flexible ultrathin polydvb/evb composite membranes for the optimization of a lactate sensor

Full Paper

Flexible Ultrathin PolyDVB/EVB Composite Membranes for theOptimization of a Lactate Sensor

Kerry Bridge,a Frank Davis,b Stuart D. Collyer,b Seamus P. J. Higsonb*a Manchester Materials Science Centre, UMIST, Grosvenor Street, Manchester, M17HS. UKb Cranfield Health, Cranfield University, Silsoe, Beds, MK454DT, UK*e-mail: [email protected]

Received: October 24, 2006Accepted: December 21, 2006

AbstractAn ultrathin composite membrane has been developed as the outer covering barrier in a model amperometric lactateoxidase enzyme electrode. The membrane was formed by cathodic electropolymerization of divinylbenzene/ethylvinylbenzene at the surface of a gold coated polyester support membrane. Permeability coefficients weredetermined for O2 and lactate across membranes with a range of polymer thicknesses. Anionic interferents (such asascorbate) were screened from the working electrode by the composite membrane. The composite enzyme electrodeshowed an increased working concentration range and extended linearity of responses in comparison to an uncoatedenzyme electrode.

Keywords: Cathodic electropolymerization, Composite membrane, Amperometric enzyme electrode,Polydivinylbenzene, Lactate

DOI: 10.1002/elan.200603765

1. Introduction

Biosensor research represents an ever growing field ofinterest, with increasing numbers of sensors beingmarketedcommercially over the last 15 years [1 – 7]. Further develop-ments in membrane technology have helped to maintain asustained interest in the area of amperometric membranebased sensors [8 – 10].When measuring whole blood samples, two problems

must be overcome: Firstly interferents that may be presentin the blood (such as ascorbate) must be prevented fromreaching the electrode since they can be oxidized and giverise to erroneous readings. Secondly physiological fluids,especially blood, also have a tendency to deposit materialssuch as proteins, usually irreversibly, onto solid surfaces.This biofouling process can diminish the response of sensors– and in some cases can passivate them completely. Oneapproach is to employ two separate functional membranes[11]. An outer membrane is provided to act as a substratediffusion limiting barrier as well as to provide favorablebiocompatibility [12]. An underlying (inner) membrane isalso employed as a permselective barrier [13] and is typicallypositioned immediately over the working electrode toscreen out interferents. Unfortunately both membranesact as additional diffusional barriers through which theenzyme substrates and/or H2O2 must traverse, so hinderingtheir passage to the enzyme or working electrode; thisfrequently increases the overall response time.This has led to research towards the development of single

multifunctional ultra thin film composite membranes [14,

15]. Membranes of this type have been shown to be capableof serving as both highly substrate diffusion limiting barrierstowards enzyme substrates, whilst also effectively screeninganionic interferents from theworking electrode via a chargeexclusion principle [16]. The usual method for formation ofcomposite membranes of this type is to deposit a homoge-neous film (ideally 100 nm thickness) of a biocompatiblepolymer onto a highly porous support membrane, whichoffers mechanical support to the structure. Amperometricglucose oxidase laminate enzyme electrodes employingsuch biocompatible compositemembranes have been foundto display shortened response times [17].It should be noted in this context that the separational

performance of a homogeneous polymer film membranerelies on its chemical properties and not on its thickness perse, whereas the rate at which the separation can occur isdependent on the thickness of the membrane. It thereforefollows that the thinner a membrane is, the better will be itsperformance also. On the other hand it should be appre-ciated that if a membrane is made too thin, it will becomeexcessively fragile and thus be rendered useless. A mem-brane of substantially reduced thickness which could offerthe required separational performance, whilst still possess-ing themechanical strength of a conventional contemporarymembrane, would therefore clearly be highly advantageous.Various methods exist for themanufacture of membranes

of this type, with one of the most applicable being that ofelectrodeposition of either a conducting or non-conductingpolymer, the use of which within biosensors has beenreviewed [18]. Electrodeposition affords the possibility of

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accurate control of the location and thickness of a depositedpolymer film. The active species e.g., an enzyme is incorpo-rated within the polymeric film [18].Previous work within our group combined the use of an

active layer such as a chemically crosslinked film of glucoseoxidase – which is either (i) directly deposited onto or (ii)laminated to a supported electrodeposited membrane at anelectrode surface. Once deposited, this active layer is thencoveredwith the permselective compositemembranewhichboth screens out interferents and offers favorable biocom-patibility. Our work initially utilized highly porous commer-cial alumina membranes as supports for ultrathin polydivi-nylbenzene/ethylvinylbenzene (DVB/EVB) or polyacrylo-nitrile films [19, 20]. TheDVB/EVB system was found to beespecially suitable, giving increased linearity of glucoseresponse, good exclusion of ascorbate and favorable bio-compatibility [19]. The alumina support membranes were,however, somewhat inflexible and brittle. The next phase ofthis work was focused towards developing this sensorfurther by increasing its mechanical flexibility and soincreasing the possible scope of application within sensorsfor practical applications. A sensor that can withstandgreater manipulation would in particular allow for theproduction of different future cell designs. This led to thedevelopment of a glucose sensor containing a permselectivemembrane based on commercial polyester membranes [21],which besides their improved physical properties displayedimproved biocompatibility, stability and accuracy in re-sponses in comparison to similar enzymeelectrodes employ-ing alumina membranes.Ever since La Roche produced the first lactate sensor,

(the “Lactate Analyzer LA640”) in 1975, research hascontinued in this area. Within clinical analysis and biotech-nology, glucose and lactate are two of the enzyme substratesthat are most often determined at this present time [22].Lactate is widely distributed within blood, muscle tissue

and body fluids and is an important parameter within bothsports medicine and clinical care [23]. For example, themonitoring of lactate levels is useful in high dependencecare units and during surgery, since the determination oflactate levels may provide early detection of clinical shockand therefore this approach helps to minimize metabolicstress [24]. The monitoring of patient lactate levels isparticularly important when conditions such as myocardialinfarction, congestive heart failure, respiratory failure,pulmonary edema, septacemia or hemorrhage have beendiagnosed [25]. These conditions result in an inadequatecardiac output, causing a decrease in blood pressure. Thelimited blood flow then causes the circulatory system todeteriorate, which in turn reduces the cardiac outputfurther, causing a vicious cycle that leads to 85% of patientswho develop cardiogenic shock not surviving [26]. The pooravailable supply of oxygen to shocked tissue diminishes theoxidative metabolism and results in acidosis. When thisoccurs the cells must obtain their energy by the anaerobicprocess of glycolysis, which causes excessive quantities oflactic acid to be released into the bloodstream. In addition tothis, the poor blood flow through the tissues limits the

removal of carbon dioxide from the blood, which reacts withwater to form very high concentrations of intracellularcarbonic acid, and so the condition acidosis. Early detectionof these excessive levels of blood lactate may thereforeprove crucial to a patientMs survival. Normal physiologicalmeasurements of lactate within whole blood range between0 – 2 mM, whilst levels of lactate found in a patient sufferingfrom this type of shock rarely exceed 20 mM [27].The use of lactate sensors within sports medicine is also

widely used to monitor fitness as well as to detect injury totissues and even thrombosis [28]. Lactate sensors also allowan athlete to monitor the success of their training bydetermining the musclesM anaerobic threshold during phys-ical exercise [23]. Actively contracting muscles obtainenergy in the form of Adenosine Triphosphate (ATP)from glucose stored in the bloodstream and the breakdownof glycogen stored in themuscles. The breakdownof glucoseinitially produces pyruvic acid, which, when mixed withoxygen is converted to carbon dioxide, water andATP. If themuscles are contracting vigorously for long periods of time,however, the circulatory system begins to become dimin-ished of oxygen. Under such anaerobic conditions, thepyruvic acid produced via the breakdown of glucose isconverted to lactic acid, which leaks out of the muscles intothe blood stream and is carried around the body.During normal cellular respiration, with only moderate

physical exertion, the enzyme lactate oxidase (LOD) acts asa catalyst for the oxidation of lactate back to pyruvate andthe flow of oxygen to muscle cells is generally sufficient toprevent this build up of lactate and so prevent cramp. If,however, the oxygen supply remains diminished, thefunctioning of the body becomes impaired and the musclesfatigue very quickly.As soon as oxygen becomes available again, the lactic acid

is reconverted to pyruvic and then into carbon dioxide,water and ATP. Given that high levels of lactate within thebloodstream are detrimental to an athleteMs performance,many undergo endurance training in an attempt to enablethe body toperformat a greater pacewith aminimal amountof lactate production. This kind of training requires themonitoring of lactate levels in blood up to concentrations of22 mM [23, 28].Lactate analysis is also widely used within the food

industry. It is useful for evaluating food freshness andstability of milk, dairy products, fruits, vegetables, sausagesand wines [28]. Lactate is a major metabolite of bacterialenergy metabolism and is produced during the anaerobicfermentation of glucose and other carbohydrates [29]. Theconversion of pyruvate to lactate by bacteria within milkproduces yoghurt and many cheeses and also helps createpickled foods, soy sauce, dough breads and even chocolate.A sensor utilizing the flexible ultrathin polyDVB/EVB-

coated membranes described earlier [21] could producereliable results for all types of lactate sensor describedabove. The advantages of this systemovermany of the othertypes of sensors reported are the control of membranethickness conferred by electrochemical deposition and theflexibility of the resultant sensor. These membranes have

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www.electroanalysis.wiley-vch.de

already been shown to exclude ascorbate interference andwhen utilized tomeasure glucose in whole blood samples, toconfer excellent hemocompatibility. Results from thesesensors have been shown to correlate with standard hospitaltests for glucose with a high degree of accuracy [21]. Theflexibility of the composite membranes, however, would bemost advantageous towards the development of a flexiblesensor for use in sport science. As described earlier, theflexibility of the sensor could allow a lactate sensor to beproduced in the form of a sticking plaster, to determinelactate levels within sweat upon the surface of an athleteMsskin.This paper therefore describes the fabrication of a range

of polyDVB/EVB-coated membranes (coated via 1, 2, 3, 5and 10 voltammetric cycles) and their incorporationwithin alactate oxidase sensor. The permeability coefficients for therange of polymer-coated membranes towards the enzymesubstrates were investigated, along with the membranesMeffects on response linearity and screening of electroactiveinterferents.

2. Experimental

2.1. Reagents and Membranes

NMN-dimethylformamide, tetrabutylammonium perchlo-rate (electrochemical grade) and divinylbenzene (70 – 85%DVB, 15 – 30% 3- and 4- EVB, containing 0.1% polymer-ization inhibitor: 4-tert-butylcatechol) were all purchasedfrom Fluka Chemicals (Dorset, England).Disodium hydrogen phosphate (Na2HPO4), sodium dihy-

drogen phosphate (NaH2PO4) and sodium chloride (NaCl)were all obtained from BDH (Poole, Dorset). Lactateoxidase from Pedicoccus species (20 – 40 units/mg solid), l-lactic acid (lithium salt) bovine serum albumin (fraction V)and glutaraldehyde (grade II, 25% aqueous solution) wereall purchased from the Sigma Chemical Company (PooleDorset, UK).Aluminum oxide Chromatography Grade was obtained

from F. S. A. Laboratory Supplies (Loughborough, UK).Granular calcium hydride was purchased from FisherScientific (Leicestershire, UK).Electrolube silver conductive paint (Electrolube Ltd,

Berkshire) was purchased from Maplins Electronics, Man-chester, UK and used for the fabrication of Au counterelectrodes.Cyclopore 0.4 mm polyester membranes, were purchased

from Whatman International, Maidstone, UK. 0.015 mmpore diameter polycarbonate membranes (Poretics Corpo-ration, Livermore,USA)were used as the lowermembraneswithin enzyme/membrane laminates [17].

2.2. Buffers and Solutions

A phosphate buffer (pH 7.4) comprising 5.28� 10�2 M Na2HPO4, 1.3� 10�3 M NaH2PO4 and 5.1� 10�3 M NaCl in

deionizedwater was used for the preparation of all solutionsfor diffusion chamber mass transport investigations andlactate calibration measurements.

2.3. Preparation of Conductive Faced MembraneElectrodes and Counter Electrodes

In order to electropolymerize the DVB/EVB monomeronto the host membranes, the uppermost surface was firstrendered electrically conductive by Au sputter coating themembranes with an Edwards S150 B sputter coater in asimilar manner as used for electron microscopy samplepreparation [16]. The gold-coated membranes were thenattached to multicore wires using fluxless solder and werethen used as working electrode assemblies for the electro-chemical preparation of thin-polymer-film composite mem-branes.Gold counter electrodeswere constructedby coating glass

slides in a similar manner and attaching multicore wires viathe application of NElectrolubeM silver conductive paint. Afurther coating of epoxy resin served to insulate the wire/electrode joint, whilst also enhancing the mechanicalstrength of the electrode.

2.4. Removal of Polymerization Inhibitor present in as-received Divinylbenzene

Prior to the polymerization of the divinylbenzene, thepolymerization inhibitor (4-tert butylcatechol) was removedfrom the as-receivedmonomer as previously described [19].It must be noted, however, that the monomer preparationconsisted of amixture of 70 – 85%divinylbenzene plus 0.1%polymerization inhibitor with the rest of solution constitut-ing 3- and 4-ethylvinylbenzene. Since no attempt was madeto separate the DVB from the EVB, the resulting polymerfilms obtained are copolymers of the two styrenic mono-mers.The as-received DVB/EVB was extracted 10 times with

5% aqueous sodium hydroxide to remove the 4-tert-butylcatechol. The extract produced was washed six timeswith high purity deionized water and then passed through acolumn of neutral activated aluminum oxide onto granularcalcium hydride. The inhibitor free divinylbenzenewas thenstored at 4 oC over calcium hydride prior to use.

2.5. Electrodeposition of polyDVB/EVB onto PolyesterHost Support Membranes

Solutions of monomer (70% v/v) in N’,N-dimethylforma-mide containing 0.12 M electrolyte tetra-n-butyl ammoni-um perchlorate were degassed for 20 minutes using purifiednitrogen prior to polymerization. The electropolymeriza-tion of DVB/EVB onto gold-coated polyester membraneswas achievedvia the samemethoddescribed earlier [21]: i.e.,by cyclically scanning the working electrode potential from

569Flexible Ultrathin PolyDVB/EVB Composite Membranes



0 V through to�4.0 Vand back to the starting potential (vs.Ag) at a scan rate of 50 mVs�1. Polymer-coatedmembraneswere produced via 1, 2, 3, 5 and 10 potential cycles.

2.6. Enzyme Laminate Fabrication

Lactate oxidase was immobilized within an enzyme lami-nate for the detection of lactate concentrations in phosphatebuffer. The laminate was prepared as follows: Lactateoxidase (40 units mL�1) and bovine serum albumin (0.1 mgmL�1) were dissolved in (pH 7.4) phosphate buffer to formaLOD/BSA solution. 6 mL of this solution was then added to

3 mL glutaraldehyde (0.5% v/v in buffer) mixed rapidly andplaced on a 1 cm2 portion of (0.015 mm pore) polycarbonatemembrane. A 1 cm2 portion of the thin-polymer-filmcomposite membrane (polymer film uppermost) was thenplaced on top of the enzyme/albuminmatrix and the enzymemembrane laminate compressed under gentle finger pres-sure for approximately 5 minutes to allow crosslinkingof theprotein and thus immobilization of the enzyme. The enzymelaminate was then positioned over the working electrode ofa modified Rank oxygen electrode [13] such that thepolymer-coated surface faced uppermost (Figure 1b).

Fig. 1. Schematic of a) a polycarbonate-DVB/EVB composite membrane. b) Side view of an enzyme laminate placed over the workingelectrode of the modified Rank Cell. c) Electrochemical cell used for enzyme electrode measurements.




2.7. Enzyme electrode glass cell for lactate determinations

A bespoke glass electrochemical cell was utilized as before[19, 20], Figure 1c, for enzyme electrode lactate determi-nations. This glass cell consisted of two halves clampedtogether. The base was constructed with a flat circularsurface, at the centre of which was a platinum workingelectrode of 2 mmdiameter. The polymer coated compositemembrane laminate was positioned over the platinumelectrode of the cell (polarized at þ650 mV vs. Ag/AgCl)and sealed inplacewith an NO-ringM to follow thedetectionofH2O2. The upper half of the cell acted as a chamber intowhich solutions and reference and counter electrodes wereintroduced. A circular hole in the base of this served toexpose the underlying working electrode to the solution. Afurther circular indentation was made in the undersidesurface of the solution well to house the O-ring. A silverelectrodewas placed vertically into themain chamber of thecell to act as a combined counter and reference electrode.The enzyme laminate was positioned over the Pt workingelectrode with the polymer-coated surface positioneduppermost.

2.8. Investigation into Solute Permeability Coefficients

Themass transport, and therefore permeability coefficients,of both oxygen and lactate across a range of ultrathinpolymer composite membranes were determined usingconventional diffusion chamber apparatus as previouslydescribed [13]. A Rank Oxygen Electrode assembly (aspreviously described [13]) was used for the determination ofoxygen levels within solution, whilst a lactate enzymeelectrode (as described above), was fabricated for thedetermination of lactate levels.

3. Results and Discussion

3.1. The Electrodeposition of PolyDVB/EVB ontoPolyester Host Support Membranes

The electropolymerization of DVB/EVB onto gold-coatedpolyester membranes was achieved via the same methoddescribed earlier [21]. Polymer-coated membranes wereproduced via 1, 2, 3, 5 and 10 potential cycles.The surface topography of polyDVB/EVB-coated poly-

ester membranes was investigated and described in ourearlier paper [21]. Membranes were found to exhibit aNpittedM topography following the first few potential cycles,due to the critical gel-point being reached earlier than on thesurface, as the concentration of soluble polymerwas presentin the pores was greater. Since themembranes incorporatedwithin this lactate oxidase sensor are identical to thoseemployed within the glucose oxidase sensor describedearlier [21], the film thickness has been calculated viacharge integration to be approximately 50 nm for a 10 layerfilm.

3.2. Enzyme Immobilization

The prototype sensor described in this chapter was designedto electrochemically quantify levels of lactate concentra-tions via the amperometric detection of hydrogen peroxideproduced by the enzyme lactate oxidase (LOD):

L� lactateþO2 �!LOD

pyruvateþH2O2

LOD has previously been shown to remain active for up to21 days (from an initial activity of 42.4 mMH2O2/min/mg toapproximately 5 mM H2O2/min/mg), when immobilizedwithin a crosslinked within a gel of bovine serum albuminwith glutaraldehyde [22]. The rigid matrix formed in thisway also adheres to both underlying and upper membranesto produce an enzyme laminate. The LOD enzymedescribed in the chapter was therefore immobilized in thisway.

3.3. The Determination of Permeability Coefficients

The Permeability coefficients, (P), for lactate and oxygenthrough polymer-coated polyester membranes were ob-tained as described above. Figure 2a depicts the permeabil-ity coefficients for lactate across a membrane coated withpolyDVB/EVB via 1, 2, 3, 5, and 10 potential cycles, whilstFigure 2b depicts the P coefficients for oxygen.The P values for lactate across polyDVB/EVB-coated

polyester membranes (Figure 2a) decreased most markedlyfollowing the first potential cycle, after which point theytend towards a plateau. This would indicate that themembrane pores become blocked at this point, however,this may not be the case since the P values for oxygenindicate that it is after the second potential that the masstransport of oxygen molecules through the membrane ismost greatly hindered (Figure 2b).The oxygen P values were found to decrease as each

successive polymer was applied and the underlying mem-brane became increasingly coated with layers of polymer.The greatest decrease in the mass transport of oxygenthrough the membrane was found to occur following theinitial first and second potential cycles, after which point theP values decrease at a reduced rate.Thedifference inP values between lactate andoxygen can

initially be explained by the larger lactatemolecule meetingincreased resistance to the diminishing pore size followingthe first potential cycle. Our earlier papers [19 – 21] describehow the smaller oxygenmoleculemay still be able to diffusethrough any small pinhole areas of the pores that remainunblocked after the first cycle, whereas the lactate begins topartition through the polymer film itself. Following theapplication of the second potential, however, both sub-strates begin to use partitioning as their mode of transport.Once all membrane pores are blocked, however, molec-

ular size may no longer be important and both substrates




must partition across the polymer barrier. We have alreadyseen that the O2 permeability decreases significantly afterthe second initial polymer layer has beendeposited and thencontinues to decrease gradually further with each successivelayer deposited, implying that partitioning becomes theincreasing mode of mass transport for O2 across themembrane, Figure 2b. The greatest decrease in lactatepermeability on the other hand, occurs following the firstpolymer coatings, Figure 2a; at this point the lactate mustnow also partition through the composite membrane.Following 10 potential cycles, the lactate permeabilitycoefficient reaches a minimum as the polymer film at thispoint is at its thickest. The gradual decrease in both lactateand oxygen permeability as each successive coating isapplied may be due to either a) an increased resistance

being offered to a larger molecule as it partitions across thepolymer, or b) a possible residual Npin – hole effectM.Earlier SEM studies [21] suggested that, although not

visible at the surface, the vast majority of the membranepores have someamount of polymer formedwithin or acrossthem after 2 potential cycles. Since it is following these firstfew potential cycles that membrane pores become blocked,successive potential cycles therefore contribute to thecumulative thickness of solid polymer formed, resulting inincreasingly lowered P values.Previous studies employing thin-polymer-film composite

membranes as the upper coveringmembranewithin glucosesensors have shown that, in this context, membranesexhibiting lower glucose/oxygen P ratios are associatedwith extended sensor response linearity ranges [19 – 21]. Ittherefore follows that lowered lactate/oxygen P ratioswould also contribute to greater linearity of response andso these values were calculated and are depicted in Fig-ure 2c.P ratio values are seen to decrease slightly following the

first potential cycle, since the larger lactate molecule firstmeets an initial increased resistance as the first electro-deposition of polyDVB/EVB decreases the pore diameter.P ratio values are then seen to rise again slightly, with thesecond potential cycle. Individual P values for both sub-strates have indicated that it is within the first two cycles thatthe polymer blocks the membrane pores. Following thesecond potential cycle, however, the P ratio values continueto decrease gradually with each successive polymer coatingapplied.Figure 2c shows that lactate/O2 P ratios fall for the

membrane coated via 5 potential cycles to values approach-ing those seen at membranes coated with one or no polymerlayers. The permeability coefficients, of course, for bothsolutes, however, are lower. TheP ratios are seen to increasefollowing the tenth potential cycle, however, a characteristicalso observed for glucose/oxygen P ratios in our previouswork on glucose sensors [21]. This was attributed to a slightdeterioration in the uniformity of the polymer-coatedmembrane surface following multiple potential cycles [21].For this reason, the prototype lactate oxidase enzymeelectrode was constructed by utilizing a polyDVB/EVB-coated compositemembrane coated via 5 potential cycles asthe upper covering barrier.

3.4. Calibration and Comparison of a Range of LactateSensors Incorporating Flexible Thin Film PolyDVB/EVB Composite Membranes

A range of lactate oxidase enzyme electrodes was con-structed, with each electrode employing a flexible compo-site membrane produced via 1, 2, 3, 5 or 10 potential cycles.Calibration curves were obtained over a concentrationrange of 0 mM to 25 mM lactate in phosphate buffer(Figure 3). All membranes served as effective limitingbarriers to the diffusion of both the enzyme substrates andthe responses to normal physiological concentrations found

Fig. 2. Permeability coefficients across polyDVB/EVB coatedthin-polymer-film composite membranes for a) lactate, b) oxygen,and c) the lactate/oxygen permeability ratios across polyDVB/EVB coated membranes.




in human whole blood (0 – 2 mM) were found to be linearfor all membranes. With each polymer coating deposited,the amperometric responses became increasingly linearizedand response times for all sensors were found to be<1 minute.

3.5. Exclusion of Electroactive Interferents Present InHuman Whole Blood

An ultrathin polyDVB/EVB composite membrane coatedvia 5 potential cycles was employed as the upper coveringmembrane in the enzyme electrode. The sensor describedwithin this chapter is initially aimed towards the detection oflactate within whole blood for use within clinical emergencysituations, e.g., to monitor the conditions of shock experi-enced by a patient. The possibility of utilizing the ultrathin-composite membranes within a sensor designed for in vivoanalyses or within whole blood in vitro, requires that themembranes permit effective screening of electroactiveinterferents that may be present in blood. In the same way,that the effect of anionic interferents such as ascorbate wereinvestigated for the glucose sensors described earlier [19 –21], the lactate sensor produced was also assessed for itspossible capabilities for screening interferents. What is ofinterest is that the exclusion cannot be solely on the basis ofcharge exclusion or lactate itself would be unable to crossthe composite membrane. Our previous work [21] hasshown that glucose passes more slowly through thesemembranes than the much smaller oxygen molecule, there-fore it appears that a combination of size and chargeexclusion minimizes the passage of ascorbate. Also forinterference to occur, ascorbate would have to cross theentire membrane to be reduced at the electrode whereaslactate will be oxidized within the membrane and producehydrogen peroxide which is uncharged.The enzyme electrode was therefore evaluated for its

ability to screen anionic interferents as described earlierdescribed earlier [19 – 21] andwas exposed to lactate both inthe absence and presence of 1 mM ascorbate. It was found

that the enzyme electrode response was not significantlyaffected by the presence of ascorbate until a concentrationof 20 mM lactate was exceeded. The response was found toincrease by 10 nA following the addition of 20 mM lactatewith ascorbate and 10 nA for 30 mM lactate with ascorbate.This increase in response may be due to the production athigh lactate concentrations of high levels of pyruvic acid andhydrogenperoxide by the enzyme. Previous researchers [30]have reported the facile reaction between pyruvate andhydrogen peroxide to give acetate, water and carbondioxide, which is acidic (pKa¼ 6.1) and in sufficiently largequantities could lower the enzyme electrodeMs net anioniccharge and allow anionic interferents to reach the workingelectrodemore freely. Since the typical concentration rangeof lactate found within blood is between 0 mM and 2 mM,these results therefore demonstrate that for physiologicallactate analyses, the sensor proves to be effective at screen-ing electroactive interferents.The membrane described here for use within a lactate

sensor employed the samemembranes described earlier [21]and SEM images depicting the surfaces of both the bareflexible polyester membrane, and the polyDVB/EVB-coated membrane following exposure to whole humanblood for 2 hours, showed that both coated and uncoatedsurfaces offered favorable hemo/biocompatibility [21].

4. Conclusions

Flexible ultrathin-polyDVB/EVB composite membraneshave been electrochemically fabricated and successfullyemployed as the outer covering barrier in a model lactateoxidase sensor for the determination of lactate.Cyclically scanning the working electrode potential

between 0 Vand �4.0 V (vs. Ag) via 1, 2, 3, 5 and 10 cyclesproduced a range of composite membranes. Diffusionchamber experiments were performed to determine thepermeability coefficients of lactate and oxygen across therange of membranes. As the pores of the underlyingpolyester membranes became increasingly blocked bypolymer, the lactate and O2 P values decreased as bothsolutes begin to partition through the polymer film to reachthe enzyme matrix.The composite membranes fabricated were employed as

the outer covering barrier in model lactate oxidase sensorsfor the determination of lactate. Lactate calibration curveswere produced for all enzyme electrodes and were found todisplay increasingly substrate diffusion limiting propertieswith increasing polymer coatings.The sensors also effectively screened against electroactive

interferents via a charge exclusion mechanism. All compo-site membranes produced linear responses for normalphysiological concentrations in human whole blood (0 –2 mM) and linearity of sensor responses was also achievedup to 25 mM in some cases. The sensor response time wasfound to be <1 minute. The underlying polyester mem-brane proved to act as a suitably biocompatible support forthe polymer film, which also displayed favorable biocom-

Fig. 3. Lactate oxidase enzyme electrode calibrations employingmembranes coated via 0 (&), 1 (� ), 2 (^), 3 (*), 5 ((&), and 10(þ) potential cycles




patibility, thereby helping minimize surface biofoulingeffects.

5. Acknowledgements

The authors would like to thank the EPSRC for a PhDstudentship forK.Bridge, theEuropeanCommission for thefunding for F. Davis within research contract NMP2-CT-2003-505485, “ELISHA” program and the Nuffield Foun-dation for further financial support.

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flexible ultrathin polydvb/evb composite membranes for the optimization of a lactate sensor

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