Sensors and Actuators B, 7 (1992) 351-355

351

Miniaturized
U. Bilitewski,

disposable biosensors
P. Riiger and R. D. Schmid

G. C. Chemnitius,

Department of Enzyme Technology, GBF (Gesellschaft ftir Biotechnologische Forschung mbH), 3300 Braunschweig (FRG)

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

Introduction Over the past two decades enzyme assays have been established as standard analytical methods for food quality control. Routinely, soluble enzymes and reagents are used in photometric tests. Biosensors containing all the required enzymes and reagents would simplify the performance of the tests and therefore reduce the costs of each single assay. Analysis using biosensors is suitable as a screening method, to be used by scientists in laboratories where the rapid processing of large numbers of samples is required and by distributors and consumers at the place of sale where the quality must be assessed immediately. Miniaturization of the sensor reduces the amount of enzyme and reagents needed. Thick film technology, a well established technology for the production of electronic circuits, provides the possibility to fabricate miniaturized and relatively low cost electrodes. Therefore biosensors based on this technology have been developed recently [l-8], most of which were designed for medical applications. In this paper we describe different sensors made in thick film technology which can be used as disposable sensors in food quality control. The manufacture of disposable biosensors should be easy and rapid so as to reduce the costs involved. Therefore two methods which allow an automation of the process were chosen for the immobilization of the enzymes. This is on the one hand by dripping the enzyme solution onto the electrode
0925-4005/92/$5.00

surface and on the other hand by screen printing of an enzyme containing ink onto the working electrodes. The latter approach results in a biosensor fabricated by only one technology; the enzyme layer is screen printed by the same process as the electrode pads, conductor lines and insulation. We selected glucose and amine concentrations as parameters to demonstrate the potential of thick film biosensors in quality control. Biogenic amines are indicators of the freshness of fish. They are generated during protein decomposition via amino acid decarboxylation following the slaughter of the fish, their concentration increasing with storage time [9]. Hence a sensitive and specific determination is required. Different approaches have already been reported to use immobilized polyamine oxidases for polyamine determination in fish and tissue samples [lo- 121. But none of the systems described so far were considered suitable for use as disposable sensors for rapid polyamine determination at the place of sale. We present the preparation of easily used, rapid, disposable sensors by immobilizing putrescine oxidase (EC 1.4.3.10) onto the working electrodes of thick film sensors. The resulting biosensors are characterized. Additionally thick film biosensors for the determination of glucose in juices were prepared. Taking the example of the glucose sensors the two immobilization methods were compared with respect to sensitivity, linear range, stability and influence of interfering substances present in real samples.
@ 1992 Elsevier Sequoia. All rights reserved

352

Experimental Reagents

For the construction of the thick film electrodes the following reagents were used: fired aluminium oxide ceramics, 10 x 10 cm for the preparation of eight sensors, silver/palladium thick film paste and protective overglaze were obtained from Du Pont (Bad Homburg, FRG). Platinum containing thick film paste was purchased from Ferro (Sindelfingen, FRG) and carbon ink was bought from Acheson Colloids Co. (Plymouth, UK). For enzyme immobilization glucose oxidase EC 1.1.3.4. from Penicilhum amagasakiense (Nagase Biochem. Ltd., Tokyo, Japan), putrescine oxidase from Micrococcus rubens (a kind gift of Amano Pharmaceutical Co., Ltd., Nagoya, Japan), 3-aminopropyl-triethoxysilane (Aldrich, Steinheim, FRG), bovine serum albumin (BSA, fraction V, Boehringer Mannheim, FRG), glutaraldehyde (25%, Merck, Darmstadt, FRG), graphite powder (Merck), tetrathiafulvalene (TTF, Fluka, Buchs, Switzerland) and polyvinylpyrrolidone (Fluka) were used. Glucose was purchased from Merck. 0.1 mol/l stock solutions of amine hydrochlorides (Sigma, Deisenhofen, FRG) in 0.1 mol/l HCl were prepared.
Sample preparation

for Hz02 detection. Therefore, these electrodes were modified with the mediator tetrathiafulvalene, TTF. The layout and fabrication of the thick film sensors comprising four working electrodes, a reference and a counter electrode have been described in earlier publications [7, 81. The reference potential of the solid state integrated Ag/AgCl reference electrode (glucose sensors) in buffer containing 0.1 KC1 was 83 mV negative of a conventional Ag/AgCl (3 M KCI) reference electrode. Hence the measurements were carried out at 520 and 600 mV ( H2 O2 detection) or 140 and 220 mV (mediator based system) versus the solid-state integrated Ag/AgCl or a conventional Ag/AgCl (3 M KCl) reference electrode, remode with a spectively, in a three-electrode four-channel potentiostat from Bank (Giittingen, FRG).
Sensors based on immobilization with glutaraldehyde

Prior to analysis samples of wines and juices were diluted with 0.1 mol/l potassium phosphate buffer pH 7.0 containing 0.1 mol/l KC]. For amine determination extracts of fish samples were prepared. Pollack was purchased from a local fish shop and stored at 4 C. Samples of 100 g were disrupted in a homogenizer (Moulinex, Kiiln, FRG). Ten grams of the homogenized fish meat were extracted in 80 ml 10% trichloroacetic acid (Merck) using an Ultra Turrax dispersing apparatus (IKA Labortechnik, Staufen, FRG). The extracts were filtered and made up to 100 ml with 10% trichloroacetic acid in a volumetric flask.
Thick Jilm sensor

For the detection of H,Oz the working electrodes were printed with platinum paste. Prior to immobilization of the enzymes by cross-linking with glutaraledhyde the platinum electrodes of some sensors were modified with 3-aminopropyltriethoxysilane (10% in phosphate buffer pH 7.0). For glucose oxidase immobilization a solution of 1.2 mg glucose oxidase, 5 mg BSA, 15.7 ~1 2.5% glutaraldehyde in 100 ~1 phosphate buffer and for putrescine oxidase immobilization a mixture of 20 ~1 of putrescine oxidase, 20 pl 10% BSA in water and 20 ~1 of 5% glutaraldehyde in water were prepared. The enzyme glutaraldehyde mixtures were dripped onto the working electrodes and were allowed to dry at room temperature for approximately 1 h. When not in use the sensors were stored at 4 C in phosphate buffer.
Sensors with printed enzyme layer

Two different detection principles were used: sensors fabricated by cross-linking the enzyme with glutaraldehyde at a platinum electrode, were based on H202 detection. Whilst, because the enzyme printing pastes contained a high proportion of graphite particles (so as to achieve sufficient adsorption of the enzyme) they acted as graphite electrodes and hence required a high overpotential

In order to obtain a good adhesion of the printed enzyme layer to the electrodes carbon paste was used for printing of the working electrodes. The enzyme ink was prepared as follows: 1 g graphite powder was mixed with 20 ml of a saturated solution of TTF in toluene. The mixture was stirred until the toluene had evaporated. Twelve mg of glucose oxidase were dissolved in 0.9 g polyvinylpyrrolidone solution ( 10% in distilled water) and 0.5 g of the TTF coated graphite

353

powder were added. After thoroughly mixing the ink was applied to the working electrodes by screen printing. The enzyme layer was dried overnight and the sensors were stored on silica gel at 4 C. Sensors without enzyme were prepared in the same way to recognize interferences of electroactive compounds in the samples.

Results and discussion Glucose sensors

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

The suitability of thick film technology for the construction of glucose sensors by immobilization of glucose oxidase by cross-linking with glutaraldehyde on platinum thick film electrodes has been shown previously [7, 81. Automation should be possible using micro-dispensers which are already used in thick film laboratories for the application of soldering pastes or adhesives. The linear range obtained with these sensors extended to 1 mmol/l glucose with a lower detection limit (signal:noise 3:l) of 1 umol/l glucose and a sensitivity of 0.2 uA/mmol/l. The shelf life of the sensors in buffer was one month at room temperature and 20 days in semi-continuous use. The standard deviation was 4.7%. Alternatively the printing of the enzyme layer on the working electrode was tested. In this case the properties of the enzyme layer, and hence the response of the sensor, depend on the one hand on the printing process, for example on the screen (material and mesh size) and printing speed, and on the other hand on the composition and viscosity of the enzyme containing ink. The thicker and denser the enzyme layer and the more hydrophilic the organic binder of the enzyme layer the more the response of the sensor is controlled by diffusion. The parameters we applied resulted in glucose sensors with linear range from 1 to 3 mmol/l glucose (Fig. 1). At glucose concentrations lower than 1 mmol/l the sensor showed the typical deviation from linearity of mediator based sensors because of the competition of oxygen and TTF as electron acceptors at small substrate concentrations. Hence the lower detection limit of glucose with the H,02 detecting system was superior. The sensitivity of the graphite based printed biosensor between 1 and 3 mmol/l glucose is 1 uA/mmol/l at room temperature. The standard deviation from one sensor to the next, printed with one batch of

concentration

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

enzyme paste, was found to be 5% over the sensors linear range. As the sensors are considered as disposable sensors the low stability on storage in solution (only 60% sensitivity remains after storage for 1 h) probably due to loss of mediator is not of great importance. The shelf life at 4 C over silica gel is at least 6 months without any detectable loss of enzyme activity and therewith much better than the shelf life of the glutaraldehyde based sensors. Table 1 shows the results of glucose determination in fruit juice and wine for both systems. The response of the non-enzyme electrode was substracted from the current output of the enzyme containing sensor. Sufficient agreement with the reference method (Boehringer test kit) was obtained for all the juices tested. In the case of wine the signal obtained by the mediator based electrode was very strongly affected by high background current and electrical noise probably due to changes in the enzyme layer because of the alcohol content of the sample.
Amine sensors

Putrescine oxidase from Micrococcus rubens is known to convert mainly putrescine but also cadaverine and spermindine [ 131. Therefore the signal obtained by the enzyme presents the combined response to several amines. All optimization procedures were performed using putrescine as the enzyme substrate. The pH dependence was determined for several buffer systems (Fig. 2). Though highest sensor response was recorded in 0.1 mol/l glycine pH 8.5, Clark and Lubs buffer, pH 8.5,

354 TABLE I. Measurement of glucose in real samples by different amperometric thick film biosensors Sample, Content of glucose (g/l) Thick film biosensor Based on H,Oz dectection Red wine Multi-vitamin juice Orange juice Orange drink Apple juice 2.25 12.7 7.40 1.23 23.7 Printed enzyme/ mediator paste 2.35 12.8 7.77 6.95 22.2 Reference method (Boehringer)

12.9 7.29 6.55 21.2

5
e

60-

0 : z

60

$ +I .z E .Y 20 0 + -\_ 40 -

oJ
1

: Immobilization : Immobilization I I
2 3 time

with prior without

&Ionization silanizotion

prior

I
4 [days]

I
5

I
6

Fig. 2. Dependence of the response of the amine sensor on pH for a putrescine concentration of 16.7 umol/l. 0: 0. I mol/l potassium phosphate, A: Clark and Lubs solution, W: 0.1 mol/l glycine, +: 0.2 mol/l borate.

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

T 1.50

1.25 T 1.00 -

% : :

OJm 0

mo
concentration

0.0 0.5 1.0 pYtl.mCie[wrl0I/Il

putrescine

[rmol/l]

Fig. 3. Calibration curve obtained with a thick film biosensor based on immobilization of putrescine oxidase with glutaraldehyde onto silanized Pt working electrodes.

was used throughout further experiments because time required to establish a stable background current was much shorter for this buffer (around 3 min compared to 15 min for glycine buffer). A typical calibration curve for putrescine is shown in Fig. 3. The linear detection range stretches from the lower detection limit of 0.06 to 200 pmol/l with a sensitivity of 2.4 nA/umol/l and a standard deviation of 7%. Figure 4 depicts the stability of the sensors, which could be improved by silanization of the platinum working electrodes prior to enzyme immobilization. Though the sensor signal is not specific to only one amine it could be used as an indicator of fish freshness, as over a storage period of one week the amine content increased from 25 to 130 ppm expressed as apparent putrescine concentration.

355

Conclusions It is demonstrated that biosensors based on thick film technology are a versatile tool for the determination of various compounds in foodstuffs. Special attention was paid to an easy and cheap fabrication of the biosensor in order to enable their use as disposable sensors for rapid screening of large numbers of samples. It is shown that the immobilization of enzymes by incorporation in a carbon paste could be an alternative to cross-linking the enzyme using glutaraldehyde.

4 C. C. Liu, Apparatus and method for sensing species, substances and substrates using oxidase, US Pafenf No. 4 655 880 (1987). 5 D. R. Matthews, E. Bown, A. Watson, R. R. Holman, J. Steemson, S. Hughes and D. Scott, Pen-sized digital 30-second blood glucose meter, Lance& 4 ( 1987) 778-779. 6 J. Kulys and E. J. DCosta, Printed amperometric sensor based on TCNQ and cholinesterase, Biosensors Biolectron., 6 (1991) lO9115. 7 U. Bilitewski, P. Riiger and R. D. Schmid, Glucose biosensors based on thick film technology, Biosensors Biolectron., 6 (1991)
369-373.

8 P. Riiger, U. Bilitewski and R. D. Schmid, Glucose and ethanol biosensors based on thick film technology, Sensors and Actuators
B, 4 (1991) 267-271.

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