polydivinylbenzene/ethylvinylbenzene composite membranes for the optimization of a whole blood...

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Polydivinylbenzene/Ethylvinylbenzene Composite Membranes forthe Optimization of a Whole Blood Glucose Sensor

Kerry Bridgeb, Frank Davisa, Stuart Collyera, Seamus P. J. Higson*a

a Institute of Biosciences and Technology, Cranfield University, Silsoe, Beds, MK45 4DT, UKb Manchester Materials Science Centre, UMIST, Grosvenor Street, Manchester, M1 7HS, UK*e-mail: [email protected]

Received: April 28, 2005Accepted: November 7, 2005

AbstractA novel ultra thin polydivinylbenzene/ethylvinylbenzene composite membrane has been developed for use as theouter covering barrier in a model amperometric glucose oxidase enzyme electrode. The composite membrane wasformed via the cathodic electropolymerization of divinylbenzene/ethylvinylbenzene at the surface of gold sputtercoated host alumina membranes, (serving solely as a mechanical support for the thin polymer film). Permeabilitycoefficients were determined for the enzyme substrates, O2 and glucose, across composite membranes formed with arange of polymer thicknesses. Due to the highly substrate diffusion limiting nature of the composite membrane, it wasfound that anionic interferents present in blood (such as ascorbate), were effectively screened from the workingelectrode via a charge exclusion mechanism, in a manner similar to previous findings within our laboratory. Theenzyme electrode showed an initial 32% signal drift when first exposed to whole human blood over a period of 2hours, after which time enzyme electrode responses remained essentially stable. Whole blood patient glucosedeterminations yielded a correlation coefficient of r2¼ 0.97 in comparison to standard hospital analyses.

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

DOI: 10.1002/elan.200503399

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]. Glucose continues to be a majortarget analyte for many sensors, with the focus of much ofthis research being driven by the growing incidence ofdiabetes within the western world [11 – 12]. Both theimmediate and long-term management of the disease relieson the ability to monitor and regulate blood glucoseconcentrations over a 0 to 30 mM concentration range. Asensor produced for this purpose must, therefore meetrequired levels of sensitivity, specificity robustness andreliability [13].Conventionalmembrane based enzymeelectrodes for use

with whole blood often employ two separate functionalmembranes [14]. The covering membrane usually acts as asubstrate diffusion limiting barrier andmust offer favorablebiocompatibility, so as tominimize sensor surface biofoulingand therefore loss of response [15, 16]. The underlying(inner) membrane is usually designed so as to be permse-lective [17] and is typically positioned immediately over theworking electrode to screen anionic interferents thatmay bepresent in the blood (such as ascorbate).Unfortunately both

membranes act as additional diffusional barriers throughwhich the enzyme substrates and / or H2O2must traverse, sohindering their passage to the enzyme or working electrodeand thus increasing the overall response time.Previous research has shown, however, that it is possible

to reduce the time taken for diffusional equilibria to beestablished by replacing the inner and outer membraneswith a single multifunctional ultra thin film compositemembrane [18, 19]. Covering membranes of this type withbiocompatible coatings, have been shown to be capable ofserving as both highly substrate diffusion limiting barrierstowards enzyme substrates, whilst also effectively screeninganionic interferents from theworking electrode via a chargeexclusion principle [20]. Composite membranes of this typeare formed from a homogeneous film (ideally 100 nmthickness) of a biocompatible polymer deposited onto ahighly porous support membrane, which offers mechanicalsupport to the structure.Amperometric glucose oxidase laminate enzyme electro-

des employing such biocompatible composite membraneshave been found to display shortened response times [21].This performance is made possible via the novel way inwhich the composite membrane acts as a bifunctionalmembrane, thereby negating the use of two conventionalmembranes.It should be noted in this context that the separational

performance of a homogeneous polymer film membrane

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relies 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, one of the most applicable being that ofelectrodeposition of either a conducting or nonconductingpolymer, the use of which within biosensors has beenreviewed [22]. Electrodeposition affords the possibility ofaccurate control of the location and thickness of a depositedpolymer film. Most films used within this field actuallyincorporated the active species e.g. an enzyme within thepolymeric film [22].A second approach is to combine an active layer such as a

chemically crosslinked film of glucose oxidase which iseither directly deposited onto or laminated to a supportedelectrodeposited membrane on an electrode. For examplepolydiaminobenzene can be deposited electrochemicallyonto a graphite electrode to modify its surface. Thiscomposite electrode may then be used as a host surface forthe deposition of a glucose oxidase/dimethyl ferrocene/glutaraldehyde crosslinked layer [23], with the final systembeing suitable for use as a glucose sensorwith a lifetimeof upto four months. Other approaches include depositingpolypyrrole onto platinum, followed by further depositionof a polypyrrole/tyrosinase composite to detect phenols[24]. Solvent cast films of poly(4-vinylpyridine-co-styrene)have been used as substrates for crosslinked glucoseoxidase/laponite composites and have been shown toeliminate interference from acetaminophen, ureate andascorbate [25]. Polymers such as cast films of Os-polyviny-limidazole [26] have been used in alcohol biosensors andelectropolymerized Azure B films in phenol sensors [27].An interesting variation of this method is the deposition ofpolydiaminobenzene from liquid crystalline solutions whichhave been shown to give films which were nanoporous andselectively excluded negative ions [28].This paper describes the use of an ultra-thin polydivinyl-

benzene/ethylvinylbenzene (DVB/EVB) film compositemembrane, with an effective diffusional barrier thicknessof approximately 100 nm, for the optimization of a glucosebiosensor for whole blood glucose determinations. Theperformance of each composite membrane as a substratediffusion limiting barrier was determined. The biocompat-ibility as well as the ability to screen anionic interferentsolutes within patient blood samples were also investigated.A model glucose oxidase enzyme electrode was alsofabricated for the determination of glucose levels in patientwhole blood samples. The results are compared to standardhospital analyses for the same patient samples.

2. Experimental

2.1. Reagents and Membranes

N ’N-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). Further purifica-tion was performed to remove the polymerization inhibitorfrom the divinylbenzene monomer (as described later); noattempt was made to separate the DVB from the EVB. Allother reagents were used as supplied.d-Glucose, disodium hydrogen phosphate (Na2HPO4),

sodium dihydrogen phosphate (NaH2PO4) and sodiumchloride (NaCl) were all obtained from BDH (Poole,Dorset).Glucose oxidase from Asperigillus niger (75% protein

150 000 units/g solid), 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. ElectroDag silver paint was purchased fromAgarScientific Ltd, for electron microscopy sample preparations.Anopore aluminamembranes with a nominal pore size of

0.1 mm were purchased from Whatman International Ltd,Maidstone, UK, and used for the preparation of thinpolymer film composite membranes. 0.015 mm pore diam-eter polycarbonate membranes (Poretics Corporation,Livermore,USA)were used as the lowermembranes withinenzyme / membrane laminates [21].

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

2.3. Preparation of Conductive Faced MembraneElectrodes and Counter Electrodes

In order to electropolymerize the DVB/EVB monomeronto the host Anopore alumina membranes, the uppermostsurface was first rendered electrically conductive by Ausputter coating the membranes with an Edwards S150 Bsputter coater in a similar manner as used for electronmicroscopy sample preparation [20]. The gold-coated

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membranes were then attached to multicore wires usingfluxless solder and were then used as working electrodeassemblies for the electrochemical preparation of thinpolymer film composite membranes.Gold counter electrodeswere constructedby coating glass

slides in a similar manner and attaching multicore wires viathe application of KElectrolubeL 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 re-moved from the as-received monomer as previously descri-bed [29]. It must be noted, however, that the monomerpreparation consisted of a mixture of 70 – 85% divinylben-zene plus 0.1% polymerization inhibitor with the rest ofsolution constituting 3- and 4-ethylvinylbenzene. Since noattempt was made to separate the DVB from the EVB, theresulting polymer films obtained are copolymers of the twostyrenic monomers.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 de-ionized water and then passed through acolumn of neutral activated aluminum oxide onto granularcalcium hydride. The inhibitor free divinylbenzenewas thenstored at 48 over calcium hydride prior to use.

2.5. Enzyme Laminate Fabrication

The enzyme laminate employed in the resulting enzymeelectrode was produced by first forming a GOD/BSA

solution comprising glucose oxidase (2560 units/mL) andbovine serum albumin (0.1 mg mL�1) dissolved in buffer.3 mL glutaraldehyde (5% v/v in buffer) was then added to6 mL GOD/BSA solution and mixed rapidly. The enzyme/glutaraldehyde mixture was then placed on a 1 cm2 portionof (0.015 mm pore) polycarbonate membrane and a second1 cm2 portion of the polydivinylbenzene composite mem-brane (polymer coating face up) placed on top. Using glassslides, the crosslinked enzyme laminate was then com-pressed under gentle finger pressure for approximately5 minutes.

2.6. Enzyme Electrode Glass Cell for Whole BloodGlucose Determinations

A bespoke glass electrochemical cell was fabricated forenzyme electrode blood glucose determinations, Figure 1.This glass cell consisted of two halves clamped together. Thebase was constructed with a flat circular surface, at thecentre of which was a platinum working electrode of 2mmdiameter. The polymer coated composite membrane lam-inate was positioned over the platinum electrode of the cell(polarized at þ650 mV vs. Ag/AgCl) and sealed in placewith an KO-ringL to follow the detection of H2O2. The upperhalf of the cell acted as a chamber into which solutions andreference and counter electrodes were introduced. Acircular hole in the base of this served to expose theunderlying working electrode to the solution. A furthercircular indentation was made in the underside surface ofthe solution well to house the O-ring. A silver electrode wasplaced vertically into themain chamber of the cell to act as acombined counter and reference electrode. The enzymelaminate was positioned over the Pt working electrode withthe polymer-coated surface positioned uppermost.

Fig. 1. Schematic of electrochemical cell.

97Polydivinylbenzene/Ethylvinylbenzene Composite Membranes



2.7. Investigation into Solute Permeability Coefficients

Themass transport, and therefore permeability coefficients,of both oxygen and glucose across a range of ultra-thinpolymer composite membranes were determined usingconventional diffusion chamber apparatus as previouslydescribed [21]. A Rank Oxygen Electrode assembly (aspreviously described [21]) was used for the determination ofoxygen levels within solution, whilst a glucose enzymeelectrode (again as previously described [21]), was fabri-cated for the determination of glucose levels.

2.8. Scanning Electron Microscopy

A bare alumina membrane and a range of polymer coatedcomposite membranes were studied using a Phillips XL30FEG Scanning Electron Microscope. Samples were mount-ed onto aluminum microscopy stages by applying a smallamount of silver conductive KElectroDagL (415 M) paintaround the sample perimeter.

3. Results and Discussion

It is known that divinylbenzene and ethylvinylbenzenemonomers electropolymerize via a classical cathodic gelpoint mechanism to produce insulating films of polyDVB/EVB [30]. For this study, the monomers (following theremoval of the polymerization inhibitor) were electro-chemically polymerized onto gold sputter coated alumina

membranes, by cyclically scanning the working electrodepotential from 0 V through to �4.0 V and back to thestarting potential (vs. Ag/AgCl) at a scan rate of 50 mV s�1.The solvent used was NLN-dimethylformamide in which0.12 Melectrolyte tetra-n-butyl ammoniumperchloratewasdissolved. For comparison, membranes (with a nominalpore size of 0.1 mm) were prepared via 1, 2, 3, 5 and 50potential cycles.The polymerization of DVB/EVB initially occurs via the

two-electron reduction of monomer at the surface of themembrane, to form an equivalent di-anion. This transfer ofelectrons then initiates the complete polymerization ofmonomer present in close proximity to the workingelectrode surface [29]. Termination of the whole process iscaused either via reaction with the cation of the supportingelectrolyte, or with proton donors such as trace waterpresent in solution [29].The area underneath each peak produced in a polymer-

ization curve can be related to the quantity of charge thatthat has passed due to a Faradaic reaction. The irreversiblepeak observed in Figure 2 is seen to diminish in size witheach successive cycle, a trend which is characteristic of theelectrochemical deposition of an insulating polymer [20].We can therefore summise that the electrochemical poly-merization ofDVB/EVBmonomer is a self-limiting processsince the polymer will, with time, first passivate and finallyinsulate the electrode surface at which it is deposited.Furthermore, by integrating the charge underneath each

polymerization curve, it is possible to estimate the cumu-lative thickness of polymer film deposited. If it is assumedvia molecular modeling that the cross-sectional dimensions

Fig. 2. Cyclic voltammograms for electrochemical deposition of polydivinylbenzene onto host support membrane (at a scan rate of50 mV s�1).

98 K. Bridge et al.



of a polyDVB/EVB monomer unit are ca. 8.06 N and ca.5.28 N and that the depth of each polymer monolayer is ca.1.5 N, then the thickness of the polymer film following 50potential cycles can be calculated as being ca. 110 nm.Figure 3a depicts the surface of a bare aluminamembrane

for comparison withmembranes coated via electropolymer-isation techniques, (Figs. 3b and c). These images showedthat themembranes coated with polyDVB/EVB via 2 cyclesor less, (Fig. 3b), display an incomplete membrane surfacecoverage with some pores still visible in the polymer film.The surface topography of the polymer coated membraneafter 50 potential cycles by contrast, (Fig. 3c), shows a nearcomplete polymer film coverage upon the host porousalumina support membrane. Figure 3d, shows a cross-section of the membrane and confirms that the polymerfilm formed on surface of the Au coating to be approx-imately 100 nm in thickness.Theprototype glucose sensor to bedeveloped in this study

is based on the amperometric detection of H2O2, produced,via the catalytic oxidation of glucose by the enzyme glucoseoxidase (Gox). The control of glucose transport to theenzymemust be carefully regulated to prevent saturation oftheGox and thus plateauing of the sensor responsewithin thedesired working concentration range. Fluctuating or lowoxygen levels must also not become the rate limiting factorfor the enzymatic reaction. Previous studies have shown thatthin-polymer-film composite membranes exhibiting lowerglucose/O2 P ratios are associated with extended linearityrangeswhen used as the uppermembrane in glucose oxidaseenzyme electrodes [31, 32]. For this reason diffusionchamber apparatus was used to determine permeabilitycoefficients for both glucose and oxygen across a range ofultra-thin-film composite membranes formed via differingnumbers of potential cycles.Figure 4a depicts permeability coefficients (P) for oxygen

across a membrane coated with polyDVB/EVB via 1, 2, 3, 5and 50 potential cycles. The P value decreases as thepolymer film progressively coats and thereby insulates thehost support membrane. Although the oxygen permeabilitydecreases markedly following the 1st and 2nd voltammetriccycle applied, the P values approach a plateau thereafter.This initial large decrease in permeability can be explainedin terms of the first few potential cycles accounting for themajority of the membrane pores becoming covered withpolymer, with further cycles simply leading to an increase inthe cumulative polymer film thickness. The fact thatincreased film thickness does not lead to further loweringof the permeability indicates that the rate limiting step is theinitial penetration of dissolved oxygen into the membranerather than the transfer across the membrane.

"Fig. 3. SEM image of the surface of an alumina membrane. a)Ssurface view of the membrane as supplied. b) Surface view of themembrane partially coated with polydivinylbenzene following 2potential cycles. c) Surface view of the membrane coated withpolyDVB/EVB following 50 potential cycles. d) A cross-section ofa membrane coated with poly DVB/EVB following 50 potentialcycles.




Glucose permeability coefficients (Fig. 4b) follow asimilar trend, with a marked decrease in P values occurringafter the application of the 1st potential cycle but then nofurther changes for samples that have been coated for 2, 3 or5 cycles. This indicates that for these films, again the ratelimiting step is the transfer of glucose between solution andthe membrane. Figure 3b has previously shown that al-though the majority of the membrane surface was coatedwith polymer after the first two cycles, it was not entirelycovered with a homogeneous polymer film, and it mighttherefore be expected that some glucose might be able to

diffuse through these pores with ease. It is only after all ofthe pores are blocked with polymer that the membrane canserve as an effective substrate diffusion limiting barrier.It is probable that glucose permeabilities are initially

lowered by the physical blocking of pores by the accumu-lation of polymer on the membrane surface. We have alsoalready observed that oxygen permeability values decreasemarkedly between the initial 1st and 2nd voltammetric cycles,implying that partitioning becomes the increasing mode ofmass transport for O2 across the membrane. Glucosepermeability values decrease initially after the 1st cycledue to the increased resistance being offered to the largermolecule as it partitions across the polymer, and following50 potential sweeps, the glucose permeability coefficientagain tends towards a plateau for similar reasons, since thepolymer film is now homogenous.The differential effect upon the glucose/O2 ratios suggests

that the diffusion rates for glucose andO2 are not in the sameratio through the membrane as they are for free diffusionthrough an open pore. Under such conditions glucose andoxygen must partition through the polymer film, wheremolecular size per semay not be overwhelmingly important.We have already seen that electron micrographs display amembrane surface with incomplete polymer coverage afterthe 2nd cycle (Fig. 3b), whereas membranes coated via 50potential cycles, (Fig. 3c) are seen to possess essentiallypore-free thin-polymer films. Since diffusion within anaqueous solution across a pore will clearly be favored byeither solute, a membrane coated via a homogeneous filmwill act as a highly substrate diffusion limiting barrier.It shouldbenoted that thepermeability coefficients forO2

always remain significantly greater than those observed forglucose reflecting the relative molecular weights of thesetwo solutes [21].Figure 4c shows that the glucose/O2P ratio observed after

50 cycles is less than that seen after 5 cycles, at a valueapproaching those seen at membranes coated with less than3 cycles. It must be remembered, however, that the finiteindividual permeability coefficient values for both solutesare also lower than those recorded after 50 cycles. It shouldalso be noted that although the lowered permeabilitycoefficients may appear quite large, the diffusional barrieris of course extremely thin. It might, therefore, be reason-able to expect rapid dynamic diffusional equilibria to bereached across the barrier when used within a biosensorapplication.A range of ultra-thin polydivinylbenzene composite

membranes produced via 1,2,3,5 and 50 potential cycleswere therefore employed in glucose oxidase enzymeelectrodes with reproducible calibration curves being ob-tained over a concentration range of 0mM through to 20mMglucose in phosphate buffer (Fig. 5). Each polymer layerserved to enhance the substrate diffusion limiting propertiesof the membranes, resulting in increased linearisation ofsensor responses. The response times for all sensors werefound to be <1 minute.An ultra-thin polydivinylbenzene composite membrane

coated via 50 potential cycles was employed as the upper

Fig. 4. Permeability coefficients across polyDVB/EVB coatedthin polymer film composite membranes for a) oxygen, b) glucoseand c) the glucose/oxygen permeability ratios across polyDVB/EVB coated membranes.

100 K. Bridge et al.



covering membrane in the enzyme electrode. The screeningof electroactive interferents present in blood was firstconsidered, as solutes such as ascorbate and urea are knownto be capable of being oxidized at working electrodespolarized at potentials of þ650 mV vs. Ag/AgCl, thusleading to erroneous responses. The highly substratediffusion limiting nature of some outer coveringmembraneshas been previously shown by us to permit the effectiveexclusion of such anionic interferents via a charge exclusionprinciple as previously described [14].The enzyme electrode was therefore evaluated for its

ability to screen anionic interferents in this way and wasexposed to glucose both in the absence and presence of1 mM ascorbate. It was found that the enzyme electroderesponse was not significantly affected by the presence ofascorbate until a concentration of 10 mM glucose wasexceeded. The response was found to increase by 10 nAfollowing the addition of 10 mM glucose with ascorbate and12 nA for 30 mM glucose with ascorbate. This thereforedemonstrates that the highly substrate diffusion limitingmembrane coated via 50 cycles does indeed serve toeffectively screen anionic interferents under conditions oflower glucose concentration in a similar manner to previousfindings within our laboratory [18, 20]. In this context wehave previously shown that under conditions of low glucoseflux, the anionic enzyme/albumin matrix prevented thepassage of anionic solutes to the underlying electrode [18]. Itappears that interferent contributions (due to a fixedconcentration) only become significant when the enzymelayer experiences a higher glucose flux. Here, it is probablethat despite ambient solution buffering, the enzyme cata-lyzed production of gluconolactone (and therefore gluconicacid) will lower the local enzyme layer pH, and theprotective barrier action of the enzyme layer will bediminished [18].The enzyme electrode utilizing an ultra thin polyDVB/

EVB film composite membrane without an inner permse-

lective barrier was then used to determine glucose levels inpatient whole blood samples. In order to monitor any signaldrift that may occur due to the initial exposure to blood, theenzyme electrode was precalibrated for the response to5 mMglucose and then exposed to pooled whole blood for 2hours. Responses to 5 mM glucose buffer samples after thistime indicated a decrease in sensor responses of approx-imately 32% – after which time the response of the sensorremained stable. Longer exposures (up to 20 hours) did notcause increased drops in sensitivity. This initial loss ofresponse on exposure to bloodmay therefore be treated as apreconditioning period. The enzyme electrode was thenrecalibrated prior to the determination of blood glucosesamples.A local hospital biochemistry laboratory supplied patient

blood samples for comparative testing, however, KblindLtesting of the blood samples were performed using theenzyme electrode. The blood glucose analyses via theenzyme electrode showed a very close correlation of r2¼0.97 when compared to the hospital laboratory analyses(Fig. 6).When dealing with repeated analyses of whole blood, the

biocompatibility of the polymer film with which it comesinto contact represents a major factor in determining thestability and therefore the reliability of the sensor. ThepolyDVB/EVB composite membrane, along with a barealumina membrane were therefore both exposed to wholeblood for 2 hours prior to observation under the SEM(Figs. 7a and b respectively). The polymer coated mem-brane was found to show enhanced biocompatibility aftersuch an exposure to whole blood in comparison to anuncoated aluminamembrane, as seen by the reduced sensorfouling at the polyDVB/EVB coated membrane.

4. Conclusions

Ultra thin polyDVB/EVB compositemembranes have beenelectrochemically fabricated and successfully employed as

Fig. 5. Glucose enzyme electrode calibrations employing mem-branes coated via 0 (^), 1 (&), 2 (~), 3 (� ), 5 (*) and 50 (þ)potential cycles.

Fig. 6. Whole blood glucose determinations for patient samplesvia enzyme electrodes employing 50 polymerizations (Y axis) incomparison to standard hospital results (X axis).




the outer covering barrier in a glucose sensor for wholeblood glucose determinations.As the pores of the underlying alumina membranes

became increasingly blocked by polymer, the glucose/O2 Pratios decreased as both solutes begin to partition throughthe polymer film to reach the enzyme matrix.The enzyme electrode fabricated was found to serve both

as a substrate diffusion limiting barrier and as an effectivemeans for screening towards electroactive interferents via acharge exclusion mechanism. The polymer layer alsodisplayed favorable biocompatibility in comparison tobare aluminamembranes, thereby helpingminimize surfacebiofouling effects.A close correlation of r2¼ 0.97 was observed between the

blood glucose determinations performed via the enzymeelectrode in comparison to standard hospital analyses.

5. Acknowledgements

The authors would like to thank the EPSRC for a PhDstudentship for K. Bridge, the BBSRC for funding for F.Davis as part of the Centre for Bioarray innovation withinthe postgenomic consortium and the Nuffield Foundation

for further financial support. We would also like to thankMr. R. Hinchcliffe of the Manchester Royal Infirmary forsupplying blood samples for comparative testing.

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Fig. 7. Scanning electron micrographs depicting the effects ofblood biofouling on a) an unmodified alumina membrane surfaceand b) a polyDVB/EVB alumina membrane coated surface.

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polydivinylbenzene/ethylvinylbenzene composite membranes for the optimization of a whole blood...

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