.
P~fc~ Lbly, a clear, transparent acrylic plastic m,q~t~ri-q-l, such as V825 acrylic 20 from Rohm & Haas, is used to mold these parts due to its strength, durability, relatively low cost and ease of proc~ ing Gasloet 26, shown in FIGS. 6 and 7, is ly~ c~lly formed from a m~tPri~l which, when held f~rmly b~n recess ~. ;...- t~ 24 and sensor 28, forms a seal around sample ch~..l~r 54 ll~ugh which the pa~sage of fluids is subst~nti~lly prevented. ~
~ypically, gasket 26 is formulated from a durable organic polymer which does not creep or flow when s~l~c,s~l, has a low durometer rating, and can be slightly hygr~scopic. Pl~fel~bly, a m~t~ l used in the fabrication of gasket 26 has a hardness of belwæll 10 and 100 on the Shore A scale; more preferably, a hardness of from about 40 to about 70 on the Shore A scale; and most preferably, a hardness of from about 45 to about 55 on the Shore A scale.
Re~ll~e gasket 26 is typically an organic polymer, it is fabricated so as not to ~138856 contain a ~lb~ l amount of any mobile extractable materials, such as pl~ctiri7P~rs~
which may leach into sensor 28. Addition~lly, as is the case for other sensor components as described above, it is i",po,l~nt the material sel~P~t~ for formation of gasket 26 be free of any species which could migrate into a sample in ch~m~r 54, affecting S ele~,ocl-~l--ir~ bur~llentc~ andtor destroying sensor cG",ponents. M~tPri~l used in the formation of gasket 26 is preferably sol~ctçd to be e-C~Pnti~lly free of mobile transition and main group metals, especially battery metals such as iron, cobalt, nickel, lead, copper, extractables, and species such as sulfides which are ~lot~rious to ~,leîell~d electrode m~t.~ri~lc, Gasket 26 is typically formed form a highly cross-linked ela~tul.. eric co,-,~oui d.
Any elastomeric m~ori~l which meets all the purity and physical la~uir~lllents listed above may serve. Most prefe.dbly, Sarlink~ 2450 elastomeric m~t~.ri~l from DSM
having a har~ness of about 50 on the Shore A scale is used to form gasket 26.
Sensor pad 30, also shown in FIGS. 6 and 7, can be formed of a m~t~.ri~l similar15 to that used to form gasket 26. Pad 30 is formed of a durable organic polymer which does not creep or flow when sll~ssed, and has a low dulu~ t~.. Preferably, a rn~tPri~l used to form pad 30 has a har~ness of between 40 and 60 on the Shore A scale. Most preferably, a silicone rubber or m~t~ori~l such as Sarlink 2450 is used to form pad 30.
According to the present invention, a sample ch~mlxr 54 of any size can be 20 fabricated. Fabric~tion of a large sample chamber may be advantageous in somecileu...~nc~s. As noted above, however, in the field of electrochPmi~l analysis of blood, it is cornnlonly desirable to pc.ro.", as many analyte analyses as possible on a very small volume of blood. Thus, acco~h~g to a p~ef~.led ~"bodiment of the present invention, it is d~i,db~e to fabricate sensor 28 with a sample ch~ f r 54 that is as small 25 as possible. Using the novel m~t~.ri~l~ and m~ho~1c of the present invention a sensor may effectively be utilized for a period of at least thirty (30) days, or the measurement of at least one thousand (1,000) blood samples having a sample charnber with a volume of less than about 10.0 ~1 (microlil~l~); and preferably, from about 3.0 to about 5.0 ~1.
Refelling now to FIGS. 9A and 9B through 11, 25, and Table I, a planar 30 ele~;l,ùchemical sensor 28 in accûrdance with a prefell~d embodiment of the present invention is shown. FIG. 9A also shows phantom outlines of sample chamber 54, inlet i`2~138856 ' ' -19-56 and outlet 58. These ~atures are shown to illustrate the relative position of electrodes 86, 88, 90, and 92 described below to the flow path of a sample to be tested. Sensor 28 includes S~ t;~lly planar ;,.llsll~t~ 62, conductive metal strips 64, 66, 68, and 70 deposil~d thereupon, and dielectric layer 72 deposited on substrate 62 so as to cover portions of col-ductive strips 64, 66, 68, and 70, while leaving portions of some of the strips uncovered-.
Sul,sl-dte 62 is formed from any s~lbsl~h~;qlly electrically in~ulqting material such as ceramic, glass, refractory, polymers or colllbinalions thereof. Formation of such an inS l1qtin~ late as a mPchqnicql support or base is common knowledge to those ofol~indl~ skill in the art. In the ~l~ rt;lled embodiment, the substrate comprises approximately 969~ alumina and apprv~ y 4% glass binder. A suitable mq~teri~l comprising the pr~fe.led composition is available from Coors Ceramic Company, Grand Junction, CO. Although in the pleÇ~led emb~imP,nt~ of the present invention a single s.ll,stlate forms the follndqtion of sensor 28, a plurality of ~ bslldtes can also be used, each s.~ppolling ~te sensor co,~l on~ and/or helping to support sensor col"poner,t~
s.~ppol~d by other s~hdt~s.
Cor.ducti~e strips 64, 66, 68 and 70 are depo~ d atop ~ t~ 62 so as to extend from a first end 74 to a second end 76 thereof in a p-ef~l.ed emb~imPn~ At first end 74, the co~ductive strips are typically deposiled so as to be wide enough to define contact pads 78, 80, 82, and 84, ~spe~ ely. At second end 76, the co~ductive strips are typically d~ ;led so as to be somewhat narrower, exposed regions of which may define electrodes, as de5c~ below.
~ofi-J.u;~ e strips 64, 66, 68 and 70 may be depos;l~d using well known thin or thick-film techniques. Typically, a co,..polJ.-d inclll-ling a metal is applied via typical thick-film scl~.-ing to sul,~lndte 62, and the applied c~ lpou-~d and substrate are the fired to sinter the active metal and to co-adhere the active metal to the :~llbSlldte. The dc~;~,odc~i~e metal may comprise any conductive metal, for exarnple, silver, platinum or gold, which is not oxi~li7~d or reduced in a ~tenlial range in which oxidation or reduction of any species to be measured occurs. Additionally, materials selected for fabrication of conductive strips 64, 66, 68 and 70 are desirably selected so as to be free of any impurities such as battery metals (electrochemically active in water) which are typically present in off-the-shelf m~teri~lc commercially available for wire bonding, soldering, or welding. See EP-A-9481090.2 or USSN 08/045,847 filed 04/09/93 which is inco~ ed herein by . fc.~
Many thick-film pastes suitable for use in the present invention are commercially available, such as a silver pastes available as product number 3571UF/Ag from Me~h, Inc., of Elverson, PA (Metech), silver chloride available as product nulllber 2539/Ag/AgCl from Metech; gold pastes available as product number PC10231/Au from Metech, and p!~tin--m paste available as product number PC10208/Pt from M~t~h.
With specific regard to c~nductive strip 66, which defines in part a working electrode 90 a pr~f~llcd material is a very high purity platinum thick-film paste.
Conductive strip 68 preferably comprises a layer of silver deposited atop substrate 62 with a layer of silver/silver chloride deposited the.cu~n in the electrode region, ~iccus~d below, to create a r~f~ ncc electrode 86. A layer of cellulose acetate is depo~;~d atop the layer of silver chloride Conductive strips 64, 66 and 70 comprise a plaliml"l thick-film paste in ~l~f~l~d embo lim~ntc.
Employment of a silver lcf~cl oe electrode is within the scope of the present invention. M~lifi~tion of the t~chin~c of the present invention with respect to voltage settingc, upon the s.ll!s~ ll;o~ of a silver lcfc.cl ce electrode for a silver/silver chloride erelc~ce electrode, would be easily made by one of or~in~y skill in the art.
At the second end 76 of S~ h; te 62, ~ ctric layer 72 is depositcd so as to cover portions of c~n~luctive strips 64, 66, 68 and 70, while leaving portions of the conductive strips uncovered so as to define reference electrode 86, counter electrode 88, working electrode 90, ~,lt~ rc~nce collccling electrode 92, and contact pads 78, 80, 82, and 84. Material ~l~d for fabrication of the diel~tric layer 72 is desirably ~1~trir~11y inclll~ting and non-porous, free of i~ ulilies which may be subject to oxidation or reduction in the poten~ial range of any species or analyte to be measured, as described above, and is further s~lected so as to be free of mobile ions that would potentially carry charge and intclrelc with the activity of any electrolyte employed in the sensor. Further, dielectric 72 is sel~t~d so as to firmly adhere to substrate 62 and conductive strips 64, 66, 68, and 70, so as to allow electrodes 86, 88, 90, and 92 to be electrically addressable, while effectively electrically in~ ting portions covered by the dielectric.

213`8856 ~qt*1iql~ such as ceramics, glass, refractory mqtPriql~, polymeric mqtPriql~ or co",binations thereof are well known as di~Pl~pctnc mqtenql~ and are suitable for use as a dielectric in the present invention. A lJ~cf~.lcd mq~,nql is commercially available as ~du~:l Number 9615, a ceramic materiâl from E.I. DuPont de Nemours & Co., Electronics Department, Wilmington, DE.
With respect to materials advantageou~ly sPle~cted for fabrir-q-tion of conductive strips 64, 66, 68, and 70, it is noted that mqtPriql sPl~tion becomes less i~ ailt in regions of the strips which define contact pads 78, 80, 82 and 84 and which CQI-n-Pct the bonding pads to regions which define electrodes. For eY-q-mple, the contact pads and regions of the co~ductive strips colln~ g them to the electrodes may be fabricated from any cQnducting material that adheres to ~l,ate 62 and that does not interfere with the electrical in~--lqtiorl function of llip1pctric layer 72. According to one embo~liment~ the contact pads and regions of the conductive strips connecting them to the electrodes are fabricated from a gold paste.
In ~lrlition to the material se1Pction p~. i.. ~, s .~ Js~ above, and as di~cuss~d with respect to se1~Pctiol- of the ~idp~ctric mq~tPriql, it is advantageous in the fabri~tion of a sensor to select materials for fabri~-q-tion of the ~ub:~Llale~ the coîlductive strips, and the ~lip1pctric layer such that good adherence is achieved between q~ q~ pnt layers, that is, d~1q..~in~;on is minimi7~Pd Sec EP-A-94810190.2 or USSN 08/045,847 filed 04/09/93.
20 If good adherence is not achieved, reference, coun~r, WOlkiflg and inlc~rclcnce coll~li~g electrodes 86, 88, 90, and 92, will not be well-defined which in one embodiment is defined by a screen used in the thick-film deposition process, anddisad~,drllag~us ~l~:l.~l--...;~try will result.
A cross-sectional side view of wolking electrode 90, taken along section line 10-10, is illustrated in FIG. 10. As described above, conductive strip 66 is depos;l~d upon S~;~llalc 62, and ~lipl~pctric layer 72 covers portions of conductive strip 66 leaving a portion uncovered to define a working electrode area. An active layer 96, compri~ing a catalytically active quantity of an en_yme immobili_ed onto pl~tini7~d carbon powder particles, is deposited upon conductive strip 66 using techniques similar to the depo~ition of conductive strips 64, 66, 68 and 70. Typically, thick-film screen printing at low tel~ tule is used to apply an active paste to conductive strip 66 in order to limit -æ-thermal damage to the enzyme, see Table I.
As noted, active layer 96 comprises an enz~.,c immobilized into an electri<-q-lly con~ucting support ~ which con~ of or c4mprisP-s a porous layer of resin-bonded carbon or gldphilG particles. The particles have intimqtely mixed therewith, 5 or deposited or adsorbed onto the surface of the individual particles prior to bonding to form the layer, a finely divided platinum group metal to form a porous, substrate layer onto which the enzyme is adsorbed or immobilized and comprising a substq tiqlly h~ t~g~l~us layer of resin-bonded carbon or graphite particles with the pl-q-tinllm group metal adsoll,~ on the carbon or E,,~hilG particles. An enzyme immobilized or adsorbed 10 onto a porous layer of resin bonded p!-tini7e~1 carbon particles is rli~los~p~ by Mullen, in U.S. Patent No. 5,160,418 and RPnnettQ et al., in U.S. Patent No. 4,970,145, both of which are inco,~~ ed by reference. The active layer 96 may alternatively be forrned by first deposiling the finely divided pl~linu... group metal, optionally preadsorbed onto or ~miYPd with finely divided carbon or gla~ilG, with or without all or some of the 15 resin binder, if used, on the surface of the electri~lly ~n~lucfive ;,~sl,ale, or conductive strip 66.
The plqtinum group metal in finely divided Pl.omPn~l form, including plqtinum, m., iridium, or rhodium, may be replaced by the co"Gsponding oxides, such as pldlinulll or palladium oxide. Th~erole, all references herein to a plqtini7~d mqt~ l are 20 to be taken as including a pla~ . group metal, as described above, and/or co"~nding oxides-conl;~ining material unless the context ,G Iuir~s otherwise.
Any suitable carbon or gldphilG powder which readily ~ the subse~luenl immobili~tion of an a~L~IIIC may be used to form the active layer. To this end, carbon pov~d~r should be used having a high density of f~lnction~l groups, such as c~l~Aylate, 25 amino and sulfur~4nt~ining groups, on the surface, as opposed to the more vitreous and glassy c~ns, which bind e IL~ S only poorly. Typically, carbon or graphite powder particle size ranges from between about 3.0 and about 50.0 nm; preferably, particle sizes range from b~ween about 5.0 and 30.0 nm.
Platinum may be deposited on the carbon particles in any convenient fashion, for30 example, vapor phase deposition, electro~hemic~l deposition, or simple adsorption from colloidal s-lspension to give platinum group metal loadings in the range of between about 0.1 to about 20.0 percent, by weight, based on the weight of carbon. Preferably, the pl~tinllm group metal loalin~ are between about 5.0 to about 15.0 percent by weight.
These limits are, however, practical rather than critical. Below about 1.0 percent platinum group metal, the output signal falls to a level which, in practical terms, is too S low to be ",eas.lr~d except by very sensitive a~paldt~ls; above about 20.0 percent, the loading of pld~inulll group metal b~com~s IJn~conomic~ with little ~dition~l benefit in terms of increased l-~sponse or sensitivity. In the l,lefell~;d technique, the carbon powder is pl~tinj7~ by the oxidative d~ornposition of a platinum compound such as chlol~latinic acid or, more p.efeldbly, a complex of plali~ ", or palladium with an 07ci~ i7~l~le ligand, in the presenoe of the carbon powder, thereby to deposit colloidal siæ p1~*num or palladium direct upon the surface of the carbon particle, in the manner taught, for example, by Petrow et al., in U.S. Patent Nos. 4,044,193 and 4,166,143, both of which are incG~ ed herein by reference. Preferably, the platinum group metal or oxide particles have a particle siæ in the range of bcl~n about 1.0 nm to about 20.0 15 nm, and most p~cfe~lbly are of a colloidal siæ in the range of between about 1.0 nm to about 4.0 nm.
The prerell~d activ. e layer ;,~ used in accor~ance with the present invention are, in fact, collllllc;lc;aliy available '~ sold under the narne PLATINUM-ON
CARBON BLACK from E-TEK, Inc., r,;...~ngh~m, MA. An enzyme, such as glucose 20 oxidase, or lactate oxidase, can be i.~ ol~ 7~d onto pl~tini7~ carbon powder particles, ple~ed by the deposition of colloidal pl ~;nl~" having a particle size of between about 1.5 to about 2.5 nm onto the carbon po. d~, having a nominal particle size of about 30.0 nm, by the oxidative deco...~ ;on of co rl~X p~ h~ sulfite acid aI) using H2O2.
In the present invention, the platinum activated carbon is treated in a pho~l,h~25 buffer formulation having a pH of about 7.5. The platinwll activated carbon is added to the buffer to neutralize any sulfuric acid present from the formation of the pl~tini7~
carbon powder particles. To the platinum activated carbon and buffer Illib~lule a co-protein, such as bovine serum albumin, is added to adsorb onto the carbon. The bovine serum albumin is added to help stabilize the enzyme, such as glucose oxidase, as 30 is known to those skilled in the art. A binder, such as a commercially available resin solution sold under product number 8101RS from Metech, is then added to the bovine 213885~

serum albumin-platinum activated carbon ~-lixlur~. The binder material, as noted above, acts to hold the cG~I~ponenls of the active layer together. To this ~ ure, a sl-rf~cPnt may be added to provide better printing flow char~cte-ri~tics when active layer 96 is screen printed upon conductive strip 66. An ~lditionq-l benefit of the surfactant is to act as a wetting agent for the sensor during use. The active layer 96 being comprised of a hydr~phobic binder becomes ~liffie~ to wet with water after it is fully dried. The surfactant f~ilitqtPS this wetup. The surfactant mqtPri~l used can be any liquidsurfactant, known to those skilled in the art, which is water soluble and exhibits a hydrophilic lipophilic balance (HLB) in the range of 12-16. Typical surfactant m~t*riql~
for use in this regard can be alkylarylpolyether alcohols, such as alk~lphe.loxypolyethoxyethanol. One such mqteriql is sold under the trademark Triton0 from Union Carbide Chemir-ql~ and Plastics Co., Inc., Danbury, CT. The plefe,ledmqt~riql for use in the present application is Triton0 X-lOO surfactant (HLB 13.5). After these co--lponenls are milled, a resin thinner may be added to adjust the active layer 96 viscosity for plilllh~g pUlpOS~S. Typically, a petroleum solvent-based resin thinner is used to bring the paste viscosity within the range of between lO,OOO to about lOO,OOO
cen~ oise. Resin thinners for this pul~03e are commercially available as product number 8101 thinner from Metech. An enzyme, such as glncQse oxidase or lactate oxi~lq~e~ is then added to the IllLl~lule, and the final paste is screen printed upon con~ ctive strip 66.
Other enL~Illes may be ~imi!qrly added to the Illi~lule to prepa~ active layers s~ific for other analytes.
It is p~f~l~d to put the active layer down last, i.e. before depositing the cover mc.llbl~le, to minimi7~ the thermal impact to the enzyme from other steps in the sensor formation, see Table I and FIG. 25.
Int~ .re~llce cGl~ec~ing electrode 92 is formed in a ~lla~m~r similar to the working electrode 9O. The int~.Ç~ence collccting electrode 92, however, includes an inactive layer (not shown) which is made using the same collll)onents and method used in a process of forming the working electrode, however, an inactive or nonreactive protein, such as the bovine serum albumin is added to the mixture of bovine serum albumin-platinum activated carbon, resin, surfactant, and thinner. As noted above, the intelrel~nce cGIl~ling electrode seNes to adjust for any interfering species, such as the .

neutral species ~et~minophen, diffusing through a semi-permeable l,-elllbldne layer 94 below) on top of electrodes 86, 88, 90, and 92.
Refe~dng now to FIG. 11, a cross-section~l side view of reference electrode 86, taken along section line 11-11, is shown. Reference electrode 86, as noted above, is S formed as a col-ductive strip 68, preferably comprising a layer of silver is deposited thereon. DiPlPctric layer 72 is depos;t~d covering a portion of conductive strip 68, while leaving a portion uncovered to define the electrode areas and contact pads. A
silver/silver chloride layer 102 is deposited upon conductive strip 68 by screen printing techniques known to those of skill in the art. Silver/silver chloride reference electrode 10 inks, such as those available as product null.bel 2359 from l~ete~h, are developed to provide a sldndar~ reference electrode utili7ing the silver/silver chloride couple.
Reference electrode stability measu~.... nts showed that over a period of several days, the potential of the refer nce electrode (86) shifted upon ~A~s~re of the sensor to whole blood. The root cause of the problem was id~Pntifi~P~ as a gradual de;-c~ in the 15 rejectiQn plo~ ies of the Illelllbl~nc (94) allowing pe~ ;on by blood pr~leins, which fouled the l~f~ nce. Cellulose acetate was chosen as a shield for the reference electrode due to its barrier l~lu~llies to pr~teins and its ability to tl~ls~ll sufficient water and electrolytes to ..~ a stable ~tential at the surface of the printed silver/silver chloride.
The choice of a prop_r solvent and cure process is critical in plt;l,a,ing a uni~l---cellulose acetate layer over the reference electrode. The solvent must have a low vapor p~S~ (high boiling point) in order to provide suffiti~p-nt screen life for the printing process to be completed. It must be col--~til,le with the printing screens, ~, not ~e~r~;le the scr_en em~ ion dudng pdll~g. The viscosity of the plepa~d paste must be relatively high, 40,000 oelllipoise to 350,000 ce.llil)oise. This m~nd~tes that the %
solids of the polymer solution be fairly high, thus the solvent must be very good for the polymer. Suitable solvents include the so called "super solvents", polar aprotic solvents such as dimethyl fo.lll~lide, dimethylsulfoxide, hexamethylphosphoramide, and 1,3-dimethyl-2-imidazolidinone (DMI) are examples of this class of solvent. One final restriction was that the solvent not be a carcinogen, mutagen, or teratogen in order that it might be handled more readily by the formulation technician and the screen printer.

The plefelled solvent is DMI.
Solutions prepared in the concentration range of from 15 to 35 grarns of cellulose acetate in 100 ml of DMI were found ^~p~hle for the pl~nting process. The pr~ft;llc;d c4r~Pnt~ation was chosen as 20 grams c~llulose acetate in 100 mL of DMI. In order to 5 rapidly dissolve the polymer, the solvent is heated to between 60 and 100C, with the pl~f~red t~.-l~,dlurt; being 95C. The polymer is added to the rapidly stirred (m~n~-tie stir bar), heated solvent (water bath with the ~ ~ldl~lre preset). It is then ll.~h~ni~ y mixed in with a sp~t~ , after which it is stirred continuously until completely dissolved.
The polymer/solvent ~ ult; (pdste) is then removed from the water bath, allowed to 10 c~ol to room te~ ~, labeled and set aside until needed for printing.
The paste is generally printed on the same day it is prepared, it can be used upto seve~l months after prepaldlion, however, p~lÇol-,-~lce of the layer gradually decreased with paste shelf life. The paste is applied in a 2 pass print after which it is allowed to level for a period of time no less than 10 111inUlei5 and no more than one hour.
It is cured in a box oven at 55C for 10 111il~Ul~5, the tem~ dlu~e of the oven is then ramped up to 100C over a 10 minute period, the curing c4nl;.~u~ for 10 ~..inut~ s more at this lelll~ldtu~e. This print mPtho-11cure cycle is crucial to the p~,Çollllance of the cellulose acetate membrane. Low cure ~---~.dtur~s do not remove sufficient solvent, while longer cures or higher cure t~ s lead to a brittle membrane which 20 del~--in~5 easily from the ~sl,dte, particularly after post-tre~tm~nt of the sensor with an anti-drying agent. P~tu~g with more passes leads to a thicker membrane, which is also prone to ~ --;n~l;on. It is ilupol~ll not to completely remove solvent, as complete removal would hinder the hydration process.
A layer of celllllose acetate 100 is applied over the silver/silver chloride layer 102 25 to protect the silver chl~ le from con~ --;n~nl~ present in blood samples that would shift the reference ~tenlial. The cellulose acetate layer 100 can be applied by a spotting technique or by a screen p,intii~g t~-hnique. If the spotting technique is used, an Asymtek XYZ table, available from Asymtek Corporation, ~rl~, CA, and known to those of skill in the art, will be used. If a screen printing deposition process is used, a 30 high viscosity solution from a high boiling solvent, such as 2-(2-ethoxyethoxy)ethylene acetate will be used.

213885~
.

Lastly, as noted above, each electrode 86, 88, 90 and 92 is covered with a glucose and oxygen-permeable membrane 94.
Me.l,bl~le 94 can be formed from cellulose ~sPt~te, polyulc;tll~e, QilioQnP
conlpoullds, and other melnbl~e ~n~tPn~lQ known to those skilled in the art such as S Nafion~ m~teri~1 available from E.I. DuPont de Nemours, Wilmington, DE. The plefe,l~d ~le.llbldne 94 is a dispersion of a polymPri7~hle silicon-cont~ining colnpound applied in an inc4rnpl~ y cured form of a sili~ one colllpound dispersed phase in a liquid carrier. The carrier is e~C~s~t;~lly insoluble in the dispersed phase and removable from the dispersion during curing. The ~liQperQj~n will dry and cure as a continllous layer, film or membrane, having a high glucoæ and oxygen permeability to function as a single me.ll~ldne in an ele~ e~ glucoæ ænsor. A single-layered, semi-permeable membrane is IiQrlo~ by Jones, in EP Patent No. 207 370 Bl which is incol~,ated herein by ,~felw~ce. The silicon con~ining compound may be dispersed in the continuous phase as an oligomer, ~.repoly,.ler, or incompletely cured polymer.
The poly~ ble silicon-con~ining cG!~pol~nd, after dis~l~ion in a c~nti~Uous phase, such as by ;ncl".~ing an emulsifier, can be cured in any known manner during removal of the continuous phase, such as by evaporation of water from a water con~;l-.JQus phase sili~ne emnlQion or dispersion, as ~lise~los~ by Johnson et al., in U.S. Patent No. 4,221,688, and Elias, in U.S. Patent No. 4,427,811, both of which are incol~,ated herein by ~erelence. Further, the dispersion of the silicon cont~ining cG".pound can include a suitable curing catalyst, or can be heat cured, so the ~ ~rQion of the pol~,n~ hle silicon~o~ ning c4,~pound is applied as a layer in the form of an incompletely cured di~ ;on and at least a portion of the carrier or continuous phase is removed from the dis~sion during final curing. The emlllQinn can consist of a dispersion of silicone latex particles and silica. Upon t;v~ofàtion of water, the silicone latex particles are cross-linked by the silica. The morphology of the res--lting membrane is polydiorgano cross-linked particles bounded by a continuum of silica or sili~tes. It is the silica phase in which analyte ~lallSpOll, i e. glucose, lactate, etc., takes place.
In accordance with one aspect of the present invention, the polym~-ri7~hle silicon~ont;lining compound is an organosiloxane, and particularly a diorganosiloxane, comprising es~e,'lially a linear species of repeating diorganosiloxane units which may -- 213885~

include small numbers of monoorganosiloxane units up to a maximum of about one unit for each lOO dior~nQsi10Y~ne units Wht;leill the polymer chain is termin~tPd at each end with siliron~bonded h~dn~,.yls.
In accol~ance with another illlpol~1t aspect of the present invention, the S pol~ e.~ble si1icQnP-cont~ining c~lllpound forming an oxygen and glucose-permeable lallC iS applied onto an electrode as an aqueous si1icone emulsion comprising a continu~us water phase and an ~nioni-~q11y st~bilized dispersed ci1icone phase whelein the silicone phase is a graft copolymer of a water soluble silicate and a hydroxyl endblocked polydio.ganosiloxane. As disclosed by Saam, in U.S. Patent N O. 4,244,849, incol~ldted herein by reference, such silicone en~1-1cions, having a pH within the range of from about 8.5 to about 12.0, are stable upon eYtende~ storage and result in a cured clasto,nelic continuous layer upon-removal of water under ambient conditions. These silicone compounds are obtained from the intçr~tion of hydroxyl endblocked polydior~nn~;10xanes and alkali metal ~ tf~s to form graft polymers ~n;Qr~ 11Y
stabilized in aqueous ernlllcionc at pH of, for ~ a rle~ 8.5 to 12Ø If stability is not , however, the pH is not critir~1- The em~lcion can be applied in layer form to manufacture the l,.e."l)ldne as soon as the co...~n~nt~ are homogeneously dispersed.
The e ~ ion Uhydroxyl endblocked polydiorg~nosi10xane" is understood to describe an ~CCf~t;~l1y linear polymer of l~ ;n~ diorganosiloy~n~ units conl~;nil1g no more than small impurities of monoGl~nos;10Y~ne units. The hydroxyl endblocked diorg~nosi1n~ne will therefore have e,~ 11y two silicon-bonded hydr~Ayl radicals per mol^cllle To impart elast~ ,.ic ~.lies to the product obtained after removal of the water from the emlll~i~n~ the polycilox~np should have a weight average rnolccul~r weight (M.,) of at lP~st 5,000. Polysiloxanes with weight average mol~ul~r weights below about 5,000 down to about 90, also are useful if the polymers form a continuous film or layer upon curing. Tensile strengths and elon~tiQn~ at break improve with increasing molecular weight, with relatively high tensile strengths and elongations obtained above 50,000 M~,. However, since in a p~felled embodiment of the invention, the cured polymers are bonded directly to an electrode, and do not undergo any severe m~.h~nic~l stress during use, high strength is not necP~ry for the polymer to be useful. The maximum M~ is one which can be emulsified or otherwise dispersed in a liquid carrier or continuous phase, such as water. Weight average molecular weights up to about1,000,000 for the incompletely cured dis~r~d polysiloxane are practical for use in the sensor of the present invention. Upon curing, there is no upper limit to the n~olecul~r weight of the m~nlbl~ne. The p,ere~ , for the polynleri7~hle dispersed siloxane is in the range of 1,000 to 700,000.
Organic radicals on useful hydroxyl endblocked polydior~nociloY~n~s can be, for example, monovalent hydroc~l,on radicals cont~ less than seven carbon atoms per radical and 2-(perfluoroalkyl)ethyl radicals corlt~ining less than seven carbon atoms per radical. Examples of monovalent hydroc~l,on radicals include methyl, ethyl, propyl, butyl, isopropyl, pentyl, hexyl, vinyl, cyclohexyl and phenyl; and examples of 2-(perfluoroalkyl)ethyl radicals include 3,3,3-trifluoropropyl and 2-(perfluolubulylmethyl). The hydroxyl endblocked polydiorganosiloxanes plerel~bly contain organic radicals in which at least 50 percent are methyl. The ~lefer,~d polydiorg~nociloY~nes are the hydroxyl endblocked polydimethylsiloxanes.
In accor~ce with one illlpO~ aspect of the present invention, the hydroxyl endblocked polydior~a~-osiloY~e is employed as an ~nio~ir~lly stabili_ed aqueousemulsion. For the p.l~poses of this embodiment "anionically stabilizedN means the polydiorg~nosiloxane is stabilized in emulsion with an anionic surfactant. The most pr~f~ .led anionically stabilized aqueous emulsion of hydroxyl endblocked polydiorganosiloxane are those prepaled by the method of anionic emulsion poly....~-.;,,lion described by Findlay et al., in U.S. Patent No. 3,294,725, hereby inc~jlpo,~ted herein by reference. Another method of pley~u~g hydroxyl endblocked polydiol~no~ nPs is d~lil,ed by Hyde et al., in U.S. Patent No. 2,891,920, also il~col~ ted herein by ~fer~. ce.
An alkali metal silicate or colloidal silica must be included in the emulsified silicone co"")o~ilion for the pre~lion of e~tende~ storage stable em~ iQn~ used in the invention. The alkali metal silicates pl~ ~ell~ d for use in the emulsions forming the oxygen and low molecular weight analyt~permeable membranes of the present invention are water soluble silicates. The alkali metal silicate is preferably employed as an aqueous solution. Aqueous silicate solutions of any of the allcali metals can be employed, such as lithium silicate, sodium silicate, potassium silicate, rubidium silicate and cesium q~P,.
The colloidal silicas are well known in the art and commercially available, and can be incllld~Pd in the dis~ ;on for increased strength and storage stability. Although any of the colloidal silicas can be used, inclutling fumed and pr~i~ d colloi~ql silicas, silicas in an aqucous m~lilJm are plefell~;d. Colloidal silicas in an aqueous m~P~ium are usually available in a stabiliæd form, such as those stabilized with sodium ion, qmmoniq or an alu--lin~l--- ion. Aqueous colloid-q-l silicas which have been stabilized with sodium ion are particularly useful for ~l-ning an emulsion because the pH requirement can be met without having to add other co~ orl~nt~ to bring the pH within the range of, for e~amrle, 8.5 to 12Ø The eAyr~ssion -~colloidal silica" as used herein are those silicas which have particle di-q-mçters of from about 0.0001 to about 0.1 micrometers.
Preferably, the particle iiqme~prs of the col1oi~l-q-l silicas are from about 0.03 to about 0.08 micrometers; most preferably, the silica particle iiqmPt~s is about 0.06 micfol--~".
The colln:~ql silica can be added to the qnioni~qlly stabilized hydroxylated polydiol~no;,;lol~-q-ne in the form of a dry powder or as an aqueous dispersion.~fe.dbly, the c~lloj1ql silica is added in the form of a sodium ion stabilized aqueous dispersion of colloidal silica, many of which are commercially available. These co-----.er~ial colloidal silicas are usually available in aqueous dispersions having between about 10.0 to about 30.0 percent, by weight, colloidal silica, and a pH between about 8.5 to about 10.5.
Aqueous solutions of sodium or pot-q~sil~m silicate are well known and are commercially available. The s~lution~ generally do not contain any signifit qnt amount of ~lise~ ulicles of amorphous silica and are co m m only r~f~llcd to as water glass.
The ratio, by weight, of silica to alkali metal oxide in the aqueous solution~ of alkali metal ~ilic~q~ s is not critical and can be varied between about 1.5 to about 3.5 for the sodium silicates, and about 2.1 to about 2.5 for the potassium ~ilic-q-~s The aqueous alkali metal silicate solutions are particularly useful in ylGpa~ing the emulsions used in the present invention because the qddition of the silicate solution often brings the pH of the emulsion within the range of about 8.5 to about 12.0 so that additional ingredients are not ne~e~s~y to adjust the pH of the emulsion. Of course, other aqueous alkali metal silicate solutions, such as those plepal~d by hydrolyzing silicon esters in aqueous aL~li 21388S~

metal hydroxide solutions, can also be employed in the present invention.
In accor~ancG with one aspect of the present invention, the polymPri7^ble silicon co~ -ni~-g co~ ound is .lis~,~ by cG~Ilbining an aqueous solution of an alkali metal silicate and the polyl,le.i~dble silicon co~ ;ni~g co~"pouild in an emulsion SQ that S a graft copolymer is formed as dispersed particles. The pr~f~led procedure forp~ g si1i~one emul~ion~ is to add the alkali metal silicate to an anionically stabili~ed aqueous em~lciol~ of one or more hydroxyl endblocked polydior~qnQ~ilnx-qn~-s, adjust the pH of the emulsion within the range of about 8.5 to about 12.0, and then age theemulsion for a period to form an el~lo",e~ic product upon removal of the water under ambient conditions. In this pr~lurG, the pH of the emulsion col~'; ;nin~ dissolved silicate and dis~l~d l~ u~-yl endblocked polydior~,qno~iloxane is il~ ~n~ to theformation of the emulsion. A pH of 8.5 to 12.0 mqintqins the aLkali metal silicate dissolved so that sllffi~ ient graft co~ol~ f~ ;on between the dissolved silicate and dispersed ~;1QY~qne occurs during removal of the carrier (e.g., water) to produce an em--l~ion capable of providing poly.--.;7-~;on, or further polymeri7-qtion~ of the silicon co~ g compound when de~iled as a layer to form a ."e "bl~ e. If the pH
is lower than the stated range, silicic acid is formed from the aL~ali metal ~ili~qte. Silicic acid is unstqble and rapidly polymeri7~c by con~en~qtion~ which can gel the e-mul~;ol-Since silicic acid formation is almost comI)!et~?y s~,~pressed at a pH of between about 10.0 to about 12.0, and the reaction bel~ dissolved alk,li metal silicate and ~ per~d silo~qnes occurs more rapidly within this pH range, this range is plef~led for emulsions CQI~t;~n;' an aL~ali metal silieqvt~.
Silicone em~ iQns plc;p~ed by silicate copol~ r~ t;on are aged at a pH range of b~l~een about 8.5 to about 12.0 for a period sllffirient to allow interaction b~w~n the dissolved silicate and the di~.~d silnYqne so that an elastomeric product is formed upon removal of the water under a,nbicnl conditions. The aging period is effectively reduced when an organic tin salt is employed in an amount between about 0.1 to about 2.0 parts, by weight, of polydiorganosiloxane. The organic tin salts expected to be useful in the emulsions include mono-, diand triorganotin salts. The anion of the tin s.lt employed is not critical and can be either organic or inorganic, although organic anions such as carboxylates are generally pr~fe.l~d. Organic tin salts that can be employed ~;

213885~

include octyltin tri~cetqtp~ dioctyltin dioctoate, didecyltin ~liqr~ptqte~ dibutyltin ~li ^etqtP, dibutyltin dibr~,llide, dioctyltin dilaurate and trioctyltin -q-rePtP The ple diorganotin dic. rbo~cylate is dioctyltin dilaurate.
The relative amounts of alkali met. l silicq~s and hydroxyl Pn-lblor1~P~d S polydiol~,~no~ xq-nP employed can vary over a co~ Prable range. ~eft;lled Plq~QmPr ~lope,lies are obt. ined when between about 0.3 to about 30 parts, by weight, silicate is employed for each 100 parts, by weight, siloxane.
In accor~ance with one aspect of the invention, an alkyl tin salt is added to the dispersion to cataly_e the curing of the final emulsion during the devo1q~i7qtion, or other removal, of the carrier to yield the cured me~,-l,l~ne. ~refe"~d salts are dialkyltin dicarboxylates such as dibutyltin ~i~retq-t~, dibutyltin dilaurate, and dioctyltin ~ilqllr~qte;
the most pl~ fe.led tin salt is dibutyltin dilaurate. The ernl-lciQn of catalyst is used in an qmount sufficiPnt to yield ~l~.~n about 0.1 to about 2.0 parts, by weight, of the alkyl tin salt for each 100 parts, by weight, of the polymeri7~1e silicon con~ ning co~ ~u~
such as polydiol~,.nos;loY~q-nP~. Larger amounts could by used, but would serve no useful p~ )os~., The dispersion of the polymeri7^' 1e silicon-contq-ining col~po~n~(s) can contain colllponenls in a broad range of crn~pntr~tiQns The pref~led c~ ncentrations will depend on the t1lir~n~P$~c of the me.l.bl~ne desired. For eY~mple, to provide a thin Pl~ctompric ",e.~ldne (20 microns) that does not form cracks as the carrier or co~tinuou~c phase e~apol~, it is best to use a dispersion having a co,llbined amount of silicate and polydior~,~n~;lox~n~P- in the range of between about 67.0 to about 160.0 parts, by weight, for each 100 parts, by weight, of carrier such as water. ~r~felled "æ",bldne; thi~ L .,f cc~c are bcl~ about 10.0 to about 100.0 microns, pr~L~dbly about 20.0 microns.
If an emulsifying agent is incol~l~ted into the colll~s;Lion to form the dispe.a;on the amount of emulsifying agent can be less than about 2.0 percent, by weight, of the emulsion. The emulsifying agent can result from neutr~li7~d sulfonic acid used in the emulsion polym~-ri7~tio~ method for the pr~pa-alion of a hydroxyl endblocked polydio,gdnoailoxane.
Anionic surf~^t~ntc are preferably the salts of the surface active sulfonic acids used in the emulsion polymeri_ation to form the hydroxyl endblocked 213885~

polydiorg. nosiloxane. The alkali metal sqlts of the sulfonic acids are prcf~
particularly the sodium salts. The sulfonic acid can be illll~trqt~P~ by liphq~ qlly s~lbs~ ~d bc- ~n~.~lfonic acids"-~kll~qlPne sulfonic acids, and diphenylether sulfonic acids, a liphatic sulfonic acids, . nd silylalkylsulfonic acids. Other anionic emulsifying S agents can be used, for example, aLl~ali metal sulforicinoleates, sulfonated glyceryl esters of fatty acids, salts of sulfonated monovalent a Icohol esters, amides of amino sulfonic acid such æ the sodium salt of oleyl methyltq~ ide, sulfonated aromatic hydr~ on~kli salts such æ sodium alpha-n~rhtl.~lP-ne monosulfonate, condPn~qtion products of naph~h~lPne sulfonic acids with formaldehyde, . nd s~llfqt~s such as qmmonium lauryl sulfate, triethqnol amine lauryl sulfate and sodium lauryl ether sulfate.
Nonionic emulsifying agents can . lso be included in the emulsion (in ~ iiti~n to the anionic emulsifying agents). Such nonionic emulsifying agents are, for Pyqmple saponins, col-~en~qvtinn products of fatty acids with ethylene oxide such æ dodecyl ether of tcl~ lene oxide, cor.~en~q~tion products of ethylene oxide and so l,ilan trio~e~, conden~tior~ products of phenolic compounds having side chains with ethylene oxide, such æ conden~q-tiol- products of ethylene oxide with isododecylphPnQl, and imine derivatives such as polyrnP;li7~ ethylene imine.
The poly~ hle silicon~on-l oun(l dispersion used to form the oxygen and glucos~-permeable ~I~e.ll~ es of the present invention may contain ~litionql ingredients to modify the pr~.lies of the dispersions, or the cured polymeric membrane products obl~in~d from the iis~ nc~ For eY~mple~ a thicl~pner may be added to modify visc~sil~ of the dis~ ion or to provide thixotropy for the dispersion. An anlifoanl agent may be added to the dispersion to reduce foaming during ylc~ation~ coating or curing in layer form.
Fillers may be added to the dispersion to lcinfol~e, extend or pigment the ~c~bl~e. Useful fillers include colloidal silica, carbon black, clay, alumina, r~lcil-m carbonate, quartz, zinc oxide, mica, titanium dioxide and others well known in the art.
These fillers should be finely divided and it may be advantageous to use aqueousdispersions of such fillers.
The filler ~lercl~ly has an average particle ~i~meter of less than about 10.0 micrometers. When the silicone emulsions are spread out for final curing to form the -213885~

oxygen and glucose-permeable membranes of the present invention, the water, or other nonsolvent carrier, e~ a~.ales, or is otherwise removed, to leave a cured oxygen and glucose-p~.",eable nle~ bldne. E~ lion of the carrier is usually complete within a few hours to about one day depen~ling on the dispersion film thickness and method of S application. Another of the illlpolldnt advantages of the present .lle.,lll,l~dne is elcr.-PllPnt adhesion to both polar and non~1~r subsl.~t~s One of the more illl~l~nl advantages of the oxygen and analyte-~.-neable Illembldnes used with the present invention, is the capability of these membranes to be bonded to an electrode activated with a suitable enzyme catalyst, such as glucose oxidase, 10 glucose dehydrogenase or lactate oYi-lq~e. In accof~dnce with one embodiment of the present invention, a co.~.pound capable of catalyzing the reaction of glucose with oxygen is incol~laled within the anode, or active layer 96, and the oxygen and glucose-permeable m n~blane 94 of the present invention is coated over the reference, counter, working (including active layer 96), and int~l~Gilce coll~;~ing electrodes 86, 88, 90, and 92.
The melllbldne m~teriql~ d~ d herein are very co...l,~l;hle with whole blood 98, have a durable surface and are highly selective to oxygen penetration so that a suffirient stoichiometric excess of oxygen ~Illledl~ the l"cll,bl~dne 94 even from whole blood.
The plefell~d materials for ll.e.,.. b.~u~e 94 are an anionically stabilized, water-basedhydroxylendblockedpolydimethylsiloxane~ u~ ,rcor.s~h-ingatleastabout 10.0 percent silica, by weight. Most ~feldbly, the ~ t~m~qr cont~in~ about 14.0 percent, by weight, cQ11oi~1~1 silica, and is c~l.. ~ially available as FC~l coating from Dow Corning, Mi-11qnd, MI. This mqt~riql is a low viscosity, filled, opaque çm~ jQn Typically, this mqteriql has a pH of about 11.0, and a viscosity of about 40,000 cp.
In another aspect of the present invention, it has been found that during dry storage of sensors 2~, membranes 94 become in.;l~singly more difficult to wetup. It is believed that residual water and other solvents, initially present in the membrane 94 after casting, e~,dpoldte during storage and cause coalescence of silicone agglomerates. This tightening of the membrane structure can decrease the sensitivity and increase the response time of the sensor toward glucose.

213885~

The sensors 28 can be post-treated to pl~enl the membrane from aging during dry storage, for eY~mp~ by preventing the me.nb~ s from fully drying with humi~ific~tion, or tre~tment with a high boiling point, water soluble, hydrophilic polymer liquid antidrying agent, such as surf~ct~nt~ or polyethylene glycols. ~r~re,dbly, a non-ionic surfactant, having a molec~ r weight of at least about 300, such as Triton~
X-lO0 surfactant, Tergitol~ 15 surfactant from Union Carbide ChPmir~l~ and Plastics Co., Inc., Danbury, CT, Tween0 20 ethoxylated so~ an esters surfactant from ICI
Surf~t~t~, Wilmington, DE, and polyethylene glycols having molecular weights bet veen about 200 and 600, are applied for post-tr~tment of sensors 28 to improve output, and response time, while minimi7ing sensor drift, upon initial start-up. The ~,er~ dm~t~ for post-tre~tm~nt is polyethylene glycol having a molecular weight of about 400.
Referring again to FIGS. 9A and 9B, while noting that a variety of sensor confi~ul~ti~ns can be advantageous in dirr~r~nt ~pp1ir~ti()n~, the following non-1imiting pr~fe.led ~ hc;orl~l s~-ifir~tiolls of a sensor 28 fabricated in accol~ance with a pl~fell~d e.llbo~ .lent of the present invention are given.
Sub ,~ldle 62 can be fabricated in a variety of shapes and sizes. According to one specific ~.~ Ç~ d emba~im~nt of the invention, s~slldle 62 is from about 0.4 inch to about 0.5 inch long; p.~f~ldbly, about 0.45 inch long. Subs~ e 62 is from about O. l5 inch to about 0.25 inch wide; p,~fe,dbly, about 0.18 inch wide. Sùllslld~ 62 is from about 0.02 inch to about 0.05 inch thick; p,~,ft;,ably, about 0.025 inch thick. Conductive strips 64, 66, 68 and 70 are each d~pos;t~xi in a thi~lrn~ss of from about lO.0 microns to about 20.0 microns; p,erel~ly, the strips are about l5.0 microns thick. Conductive strips 64, 66, 68, and 70? at end 76 of the sensor, are from about O.Ol inch to about 0.03 inch wide, pmre.dbly about O.Ol wide. CQnt~^-t pads 78, 80, 82, and 84 at end 74 of the sensor, are from about 0.025 inch to about 0.05 inch wide, pleÇ~l~bly about 0.03 inch wide.
Dielectric layer 72 is ~rerelal)ly deposited in a thickne~ss of from about lO.0 microns to about 50.0 microns, pr~felably about 20.0 microns thick. Thickness values are given after firing or curing.
Portions of the conductive strips are exposed to define the reference electrode 86, 21~8856 counter electrode 88, working cle~t.~de 90, and in~.rerence collccling electrode 92.
The eA~osed surface area for the ~ereleilce electrode 86 is about 0.00015 inch2, and for the count~r cle~;tl~de 88 is about 0.00022 inch2. The working and illt~lre.e.~ce coll~cting el~tlodes 90, 92, each have surface areas of about 0.00038 inch2. These eYI-ose~surface area ~limension~l ~p~xifir~tion~ do not take into consider~tion surface area due to the edges of the electrodes, defined by the thirl~nP~ of the electrodes as depo~;led or the ~;ly of the layer. Such edge ~im~n~ionC are minim~l relative to the overall electrode areas. However, the e Apos~d surface area s~-ifi~tion are thus somewhat approxim~te.
A cell~-lose acetate layer 100 is applied over the silver/silver chloride layer 102 of the reference electrode 86, which was depo~iled over the e Aposed portion of conductive strip 68. The cellulose acetate layer 100 protects the silver chloride from cont~min~t~s that would shift the lert;~ence ~)otenlial.
The active and inactive layers are then applied over the exposed portions of cond~ctive strips 66 and 70, fo,llling the working and int~,Ç~ r~nce co"~ling electrodes 90, 92, ~s~;ti~ely.
Cover l,.~.l-b,ane 94 is then de~ ~ p~f~.~bly spun-cast, to a total thic~n~
from about 5.0 microns to about 50.0 microns, preferably from about 10.0 microns to about 20.0 microns. The cover or p,otecti~e me..-b,~e 94 is preferably applied in layers to enable thin overall ~hi~ l~n~s with r~u~d perm~ility char^^t~vri~tics.
The present invention will be further illus~led by the following examples which are int*nded to be illust-rative in nature and are not to be construed as limiting the scope of the invention.

EXAMPLE I
Referring to FIGS. 9 and 9B and 25 and Table I, one suitable construction of a solid state, planar glucose sensor 28 incluAing the col.~onents and design subs~ 1y in acconlance with an aspect of the present invention is provided by the following combination of elem~nt~.
A par~al assembly of planar glucose sensor 28 having substrate 62 and conductive metal strips 64, 66, 68 and 70 was fabricated in accordance with a method of the present invention on a 0.025 inch thick, 0.18 inch by 0.45 inch, electrically -nonconducfin~ s~lldte 62 comprising appr~im~tely 96% alumina and approxim~tely 49~i glass binder, available from Coors Ceramic Company, Grand Junction, CO. Portions of conductive strips 64, 66, 68 and 70, as well as contact pads 78, 80, 82 and 84, were de~o~iled onto the ~ dle using a screen printing technique, with a 10.0 micron 5 emulsion of gold condiJc.tor paste, available as product number PC10231 from Metech, Inc., Elverson, PA. A st~inless steel screen having a 325 mesh pattern WdS used to screen print the gold paste onto substrate 62. Conductor strips 64, 66 and 70 were co~n~c~ with p!~tinllm upper portions, as the cond-lctive strips were continlJ~Pd toward second end 76. These strips were fabricated by screen printing a 10.0 micron emulsion high purity platinum conductor paste, available as product number PC10208 from Metech, onto the s.ll,s~ e~ A screen similar to that described above was used to deposit the plalinu,l, conductor composition. Conductor strip 68 was ~imil~rly continued toward second end 76 by applying a 10.0 micron emlllQiQIl silver cor~ductor paste, available as product nu",bcl 3571UF from lU~Pt~ch, onto the sub~ . The 325 mesh screen made of s~inlpss steel wire was used to screen print the sUver conductor paste. A 10.0 micron çmUlQ;on silver/silver chloride l~,f~r~ electrode ink, available as product nu,l,ber 2539 from Metech, was ~ul)~uenlly screen printed over a portion of conductive strip 68 at end 76, covering an area of c~nductive strip 68 at least as large as, and prt;f~bly larger than, the area of conductive strip 68 to be ~A~sed by ~lielectric layer 72 to define ler~lcnc~ electrode 86. Lastly, a cellulose acetate layer 100, available as product number 18095-5 from Aldrich Chtomic~l Co., Milwaukee, WI, was screen printed over the silver/silver chlori~le n fe~nc~ electrode 86. This layer is applied over the reference ele~ de to protect the silver chlori~le from cor~ ;on that could shift the reference polenlial.
In this eY~mp'e a BTU 7 zone furnace with a 3 zone dryer, from Fast Fire of Billerica, MA, was used in firing the inorganic pastes. Firing was c~rried out per the manufacturer's recomm~nd~tions, ramped to the peak c~nditions. The gold collductQr paste was fired at 850C for a 10 minute peak, the pl~timlm conductor paste was fired at 750C for a 13 minute pealc, and the silver conductor ink was fired at 750C for a 10 minute peak.
Conductive strips 64, 66, 68 and 70 were deposited on substrate 62 so as to be 0.01 inch wide at end 76; contact pads 78, 80, 82 and 84 were deposited on substrate 62 so as to be 0.03 inch wide, and 0.8 inch long at end 74.
A ~ pl~pctlic mqtPri~l 72, available as pr~lucl n ~ b~ 9615 from DuPont Electronics, Wilmington, DE, was screen printed as a 15.0 micron emll1cion over a large portion of sensor 28, ey~en~ing from second end 76 to contact pads 78, 80, 82 and 84.
A 325 mesh screen made of st~inlp-cc steel was used for the screen printin~ process. The ic was fired at 750C for a 10 minute peak. As noted above, portions of col-ductive strips 64, 66, 68 and 70 were not covered by dielectric 72, exposing their electrode areas.
Silver/silver chloride is applied at 75C for 30 minutes.
Cellulose acetate is applied at 55C for 10 ~llinu~s, ramped to 100C for 10 in.l~s, and then 10 minutes at 100C (30 minute cure time).
An active layer 96, comrricing a catalytically active quantity of glucose oxidase, available from Biozyme Labol~lt~lies ~n~ ;on~l, Ltd., San Diego, CA, immobilizedonto ~1~I;ni7~ carbon powder particles, available from E-TEK, Inc., Fr~min~h~m, MA, was de~;t ~ upon cor ducfive strip 66 to form woll~ing electrode 90 also using a thick film screen plillth~g technique. An inactive layer, col.~ ;n~ an inactive protein, such as bovine serum albumin; sold under the trademark Pentex~ bovine albumin from Miles, Inc., K~nl~l~ee, IL, immobilized onto p!~tini7~d carbon powder particles, available from E-T~, was deposiled upon cond~ctive strip 70 to form h~tclÇe~nce correcting electrode 92 using similar thick film screen plh thlg techniques. The fabric~tion of the active and inactive layers is desc- ;Ixd in further detail in FY~mr~
After the cq~ ctive strips, ~ ~tric Layer, and do~l.odcs are deposited onto s~lbsl~ e 62 and the conhct are m~cl~cd to pl~ electrode '~huntin~ a cover membrane 94 is spun-cast over the electrode area of the sensor. An ~nior~ ly stabili_ed, water-based hydroxyl endblocked polydimethylsiloxane elastomer, comprising about 14 percent, by weight, colloidal silica, commercially available as Fabric Coating (FC)~l from Dow Co~ning, ~i~ n~, MI, was applied to the sensor 28 using a spin-ca-sting technique. An IVEK laboratory pump and an Integrated Technologies P 6000 spin coater were used to apply the cover membran~94 in multiple layers over the sensor. The first layer was applied by complete flooding of the wafer with the membrane 213885~

ela~o...er material. The Integrated Technologies P~000 spin coater was then activated to a spin speed of 7,000 rpm, and a spin time of 90 s~n~l~. After the spinning was co---ple~cd, the first layer was allowed to dry for 15 minut~s The sensor 28 was then spun again at 7,000 rpm, and the second layer of the me~b~a~le material was applied.
S Two ~litionql layers were applied using the spin/flood technique used to apply the second layer, allowing 15 minutes bel~een casting each layer. After all four layers have been cast, the ~ lbl~e 94 was cured overnight at room l~ ...e in a dust-free envirorm~nt The tot~ thickness of the multiple layers of membrane 94, after curing, is approximately 20.0 microns.
EXAMPLE II
Active layer 96 was plGpa~ed for use in working electrode 90 (as noted in Fxqmrle I). The active layer 96, for a glucose sensor, primqrily includes a catalytically active 4ualllily of glucose oxidqcP~ available from Biozyme Labold~lies, immobilized onto pl~-;";,~ carbon ~wder particles, available from E-TEK, and the particles are di~llibut~ s.~bs~ lly unifollllly tll~u~ ou~ the layer.
About 3.15 grams.of plqtini7~d carbon powder particles, Vulcan0 XC-72 carbon black, available from Cabot Col~ldtion, Roston, MA, plG~ed by the deposition of colloidal pl~l;nll.,, (particle size between about 1.5 to 2.5 nm) onto the surface of the carbon po~dcr (nominal particle si_e about 30 nm) by oxidative decomposition of COIllp'~ pldlin~llll sulfite acid (II) using H2O2, were treated in a phosphate buffer to neutralize any residual sulfuric acid present. The phosrhqt~ buffer also incll)~es a microbicide, sold under the tr~P-mq-lc Kathon0 CG microbicide of Rohm & Haas Corp., PhilqA~o-lphiq~ PA. The buffer was pr~ d by adding 11.499 grarns sodium phosph~te, dibasic (Na2HPO4), 2.898 grams sodium phosph~te, monobasic monohydrate (NaH2PO4nH20), and 1.0 gram of the Kathon0 CG microbicide to 1.0 liter of tli~tilled water. The buffer formulation was tested using a pH meter and electrode, to have a pH
of 7.5. Approximately 100 ml of the pho~ te buffer was added to the 3.15 grams of pl~tini7f~d activated carbon, and was mixed for 7 days. The buffer was replaced after the first 3 days of mixing by allowing the ~ ~ activated carbon to settle, decanting off 60 ml of the used buffer, and replacing it with 100 ml of fresh buffer. The ~ ule was 213885~

~o-then vacuum filtered after the 7 days of mixing, and the neutralized carbon was washed while under vacuum filtration using 100 ml of buffer. The vacuum was ~ in~l for about 15 to 20 seconds after the bulk of the buffer had been pulled l~l~oug~ the carbon to slightly dry the carbon and improve h~nrlling of the m~t~riql S The pl~l;ni7~d activated carbon (PAC) was then mixed with 625 mg of Pentex~
bovine serum albumin (BSA). The 625 mg of BSA was first added to a flask Con~ ing the PAC and an additional 40 ml of buffer. The BSA and PAC were gently mixed with a labo,~t~.~ rotator and allowed to sit for 1/2 hour to permit the BSA to dissolve. The ule was again gently mixed overnight at a speed setting of 3.5 for ~pn)~ t~ly 18hours at room t~.llpel~lul~ . The BSA-PAC ~ clur~ was then vacuum filtered and washed under the vacuum filtration with 100 ml of buffer. Again, the vacuum was applied for about 20 seconds after the bulk of the buffer was pulled through the BSA-PAC to dry the BSA-PAC to ~tween about 60 to 70 percent moisture. The BSA-PAC was then refrigerated for future use in the active and inactive layer inks for screen printing.
The active layer ink was forrnl~lqt~ by adding 5.0 grams of a binder resin, available as product ~.~...~. 8101 RS from lUetech, to 2.0 grams of the BSA-PAC (as pr~ared above). To this u~ lU~, 0.25 gram of Triton~9 X-100 s--rf~tq-nt was added as a ~lillling flow aid and wefflng agent for the layer. The llli~lure was then milled using a standard paint industry three roll mill. 1.0 ml of AlbessoT thinner, available from Metech as 8101 RS thinner, was added to the ~ni~lur~, after the first milling was completed to adjust the viscosity of the paste for printing pU1~03eS. The ~ixlure was then milled for a second period. Lastly, 0.4 gram of g1ucose oYiflq~ available from Biozyme I~,~to,ies, was added and milled into the ll~clule. The active paste was then screen-printed onto con~lucfive strip 66 electrode portion to form working electrode 90.
EXAMPLE III
An inactive layer ink, used to form the intel~reilce cGIlec~ g electrode 92, wasformulated using the procedure set forth in Example II. The inactive layer, however, does not include any catalytically active quantity of an enzyme such as glucose oxidase.
The inactive layer ink was prepaled by milling 5.0 grams of binder resin with 2.0 grams of BSA-PAC (as p,epaled in Example Il), 0.25 gram of Triton~ X-100 surfactant and 1.0 21388S~

ml of AlbessoT (8101 RS) thinner. To this ~ lure an additional 0.4 gram of Pentex~
BSA was added and milled. Inactive layer paste was then screen-printed onto conductive strip 70 ~ de portion to form i.,~l~er~-ce co~ ing electrode 92.

EXAMPLE IV
R~fe,ling again to FIGS. 1 through 8, one suitable construction of a sensor package 10 including the CGlll~)one:llls and design ~lbs~hl;~lly in acco~ance with an aspect of the present invention-is provided by the following combination of r1e."~
Sensor package 10 is molded of V825 acrylic plastic, available from Rohm &
Haas Corp., and inch)~Ps an open back J-body, having a width of about 0.5 inch, a main body 14 length of about 1.535 inches, and a thiclrness of about 0.37 inch. A handle 12, or gate portion, extends from the main body for aiding the insertion or removal of the sensor p~ age 10 into or from an in~ ",ent. The package 10 includes a housing 20having a ~.ll.s~ lly oval-shaped recess 22 formed therein. The recess has a length of about 0.1 inch and a width of about 0.065 inch. The recess incl~ldçs an outer perim~te~
24 and a paS~g~,~.dy 18 made up of an inlet 56 and outlet 58. The passageway enters and exits recess 22 lengthwise. Passageway 18 has a s-~b~ lly circular cross-section and a ~i~metor of app~,~ Ply 0.03 inch. As shown in FIGS. 5-8, a velocity colllpe.l~lol or bump 60 is provided in the recess 22. Velocity co",pensator 60 traverses the width of recess æ, and is ap~,o~ n~l~ly 0.065 inch in length and about 0.04 inch in width. The velocity co...l~nC-~r 60 is a bump-like pr~ ion in passageway 18 which has a radius of about 0.02 inch. The velocily co...~ or reduces the int~rn~l volume of the sample ~`,h~.ber and creates a cross ~I;ollql area close to the inlet 56 and outlet 58 1;~,... ~.~. A gasket 26 is then provided to oor~ t, and form a seal ~-, thehou~ing recess ~.;-n~, 24 and a sensor 28 (as pç~ ed in Example I). Gasket 26 ismade from SarlinkT 2450 elastomer having a hardness of about 50 on the Shore A scale.
Gasket 26 is square-shaped, having sides of about 0.17 inch. Gasket 26 further includes a s~ nli~lly oval-shaped opening having a length of about 0.1 inch and a width of about 0.064 inch.
Gasket 26 is approximately 0.014 inch thick at its central cavity portion and approximately 0.05 inch thick at two outer sides along the length of the gasket opening.

`- 213885~

These thicker surfaces allow the gasket to fit around the holl~ing recess pprimet~pr~ while also allowing the recess 22 to be open to the sensor electrode area to form a sensor sample chal.ll~r.
As noted in Example I a solid state, planar electrochemi~l sensor is formed on S a ceramic ;,~s~,~te of about 0.025 inch thi~nP-c~, and 0.45 inch length and 0.18 inch width. The sensor 28 is placed upon a base pad made of a silicone rubber m~tPri~l having a hardness of between about 40 to 60 on the Shore A scale. The pad has a length of about 0.5 inch and a width of about 0.227 inch. The base pad 30 has a total thi~-~nP~
of about 0.058 inch, including a series of transverse pn~lrua,ons on the rear side thereof which extend about 0.015 inch from the base pad rear surface and are spaced about 0.1 inch apart. The base pad 30 also incllJdes a central lc~:~n~ular-shaped cavity on the opposile side thereof for receipt of sensor 28. The cavity is about 0.45 inch long and about 0.185 inch wide.
Lastly, a contact lead frame 32 is provided to c~nnPrl sensor 28 to an instrument which can measure and convert the current to det~,lline the glucose (or lactate)concentration in the ~mpl~ Lead frame 32, also shown in FIGS. 3 and 4, includes four leads 34, each appro~im~t,P-ly 0.041 inch wide at a base end and about 0.026 inch wide at the lead cont~t~ 50. The leads are a~pn"~im~t~Ply 1 inch in length and appro~im~Ply 0.01 inch thick. The leads 34 are made from a BeCu alloy m~teri~l, which is nickel plated to a thickness of between about 40 to 80 microin~hes, and gold plated with a microelectronic grade gold plate material to a thi~l~nP-ss of between about 20 to 50 microinches thi~P~
The lead frame 32, also molded of V825 acrylic plastic, inrll~des the leads secured to a base 36 at a first end portion 38, and a sensor recess 40 at a second end portion 42. The sensor recess 40 is about 0.042 inch deep, appr~im~tPly 0.5 inch in length, and about O.225 inch in width, for receipt of the base pad 30 and sensor 28.
Lead frame 32 incllldes a second rectangularly-shaped recess 46 that is about 0.06 inch deep, about 0.296 inch in length, and about 0.085 inch in width. The second reoess 46 is for receipt of a stabilizer bar 44, which aligns the leads 34 with the sensor contact pads (described above). The stabilizer bar 44 is a ~ lar-solid shaped piece, also molded of the V825 acrylic plastic material from Rohm & Haas Corp. The stabilizer bar is 213885~

appl~Ail..ately 0.29 inch in length and 0.07-5 inch in width and height.
After the base pad 30 and sensor 28 are placed into the sensor recess 40, leads 34 are bent around frame 32 until leads 34 come into contact with the sensor, and the stabilizer bar 44 is secured in recess 46. The lead frame is ~lJr~Aim~tp-ly 1.147 inches S ir~ length and about 0.395 inch in width. After the co---pone.,l~ incluAing the gasket 26, sensor 28, base pad 30 and contact lead frame 32 are assembled, the hou~ing and lead frame are secured together by an ultrasonic weld around the outer periphery of the contact lead frame. Four insl,~n~.ellt electrical contact surfaces 52 are exposed after the sensor package 10 is assembled. The contact surfaces are spaced between three dividers 10which extend past lead frame first end 38 about 0.064 inch, and are about 0.1 inch long and 0.033 inch wide. Instrument contact surfaces 52 have about 0.1 inch exposed for electrical contact with an instrument.

EXAMPLE V
A planar glucose sensor, constructed s~lbs~ y in accordance with EXAMPLES I-IV, was evaluated with whole blood, and the rel~tion~hir ~lween glucose con~..l.~lion in mg/dl and sensor current in n~no~mreres (nA) was plotted as shown in FIG. 12. One of the significant fealules of the sensor, as graphically illustrated in FIG.
12, is the linear relationship of ~lucose conc~ntration to sensor current. It is believed 20 that the sensor membrane 94, being both glucose and oxygen-permeable, allows a stoichiometric excess of oxygen to glucose to permeate the -le,llb,~e from whole blood r~sulting in the linear relationship from the low end to the high end of the graph.
A similar sensor was evaluated to dt;~,llline the response to lactate in whole blood. The sensor used was ~ 11Y equivalent to that constructed in EXAMPLES25 I-IV, with the exception of the use of lactate oxi~ce instead of glucose o~ ce. The relationship b~l~.~n the lactate concentration in mmole./L and sensor current innanoamperes (nA) was plotted in FIG. 13. One of the significant fealulcs of the sensor, as graphically illustrated in FIG. 13, is the linear relationship of lactate concentration to sensor current. Once again, it is believed that the membrane, being both lactate and 30 oxygen-permeable, allows a stoichiometric excess of oxygen to lactate to permeate the membrane from whole blood resulting in the linear relationship from the low end at about 213885~

~4-l.00 mmoles/L to the high end of the graph at about 20.0 mmoles/L lactate.

EXAMPLE VI
To determine the effect of the in~.rer~nce cGl~ g electrode 92, a glucose sensor l~ponse to glucose c4~c~ ;0n, with and without the coll~;ling electrode ~rp1i~ was recorded as gr~rhic~11y illustrated in FIG. 14. Electrode 92 is provided to adjust for any int~l~e,i.lg ~cies, such as the neutral species ~cet~minophen, which can diffuse through the sensor's semi-permeable membrane 94.
In this c,.a n?le, l.0 mmole/L of an int~rt;ling ~ slz~nce (~l~t~minophen) was added to a series of blood s~ es~ covering a range of glucose concentrations up to 500.0 mg/dl. As noted, the data are shown in FIG. 14 with and without the correcting electrode applied. Without the coll~ing electrode, there is ap~lv~im~te1y a 65.0 mg/dl positive offset from the case where the coll~ling electrode is applied. In other words, if the int~.r~.~noe c~ll~cling electrode is not used, a mean error of + 65.0 mg/dl is oblained over the range of glucose conc~nt-~tion~. This is enough to cause a normal blood glucose level of about 82.0 mg/dl to read outside of the normal range, to about 147.0 mg/dl if left uncoll~;led.
The glucose sensor respon~e with the coll~;ling electrode applied shows ey~c~ ntcoll~ ldlion with the ideal correction.
EXAMPLE VII
To de~,-lline the liÇeli.l,c of the present electrochemic~1 sensors, a glucose sensor, constructed s~ s~ lly in acco~ce with EXAMPLES I-IV, was used over an eYtçn~ s~ ling period, and for a large number of ~mp'es The sensor was tested over a period of sixty-nine (69) days, wl.e.~in a total of two thousand two hundred fifty (2,250) samples were ev~ t~d. The current in nA for an aqueous solution having aglucose concentration of 180 mg/dl was measured at various test points over the sixty-nine (69) day period. As shown in FIG. 15, the present glucose sensor provides aresponse over lO nA for a period of at least sixty-nine (69) days, and/or at least two thousand two hundred fifty (2,250) samples.

213885~

EXAMPLE VIII
To determine the effect of sensor post-tre~tm~nt with surfactant on the initial pelrol...ance after a storage period, sensors were tested and the rel~tio~hip between glucose concentration from about 83.0 mg/dl to about 470.0 mg/dl and sensor current in S nanoamperes (nA) was recorded in FIG. 18. A first sensor was post-treated with Triton0 X-100 (as noted above) while a second sensor was not post-treated. The sensors were stored one week at room ~.ll~dlule prior to the present ev~ tion. The w~ll~led sensor exhibits a low and non-linear l~nse to the glucose conccnt,dtion (as shown more clearly in the exploded portion of the graphical illustration shown in FIG. 18, the 10 r~sponse of the wlll~;d~d sensor exhibits sensor drift past about 200.0 mg/dl glucoæ).
This is the result of slow wetup caused by the membrane drying out during storage. On the other hand, the treated sensor exhibits a linear, fully wetup response after only one hour of wetup.
A variety of surf~ct~nts were evaluated to determine the effect of ænsor 15 post-~ fnt with a surfactant on the initial pe,rol"~ce of the sensor. An aqueous sample having a glucoæ conc~t~dlion of about 180.0 mg/dl was tested with five glucose sensors. The first ænsor had no post-tr~tm~nt, and the r~ ining sensors were se~ately post-treated with Triton0 X-100 surf~t~nt, Tergitol~ 15 surfactant from Union Carbide Chemic~l~ and Plastics Co., Inc., Danbury, CT, Tween~ 20 ethoxylated solbil~n esters surfactant and polyethylene glycol having a molec~ r weight of about 300. The sensors were tested and the rel~tion~hir bel~oen the post-tre~tm~ t and the ænsor current in n~no~ f r~s (nA) was plotted in FIG. 17. In the ~hænce of any post-tre~tmtont, glucose sensors become difficult to wetup, as evidenc~ from the low r~sponse obærved with un~l~ted sensors. This effect is the result of the membrane drying out during storage. Tre~tmelt of the ænsor with an antidrying agent, such as the surf~t~nt~ utilized herein, more than doubled the sensor current output.

EXAMPLE IX
To d~ uine the effect of membrane thickness on the linearity of a sensor's r~s~onse, a thin membrane 2-layer (about 10.0 microns), a thick membrane 2-layer (about 22.0 microns)t and a 4-layer membrane (about 22.0 microns) were separately evaluated.

2 1 3 8 8 S ~

The sensors were tested and the re1~tion~hir between the ~luc4se c4nce-ntration in an aqueous solution in mg/dl and sensor current in n~o~mreres (nA) was plotted in FIG.
19. The multi-layer ~el~bl~es were all p~ ed from anionically stabilized, water-based hydroxyl endblocked polydimethyl~ilox~ne elastomer c~l-t~ining about 14.0 S percent by weight colloidal silica, commercially available as FC-61 coating from Dow Corning, MiAl~nd, MI. As can be observed from FIG. 19, multiple layers improve sensor ~ G Ço"-,ance as evidenr~d by a linear ,~ ~.~nse. The thick 2-layer me",bldnes of the same thickness (about 22.0 microns) as the 4-layer membranes exhibit higher output and a non-linear response to glucose. The 4-layer membrane provides improved 10 ~lro"~ ce due to the elimin~tion of membl~dne defects. The 2-layer membrane has me",bldne defects which allow an excess of gll~c4se, with respect to oxygen, to pass through the membrane thereby accounting for the non-linearity of the response, as well as the higher output.
A similar evaluation was pe.~""ed CGIIIp~illg a 2-layer (about 11.0 microns) and a 4-layer (about 18.0 microns) spin-cast membrane of FC-61 coating rn~eri~l The sensors were tested and the relationship between the ~ c~se c4nc~ntr~tiQn~ ranging from about 69.0 mgtdl to about 487.0 mg/dl, and sensor current in nanoamperes (nA) was plotted in FIG. 20. Again, the 2-layer spin-cast membrane compriced defects which allowed an excess of glucose with respect to oxygen to permeate the membrane, which 20 resulted in higher output and a non-linear r~onse.
A 2-layer (about 10.0 microns) spin-cast membrane was also colllpared to a stenciled membrane (about 65.0 microns)- to de~l"~ine an effective membrane thir~ne-s~.
The ~,.e",~anes were comprised of the cGIllll~clcially available FC-61 coating m~t.ori~l (as noted above). The sensors were tested and the rel~tion~hir between the glucose 25 concenl,dlion, up to about 500.0 mg/dl, and the sensor current in n~no~mreres (nA) was plotted in FIG. 21. The thick, stenr-il~ membranes exhibit slow wetup as evidenced by the non-linear glucose concentration response and the low output. It was observed that membranes with a thickness of 65.0 microns are too thick to provide for a useful glucose response. Note the positive deviation from a linear response of the stenciled film in the 30 inset graph. Moreover, the thick stenciled membranes had a slow response time of greater than about 60 seconds. The 2-layer, spin-cast membrane exhibits high output and 21~885~

non-linear response (as described above).

EXAMPLE X
To detel",ille the effect of incoll,ol~d~ing a surfactant in the p!~tini7~d activated carbon (PAC) material on p~.çu~ ance versus storage, a glucose sensor recpol-se was evaluated with no surfactant in the PAC after one day and after 21 days in storage at room ~.~ lure. The sensors were tested and the relationship between glucose co~n~.a~ion, up to about 500.0 mg/dl, and sensor current in nano~mreres (nA) wasplotted in FIG. 22. As shown in FIG. 22, the sensor output degrades over time if a surfactant, such as Triton~ X-lO0, is not added to the PAC material. FIG. 16 shows the same effect as FIG. 22 but with the optimi_ed multi-layer membrane.
FIG. 23 is a graphical illustration of the glucose concentration, up to about 500.0 mg/dl, and sensor current in n~no~mr~res (nA) wherein the glucose sensors include Triton~ X-lO0 surfactant in the PAC m~ten~1. The ~ ition of the surfactant to the PAC, lS active and inactive layers, aids in sensor wetup of aged sensors. The addition of the s lrf~rt~nt in the PAC material provides for equivalent ~,fo~ ce in new and agedsensors.

EXAMPLE XI
To determine the effect of adding a sllrfact~nt to membrane 94 on the sensors ~.ro""~ce, glucose sensors were tested and the relationship between glucose conce,~ lion, up to about 500.0 mg/dl, and sensor current in n~no~mreres (nA) was plotted in FIG. 24. The sensor membrane 94, comprised essenti~11y of the commercially available FC-61 coating m~teri~l (as d~,ibed above) was applied to se~le sensol~, one sensor also includ~ a surfactant m~t~r~ Makon~ lO surfactant available from Stepan Co., Northfield, IL. Both membranes were 2-layer, spin-cast membranes about ll.0 microns thick. The addition of a surfactant in the me",bldne provides improved wetup and higher rc~onsc to glucose concentration. This effect can be minimi7~d,although not elimin~t~d if membranes are post-treated with an antidrying agent, as shown in FIGS. 17 and 18.
Although particular embodiments of the invention have been described in detail , ~8-for pUl~)O~S of illustration, various mo lifications may be made without departing from the spirit and scope of the present invention. Design conQi~lerations may alter the configuration of the sensor and/or the sensor package to optimize the efficiency of certain applications and minimi7~ the cost ~ ted with the production and use thereof.
S Accoldingly, this invention is not to be limited except by the appended claims.

TABLE I

SCREEN PRINTED LAYERS
INK OVEN/FURNACE TE~IP./REClPE

Ag/AgC1 OVEN 75C 30 MINU~S
6 CELLULOSE ACETATE OVEN 55C 10 MIN[~S

MINUTES
7 BSA-PAC (INACIIVE) OVEN 55C 20 MIN~
8 GLUCOSE OXIDASE (ACTIVE) OVEN 55C 20 MINI~S

Claims (58)

1. A solid state, multi-use electrochemical sensor comprising:
an electrically nonconductive substrate;
a working electrode, including an electrically conductive material adhered to a portion of said substrate, a first portion of said conductive material being covered with an electrically insulating dielectric coating, a portion of said conductive material being covered with an active layer comprising a catalytically active quantity of an enzyme carried by platinized carbon powder particles, wherein said particles are distributed throughout said active layer;
a counter electrode, including a second electrically conductive material adheredto a second portion of said substrate, a portion of said second conductive material being covered with an electrically insulating dielectric coating, and at least one portion of said second conductive material remaining uncovered by said electrically insulating dielectric coating;
a reference electrode, including a third electrically conductive material adhered to a third portion of said substrate, a portion of said third conductive material being covered with said electrically insulating dielectric coating, and at least one portion of said third conductive material remaining uncovered by said electrically insulating dielectric coating; and a a semi-permeable membrane covering said working electrode.

2. The sensor of claim 1, wherein said substrate is planar and comprises a material selected from the group consisting of ceramics, glasses, refractories and combinations thereof.

3. The sensor of claim 1, wherein said conductive material comprises a composite admixed with a glass binder.

4. The sensor of claim 1, wherein said conductive material comprises a thick-film paste comprising a metal selected from the group consisting of silver, gold, and platinum.

5. The sensor of claim 4, wherein said electrically conductive material comprises a high-purity, thick-film platinum paste.

6. The sensor of claim 1, wherein said second electrically conductive material comprises a thick-film paste comprising a metal selected from a group consisting of silver, gold and platinum.

7. The sensor of claim 1, wherein said dielectric coating comprises a material selected from the group comprising of ceramics, glasses, polymers, andcombinations thereof.

8. The sensor of claim 1, wherein said covered portions of said electrically conductive material are defined according to a thick-film silk-screening technique.

9. The sensor of claim 1, wherein said semi-permeable membrane is permeable to low molecular weight analyte and comprising a mixture of silicone and silica.

10. The sensor of claim 1, wherein said semi-permeable membrane is an anionically-stabilized, water-based hydroxyl endblocked polydimethysiloxane elastomer comprising about 10.0 percent silica, by weight.

11. The sensor of claim 1, wherein said semi-permeable membrane is an anionically-stabilized, water-based hydroxyl endblocked polydimethysiloxane elastomer comprising about 14.0 percent silica, by weight.

12. The sensor of claim 1, wherein said third electrically conductive material has printed over it a silver/silver chloride thick-film paste.

13. The sensor of claim 12, wherein said third electrically conductive material has deposited over it cellulose acetate.

14. The sensor of claim 1, wherein said sensor further comprises an interference correcting electrode, including an conductive material adhered to a portion of said substrate, a first portion of said conductive material being covered with said electrically insulating dielectric coating, and a second portion of said conductive material being covered with an inactive layer comprising an inactive protein immobilized onto platinized carbon powder particles, wherein said particles are distributed substantially uniformly throughout said inactive layer.

15. The sensor of claim 1, wherein said electrochemical sensor is a glucose sensor, and said enzyme is glucose oxidase.

16. The sensor of claim 1, wherein said electrochemical sensor is a lactate sensor, and said enzyme is lactate oxidase.

17. The sensor of claim 1, wherein said sensor is adapted to effectively operate for the lesser of one thousand (1,000) uses or thirty (30) days.

18. The sensor of claim 1, wherein said sensor is adapted to effectively operate for the lesser of two thousand (2,000) uses or sixty (60) days.

19. A solid state, multi-use glucose sensor, comprising:
an electrically nonconductive substrate;
a working electrode, including an electrically conductive material adhered to a portion of said substrate, a first portion of said conductive material being covered with an electrically insulating dielectric coating, and a second portion of said conductive material being covered with an active layer comprising a catalytically active quantity of glucose oxidase immobilized onto platinized carbon powder particles, wherein said particles are distributed substantially uniformly throughout said active layer; and a counter electrode, including a second electrically conductive material adheredto a second portion of said substrate, a portion of said second conducive material being covered with said electrically insulating dielectric coating, and at least one portion of said second conductive material remaining uncovered by said electrically insulating dielectric coating;
a reference electrode, including a third electrically conductive material adhered to a third portion of said substrate, a portion of said third conductive material being covered with said electrically insulating dielectric coating, and at least one portion of said third conductive material remaining uncovered by said electrically insulating dielectric coating; and a glucose and oxygen-permeable membrane covering said electrodes.

20. The sensor of claim 19, wherein said silicone membrane is an anionically-stabilized, water-based hydroxyl end blocked polydimethysiloxane elastomer comprising about 14.0 percent silica, by weight.

21. The sensor of claim 18, wherein said third electrically conductive material comprises a silver/silver chloride thick-film paste.

22. The sensor of claim 18, wherein said sensor further comprises an interference correcting electrode, including an electrically conductive material adhered to a portion of said substrate, a first portion of said conductive material being covered with said electrically insulating dielectric coating, and a second portion of said conductive material being covered with an inactive layer comprising an inactive protein immobilized onto platinized carbon powder particles, wherein said particles are distributed substantially uniformly throughout said inactive layer.

23. A sensor package, comprising:
a housing having a recess formed therein, said recess having a perimeter and at least one passageway connected to said recess;
a solid state, planar electrochemical sensor underlying said recess, a gasket contacting said recess perimeter and said solid state, planar electrochemical sensor to form a seal therebetween, wherein said housing, gasket and sensor define a sample chamber.

24. The sensor package of claim 23, wherein said package further comprises a contact lead frame assembly including the plurality of leads secured to said frame at a first end and a recess for supporting said electrochemical sensor at a second end thereof.

25. The sensor package of claim 23, wherein said contact lead frame further comprises a stabilizer bar secured to said plurality of leads to maintain each lead in predetermined position and aligning said leads with contact pads on a surface of said electrochemical sensor.

26. The sensor package of claim 24, wherein said contact lead frame assembly further comprises a recess in said housing for receipt of said stabilizer bar when said plurality of leads are properly positioned within said package.

27. The sensor package of claim 23, wherein said package further comprises a sensor pad for supporting said sensor in said recess.

28. The sensor package of claim 23, wherein said sample chamber further comprises a velocity compensator.

29. The sensor package of claim 28, wherein said velocity compensator is a molded part of said housing facing said sensor.

30. The sensor package of claim 25, wherein said sample chamber has a volume of from about 3.0 to about 5.0 microliters.

31. A method for forming a solid state, multi-use electrochemical sensor, comprising:
selecting an electrically nonconductive substrate;
depositing an electrically conductive material onto a portion of said substrate;depositing an active layer over a portion of said conductive material to form a working electrode, wherein said active layer comprises a catalytically active quantity of an enzyme immobilized onto platinized carbon powder particles, which are distributed substantially uniformly throughout said active layer; and depositing a second electrically conductive material onto a second portion of said substrate to form a counter electrode;
depositing a third electrically conductive material onto a portion of said substrate to form a reference electrode; and depositing a semi-permeable membrane over said electrodes.

32. The method of claim 31, wherein said semi-permeable membrane is permeable to a low molecular weight analyte and comprising a mixture of silicone and silica.

33. The method of claim 31, wherein said semi-permeable membrane is anionically-stabilized water-based hydroxyl end blocked polydimethysiloxane elastomer comprising about 14.0 percent silica, by weight.

34. The method of claim 31 further comprising the step of depositing a fourth electrically conductive material onto a portion of said substrate;
depositing said electrically insulating dielectric material over a portion of said fourth conductive material; and depositing an inactive layer over a second portion of said fourth conductive material to form interference correcting electrode, wherein said inactive layer comprises an inactive protein immobilized onto platinized carbon powder particles, which are distributed substantially uniformly throughout said inactive layer.

35. In planar electrodes for use in glucose or lactate determinations in vitro, said electrodes each having an insulating base layer, a conductive layer, an overlying active layer and an outer protective membrane permeable to oxygen and glucose or lactate, the improvement comprising:
said active layer comprising an enzyme reactive with one of glucose or lactate, and platinized carbon powder particles, whereby said active layer is capable of causing formation of H2O2 in amounts proportional to the amount of said one glucose or lactate when said one of glucose or lactate is exposed to said active layer; and and said outer protective membrane comprising a silicone compound having an additive incorporated therein for enabling transport of said oxygen and one glucose or lactate therethrough whereby said electrode enables rapid and accurate determination of said one glucose or lactate concentration.

36. The electrode of claim 35, wherein said silicone comprises a hydroxyl end blocked polydimethylsiloxane elastomer.

37. The electrode of claim 35, wherein said membrane has a plurality of thin layers.

38. The electrode of claim 37, wherein said membrane has four layers and a total thickness of less than about 65.0 microns.

39. The electrode of claim 35, wherein said membrane additive includes at least about 10.0 percent silica, by weight.

40. The electrode of claim 35, wherein said membrane comprises an anionically stabilized, water-based hydroxyl endblocked polydimethylsiloxane elastomer comprising about 14.0 percent silica, by weight.

41. The electrode of claim 35, wherein said membrane is post-treated with an anti-drying material to prolong the storage life or wet-up of said electrode.

42. The electrode of claim 41, wherein said anti-drying material is a high boiling point, water soluble, hydrophilic polymer liquid anti-drying agent.

43. The electrode of claim 41, wherein said anti-drying material is polyethylene glycol having a molecular weight of between about 200 and about 600.

44. In a sensor having a plurality of electrodes positions in a sample chamber through which a fluid to be tested is flowed, said sample chamber having a flow path, a sample inlet and outlet each having a cross-sectional area less than thecross-sectional area of a portion of said chamber, the improvement comprising:
a velocity compensator comprising a structural barrier mounted in said flow pathbetween said inlet and outlet to reduce said cross-sectional area of said chamber in said flow path and to substantially maintain stability in fluid velocity when flowing through said chamber.

45. The sensor of claim 44, wherein said velocity electrodes or is positioned to extend towards said electrodes without obstructing fluid flow over said electrodes.

46. The sensor of claim 44, wherein said chamber is a molded plastic chamber and said velocity compensator is an integral extension thereof positioned to partially obstruct said flow path.

47. In a sensor mounted in a housing and having a plurality of electrical contacts spaced close to each other, and a plurality of elongated axially extending electrical leads connected to said contacts, the improvement comprising:
said leads being spaced apart by a stabilizer bar attached to said leads and which positively positions said leads in proper position to electrically contact said electrical contacts.

48. The sensor of claim 47, wherein said leads are resilient and are urged into contact with said contacts by said stabilizer bar.

49. The sensor of claim 48, wherein said leads are attached to a lead frame base which provides a contact area for connection of said leads to an electrical testing system.

50. The sensor of claim 49, wherein said lead frame base mounts a plurality of electrodes and forms a part of said housing, said leads comprising spring tips being attached to said contacts.

51. A sensor comprising a semi-permeable membrane for contacting a test sample, said membrane being treated with a high boiling point, water soluable hydrophilic polymer liquid anti-drying agent so that the sensor storage life or wet-up is prolonged.

52. The membrane of claim 50, wherein said anti drying agent comprises a material selected from the group consisting of surfactants or polyethylene glycols.

53. A method for prolonging the storage life or wet-up of an electrochemical sensor including a semi-permeable membrane, said method comprising treating the semi-permeable membrane with a high boiling point, water soluble, hydrophilic polymer liquid anti-drying agent.

54. An electrochemical sensor comprising.
an electrically nonconductive substrate;
an interference electrode, including an electrically conductive metal adhered toa portion of said substrate, a first portion of said conductive material being covered with an electrically insulating dielectric coating, and a second portion of said conductive material being covered with an inactive layer comprising an inactive protein immobilized onto platinized carbon powder particles, wherein said particles are distributed substantially uniformly throughout said inactive layer; and a semi-permeable membrane covering said interference electrode.

55. A process for forming a paste for an electrochemical electrode comprising:
(a) mixing platinized carbon powder particles with a neutral buffer;
(b) mixing a protein with the buffered activated carbon of step (a) to form a protein-platinized activated carbon composition;
(c) mixing a binder resin with the protein-platinized activated carbon composition of step (b);
(d) milling the mixture of step (c); and (e) mixing either an enzyme or an inactive protein to the milled product of step (d) to form a paste.

56. A paste for an electrochemical electrode comprising:
(a) platinized carbon particles, (b) protein;
(c) resin binder; and (d) either an enzyme or an inactive protein.

57. A process for forming a cellulose acetate paste for an electrochemical electrode comprising:
mixing cellulose acetate with a low vapor pressure solvent to form a paste with a high viscosity.

58. A cellulose acetate paste comprising:
(a) cellulose acetate; and (b) a low vapor pressure solvent.