ArQwTIcA

CHIMICA
ELSEVIER

ACTA
Analytica Chimica Acta 317 (1995) 259-264

Development of biosensors based on an electrolyte isolator semiconductor ( EIS) -capacitor structure and their application for process monitoring Part I. Development of the biosensors and their characterization
C. Menzel a, T. Lerch a, T. Scheper , K. Schiigerl ag *
a Institute for Technical Chemistry, University of Hannover, Callinstr. 3, D-30167 Hannover, Germany
b Institute for Biochemistry, University of Miinster, Miinster, Germany

Received 24 April 1995; revised 27 June 1995; accepted 12 August 1995

Abstract
isolator semiconductor (EIS) chips consisting of Simple non-structured fluoride sensitive electrolyte Si/SiO,/Si,N,/LaF, layers were used as transducers for measuring the H,O, concentration by peroxidase (POD) and combined this H,O, sensor with various oxidases, which form H,O, in stoichiometric amount. The concentrations of the following analytes were measured with this system: glucose with glucose oxidase (GOD) and POD, maltose with amyloglucosidase (AGLU), mutarotase (MUT), GOD and POD, amylose with /3-amylase (Amyl), AGLU, MUT, GOD and POD, sucrose with invertase (WV), MUT, GOD and POD, ethanol with alcohol oxidase and POD, sulfite with sulfiteoxidase (SOD) and POD, xanthine with xanthineoxidase (XAD) and POD, phosphate with nucleoside-phosphorylase (NP), XOD and POD. The required enzymes were coimmobilized on the surface of the transducer (pF-EIS-CAP), mounted into a flow cell, equipped with a reference electrode, integrated into a flow injection analyser (FIA) system and operated with the CAFCA (Computer Assisted Flow Control and Analysis) automation programme. Keywords: Biosensors; Semiconductors; Process monitoring; Flow injection

1. Introduction In the preceding paper [l] on a new type of enzyme sensor based on the electrolyte isolator semiconductor (EIS) capacitance structure was reported, which is marked by a very simple structure. The unstructured EIS-CAP 4 X 4 mm chip consists of a pH-sensitive 70 nm Ta,O, layer (on the top), 20 nm

* Corresponding author. 0003-2670/95/$09.50

Si,N,, 50 nm SiO, on p-Si substrate (10 0 cm), which is contacted to an Al-plate. These EIS-layer structures form a plate capacitor with variable capacitance depending on the pH value of the contacting liquid. The flatband capacitance of the EIS-CAP chip is shifted along the voltage axis as a pH value is changed. By means of the C/V instrument in a constant charge mode the capacitance is kept constant and the voltage change is measured as a function of the pH value. The calibration curve yielded a slope of 57 mV, which corre-

0 1995 Elsevier Science B.V. All rights reserved

SSDI 0003-2670(95)00419-X

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H,O, + 4-fluoroaniline
POD + F-

+ H,O + (polymers of aniline derivatives) (3)

The enzymes GOD and POD are coimmobilized on the surface of the pF-EIS-CAP by glutardialdehyde.

Fig. 1. Encapsulated pF-ESCAF

sensor

3. Characterization of the biocomponents The following enzymes were applied: Peroxidase (POD) type II (EC 1.11.1.7), Aspergillus niger (Sigma), pH optimum 5-7.5. Alcoholoxidase (AOD) (EC 1.1.3.131, Pichia pastoris (Sigma), pH optimum 7.5. primary alcohol + 0, -+ aldehyde + H,O, Sulfiteoxidase
AOD

sponds to the expected value according to the Nernst equation. The EIS-CAP chip was encapsulated by sealing it with Viton O-rings (Fig. 1). On account of the strong dependence of the voltage signal of the pH-sensitive EIS-CAP on the buffer capacity of the contacting liquid, the upper layer was replaced by 100 nm LaF,. This fluoride sensitive EIS-CAP chip has a slope of 58 mV, which corresponds to the slope of the fluoride electrodes (56-60 mV>. The EIS-CAP chips were manufactured by the Institute of Semiconductor Technology and Materials for Electrotechnique of the University of Hannover.In the following the biosensors based on this pF-sensitive FIS-CAP transducer are characterized and applied for process monitoring.

(4)

(SOD) (EC 1.8.3.1), Cucurbita

species (Sigma), pH optimum 6.

SO;- + H,O + 0, zDSO;-

+ H,O,

(5)

Xanthineoxidase (XOD) (EC 1.1.3.22), Cowmilk (Sigma), pH optimum 8.2. xanthine + H,O + 0, + urea + H,O,
XOD

(6)

Glucoseoxidase (GOD) (EC 1.1.3.4), Aspergillus 2. pF-EIS-CAP as transducer for biosensors The application of the pF-EIS-CAP as transducer for biosensors is based on the following reaction, which is catalysed by the enzyme peroxidase (POD): H,O, + 4-fluoroaniline
POD + F-

niger (Sigma), pH optimum 5-6.

Mutarotase (Mut), Porcine kidney (Sigma) o-D-glucose y @D-glucose (7)

~Arnylase (&Amyl) (EC 3.2.1.2), Plant seed (Sigma), pH optimum 6.

P-Amy1

+ (other products)

(1)

amylose

maltose

(8)
AS-

The concentration of the F- ion formed is proportional to the amount of H,O, formed and can be detected by the pF-EIS-CAP. Each enzyme which forms H,O, in stoichiometric amount can be combined with this transducer; e.g. the conversion of glucose with glucose oxidase (GOD) in gluconolactone/gluconic acid: glucose + 0,
GOD

pergillus

Amyloglucosidase (AGLU) (EC 1.10.3.3), niger Merck, pH optimum 6.

AGLU

maltose +

o-D-glucose + @D-glucose

(9)
utilis

4 H,O, + gluconolactone

(2)

Invertase (INV) (EC 3.2.1.26), Candida (Sigma), pH optimum 3.5-5.5. INv sucrose --, o-D-glucose + p-D-fructose

(10)

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Chimica Acta 317 (1995) 259-264

261

a -D-glucose is converted to P-o-glucose according to Eq. (7) and the glucose is detected by reactions (2) and (3).

4. Preparation of the enzyme membranes and their characterization The lyophilized enzymes were dissolved in 0.85% NaCl solution(1 mg enzyme per 1.5 ~1 NaCl solution) and 5 ~1 solution was put on the cleaned 12.6 mm2 pF-EIS-Cap surface and dried in. The enzyme layer was immobilized with 25% glutardialdehyde for 15-20 min and rinsed with buffer solution. The average thickness of the enzyme layers amounted to 16,000 nm. The activity of the enzyme membranes was determined by photometric tests (e.g., 89 mU cm -2 POD and 23 mU cmp2 GOD). The calibration of the pF-EIS-CAPS was carried out in 100 ml 1 M acetate buffer (total ionic strength adjustment buffer, TISAB) and with 0.1 M (1 ml in 100 ml buffer) p-fluoroaniline (Fig. 2). The best performances were found with a POD to oxidase ratio more than three. The measuring range of the fluoride sensors should be in the limits between pH 4 and 8. At lower pH the signal decreases due to a complex formation and at higher pH the signal is impaired by the OH- ion concentration. On account of the various pH optima of applied enzymes, one would expect different optima of the biosensors. However, all of them had the same pH optimum (pH 51, which corresponds to the pH optimum of the POD. As expected, the biosensor signals depend only

-probe

---probed,uolved1 mM bdhr. pt, 5 in diuold 10 bV.r. pH mM 1

t

Glucose

(g/l1

Fig. 3. Sensor signal as a function of the glucose concentration at various buffer concentrations and pH values. TlSAR 0.17 M NaCl, 0.1 M p-fluoroaniline (pFA1, pH 5.

slightly or not at all on the buffer capacity (Fig. 3). In order to stabilize the signals, the ionic strength was increased by using 0.17 M NaCl. The reproducibility of the production of the biosensors and their stability within 14 days was satisfactory. The decrease of the sensor signal after 15 days is due to the gradual deactivation of POD. On account of the formation of byproducts during the oxidation of 4-fluoroaniline and their deposition on the sensor surface, the flow along the sensor surface is absolutely necessary to free the surface from the deposits. An optimal stirrer speed of 150 rpm was obtained for the calibration. The optimal temperature of the POD is in the range of 20-25C. However, the temperature effect on the biosensor signal is very slight. Above lo- mol fluoride concentration an inhibition of the POD was observed. 20 mM 4-fluoroaniline is needed to obtain the maximum sensor signal. The simultaneous measurements with a biosensor and a fluoride sensor (without enzyme membrane) indicated, that with the above mentioned analytes no signal was obtained with the fluoride sensor alone.

5. Characterization of the biosensors

Fig. 2. Layout of the calibration set-up. (1) x/t recorder, (2) digital multimeter, (31 C/U power supply, (4) Ag/AgCl reference electrode, (5) EIS-CAP sensor, (6) pH electrode, (7) pH meter, (8) thermostated vessel, (9) magnetic stirrer.

Based on the investigations of Wollenberger et al. [2], De Groot et al. [3] and Watanabe et al. [4] a phosphate sensor was developed based on the following enzymatic reaction with nucleoside phospho-

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4
cm0 0.05 0.10 0.15

1
0.20

KH2P0,

[mol/l]

Ethanol

[gfl]

Fig. 4. Calibration diagram of the phosphate sensor (0.1 M imidazole buffer, 0.1 M NaCI, 0.001 M inosine, 0.1 M pFA, pH 5).

Fig. 6. Calibration diagram of the ethanol 0.17 M NaCl, 0.1 M pFA, pH 5).

sensor (1 M TISAB,

70 ,

rylase (NP) in the first step and with xanthineoxidase (XOD) in the second step: inosine f phosphate 4 ribose-l-phosphate + hypoxanthine
XOD

(11) (12)

hypoxanthine + 20,

--) urea + 2H,O,

2 mol H,O, is formed per 1 mol phosphate. NP, XOD and POD were coimmobilized. The sensor remained stable during 21 days storage at 4. In Fig. 4 the calibration graph is shown. In absence of phosphate the same sensor measures the hypoxanthine concentration according to Eq. (12). In Fig. 5 the corresponding calibration curve is shown. The determination of the ethanol concentration is based on the Eqs. (4) and (1) (see Fig. 6).

Fig. 7. Calibration diagram of the sulfite sensor (1 M TISAB, 0.17 M NaCl, 0.1 M pFA, pH 5).

The determination of sulfite concentration was performed by Eq. (5) (see Fig. 7). Starch consists of 80% water insoluble amylopectine and 20% of water-soluble amylose. By enzymatic determination according to Eqs. (8) (9), (2) and (3) only the water soluble amylose can be

Fig. 5. Calibration diagram of the hypoxanthine sensor (0.1 imidazole buffer, 0.1 M NaCl, 0.001 M inosine, 0.1 M pFA, pH 5).

Amylose

[g/l]

Fig, 8. Calibration diagram of the amylose 0.17 M NaCl, 0.1 M pFA, pH 5).

sensor (1 M TISAB,

C. Menzel et al. /Analytica

Chimica Acta 317 (1995) 259-264 reference electrode

263

1,
00

,
0.2 Cl.4

,
0.s

I
0.8

,
10

S-Cap
I

sucrose[g/l]
Fig. 9. Calibration diagram of the sucrose 0.17 M NaCl, 0.1 M pFA, pH 5). sensor (1 M TISAB, Fig. 10. Flow cell with the Bio-pF-EIS-CAP reference electrode. sensor and Ag/AgCl

detected. The required enzymes were coimmobilized. Fig. 8 shows the calibration graph for amylose. The sucrose concentration was determined by Eqs. (191, (7) (2) and (3). The necessary enzymes were coimmobilized. In Fig. 9 the calibration graph is shown.

6. Integration of the sensors into the FIA system The enzymes required for the analysis were coimmobilized by glutardialdehyde on the transducer sur-

face and the biosensor is mounted into a flow cell together with an Ag/AgCl reference electrode (Fig. lo), integrated into a flow injection analysis (FJA) system (EVA-line, Eppendorf Netheler and Hinz, Hamburg) and operated with the automation programme CAFCA, which was developed by Hitzmann et al. [5], and a PC (486 DX> (Fig. 11). The control was performed by commercial interface cards which were supported by the CAFCA. The evaluation of the FIA peaks was performed by five different meth-

carher
Fig. 11. Layout of the on-line FL4 setup.

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Chimica Acta 317 (1995) 259-264

ods and the most suitable one was applied for the particular system. Under standard conditions the peak height gave the best results, but in case of disturbances other methods were used as well, according to the expert system developed by Brandt and Hitzmann [6]. The injection time and the carrier flow rate have a considerable effect on the signal height. For each of the analytes the optimal conditions were evaluated. However, the injection time was limited to < 15 s, and the flow rate to > 1 ml min-.

function of the fluorine concentration. The combination of pF-EIS-CAP with various oxidases, which produce H,O,, yielded biosensors for the detection of glucose, maltose, amylose, sucrose, ethanol, hypoxanthine, phosphate and sulfite. These biosensors were mounted together with a reference electrode in a flow cell, integrated into a flow injection analysis (FL41 system and operated with a automation programme. On account of the inexpensive pF-EIS-CAP chip, this is the most inexpensive high quality biosensor known at present. The application of these biosensors for bioprocess monitoring will be treated in Part II.

7. Comparison of the pF-EIS-CAP with pF-FETs Moritz [7], Hintsche et al. [8-101 and Dransfeld et al. [ll] developed a fluoride sensitive field effect transistor (pF-FET). They coimmobilized the enzymes POD/GOD on the multigate-FET by polyurethane [8] and combined it with a pH-FET as pseudo reference electrode and with 4-pentafluorophenol, which yielded F- ion, analogue to reactions (2) and (3). Th e properties of this POD/GOD-FET (slope = 45.5 mV/log Cal, 0.02-0.5 mol 1-l linear range and 10 days life time) were practically identical with those of POD/GOD-EIS-chip. Because the EIS chip is not structured in contrast to the FET, its price is lower and its encapsulation is simpler than those of FETs. Acknowledgements The authors gratefully acknowledge the financial support from the German Scientific Foundation (DFG) and the Federal Ministry of Research and Technology (BMFT Project No. 0310087A)

References
[l] M. Beyer, C. Menzel, R. Quack, T. Scheper and K. Schiigerl, Biosensors Bioelectronics, 9 (1994) 17-21. [2] U. Wollenberger, F. Schubert and F. Scheller, Sensors Actuators B, 7 (1992) 412-415. [3] H. De Groot, H. De Groot and Th. Noll, Biochem. .I., 229 (1985) 255-260. [4] E. Watanabe, H. Endo and K. Toyama, Biosensors, 3 (1988) 581-587. [5] B.Hitzmann, F. Lammers, B. Weigel and A. van Putten, BIOforum, 16 (1993) 450-454. [6] J. Brandt and B. Hitzmann, Anal. Chim. Acta, 291 (1994) 29-40. 171 W. Moritz, I. Meierhofer and L. Mueller, Sensors Actuators, 15 (1988) 211-219. [S] R. Hintsche, G. Neumann, I. Dransfeld, G. Kampfrath, W. Hoffmann and F. Scheller, Anal. L&t., 22 (1989) 2175-2190. [9] R. Hintsche, I. Dransfeld, F. Scheller, M.T. Pham, W. Hoffmann, J. Hueller and W. Moritz, Biosensors Bioelectron., 5 (1990) 327-334 [lo] R. Hintsche and I. Dransfeld, Anal. L&t., 23 (1990) 451-460. [ill I. Dransfeld, R. Hintschse and F. Scheller, Fresenius Z. Anal. Chem., 333 (1988) 23-25.

8. Conclusions A simple EIS-chip consisting of pSi/SiO,/Si,N,/LaF, layers, which changes its capacitance depending of the fluorine concentration in the contacting liquid, was used as pF transducer (pF-EIS-CAP) together with peroxidase (POD) as receptor and 4-fluoroaniline as fluorine source for the determination of H,O, concentration. The 4channel power supply of the sensor was operated in constant charge mode and yielded the voltage shift of the flatband capacitance of the sensor as a