A glucose biosensor with enzyme-entrapped sol–gel and anoxygen-sensitive optode membrane†

Xiaojun Wu, Martin M. F. Choi* and Dan Xiao‡

Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR,China. E-mail: mfchoi@net1.hkbu.edu.hk

Received 21st June 1999, Accepted 8th November 1999

An optical biosensor for the continuous determination of glucose in beverages based on the canalisation of glucoseoxidase into a sol–gel is presented. The enzyme was entrapped within a glass matrix by the sol–gel method. Thematrix was ground to a powder form and packed into a laboratory-made flow cell. This minireactor was positionedin a spectrofluorimeter connected to a continuous sample flow system. An oxygen-sensitive optode membrane wasfabricated from tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) didodecyl sulfate adsorbed on silica gelparticles and entrapped in a silicone-rubber film. The membrane was situated against the wall of the flow cell tosense the depletion of oxygen content upon exposure to glucose. The change of the luminescence intensity of theoptode membrane can be related to glucose concentration. The effects of temperature and pH on the response ofthe biosensor were investigated. Storage, stability and repeatability of the biosensor were also studied in detail.The analytical range of the biosensor was from 0.06 to 30 mmol dm23 glucose and the time taken to reach asteady signal in a flowing solution was 5–8 min. The detection limit was found to be 6 mmol dm23. Commonmatrix interferents such as fructose, galactose, lactose, raffinose, rhamnose, stachyose, sucrose and othercomponents in beverage samples showed no interference. The glucose biosensor has been successfully applied tothe determination of glucose contents of beverage samples.

Introduction

In the past two decades, huge efforts in academe and inindustrial laboratories have been devoted to developing bio-sensors for an array of analytes.1 However, after the expenditureof an enormous amount of effort, only a few biosensor systemshas been successfully commercialised. The growing demand fora more practical and reliable test has been spurring on thedevelopment of biosensor devices. In the clinical diagnosis andfood industries, a glucose sensor is considered to be verydesirable for analysis and process control. Clark reported thefirst successful electrochemical biosensor using immobilisedglucose oxidase in conjunction with an oxygen electrode over40 years ago.2 Since then, the literature reporting glucosebiosensors has been accumulating at a very high rate. Themajority of them are electrochemical glucose biosensors.Among them, the most famous practical device for thedetermination of blood glucose content was developed byYellow Springs Instruments in the early 1970s.3 Most of theelectrochemical biosensors are based on measuring H2O2

(which is generated from the oxidation of glucose by oxygen) orthe response to other electroactive species (which are previouslyadded to the system and react with the product of the reactionbetween oxygen and glucose). These biosensors are verysensitive to the presence of glucose. However, a very compli-cated biosensor system is often specifically designed toovercome the interferents in different biological samples. Thecomponents of the sample that would be tested have to beknown initially in order to correct any error in measurement.The serious drawbacks of these types of devices are that thesensing layer is so delicate that it has to be regeneratedfrequently; and the weak electrical signal of the device cannot

withstand electric and magnetic interferences, especially in aharsh working environment.

A sol–gel matrix has been proved to be a very useful solidsupport for the immobilisation of enzymes as it can retain theenzyme activity and is considered to be the best way ofimmobilising glucose oxidase to date.4 Unfortunately, electro-chemical-based biosensors are not easy to adapt to the sol–geltechniques for the fabrication of their glucose sensing layerbecause the sol–gel matrix does not provide good electricalconductivity.

Optical biosensors have been developing very rapidly sincethe mid-1970s.5 The promising features of these devices areoften related to simple sensor design, easy operation, freedomfrom electric and magnetic interference and suitability forin situ or on-line remote monitoring. However, most opticalbiosensors developed so far are not as sensitive as theelectrochemical biosensors. In addition, they also suffer frominterference from some species in biological samples. Thisdrawback makes the optical biosensor device very complicatedin design in order to reduce the effects of interferences. Anothermajor problem is that these biosensors are not robust. As aresult, the real potential of optical biosensors is seldomrealised.

For biosensor application, a sol–gel matrix encapsulated withglucose oxidase has been well studied in recent years.4,6,7 Theencapsulated glucose oxidase exhibits excellent characteristicsin terms of activity, lifetime and optical transparency. In thispaper, we report an improved technique for the fabrication of asol–gel glucose biosensor. The enzyme was initially entrappedwithin a glass matrix by a sol–gel method.4 The matrix was thenground to a powder form and was packed into a flow celltogether with an oxygen-sensitive optode membrane previouslypositioned in the flow cell. In this way, a flow-through systemwas set up for the determination of glucose. The optodemembrane was fabricated from tris(4,7-diphenyl-1,10-phenan-throline)ruthenium(II) didodecyl sulfate [Ru(dpp)3(DS)2] ad-sorbed on silica gel particles and entrapped in a thick silicone-

† Presented at the Fifth Asian Conference on Analytical Sciences, XiamenUniversity, Xiamen, China, 4–7 May 1999.‡ Visiting scholar on leave from the College of Chemistry and ChemicalEngineering, Hunan University, Changsha, China.

This journal is © The Royal Society of Chemistry 2000

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rubber film. The change in the luminescence intensity of theoptode membrane can be related to glucose concentration.8 Inthis design, the biosensor exhibited an extremely long lifetimeand showed high sensitivity to glucose. Other favourableattributes of our work are (1) the development of a verysensitive and extremely stable optical oxygen transducer, (2) thefabrication of highly active glucose oxidase entrapped in sol–gel matrix and (3) the assembly of a more practicable flow-through bed system consisting of sol–gel powder with an almostinterference-free biosensor system. The effects of temperatureand pH on the response of the glucose biosensor wereinvestigated. The properties of storage, stability, repeatability,response time and interference of the biosensor were alsostudied in detail. We successfully applied the proposed methodto determine the glucose content of some beverage samples.

Experimental

Materials

Glucose oxidase (EC 1.1.3.4. from Aspergillus niger) with aspecific activity of 25 000 units per gram of solid, glucosestandard solution (0.10 g cm23) and b-D-glucose were obtainedfrom Sigma (St. Louis, MO, USA), 4,7-diphenyl-1,10-phenan-throline, ruthenium(II) chloride pentahydrate and tetraethylorthosilicate (TEOS) from Aldrich (Milwaukee, WI, USA) andsodium dodecyl sulfate (SDS) from Riedel-de Haen(Seelze, Germany). Tris(4,7-diphenyl-1,10-phenanthroline)-ruthenium(II) didodecyl sulfate dye ion pair [Ru(dpp)3(DS)2]was synthesised and purified as described in the literature.9Silica gel particles (60 Å, 50 mm) were obtained from Matrex(Merck, Darmstadt, Germany). The one part silicone sealantSELLEYS (Selleys Chemical, Padstow, NSW, Australia), waspurchased from a local supermarket. All other reagents were ofanalytical-reagent grade and used without further purification.The buffer solution for preparing glucose standards was 0.05mol dm23 sodium phosphate solution (pH 7.0). All solutionswere prepared with de-ionised (DI) water.

Preparation of oxygen-sensitive optode membrane

A 50 mg amount of Ru(dpp)3(DS)2 was dissolved in 10 cm3 ofacetone and 50 cm3 of ethanol. This solution was mixed with 2.0g of silica gel particles, stirred for 2 h at 40 °C, cooled andfiltered. The silica gel particles were washed with 60 cm3 of DIwater three times, then dried for 6 h at 110 °C. A 0.1 cm3 portionof this oxygen indicator adsorbed on silica gel was mixedthoroughly with about 0.3 g of silicone sealant. By the spreadingmethod the mixture was stuck tenaciously to the surface of aglass plate or a transparent film to form a silicone-basedoxygen-sensitive film. It was left at 55 °C for 24 h to cure. Thethickness of the oxygen sensing layer was estimated to beapproximately 100 mm.

Preparation of enzyme-doped silica gel powder

A 10.4 g amount of TEOS, 1.8 g of water, 4.6 g of ethanol and30 mm3 of 0.1 mol dm23 HCl were mixed and then stirred usinga magnetic stirrer at room temperature for about 8 h to preparea clear stock sol–gel solution. A 3.0 cm3 aliquot of the stock sol–gel solution was placed in a small vial and stirred under vacuumfor 20 min in order to evaporate most of the ethanol. The pH ofthe solution was adjusted to about pH 4.5 by adding 20 mm3 of20 mmol dm23 sodium phosphate buffer (pH 7.4) (solution A).In a separate small vial, 18 mg of glucose oxidase and 0.250 cm3

of 20 mmol dm23 sodium 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonate buffer solution (pH 7.5) were mixed, then

solution A was added. A vacuum was applied to the stirredmixture until a gel was formed. The gel was rinsed with 2 cm3

of water three times. The gel was allowed to dry at 4 °C for 6 d.The dried gel was collected and ground to a powder form.Unless stated otherwise, this gel was used for most studies.

Assembly of sensing system

The laboratory-made flow-through cell used in this work wasmachined from stainless steel and had a chamber volume ofapproximately 0.45 cm3 (Fig. 1). An oxygen sensing film plusa blank glass plate were positioned as the window of the flowcell. The enzyme-doped silica gel powder was subsequentlypacked into the flow cell to form a small packed flow bedresulting in a minireactor or biosensor ready for glucosesensing. This minireactor was situated in a spectrofluorimeter inconjunction with a continuous sample flow system. When theglucose biosensor was not in use, it was stored at 4 °C.

Instrumentation

Fluorescence intensity was measured on a Perkin-Elmer(Beaconsfield, Bucks., UK) LS-50B spectrofluorimeter whichwas controlled by FL WinLab software. The fluorescenceemission intensity at 602 nm was collected at an excitationwavelength of 460 nm. All measurements were made with 3 nmbandwidths for both the emission and excitation mono-chromators. For gas-phase measurements, oxygen and nitrogenwere mixed and flowed via mass flow controllers [Read Out &Control Electronics 0154 (Brookes Instrument BV, Vee-nendaal, The Netherlands)] directly to the sealed oxygensensing flow-through cell. All measurements were performed inair-saturated buffer solutions. Using a MasterFlex C/L Model77120-62 (Cole-Parmer Instrument Co., Chicago, IL, USA)peristaltic pump, the air-saturated buffer or the air-saturatedglucose solutions were pumped through the flow cell at a typicalflow rate of 1.0 cm3 min21. Unless stated otherwise, allfluorescence measurements were made under batch conditionsat 20 ± 2 °C and at a pressure of 101.3 kPa.

Results and discussion

Response behaviour of oxygen transducer

An oxygen sensing film acting as a transducer was employed tomeasure the rate of oxygen consumption in the enzymaticoxidation of glucose. The optical sensing is based on collisionquenching of the fluorescence of Ru(dpp)3(DS)2 molecules byoxygen molecules.10,11 Hence the biosensor response composedof a dynamic balance in the diffusion of glucose into the silica

Fig. 1 Schematic diagram of the flow-through cell packed with sol–gelpowder and an oxygen optode membrane. (1) Stainless steel cell body; (2)sol–gel powder; (3) oxygen optode membrane; (4) transparent glass plate;(5) sample inlet; (6) sample outlet; (7) excitation light beam; and (8)emission light beam.

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gel powder and oxygen into the silicone-rubber film, andconsumption of oxygen in the enzymatic reaction, resulting in asteady-state decreased oxygen level and, consequently, anincrease in fluorescence intensity. Quenching can be quantifiedby intensity quenching measurements. The oxygen quenchingprocess is described by the well-known Stern–Volmer equa-tion:9,11–13

I0/I = 1 + K·pO2

where I is the fluorescence intensity, the subscript 0 denotes theabsence of oxygen, K is the Stern–Volmer constant and pO2 isthe partial pressure of oxygen. A plot of I0/I versus the partialpressure of oxygen should give a straight line with a slope K andan intercept of unity on the ordinate. Fig. 2 shows the curvedStern–Volmer plot of an oxygen sensing film on exposure tovarious oxygen concentrations. The curvature was attributed tothe distribution of slightly different quenching environments forthe Ru(dpp)3(DS)2 complex, particularly when quenchingoccurs in a solid matrix.9 The emission spectrum ofRu(dpp)3(DS)2 of the oxygen sensing layer was very similar tothat in the literature.14 The fluorescence intensity showed a verybroad dynamic range for both gaseous and dissolved oxygenmeasurements, which was more than 20- and 6-fold, as shownin Fig. 2 and 3, respectively. Certainly, the sensitivity ofresponse to dissolved oxygen is far better than that in theliterature;11–16 95% of the steady forward and reverse responsescan be reached within 25 s. The concentration of Ru(dpp)3(DS)2

had a great effect on the fluorescence intensity. The fluores-cence intensity reached a maximum when the amount ofRu(dpp)3(DS)2 was at 5.2 mg per gram of silica gel particles. Athigher concentrations of Ru(dpp)3(DS)2 the fluorescence in-tensity decreased significantly, which strongly suggests that ahigh content of Ru(dpp)3(DS)2 may cause self-quenching.9 Thecompound in the silicone-rubber film was extremely stable andcould be stored for a long period ( > 1 year) without anydegradation.

Sol–gel enzyme bed

Proteins entrapped in nanometre-scale cages of sol–gel formedby the cross-linking of silicone and oxygen units in a sol–gelprocess represent a convenient, flexible and efficient im-mobilisation technique for enzymes which can retain theirbiological function in both aged gels and xerogels. It providesan efficient design that restricts the movement of the encapsu-lated recognition molecule and inhibits their intermolecularinteraction but allows free permeation of small analytemolecules.17,18 This study was performed on xerogels. Theglucose oxidase to be encapsulated is added to the sol solution,which is first subjected to vacuum to remove most of the ethanolbecause solvents such as methanol and ethanol have been foundto denature enzymes, resulting in decreased enzymatic activ-ity.

The sol–gel procedures are apparently not detrimental toprotein stability and resulted in optically transparent solids thatare chemically, thermally and dimensionally stable immobilisedproteins. The enzyme activation level is easily adjusted simplyby changing the amount of enzyme. One of the main advantagesof our biosensor is that the sol–gel takes the form of powder andit increases the contact surface area of the biosensor with thesolution, which can assist more analytes and products to diffuseinto or out of the sol–gel cages. The powder packed in a flow-through bed seems to be more suitable for real application in aflow-through analysis. The packed bed was very stable. It canbe stored for over 10 months at 4 °C without apparently losingthe enzyme activity. Even when kept under ambient conditionsfor 5 months, the enzyme activity does not decrease by morethan a few per cent.

Dynamic range

The sensing scheme includes the use of glucose oxidase, anenzyme catalysing the oxidation of glucose by the dissolvedoxygen in the analytical solution. Oxygen has a relatively lowsolubility in water, the concentration of oxygen in water inequilibrium with air being only 9.2 ppm at 20 °C and standardatmospheric pressure.11 The changes in pO2 are detected viaquenching of the fluorescence intensity with the oxygen sensingfilm. The decrease measured in the oxygen partial pressurewhen glucose is oxidised by the enzyme gives an indirectindication of the glucose concentration. The fluorescenceintensity, due to oxygen consumption, hence increases andreaches a plateau, the variation being proportional to the glucoseconcentration over a wide range. The magnitude of theanalytical signal of the glucose biosensor is therefore deter-mined by the oxygen quenching constant, the oxygen concen-tration and the glucose concentration inside the oxygen sensingmembrane. The response behaviour of the oxygen sensor, theconcentration of oxygen in the analytical solution, the amountor activity of glucose oxidase in the sol–gel powder, thetemperature of the biosensor system and the flow rate ofanalytical solution are factors which can strongly affect theworking range of the glucose biosensor. A typical calibrationcurve for this biosensor in a flow analysis system is displayed inFig. 4. The relative signal change is defined as:13

Rs = (Itest)/(Ibaseline)

where Itest and Ibaseline represent the detected fluorescencesignals from the biosensor exposed to glucose solution andbuffer solution, respectively. The maximum Rs measured withthis device increases over fivefold when the biosensor waschanged from an air-saturated buffer solution to an air-saturated30 mmol dm23 glucose solution. In terms of both relative signalchange, Rs and detection limits, our glucose biosensor showssignificant improvements over other optical biosensors of thiskind.5,6,8 The most sensitive and linear working range is

Fig. 2 Stern–Volmer curve of the oxygen sensing film at excitation andemission wavelengths of 460 and 602 nm when subjected to various oxygenconcentrations.

Fig. 3 Response curves for the oxygen sensing film cycled between (1)oxygenated water and (2) deoxygenated water.

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0.06–2.0 mmol dm23 glucose (Itest/Ibaseline = 1.0296[glucose] +0.9968; r2 = 0.995). Even though the sensitivity of thebiosensor decreases above 2.0 mmol dm23 glucose, the wholeworking range of the biosensor can still function between 0.06and 30 mmol dm23 glucose (Fig. 4) when a calibration curvehas initially been prepared that covers this range of glucoseconcentration. The biosensor performance is not very sensitiveto variations in flow rates from 0.8 to 1.2 cm3 min21. Increasingthe flow rate resulted in an increased glucose saturationconcentration. On the other hand, increasing the activity of theglucose oxidase in the sol–gel powder can lead to a lowerdetection limit and a lower glucose concentration saturationsignal. It is noteworthy that the dynamic working range anddetection limit of the biosensor can be modulated by controllingthe relative amount of glucose oxidase entrapped in the sol–gelpowder or the amount of sol–gel packed in the flow cell. It canfit the multiple requirements of an analytical method. In thiswork, a highly sensitive method for determination of glucosehas been realised with a detection limit of 6 mmol dm23 glucose.The lowest detection limit obtained was 1 mmol dm23 when theflow system was in the stop flow mode.

Effect of pH

The effect of pH was studied over the range 4.5–8.0. Thebiosensor was subjected to a 4.0 mmol dm23 glucose standardusing phosphate buffer solutions of various pH. The resultsshowed that the optimum pH value is about 6.0. It was foundthat pH changes apparently do not affect the dynamic workingrange of the biosensor. It is anticipated that the biosensor willwork satisfactorily in the pH range 5.0–8.0.

Effect of temperature

It is well known that the analytical performance of bothenzyme-immobilised silica gel and oxygen transducers is highlysensitive to variations of temperature. The unquenched excited-state lifetimes of ruthenium(II)–diimine complexes at differenttemperatures were 5.8 (0 °C), 5.9 (25 °C), 4.8 (38 °C) and 3.3ms (60 °C).12 Higher temperatures would result in a significantdecrease in lifetime and fluorescence intensity yield and alsodecrease the Stern–Volmer quenching constant of the ruth-enium(II) complex, resulting in a decrease in the sensitivity ofthe glucose biosensor. However, raising the working tem-perature has a counteracting effect on the biosensor. Theactivity of an encaptured enzyme is governed by the kinetics ofthe enzymatic reaction. The reaction rate will be increased byraising the working temperature. Therefore, a study of the effect

of temperature on the minireactor was carried out over the range15–40 °C. The signal sensitivity depended strongly on thetemperature of the biosensor analytical system, as expected. Thefluorescence intensity changed from 986 to 780 units when incontact with 100% nitrogen as the temperature increased from18 to 40 °C whereas the intensity shifted from 48 to 31 unitswhen the temperature increased from 18 to 40 °C on exposureto 100% oxygen. The dynamic working range of the biosensorwas reduced and the signal saturation point was reached earlierwhen the working temperature increased. However, the re-sponse rate of the biosensor increased sharply at higher workingtemperatures. The possible reasons are that the enzyme canacquire higher activity at higher temperatures and subsequentlygive a more pronounced signal change with oxygen consump-tion at a faster rate in the enzymatic oxidation of glucose.Although the analytical sensitivity was higher at around 40 °C,for practical purposes temperatures lower than 40 °C arerecommended so as to prolong the lifetime of the biosensorsince enzyme can easily be denatured at high temperature.

Response time, repeatability and stability of glucosebiosensor

In this study, the response time is defined as the time taken toobtain a full steady state signal when the biosensor is in contactwith an air-saturated buffer solution and then switches to aknown concentration of air-saturated glucose standard solution.The response time depends on the enzyme activity, the workingtemperature, the flow rate of the analytical solution, the particlesize of the sol–gel powder and the thickness of the oxygensensing indicator layer. When the oxygen sensor material is notdispersed into a silicone-rubber film, it can be used to detectgaseous oxygen with a very fast response time ( < 1 s). When itis dispersed in a silicone-rubber film a few micrometres thick, itcan be employed to determine dissolved oxygen in solution witha response time of ~ 1 min. As a result, the response time of ourglucose biosensor will be mainly determined by the enzymaticreaction rate of the enzyme and the analyte. A thicker oxygenindicator layer such as about 100 mm can also be employed as anoxygen transducer for the fabrication of our glucose biosensor.The typical time response curve depicted in Fig. 5 exhibitsexponential-like behaviour. The response times of the biosensorwere from 5 to 8 min. The relatively short response times areattributed to the increase in the surface area of the sol–gelpowder, which enhances the exposure of the glucose oxidase tothe analyte with a concomitant effect on the biosensor forreaching a fast steady-state signal. Moreover, if an enzymeentrapped in the sol–gel matrix has a higher activity, theresponse time should also be further shortened.

Fig. 5 displays the signal changes of the biosensor when it isexposed to concentration step changes from 0.010 to 10

Fig. 4 Calibration curve for the glucose biosensor at various glucoseconcentrations. The inset displays the linear regression curve for lowerglucose concentrations from 0.060 to 2.0 mmol dm23.

Fig. 5 Response time and reversibility of the glucose biosensor atexcitation and emission wavelengths of 460 and 602 nm when subjected tovarious concentrations of glucose using pH 7.0 phosphate buffers (0.05mol dm23). (1) 0.010; (2) 0.10; (3) 0.50; (4) 1.0; (5) 5.0; and (6) 10mmol dm23.

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mmol dm23 of glucose in 0.05 mol dm23 phosphate buffer atpH 7.0. It demonstrates that the biosensor exhibits a verydesirable analytical feature of excellent repeatability althoughonly two repeats have been performed. The long-term stabilityof the sensor was tested over a 280 d period. When the biosensorwas stored in a refrigerator at 4 °C and measured intermittently,the relative signal change of the biosensor on exposure to 5.0mmol dm23 glucose was found to be above 73% of its initialvalue over this period. The good stability of the glucosebiosensor can be explained by two possible reasons. First, mostof the positive charges at the channel entrance are balanced bythe negative charges of the nearby acidic residues. Hence thereis no detrimental electrostatic interaction with anionic sites ofthe immobilising matrix with the enzyme; as a result, the

enzyme is stabilised upon immobilisation in hydrated silica.19

Second, the cavity in the sol–gel is tailored to the size and shapeof the glucose oxidase. The bottleneck effect of the silica sol–gel prevents the enzyme from leaking.18–20 The sol–gel powderis so strong that it can withstand any slightly striking, pressingand liquid flow pressure without causing cracking and leachingof the enzyme.

Interference test

The interference test was mainly performed in two parts. Thepurpose of the first part was to investigate the effects of somecommon substances on the quenching response of the oxygen

Table 1 Effect of potential interferents on the oxygen transducer and glucose biosensor

Signal change for oxygen transducer Signal change for glucose sensor

Concentration/ Air-saturated 2% Na2SO3 1.25 mmol dm23

Interferent mmol dm23 DI water solution No glucose glucose

Saccharose 20 No No No NoFructose 20 No No No NoLactose 20 No No No NoGalactose 20 No No No NoRaffinose 20 No No No NoRhamnose 20 No No No NoStachyose 20 No No No NoNaCl 100 No No No NoKCl 20 No No No NoMgSO4 5 No No No NoCaCl2 Saturated No No No NoCuCl2 Saturated No No No ~ 9% decreaseFeCl3 2 No No No NoSodium citrate 10 No No No NoLauryl sulfate 1.5 No No No NoAscorbic acid 10 No No No ~ 3% increaseCTMABa 2 No No No NoVitamin E (in 2

mM CTMABa) 3 No No No NoSodium benzoate 50 No No No NoCaffeine 2 No ~ 1% decrease ~ 1% decrease ~ 3% decrease

a Cetyltrimethylammonium bromide.

Table 2 Results of the glucose assay and the recovery test on beverage samples

Beverage(source)

Glucose contenta/mmol dm23 RSD (%)

Glucose added/mmol dm23

Glucose foundb/mmol dm23 Recovery (%) RSD (%)

Sprite 61.0 3.22 50.0 48.34 96.7 2.31(Hong Kong) 100.0 98.02 98.0 3.74

150.0 149.2 99.3 2.98Sprite 50.1 3.12 50.0 46.86 93.7 3.17(China) 100.0 100.5 101 2.88

150.0 151.4 101 4.45Cola 185 1.32 50.0 48.86 97.7 6.83(Hong Kong) 100.0 101.4 101 4.74

200.0 201.4 101 3.31Pocari Sweat 72.5 1.74 50.0 51.7 103 2.06(Japan, 1998) 100.0 99.1 99.1 3.21

200.0 199.2 99.6 2.23Pocari Sweat 96.7 0.59 50.0 50.26 101 2.63(Japan, 1999) 100.0 100.14 100 2.59

200.0 198.8 99.4 3.33Striker 153 2.21 50.0 52.44 105 1.98(Japan) 100.0 103.7 104 3.04

200.0 201.4 101 1.74Gatorade 105 1.39 50.0 45.52 91.0 5.01(USA) 100.0 97.08 97.1 1.72

200.0 198.8 99.4 3.97Lucozade 299 1.76 50.0 45.56 91.1 4.78(UK) 100.0 97.36 97.4 3.58

200.0 197.4 98.7 4.65a The glucose content is an average of seven tests, determined with the glucose biosensor. b An average of five tests.

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sensing film. It was carried out with two groups of solutions.The first group of air-saturated solutions consisted of somepotential interferents. The second group of solutions consistedof some potential interferents together with 2% Na2SO3. Theresults showed that there were no significant signal changes ofthe oxygen sensing film on exposure to the interferents.However, caffeine caused a slight interference (Table 1).

The purpose of the second part was to examine the effect ofthese interferents on the response of the glucose biosensor. Itwas evaluated with two groups of solutions. The first group ofsolutions contained only interferents in 0.05 mol dm23

phosphate buffers. The second group of solutions consisted ofinterferents and 1.25 mmol dm23 glucose in 0.05 mol dm23

phosphate buffers. The results are given in Table 1. It is foundthat most potential interferents did not give any significantinterference on the response of the glucose biosensor. However,in the presence of glucose, CuCl2 can give some interference onthe response of the glucose biosensor. It was interesting to findthat the interference from CuCl2 could be diminished from 9 to4% if the test solution also contained 10 mmol dm23 ascorbicacid. In the presence of glucose, ascorbic acid and caffeine hada slight interference on the response of the glucose biosensor.However this interference was reduced to nearly zero if glucosewas absent from the tested solutions. In brief, we were confidentthat most substances often found in soft drink samples did notexhibit significant interference on the determination of glucoseusing our proposed biosensor method.

Glucose determination in beverage samples

Eight beverage samples of seven brands produced in variousplaces were bought from local supermarkets and used as our testsamples. The pH of a 10.0 dm3 aliquot of each sample solutionwas adjusted to about 7.0 by addition of a small volume of 0.2mol dm23 Na2HPO4 solution. It was then diluted with suitablephosphate buffer to yield a test sample solution of pH 7.0. Theglucose contents in the samples were determined by the glucosebiosensor (Table 2). The relative signal change of each samplesolution was measured and compared with that of a set ofglucose standard solutions. The recovery tests for glucose wereperformed by adding various amounts of glucose to the samplesolutions. The amounts of added glucose were then evaluated byusing our glucose biosensor. All sample solutions were air-saturated before testing. The results of the recovery of thesamples are summarised in Table 2. The recovery testsdemonstrate that the glucose biosensor offers an excellent,accurate and precise method for the determination of glucose inbeverages with almost no effect of interferences from common

matrix substances or components in the beverage samplesolution. The glucose biosensor is characterised by good long-term stability, selectivity, very high sensitivity, a broad dynamicworking range and a relatively fast response.

Acknowledgement

The work described in this paper was partially supported by agrant from the Research Grants Council of the Hong KongSpecial Administrative Region, China (project No. HKBU2058/98P).

References

1 J. Janata, M. Josowicz, P. Vanysek and D. M. DeVaney, Anal. Chem.,1998, 70, 179R.

2 L. C. Clark Jr., Trans. Am. Soc. Artif. Int. Org., 1956, 2, 41.3 E. Magner, Analyst, 1998, 123, 1967.4 Q. Chen, G. L. Kenausis and A. Heller, J. Am. Chem. Soc., 1998, 120,

4582.5 M. C. Moreno-Bondi, O. S. Wolfbeis, M. J. P. Leiner and B. P. H.

Schaffar, Anal. Chem., 1990, 62, 2377.6 U. Narang, P. N. Prasad, F. V. Bright, K. Ramanathan, N. D. Kumar,

B. D. Malhotra, M. N. Kamalasanan and S. Chandra, Anal. Chem.,1994, 66, 3139.

7 S. A. Yamanaka, F. Nishida, L. M. Ellerby, C. R. Nishida, B. Dunn,J. S. Valentine and J. I. Zink, Chem. Mater., 1992, 4, 495.

8 W. Trettnak, M. J. P. Leiner and O. S. Wolfbeis, Analyst, 1988, 113,1519.

9 I. Klimant and O. S. Wolfbeis, Anal. Chem., 1995, 67, 3160.10 J. N. Demas and B. A. DeGraff, Anal. Chem., 1991, 63, 829A.11 C. McDonagh, B. D. MacCraith and A. K. McEvoy, Anal. Chem.,

1998, 70, 45.12 J. R. Bacon and J. N. Demas, Anal. Chem., 1987, 59, 2780.13 B. D. MacCraith, C. M. McDonagh, G. O’Keeffe, E. T. Keyes, J. G.

Vos, B. O’Kelly and J. F. McGilp, Analyst, 1993, 118, 385.14 R. C. W. Lau, M. M. F. Choi and J. Lu, Talanta, 1999, 48, 321.15 B. Meier, T. Werner, I. Klimant and O. S. Wolfbeis, Sens. Actuators

B, 1995, 29, 240.16 E. R. Carraway, J. N. Demas, B. A. DeGraff and J. R. Bacon, Anal.

Chem., 1991, 63, 337.17 A. K. Williams and J. T. Hupp, J. Am. Chem. Soc., 1998, 120,

4366.18 B. C. Dave, B. Dunn, J. S. Valentine and J. I. Zink, Anal. Chem.,

1994, 66, 1120A.19 J. Heller and A. Heller, J. Am. Chem. Soc., 1998, 120, 4586.20 D. Avnir, S. Braun, O. Lev and M. Ottolenghi, Chem. Mater., 1994,

6, 1605.

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