potentiometric ion selective electrodes

7/30/2019 potentiometric ion selective electrodes

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Talanta,Vol. 36, No. I/2, pp.271-278, 1989 0039.914oja9 3.00+ 0.00Printed in Great Britain. All rights reserved Copyright 0 1989Pergamon Press plc

POTENTIOMETRIC ION- AND BIO-SELECTIVEELECTRODES BASED ON ASYMMETRIC

CELLULOSE ACETATE MEMB~N~SGEUN SIG CHA and MARK E. MEYERHOFF*

Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, U.S.A.(Received 6 lriay 1988. Accepted 14 Jufy 1988)

Summary-The potentiometric response properties of ammonium-, carbonate-, and proton-selectiveelectrodes prepared by incorporating appropriate neutral carriers within novel asymmetric celluloseacetate membranes are reported. The membranes are formed by first casting a thin layer of cellulosetriacetate without carrier, hydrolyzing one side of this film with base, and then on the other side castinga second layer of cellulose triacetate containing the membrane active components. The resultingasymmetric ion-selective membranes function equivalently, in terms of selectivity and response slopes,to non-asymmetric cellulose triacetate membranes and conventional poly(viny1 chloride)-based mem-branes. The hydrolyzed surface of the asymmetric membranes can be activated in aqueous solution withcarbonyldiimidazole for the direct immobilization of proteins on the surface of the membranes, withoutloss in potentiometric ion-response. As an example, the immobilization of urease on the surfaces ofammonium- and carbonate-selective membranes yields potentiometric bio-selective urea-probes withdesirable dynamic response properties.

Over the last two decades, a variety of potent~ome~~cion- and gas-selective membrane electrodes have beenused as detection elements to prepare novel bio-selective electrodes. Such bio-electrodes are gener-ally fabricated by immobilizing appropriate biologi-cal species (e.g., enzymes, intact cells, antibodies, etc.)in a layer adjacent to the sensing membrane of theelectrodes. Often, this process involves covalentlyattaching the bioreagents to a secondary membranewhich can then be mounted on the ion- or gas-sensingmembrane. Alternatively, the bioreagents can becrosslinked or entrapped within a gel layer adjacentto the sensing surface. However, the adhesiveness,thickness, and enzyme-loading factor of these layersare critical to the performance (including responsetimes and stability) of the resulting devices. Inthis paper, we report on the development of a newtype of ion-selective polymeric membrane to whichenzymes and other biospecies can he. covalentlyattached directly, for the purpose of preparing poten-tiometric bio-selective electrodes with improved re-sponse properties.Interest in using solvent/polymeric membrane elec-trodes as the sensors in bio-electrodes arises from thehigh ion-selectivities, rapid response times (comparedto gas-sensors), and ease of fabrication (for dispos-able sensors) associated with these devices. Tradi-tionally, the most analytically useful membranes areprepared by incorporating electrically neutral carriermolecules into plasticized poly(viny1 chloride) (PVC)membranes.9-2 Direct covalent attachment of bio-*Author to whom all correspondence should be addressed.

reagents to the surfaces of such membranes wouldrequire the incorporation of functional groups, eitherin the form of membrane-soluble lipophilic additivesor derivatized polymeric materials. However, suchchanges in membrane composition can result inloss of ~tentiome~c ion-~l~ti~ty.~13 Recently,various groups have demonstrated that carboxylated-PVC can be used as a replacement for regular PVCwithout decreasing the selectivity of neutral carrier-based membranes.146 Moreover, certain enzymes orcrosslinked enzyme gels have been shown to adheretightly, albeit non-covalently, to the surfaces of thesecarboxylated membranes.**One of our long-term goals has been to devisemethods for covalent attachment of very thin films,even monolayers, of bioreagents directly to the sur-face of polymeric ion-selective electrodes (ISEs)without altering the ion-response of the electrodes.Although some success has been achieved by usingaminated-PVC as a membrane matrix,16 we nowdescribe a new approach involving asymmetric plas-ticized cellulose acetate membranes. Though ISEs forcalcium, based on cellulose acetate membranes, havebeen described previously,9 the use of cellulose ace-tate in the fabrication of solvent/polymeric electrodesis not widespread, nor have there been any attemptsto use such membranes to prepare biosensors.In the proposed method, one side (the sample side)of a thin layer of cellulose triacetate cast withoutactive membrane components is hydrolyzed withbase. A second layer of cellulose acetate containingthe appropriate neutral carrier and plasticizer is thencast on the other side of the cellulose triacetate film

271


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GEUN SIG CHA and MARK E. MEYERHOFF

hydrolysisin IM NaOH OH OH OH

I thylenediaminein 1 M Na,CO,. pH 100 0 0t=o &o &ot&-I th iH6H2 bH2 tH2bH2 tH2 bH2ItH2 rjH2 rjH2

CTA/plasticirer/neutral carrierin CH&I#ZHCI, (2nd layer)

~ glutaraldehyde/enzyme ~? ? ?c=o C=O c.=o in 0.05 M phosphate, pH 7I$H +H 6,92 92 +2$=2 $H2 F2NH2 NH2 NH2

CTAIplasticizerl in CH,C(,/CHCI,neutral carderI

(2nd layer)

l/jjjqqOH OH OH

CDIactivationI

enzyme

I

in 0.1 M PhosphatepH .8

Ion-selective72embraneenzyme enzyme enzymeFig, 1.General schemes used to prepare enzyme-immobilized asymmetric ion-selective cellulose triacetatemembranes; (A) the direct CD! method for the hydroxylated membrane; (B) the glutaraldehyde methodfor aminated membrane.(see Fig. 1). Ammonium-, carbonate-, and pH-selective membranes serve as models for these in-vestigations. These membranes are prepared withnonactin, trifluoroacetyl-p-butylbenzene, and tri-dodecylamine, respectively. The hydroxyl groups onthe hydrolyzed surface of the membrane can beactivated in aqueous solution with carbonyl-diimidazole (CDI). Three different methods can thenbe used to attach enzymes or other proteins directlyto the surface of the ion-selective electrodes. The

relative advantages and disadvantages of each attach-ment method are demonstrated by evaluating theperformance of a number of urea-selective electrodesprepared with immobilized urease.

EXPERIMENTALApparatus

The external reference electrode was a Fisher double-junction Ag/AgCl electrode with an outer cracked-beadjunction. All membranes were mounted in Phillips electrode


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Asymmetric cellulose acetate membranes 273bodies (IS-561) (Glasblaserei Miiller, Zurich). For poten-tiometric measurements of ion-responses, the ISEs andexternal reference electrode were connected through high-imoedance amnlifiers to a Zenith Z-100 PC computerequipped with ^a Data Translations (DT2801) analog-to-digital converter. The potentiometric response of enzymeelectrodes was measured with a Fisher Model 620 pH/mVmeter and recorded on a Fisher Recordall Series 5000strip-chart recorder.Reagents

Cellulose triacetate (CTA), 1,l -carbonyldiimidazole(CDI), bis(Zethylhexy1) sebacate and nonactin wereobtained from Fluka (Ronkonkoma, NY), and dipentylphthalate, tridodecylamine, and sodium tetraphenylboratewere products of Eastman Kodak (Rochester, NY).Trifluoroacetyl-p-butylbenzene (TFABB) was purchasedfrom Specialty Organics, Inc. (Irwindale, CA), trido-decylmethylammonium chloride from Polysciences, Inc.(Warrington, PA), 1,1,2,2,-tetrachloroethane from AldrichChemical Co. (Milwaukee, WI) and urease (Type VII, fromjack beans) from Sigma Chemical Co. (St. Louis, MO).All other chemicals used were analytical-reagent grade.Standard solutions and buffers were prepared withdemineralized water.

Preparati on of vari ous ion - and bio-selective membranesFigure 1 summarizes the schemes used to prepare asym-metric ion-selective CTA membranes with immobilized en-zymes. Details of these methods are described below.Hydroxyl ated or aminated cell ul ose tri acetate membranes.A thin membrane (e.g., 50 pm) was prepared by dissolving100 mg of CTA in a mixed solvent (1.5 ml of methylenechloride, 0.5 ml of chloroform and 0.5 ml of 1,1,2,2-tetra-chloroethane) and casting the mixture in a 36-mm (id.) glassring placed on a glass plate. The solvent was allowed to

evaporate for 2 days. Acetyl groups on the bottom side ofthe membrane were then hydrolyzed by floating the mem-brane on 1M sodium hydroxide for 4.5 hr. After washingwith distilled water, the membrane was air-dried.To introduce amino groups onto the membrane, thehydroxyl groups (resulting from the hydrolysis procedure)of the membrane were activated with CD1 by immersing thebottom side of the membrane in 20 ml of cold distilled water(4) and adding an excess of l,l-carbonyldiimidazole(about 50 mg) in 10-15 mg portions over a 15-min period.After brief washing with cold water, the membrane wasimmersed in a 12.5% (w/v) ethylenediamine solution in 1Msodium carbonate buffer @H 10) for 3 hr at room tem-perature. After extensive washing with water, the membranewas air-dried.Asymmetri c cellul ose tri acetate ion-selective membranes.The hydroxylated or aminated membranes were placed ona glass plate with the derivatized surface face down. Theunmodified upper surface of the membrane was then pre-treated for 30 min with 1.2 ml of methylene chloride insidea 22-mm (i.d.) glass ring placed on top of the membrane.During this period, the membrane was kept inside a desic-cator to reduce evaporation of the methylene chloride. Amixture containing the appropriate amounts of membranecomponents, depending on which ion-selective membranewas being prepared, was then put inside the glass ring. Inall cases, 35 mg of CTA was utilized with a mixed solventof 0.8 ml of methylene chloride and 0.8 ml of chloroform.Additional components (i.e., plasticizer, neutral carrier, orlipophilic additive) for each ion-selective system were asfollows: 6 mg of nonactin and 120 ~1 of dipentyl phthalatefor NH:; 100 ~1 of bis(Zethylhexy1) sebacate, 8.3 mg oftrifluoroacetyl-p-butylbenzene, and 3.1 mg of tridodecyl-methylammonium chloride for CO:-; 155 ~1 of dipentylphthalate, 20 mg of tridodecylamine, and 12 mg of sodiumtetraphenylborate for H+. Again, the mixed solvent wasallowed to evaporate very slowly by keeping the membrane

in a vacuum desiccator with the tap only slightly open. Inthis manner, the two membrane layers were fused into asingle asymmetric membrane.Smaller disks were punched from these membranes andmounted in Phillips electrode bodies with the derivatizedside facing the outer sample solution. The inner fillingsolution varied, denending on which ion-selective membranewas being evalu&ed (ClM NH&l for NH.,+-selectivemembranes: O.lM NaH,PD./O.lM Na,HPO,IO.OlM NaClfor CO:--selective membranes; 0.02& NaH,PO,/O.O3MNa,HPO,/O.OlSM NaCl for H +-selective membranes). Allpotential measurements were made at room temperature.,with 200 ml of sample solution, after conditioning themembranes overnight.

Dir ect i mmobilization of urease on hydroxylated asym-metric ion-selective membranes. Hydroxylated membranesmounted in electrode bodies were immersed in cold waterand activated by adding an excess of l,l-carbonyl-diimidazole as described above for amination. After briefwashing of the membranes with cold water, 10 ~1 of ureasesolution [1 mg of urease (615 pmole units/mg) per 10 ~1 ofO.lM sodium phosphate/0.5M sodium chloride buffer @H7.8)] were applied directly to the surface of the membranes.The reaction was allowed to continue for 12 hr at 4. Themembranes were thoroughly washed with 0.05M Tris-HClbuffer (PH 7.2) to remove unbound enzyme and blockunreacted active groups.

Immobil ization of urease on aminated ion-selective mem-branes. For the one-step glutaraldehyde method, 10 ~1 ofurease solution (1 mg of urease per 10 ~1 of 0.05M sodiumphosphate buffer, pH 7.0) and 4.5 ~1 of 2.5% glutaraldehydesolution in the same buffer were sequentially applied to theaminated surface of the ion-selective membrane mounted inan electrode body.For the two-step glutaraldehyde method, the membranewas first activated in 2.5% glutaraldehyde solution for 5min. After a brief washing of the membrane, 10 ~1 of uneasesolution were applied directly to its surface.After the coupling reactions for 12 hr at 4 the membraneswere thoroughly washed with Tris-HCl buffer to remove anyexcess of glutaraldehyde and unbound enzyme and stored inbuffer at 4 when not in use.Evaluating potentiometri c response of ion-selective andenzyme electrodes

The potentiometric responses of various CTA membranesto ions, and corresponding enzyme electrodes to urea, wereevaluated with different background electrolytes, dependingon which ion-selective system was being examined (O.OSMTris-HCl, pH 7.2 for NH: or urea response with anNH,+-selective membrane; O.lM Tris-H,SO,, pH 8.75 forCO{- response or pH 8.4 for urea-response with aCOf--selective membrane: 11.4mM boric acid/6.7mM citricacid/lO.OmM NaH,PO, for pH response, or O.OOlM Tris-HCl/O.lM NaCl, pH 7.0 for urea response with a proton-selective membrane). The calibration plots were obtainedfrom additions of standard inorganic salt or urea solutionsto 200 ml of background electrolyte at room temperature.The solutions were magnetically stirred throughout and thesteady-state or equilibrium potentials were recorded.

RESULTS AND DISCUSSION

A scanning electron micrograph of the cross-section of a typical asymmetric ion-selective celluloseacetate membrane is shown in Fig. 2. It can be seenthat the membrane consists of two fused layers; adense ion-selective plasticized layer covered with athin modified layer (upper layer). The hydrolyzed


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GEUHSIG CHA and MARK E. MEYERHOFF

Fig. 2. Scanning electron micrograph showing the cross-section of a typical asymmetric ion-selectivecellulose triacetate membrane.

hydrophilic thin layer (approximately 5 pm in thick-ness) is immiscible with organic solvents (plasticizers)and can be used for covalent immobilization ofbiomolecules such as enzymes, by CDI-activationmethods. Although the initial studies were focused onthe direct hydrolysis of homogeneous plasticizedCTA membranes, this approach was abandoned sincea much longer period (more than 5 days) is requiredto produce a sufficient number of surface hydroxylgroups and the critical membrane active components(e.g., nonactin) may be degraded during thishydrolysis period.Previously, organic solvents (e.g., dioxan oracetone) have been used for the CD&activation of thehydroxylic polysaccharide matrices (e.g., crossIinkedagarose or dextran. cellulose, et~.).~~* Un-fortunately, these organic solvents wifi dissolve the

membrane active components (i.e., neutral carrier orplasticizer) and may, in fact, deform the ion-selectivemembranes. Therefore, it was critical to find anactivation method which would work in aqueoussolutions. In subsequent experimens, we found thatthe CDI-activation can be done in aqueous solutionprovided that a relatively large amount of CD1 isused. Indeed, after coupling of ethylenediamine to thehydrolyzed surface through aqueous CDI-activation(as described in the Experimental section), treatmentof the membrane with trinitrobenzenesulfonate re-agent (TNBS)23 gave an immediate orange-red color,indicating that the surface of the membrane had beenaminated to a significant degree. Such an experimentprovided clear evidence that the hydrophilic layer ofthe asymmetric membranes could be derivatized andused as a site for later attachment of biomolecules.


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Asymmetric cellulose acetate membranes 275

-180L

--7 -5 -3 -1log [NH;1 ~4)

Fig. 3. Potentiometric ammonium response of various cellu-lose triacetate-based membranes doped with nonactin: (A)hydroxylated asymmetric membrane; ( X) aminated asym-metric membrane; (0) unm~ified cellulose triacetatemembrane.

Potentiometri c ion response and selectivi ty ofasymmetric membranes

The potentiomet~c response characteristics of thevarious asymmetric cellulose acetate based ion-selective membranes were evaluated and are shown inFigs. 3-5 and Table 1. As can be seen, the asymmetricammonium-selective membranes exhibit little or nochange in response slope or seIectivity comparedto the unmodi~ed cellulose triacetate membrane(Fig. 3). The response slope (57 mV per decade overthe range from lo- to IO-M NH: ) and the highselectivity for NH: relative to several other cations(Table 1) were essentially the same as those forPVC-based membranes.2*6Figure 4 illustrates the ~tentiome~c response oft~fluoroacetyl-p-butyl~~ne (TFABB)-Dodd cel-lulose acetate membranes to carbonate and severalother anions. Again, asymmetric modification (toform hydroxyl groups) had little or no effect on theresponse characteristics of the membrane. The re-

150 -

Psw 100 -

-5 -4 -3log [anion] tM)

-2

Fig. 4. Potentiometric response of a typical TFABB-dopedhydroxylated asymmetric membrane to various anions in abackground of O.lM Tris-HrSO,, pH 8.75: (A) Cl-; (x )NO,; (0) total CO, (predominantly carbonate andhydrogen carbonate); (V) salicylate.

600

400s6W

200

0~ I I I2 4 6 8 10PH

Fig. 5. Potentiometric pH response of a typical TDDA-doped hydroxylated asymmetric membrane. The pH wasadjusted by the addition of sodium hydroxide to a bufferconfining 11.4mM boric acid, 6.7mh4 citric acid, andIO.OmM NaH,PO,, and monitored with a glass membraneelectrode.sponse slope and selectivity (or lack of it with respectto salicylate) observed are essentially the same asthose found for PVC-based membranes previouslyreported by Greenberg and Meyerhoff. Similar re-sults were obtained with proton-selective asymmetricmembranes prepared with tridodecylamine as themembrane active component (see Fig. 5). Althoughthe effect of surface hydroxyl or amine groups on theselectivity of the pH-sensing system was not studiedin detail, the fact that nearly Nernstian pH-responsewas observed between pH 5 and 10 in the presence of1OmM Na + , suggests that the high selectivity relativeto alkali-metal cations is preserved when the asym-metric membrane is used.Response c~ara~terist~~s of bi o-electrodes F reparedwith asymmetr ic ion select& e membranes

All three ion-selective membranes (NH: , CO:-,pH) were evaluated as sensing elements in bio-electrodes. As a model system, we chose to immo-bilize urease on each of the asymmetric membranes,using a variety of procedures. Urease catalyzes thereaction

(NH2)*C0 f 2H,0+2NH: + CO2 + 20H-Thus, each of the products can be detected directly orindirectly by one or other of the asymmetric ion-selective membranes. In the case of the CO:- sensor,buffering the sample solution at pH 8.4 allows a fixedfraction of the CO, present to be in the form ofcarbonate anions, detectable by the membrane dopedwith TFABB.

Initial experiments involved the covalent immo-bilization of urease on asymmetric ammonium-selective membranes. In one case, the urease wasattached directly to the hydroxylated membrane bythe aqueous CDI-activation method. In further stud-ies, the urease was coupled to the surface of amine-derivatized membranes by either a one- or two-step


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276 GEUNSIGCHA and MARK E. MEYERHOFFTable 1. Selectivity coefficients of nonactin-based membranes prepared in variousmatrices

tog&.,*

60 -

O-

3g -60 -w-120 -

-180 -

Membrane matrix Li+ Na+ K+ Mg*+ Ca?+ (CH,),N+ Hi4X-A -4.7 -2.9 -0.9 -3.2 -5.0 -3.7 -4.3CTA-OH -4.5 -2.9 -0.9 -3.2 -4.8 -3.1 -4.3CTA-NH2 -4.5 -2.9 -0.9 -3.2 -4.9 -3.7 -4.2PVCf -4.5 -2.9 -0.9 -2.9 -5.0 -3.7 -5.0*Determined by the separate solution method with 0.05M Tris-HCl buffer (pH 7.2) asthe background electrolyte. Values are the average for several membranes.*From Ma ef al.b

A

:_;:i

B

I A-, I I-7 -5 -3 - 1

log [urea] Uvl)Fig. 6. Typical calibration curves for the urea electrodesprepared by using asymmetic ammonium-selective cellulosetriacetate membranes: (A) ureas~minated membrane pre-pared by the one-step glutaraldehyde method; (B)unease-hydroxyfated membrane prepared by the direct CD1activation method.glutaraldehyde procedure.25 With the one-step glu-taraldehyde method, a thin enzyme layer was formedon the surface of the membrane by crosslinkingbetween enzymes. In all cases, however, immobilizingthe enzyme had little or no effect on the responseslopes and selectivity of the resulting membranestoward ammonium ions (not shown).

Typical calibration plots for the urea electrodesprepared by using asymmetric ammonium-~l~tivemembranes are shown in Fig. 6. The urease-aminatedmembrane prepared by the one-step glutaraldehydemethod displayed a wide linear response with a slopeof 57 mV per decade in the range from lo- to lo-Murea (curve A). However, the response was reducedwhen the urease was immobiIized on the hydroxylated membrane in conjunction with direct CDI-activation (a slope of 44 mV per decade for10-5-10-2M urea; curve B). Analogous results wereobtained for the urease-aminated membrane pre-pared by the two-step glutaraldehyde method (notshown). The degree of urea response is stillsignificant, considering that the enzyme is directlyattached to the surface of the membrane as a mono-layer, without crosslinking networks between en-zymes. It is well known that the steady-state potentialof enzyme-electrodes can be affected by the amount

of immobilized enzyme. c8 Thus, in the case of ureaseimmobilized directly on CDI-activated membranes,or by the two-step glutaraldehyde method involvingaminated membranes, the enzyme loading levelsappear to be s~gni~cantly lower than those observedwith a thicker layer of crosslinked urease.Owing to an extremely fast mass transfer of speciesbetween the external bulk solution and the ion-selective surface, the response of the monolayerenzyme membranes was almost instantaneous uponadditions of urea to the sample (see curve A inFig. 7). The noise on such traces results from theeffects of magnetic stirring, causing rapid fluctuationsin the steady-state ammonium ion activities inthe very thin monolayer of enzyme adjacent to theion-selective membrane. However, more stablesteady-state potentials were obtained with the thicker

10-3M

(A) 1o-3 M (Bl

Fig. 7. Typical strip-chart recordings showing dynamicresponse of urea electrodes prepared by using asymmetricammonium-selective cellulose triacetate membranes: (A)urease-aminated membrane prepared by the one-step glu-taraldehyde method; (B) ureasehydroxylated membraneprepared by the direct CDI- activation method.


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278 GEUN SIGCtu and MARK E. MEYERHOFFlayer membrane in the range from lo-$ to lo-*A4urea (total change 125 mV), whereas bio-electrodesprepared with monolayer enzyme coverage exhibitedrelatively poor response owing to the rapid transferof H + out of the ultrathin enzyme-layer (i.e., it is notpossible to sustain a steady-state pH change adjacentto the pH-sensing membrane.)

5. G. G. Guilbault. Kodak of Enzymur ic h4ethorl s ofAnufysis, Chap. 5. Dekker, New York, 1976.6. P. W. Carr and L. D. Bowers, Immobil ized Enzymes nAnalytical and Clinical Chemistry, Chap. 5.Wiley-Interscience, New York, 1980.7. G. G. Guilbault and J. G. Montalvo, J. Am. Chem.Sot., 1970, 92, 2533.8. J. E. Brady and P. W. Carr, Anal . Chem., 1980,52,977.9. J. D. R. Thomas, Anal. Chim. Acfa, 1986, 180, 289.10. P. Oanenfuss. W. E. Morf. U. Gesch, D. Ammann, E.Prets% and W. Simon, ibid., 1986, 180, 299.11. W. Simon. E. Pretsch. W. E. Morf. D. Ammann. U.Gesch and 0. Dinten,Analyst, 1984, 109, 207.12. D. Ammann. W. E. Morf. P. Anker. P. C. Meier. E.

In summary, we have described the preparationand performance of a new type of asymmetric ion-selective membrane based on cellulose acetate. Thepotentiometric response of these membranes towardions is essentially the same as that of conventionalplasticized PVC membranes. More importantly, thehydroxylat~ side of the membranes can be used toimmobilize bioreagents covalently by a variety ofreaction schemes without affecting the ionic response 13.or selectivity of the membranes. While immo-bilization of the enzyme urease in thin crosslinked 14,films results in urea-selective bio-electrodes with agreater degree of enzyme loading, and thus larger Is.urea responses (regardless of the base ion-sensor), theability to immobilize thin monolayers of biological 16reagents on the surfaces of functional ISEs by the 17.direct CD1 method may prove extremely valuable inthe design of other types of bio-electrode systems; g.e.g., disposable immunoelectrodes, etc. Work in this 19.direction is in progress. 20.Ackno~Zedgeme~ts-The authors wish to thank Mr. GlenH. Boiling for assistance in taking scanning electron micro- 21.graphs. This work was partially supported by the NationalScience Foundation (CHE-8506695) and the National 22.Institutes of Health (GM-28882). 23._.REFERENCES

Pretsch and W. Simon, I&-Se/e& EI ectrode iev.,1983, 5, 3.D. Ammann, I on-Selective M icr oelectrodes; Pri nciples,Design, and Application, Chap. 4. Springer-Verlag,Berlin, 1986.T. Satchwill and D. J. Harrison, J. Efectroa~af.Chem.,1986, 202, 75.E. Lindcr, E. Graf, Z. Niegreisz, K. T&h, E. Pungorand R. P. Buck, Anal. Chem., 1988, 60, 295.S. C. Ma, N. A. Chaniotakis and M. E. Meyerhoff, ibid.,1988, 60, 2293.S. J. Pace, Eur. Par. Appl., EP 138150, 1985; Chem.Abstr., 1985, 103, 19426k, 1985.J. Anzai, M. Shimada, T. Osa and C. Chen, Bull. Chem.Sot. Japan, 1987, 60, 4133.T. Okada, H. Sugihara and K. Hiratani, Anal. Chim.Acta, 1986, 186, 307.G. S. Bethell, J. S. Ayers, W.S. Hancock and M. T. W.Hearn, J. Bi oI. Chem., 1979, 254, 2572.M. T. W. Hearn, G. S. Bethell, J. S. Ayers and W. S.Hancock, J. Chr omatog., 1979, 185, 46j.G. S. Bethell, J. S. Ayers, M. T. W. Heam and W. S.Hancock, ibi d., 1981, 219, 361.P. Cuatrecasas, J. Biol. Chem., 1970, 245, 3059.24. J. A. Greenberg and M. E. Meyerho~, Anal. Chim.Acfu, 1982, 141, 57.

Chemistry, H. Freiser (ed.), Vol. II, Chap. 1. PlenumPress, New York, 1980.

I . M. A. Arnold, Zon-Selective El ectrode Rev., 1986,8,85.2. G. G. Guilbault and J. M. Kauffmann, Biotechnol. 25. J. F. Kennedy, B. Kalogerakis and J. M. S. Cabral,Enzvme M icr ob. Technot.. 1984. 6, 127.Appl . Bi ochem., 1987, 9, 95. 26. G. ?r. Guilbault and G. Nagy, Anal. Chem., 1973, 45,3. M. Mascini and G. G. Guilbault. Bioserzsors. 1986. 2. 417.147. 27. R. M. Ianniello and A. M. Yacynych, Anal. Chim. Acta,4. R. K. Kobos, in Zen-Selective El ectrodes n Anaiyr icai 1983, 146, 249.

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