1992 - miniaturized disposable biosensors

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Sensors and Act uat ors B , 7 (1992) 351-355 351

Miniaturized disposable biosensorsU. Bilitewski, G. C. Chemnitius, P. Riiger and R. D. SchmidDepartment of Enzyme Technolo gy, GBF (Gesell schaft ft ir Bi ot echnologische Forschung mbH ), 3300 Braunschweig (FRG)

AbstractMiniaturized amperometric thick film biosensors for applications in food quality control are described. Their easeof fabrication and relatively low cost facilitates their use as disposable sensors. As an alternative to theimmobilization of the enzyme by cross-linking with glutaraldehyde, screen printing technology was used to apply theenzyme layer, a graphite/TTF based paste, to the thick film electrodes. Examples are presented of the determinationof biogenic amines as an indicator of the freshness of fish, and of glucose in fruit juice and wine.

IntroductionOver the past two decades enzyme assays have

been established as standard analytical methodsfor food quality control. Routinely, soluble en-zymes and reagents are used in photometric tests.Biosensors containing all the required enzymesand reagents would simplify the performance ofthe tests and therefore reduce the costs of eachsingle assay. Analysis using biosensors is suitableas a screening method, to be used by scientists inlaboratories where the rapid processing of largenumbers of samples is required and by distributorsand consumers at the place of sale where thequality must be assessed immediately. Miniaturiza-tion of the sensor reduces the amount of enzymeand reagents needed.

Thick film technology, a well established tech-nology for the production of electronic circuits,provides the possibility to fabricate miniaturizedand relatively low cost electrodes. Thereforebiosensors based on this technology have beendeveloped recently [l-8], most of which were de-signed for medical applications.

In this paper we describe different sensors madein thick film technology which can be used asdisposable sensors in food quality control. Themanufacture of disposable biosensors should beeasy and rapid so as to reduce the costs involved.Therefore two methods which allow an automa-tion of the process were chosen for the immobi-lization of the enzymes. This is on the one hand bydripping the enzyme solution onto the electrode

surface and on the other hand by screen printingof an enzyme containing ink onto the workingelectrodes. The latter approach results in a biosen-sor fabricated by only one technology; the enzymelayer is screen printed by the same process as theelectrode pads, conductor lines and insulation.We selected glucose and amine concentrationsas parameters to demonstrate the potential ofthick film biosensors in quality control.

Biogenic amines are indicators of the freshnessof fish. They are generated during protein decom-position via amino acid decarboxylation followingthe slaughter of the fish, their concentration in-creasing with storage time [9]. Hence a sensitiveand specific determination is required. Differentapproaches have already been reported to useimmobilized polyamine oxidases for polyamine de-termination in fish and tissue samples [lo- 121. Butnone of the systems described so far were consid-ered suitable for use as disposable sensors forrapid polyamine determination at the place of sale.We present the preparation of easily used, rapid,disposable sensors by immobilizing putrescine oxi-dase (EC 1.4.3.10) onto the working electrodes ofthick film sensors. The resulting biosensors arecharacterized.Additionally thick film biosensors for the deter-mination of glucose in juices were prepared. Tak-ing the example of the glucose sensors the twoimmobilization methods were compared with re-spect to sensitivity, linear range, stability and in-fluence of interfering substances present in realsamples.

0925-4005/92/$5.00 @ 1992 - Elsevier Sequoia. All rights reserved


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ExperimentalReagents

For the construction of the thick film electrodesthe following reagents were used: fired aluminiumoxide ceramics, 10 x 10 cm for the preparation ofeight sensors, silver/palladium thick film paste andprotective overglaze were obtained from Du Pont(Bad Homburg, FRG). Platinum containing thickfilm paste was purchased from Ferro (Sindelfingen,FRG) and carbon ink was bought from AchesonColloids Co. (Plymouth, UK). For enzyme immo-bilization glucose oxidase EC 1.1.3.4. from Penicil-hum amagasakiense (Nagase Biochem. Ltd.,Tokyo, Japan), putrescine oxidase from M i crococ-cus rubens (a kind gift of Amano PharmaceuticalCo., Ltd., Nagoya, Japan), 3-aminopropyl-tri-ethoxysilane (Aldrich, Steinheim, FRG), bovineserum albumin (BSA, fraction V, BoehringerMannheim, FRG), glutaraldehyde (25%, Merck,Darmstadt, FRG), graphite powder (Merck), te-trathiafulvalene (TTF, Fluka, Buchs, Switzerland)and polyvinylpyrrolidone (Fluka) were used. Glu-cose was purchased from Merck. 0.1 mol/l stocksolutions of amine hydrochlorides (Sigma, Deisen-hofen, FRG) in 0.1 mol/l HCl were prepared.Sample preparationPrior to analysis samples of wines and juiceswere diluted with 0.1 mol/l potassium phosphatebuffer pH 7.0 containing 0.1 mol/l KC].For amine determination extracts of fish sam-ples were prepared. Pollack was purchased from alocal fish shop and stored at 4 C. Samples of100 g were disrupted in a homogenizer (Moulinex,Kiiln, FRG). Ten grams of the homogenized fishmeat were extracted in 80 ml 10% trichloroaceticacid (Merck) using an Ultra Turrax dispersingapparatus (IKA Labortechnik, Staufen, FRG).The extracts were filtered and made up to 100 mlwith 10% trichloroacetic acid in a volumetric flask.Thi ck Ji lm sensorTwo different detection principles were used:sensors fabricated by cross-linking the enzymewith glutaraldehyde at a platinum electrode, werebased on H202 detection. Whilst, because the en-yme printing pastes contained a high proportionof graphite particles (so as to achieve sufficientdsorption of the enzyme) they acted as graphitelectrodes and hence required a high overpotential

for Hz02 detection. Therefore, these electrodeswere modified with the mediator tetrathiafulva-lene, TTF.

The layout and fabrication of the thick filmsensors comprising four working electrodes, a ref-erence and a counter electrode have been describedin earlier publications [7, 81.The reference potential of the solid state inte-grated Ag/AgCl reference electrode (glucose sen-sors) in buffer containing 0.1 KC1 was 83 mVnegative of a conventional Ag/AgCl (3 M KCI)reference electrode. Hence the measurements werecarried out at 520 and 600 mV ( H2 O2 detection) or140 and 220 mV (mediator based system) versusthe solid-state integrated Ag/AgCl or a conven-tional Ag/AgCl (3 M KCl) reference electrode, re-spectively, in a three-electrode mode with afour-channel potentiostat from Bank (Giittingen,FRG).

Sensors based on immobil izat ion w i t hglutaraldehydeFor the detection of H,Oz the working elec-trodes were printed with platinum paste. Prior to

immobilization of the enzymes by cross-linkingwith glutaraledhyde the platinum electrodes ofsome sensors were modified with 3-aminopropyl-triethoxysilane (10% in phosphate buffer pH 7.0).For glucose oxidase immobilization a solution of1.2 mg glucose oxidase, 5 mg BSA, 15.7 ~1 2.5%glutaraldehyde in 100 ~1 phosphate buffer andfor putrescine oxidase immobilization a mixture of20 ~1 of putrescine oxidase, 20 pl 10% BSA inwater and 20 ~1 of 5% glutaraldehyde in waterwere prepared. The enzyme glutaraldehyde mix-tures were dripped onto the working electrodesand were allowed to dry at room temperature forapproximately 1 h. When not in use the sensorswere stored at 4 C in phosphate buffer.

Sensors w i t h pri nt ed enzyme layerIn order to obtain a good adhesion of theprinted enzyme layer to the electrodes carbonpaste was used for printing of the working elec-trodes. The enzyme ink was prepared as follows:1 g graphite powder was mixed with 20 ml of asaturated solution of TTF in toluene. The mixturewas stirred until the toluene had evaporated.Twelve mg of glucose oxidase were dissolved in0.9 g polyvinylpyrrolidone solution ( 10% in dis-tilled water) and 0.5 g of the TTF coated graphite


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powder were added. After thoroughly mixing theink was applied to the working electrodes byscreen printing. The enzyme layer was driedovernight and the sensors were stored on silica gelat 4 C. Sensors without enzyme were prepared inthe same way to recognize interferences of elec-troactive compounds in the samples.

Results and discussionGlucose sensorsThe suitability of thick film technology for theconstruction of glucose sensors by immobilizationof glucose oxidase by cross-linking with glu-taraldehyde on platinum thick film electrodes hasbeen shown previously [7, 81. Automation shouldbe possible using micro-dispensers which are al-ready used in thick film laboratories for the appli-cation of soldering pastes or adhesives.The linear range obtained with these sensorsextended to 1 mmol/l glucose with a lower detec-tion limit (signal:noise 3:l) of 1 umol/l glucose anda sensitivity of 0.2 uA/mmol/l. The shelf life of thesensors in buffer was one month at room tempera-ture and 20 days in semi-continuous use. Thestandard deviation was 4.7%. Alternatively theprinting of the enzyme layer on the working elec-trode was tested. In this case the properties of theenzyme layer, and hence the response of the sen-sor, depend on the one hand on the printingprocess, for example on the screen (material andmesh size) and printing speed, and on the otherhand on the composition and viscosity of theenzyme containing ink. The thicker and denser theenzyme layer and the more hydrophilic the organicbinder of the enzyme layer the more the responseof the sensor is controlled by diffusion. Theparameters we applied resulted in glucose sensorswith linear range from 1 to 3 mmol/l glucose (Fig.1). At glucose concentrations lower than 1 mmol/lthe sensor showed the typical deviation from lin-earity of mediator based sensors because of thecompetition of oxygen and TTF as electron accep-tors at small substrate concentrations. Hence thelower detection limit of glucose with the H,02detecting system was superior. The sensitivity ofthe graphite based printed biosensor between 1and 3 mmol/l glucose is 1 uA/mmol/l at roomtemperature. The standard deviation from onesensor to the next, printed with one batch of

0 1 2 3 4 5 6 7 0concentration glucose [mmol/l]

Fig. I. Calibration curve obtained with a thick film biosensor whichis based on a printed TTF modified graphite/glucose oxidase layer.

enzyme paste, was found to be 5% over the sensorslinear range. As the sensors are considered asdisposable sensors the low stability on storage insolution (only 60% sensitivity remains after stor-age for 1 h) probably due to loss of mediator isnot of great importance. The shelf life at 4 C oversilica gel is at least 6 months without any de-tectable loss of enzyme activity and therewithmuch better than the shelf life of the glutaralde-hyde based sensors.

Table 1 shows the results of glucose determina-tion in fruit juice and wine for both systems. Theresponse of the non-enzyme electrode was sub-stracted from the current output of the enzymecontaining sensor. Sufficient agreement with thereference method (Boehringer test kit) was ob-tained for all the juices tested. In the case of winethe signal obtained by the mediator based elec-trode was very strongly affected by high back-ground current and electrical noise probably dueto changes in the enzyme layer because of thealcohol content of the sample.Ami ne sensorsPutrescine oxidase from M icrococcus rubens isknown to convert mainly putrescine but also ca-daverine and spermindine [ 131. Therefore the sig-nal obtained by the enzyme presents the combinedresponse to several amines. All optimization pro-cedures were performed using putrescine as theenzyme substrate. The pH dependence was deter-mined for several buffer systems (Fig. 2). Thoughhighest sensor response was recorded in 0.1 mol/lglycine pH 8.5, Clark and Lubs buffer, pH 8.5,


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TABLE I. Measurement of glucose in real samples by different amperometric thick film biosensorsSample, Content of glucose (g/l)

Thick film biosensor Reference method(Boehringer)Based on H,Oz Printed enzyme/dectection mediator paste

Red wine 2.25 2.35Multi-vitamin juice 12.7 12.9 12.8Orange juice 7.40 7.29 7.77Orange drink 1.23 6.55 6.95Apple juice 23.7 21.2 22.2

Fig. 2. Dependence of the response of the amine sensor on pH for aputrescine concentration of 16.7 umol/l. 0: 0. I mol/l potassium phos-phate, A: Clark and Lubs solution, W: 0.1 mol/l glycine, +: 0.2 mol/lborate.

T1.50

1.25 T1.00 -

%::

0.0 0.5 1.0pYtl.mCie wrl0I/Il

OJmmoconcentration putrescine [rmol/l]

Fig. 3. Calibration curve obtained with a thick film biosensor basedon immobilization of putrescine oxidase with glutaraldehyde ontosilanized Pt working electrodes.

5Z 60-e0:z 60$+I -\_.z 40 -E.-Y

20 -0 : Immobilization with prior &Ionization+ : Immobilization without prior silanizotion

oJ I I I I I1 2 3 4 5 6time [days]

J

Fig. 4. Stability of amine sensors. Percentage of the initial slope ofthe linear range of the calibration curve.

was used throughout further experiments becausetime required to establish a stable backgroundcurrent was much shorter for this buffer (around3 min compared to 15 min for glycine buffer). Atypical calibration curve for putrescine is shown inFig. 3. The linear detection range stretches from thelower detection limit of 0.06 to 200 pmol/l with asensitivity of 2.4 nA/umol/l and a standard devia-tion of 7%.Figure 4 depicts the stability of the sensors,which could be improved by silanization of theplatinum working electrodes prior to enzyme im-mobilization.Though the sensor signal is not specific toonly one amine it could be used as an indicator offish freshness, as over a storage period of one weekthe amine content increased from 25 to 130 ppmexpressed as apparent putrescine concentration.


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ConclusionsIt is demonstrated that biosensors based on

thick film technology are a versatile tool for thedetermination of various compounds in foodstuffs.Special attention was paid to an easy and cheapfabrication of the biosensor in order to enabletheir use as disposable sensors for rapid screeningof large numbers of samples. It is shown that theimmobilization of enzymes by incorporation in acarbon paste could be an alternative to cross-link-ing the enzyme using glutaraldehyde.

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equipment and sensor electrodes therefor, EP 0 127 958 A2 ( 1984).2 H. A. 0. Hill, J. E. Frew, M. R. Ball and M. J. Green, Assay for

cholesterol and derivatives therefor, EP 0 230 786 Al (1987).3 L. A. Sherlock and M. J. Green, Creatinine assay, UK Patent No.

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C. C. Liu, Apparatus and method for sensing species, substancesand substrates using oxidase, US Pafenf No. 4 655 880 (1987).D. R. Matthews, E. Bown, A. Watson, R. R. Holman, J. Steemson,S. Hughes and D. Scott, Pen-sized digital 30-second blood glucosemeter, Lance& 4 ( 1987) 778-779.J. Kulys and E. J. DCosta, Printed amperometric sensor based onTCNQ and cholinesterase, Biosensors Biolectron., 6 (1991) lO9-115.U. Bilitewski, P. Riiger and R. D. Schmid, Glucose biosensorsbased on thick film technology, Biosensors Biolectron., 6 (1991)369-373.P. Riiger, U. Bilitewski and R. D. Schmid, Glucose and ethanolbiosensors based on thick film technology, Sensors and ActuatorsB, 4 (1991) 267-271.A. Askar and H. Treptow, in Biogene Ami ne in Lebensmitteln ,Eugen Ulmer GmbH & Co., Stuttgart, 1986.S. Todoriki, M. Tajima and M. Senda, Enzymatic oxygen electrodemethod for determination of polyamines in foods, Anal. Sci., 4(1988) 583-585.R. Gasparini, M. Scarpa, M. L. Di Paolo, R. Stevanato and A.Rigo, Amino oxidase amperometric biosensor for polyamines,Bioelectrochem. Bioenerg., 25 (1991) 307-315.R. Stevanato, B. Mondovi, S. Sabatini and A. Rigo, Spectropho-tometric assay for total polyamines by immobilized amine oxidases,Anal. Chim. Acta, 237 (1990) 391-397.H. Yamada, Putrescine oxidase (Micrococcus rubens), in H. Taborand C. White Tabor (eds.), Methodr of Enzymology, Vol. 17B,Academic Press, New York, 1971, pp. 726-730.

1992 - miniaturized disposable biosensors

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