Chemical and biochemical sensor array for two

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Sensors and Actuators B, 18-19 (1994) 229-234

Chemical and biochemical sensor array for two-dimensional imaging of analyte distributions H. Meyer*, H. Drewer, J. Krause and K. Cammann Institut fUr Chemo· und Biosensorik e. v., Lehrstuhl fUr Analytische Chemie,

Westfiilische Wzlhelms-Universitiit Munster,

Wzlhelm-Kkmm-Strasse 8, D-48149 Munster (Germany)

R. Kakerow, Y. Manoli, W. Mokwa and M. Rospert Fraunhofer-Institut fUr Mikroelektronische Schaltungen und Systeme, Finkenstrasse 61, D-47057 Duisburg (Germany)

Abstract A monolithic sensor chip containing a sensor array of 400 individually addressable platinum microelectrodes has been fabricated. This sensor array can be used in potentiometric mode as well as in amperometric mode for redundant measurements with signal averaging, multi-analyte measurements and imaging of analyte distnoutions. The sensor array is coated with an ammonium-selective PVC membrane for creating a chemical sensor array and, additionally, with a urease membrane for creating a biosensor array. Thus, two-dimensional imaging of the distributions of ammonium and urea can be obtained.

1. Introduction

In the last 20 to 30 years many chemical and biochemical sensors have been constructed for selective measurement of a variety of different analytes. All of them use the same principle: a selective membrane is responsible for analyte recognition and a sensitive transducer transforms this recognition into a measurable signal [1, 2]. But the usual chemical and biochemical sensors only provide information about the total concentration of the bulk solution. None of them is able to indicate an analyte distribution over a two-dimensional area. Sometimes, e.g., for the measurement of the local concentration of potassium or oxygen on organ surfaces in hospitals, this would be very useful [3]. In order to overcome this shortcoming, the presented sensor array has been constructed. This sensor array, as the main part of a monolithic sensor chip, has been fabricated in a modified CMOS process [4, 5]. It consists of 400 individually addressable microelectrodes. Imaging of the spatial distribution of an analyte is realized by reading out the individual electrode signals successively. In addition to this imaging, the sensor array may also be used for measuring the bulk concentration of analyte solutions. Then, measurements will become redundant and signal averaging is possible. Moreover, individual electrodes can be modified with different analyte-se-

lective membranes, thus resulting in a single-chip multianalyte sensor array.

2. Experimental set-up and chip layout

The experimental set-up used for all the presented measurements is shown in Fig. 1. The monolithic sensor chip was always arranged in a horizontal position, the sensor array facing up. Chemical sensing or biosensing was performed using an ion-selective membrane or biomembrane directly coated onto the sensor array. 300 J.Ll of the appropriate sample solution were pipetted onto the ion-selective membranelbiomembrane, completely covering the selective membrane. All reagents used for the membranes and solutions were at least ACS grade. An Ag/AgCl/3M KCI electrode was dipped into the sample solution as reference. In order to avoid interferences from potassium, a double liquid junction was used. Usually, the double liquid junction was filled

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with the 0.1 M Tris(hydroxymethyl)aminomethane buffer solution, adjusted to pH 7.0 with HCl (Tris-HCl (7.0». A multimeter was used to read the analog output signals of the sensor chip. The read-out data were collected on a personal computer using an IEEE card. The computer was also connected directly to the sensor chip by a digital I/O card. Thus, the computer could be used as an external clock, representing the switching rate while scanning the sensor array. The operational software was self-made, while the data-processing software was commercially available. The power supply (not depicted here) was adjusted to ±5,3 V. In addition to the sensor array, the sensor chip contains some other components. Figure 2 shows a block diagram of the monolithic sensor chip. Bondpads are used for connecting the individual lines on the chip to the peripheral instruments via bondwires. The control unit is needed for individually addressing the rnicroelectrodes. It consists of a horizontal and a vertical shift register and a control logic. The two large electrodes beside the sensor array and the vertical shift register, respectively, can be processed as platinum electrodes or silver electrodes, providing the possibility ofusing them as on-chip counter or reference electrodes. Thus, amperometric measurements will become more accurate [6]. The sensor array consists of 400 sensor cells arranged in a square matrix of 20 rows and 20 columns. Each sensor cell has a size of 0.5 mm X 0.5 mm, resulting in a sensor array size of 1 cm X 1 ern. Every sensor cell contains a platinum working electrode, a read-out amplifier and a sensor cell control unit. The size of the platinum electrode is 50 JLm X 50

JLm. Thus, the ratio of electrode distance to electrode

size is 10. The read-out amplifier converts the electrode potential to an analog output current. In order to minimize the power consumption and the resulting increase of temperature, only the read-out amplifier of the currently selected sensor cell is switched on. The amplification of the amplifier is about 1 mAN with an input impedance of more than 5 GU. The sensor cell control unit switches between the potentiometric mode, amperometric mode or test mode. In the test mode the selected sensor cell is directly connected to a test input, enabling this read-out amplifier to be calibrated. Figure 3 shows the schematic technological cross section of the sensor chip, the cross sections of four different components of the chip being combined in one Figure. On the left the cross sections of an nchannel transistor as well as a p-channel transistor, the two basic elements of the CMOS technology, are shown. On the right the cross sections of the two different types of electrodes are depicted. Transistors and electrodes are fabricated successively. First, the complete analog and digital circuits including the aluminium lines are produced. Secondly, passivation layers consisting of silicon dioxide and silicon nitride are deposited. This passivation is necessary in order to protect the electronic circuits against humidity while working in liquids. Thirdly, electrodes are structured into the passivation layers by the use of the lift-off technique. Last of all, the sensor chip is bonded on a substrate and encapsulated by a two-component epoxy adhesive for electrical isolation while working in liquid samples.

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3. Results and discussion

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3.1. Read-out amplifier calibration The sensor chip can be used in three different modes: potentiometric mode, amperometric mode and test mode. In the test mode, the amplifier of the selected sensor cell is switched directly to a test input. By applying a voltage to the test input and reading the analog output signal, the read-out amplifier is tested. Figure 4 shows a complete scan for a 500 mV test voltage. The read-out amplifiers of the individual sensor cells show a mean variation: the mean of the scan is 0.527 rnA and the standard deviation is 0.008 rnA (1.5 %). The mean variation results from layer inhomogeneities generated during the fabrication process. The inhomogeneities are statistically distributed over the chip area and are time constant. Thus, the mean variation of the read-out amplifiers is time constant, too. If needed, e.g., for imaging of spatial distributions of an analyte concentration, the mean variation can be de-

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termined separately and subtracted from the imaging data. Alternatively, a background data set may be used for subtraction. In this way the mean variation of the read-out amplifiers is corrected as well. By applying different voltages to the test input and reading the corresponding analog output signals, a calibration of the read-out amplifiers is possible. In Fig. 5 each point represents the mean value of 400 analog output signals. Figure 5 demonstrates a linear amplification from 0.0 to 1.0 V with 1.038 mAN.

3.2. Chemical sensor array For creating an ammonium-selective sensor array, the sensor chip was coated with a nonactin-doped PVC membrane [7, 8]. 5.5 mg nonactin, 366 mg PVC and 720 mg dibutyl sebacate were dissolved in 10 ml tetrahydrofuran. The chip was dipped into this membrane cocktail for 5 s and then air dried. This procedure was repeated twice. Thereafter, the chip was dried for 30 min at 50 °e. Before the first use of the chemical sensor array, the nonactin-doped PVC membrane was conditioned in a solution of 10- 2 M ~Cl for 30 min.

For calibration of the chemical sensor array, solutions of NH4CI of different concentrations in 0.1 M TrisHCI (7.0) were used. As Fig. 6 shows, a linear calibration curve was obtained from 10- 5 to 10- 2 M NH4CI with a slope of 57 mV per concentration decade. In order to demonstrate a two-dimensional imaging of ammonium distributions over the sensor chip area, a simple experiment was performed: the ammoniumselective membrane was covered with 0.1 M Tris-HCl (7.0) and the double liquid junction was filled with a solution of 1 M N~Cl in 0.1 M Tris-HCl (7.0). After contact of the double liquid junction with the buffer solution on the chip, NH..Cl diffused from the double liquid junction through a frit into the buffer solution on the chip. The resulting lateral diffusion of ammonium was obselVed by successively scanning the chemical sensor array. Figure 7(a}-(c) shows scans started 4, 7 and 19 min after the double liquid junction was contacted to the buffer solution on the chip. Figure 7demonstrates that the presented chemical sensor array is able to image the distribution of ammonium in a square area of 1 em X 1 em with a resolution of 0.5 mm.

3.3. Biosensor array For creating a biosensor anay, the chemical sensor array was modified according to the following procedure: 250 units urease (BC 3.5.1.5, derived from jack beans) and 7.5 mg BSA were dissolved in 100 J.LI 0.1 M TrisHCl (7.0). A small amount of undissolved material was separated using a centrifuge. 50 pol of the solution were mixed with 50 ILl 2.5 % glutaraldehyde solution and dropped onto the ammonium-selective PVC membrane of the chemical sensor array. Within 15 min this mixture reacted to form a homogeneous gel membrane. The membrane was dipped into 0.1 M Tris-HCI (7.0) for one hour in order to wash out the excess glutaraldehyde. Then the resulting biosensor array was calibrated by pipetting urea solutions of different concentrations in 0.1 M Tris-HCl (7.0) onto the urease membrane and always starting the corresponding scan 10 min later.

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As Fig. 8 shows, a linear calibration curve was obtained from 10- 4 to 10- 2 M urea with a slope of 56 mV per concentration decade. When the same urea solutions were pipetted onto the chemical sensor array without the urease membrane, no significant response was observed. For imaging the spatial distribution ofurea, the urease membrane was covered with 0.1 M Tris-HO (7.0), whereas the double liquid junction was filled with a solution of 1 M urea in 0.1 M Tris·HCI (7.0). Figure 9(a)-(c) shows scans started 7, 10 and 19 min after the double liquid junction was contacted to the buffer solution on the chip. The same experiment was carried out using the chemical sensor array witbout a urease membrane, but apart from the background, no response was observed.

4. Conclusions The presented monolithic sensor array can be used for imaging two-dimensional distributions of analyte concentrations. Due to its dimensions, tbis array enables imaging to be carried out over an area of 1 em x 1 em with a resolution limit of 0.5 mm. The sensor array can still be used for imaging experiments when it is completely coated with an ion-selective membrane and a biomembrane. This enables selective sensing and imaging to be combined. An alternative to tbe complete coating of the sensor array would be a local coating of the individual platinum microelectrodes only. This may be realized by the use ofa micf(Hjispensing machine or by the use of pbotolithograpby, which is already used for the fabrication of the monolithic sensor chip. Thus, modification of individual microelectrodes with different analyte-selective membranes should be p0ssible, resulting in a multi-analyte sensor array. Last but not least, amperometric measurements similar to the presented potentiometric measurements, demonstrating the possibilities of the sensor array, should be carried out. If comparable good results are

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achieved in the amperometric mode, imaging of a large variety of different analytes will be possible.

Aclmowledgement

We thank the Bundesministerium fUr Forschung und Technologie (BMFr) for financial support (Grant No. 322-4003·0319508A).

References 1 F.W. ScheUer, R. Hintsche, D. Pfei1fer, F. Schuben, K. Riedel and R. Kindervater, Biosensors: fundamentals, applications and trends, Sensors and .Aetuotors B, 4 (1991) 197-206. 2 K. Cammann, U. Lemke. A. Rohen, J. Sander, H. Wilken and B. Winter, Chemical sensors and biosensors - principles and applications, AnBn'. Chern., Int. Ed. Engl., 30 (1991) 516-539. 3 J. Hauss, K. Schonleben and H.-U. Spiegel, Tht:rdpy Control by Observations of TISSUe-pO?, Verlag Hans Huber, Bern, 1982. 4 P.E. Allen and D.R. Holberg, CMOS .Analog Cireuit Design, Holt, Rinehart and Winston, New York, 1987. 5 P.R. Gray and R.G. Meyer, MOS operational amplifier design - a tutorial overview, IEEE J. Solid~State Cire.• SC-17 (1982) 969-982. 6 B. Ross, K. Cammann, W. Mokwa and M. Rospert, Ultramicroelectrode arrays as transducers for new amperometric oxygen sensors, Sensors and .ActuDton B, 7 (1992) 758-762. 7 K. Cammann, Worlcing wah Ion-&ketivt Electroths. Springer. Berlin, 1979.

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8 S.B. Butt, Development of a potentiometric ammonia-gassensor and a urea-biosensor on the basis of a polymer

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membrane, Ph.D. Thesis, Miinster, Germany, 1990.

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Biographies

Heinrich Meyer was born in Cloppenburg, Germany, in 1965. He received the diploma in chemistry at the Westfalische Wilhelms~Universitat of Munster in 1991. Thereafter, he started a Ph.D. as a member of a scientific group at the Universitat of Munster working in the field of chemical and biochemical sensors. ,.....,

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Heinz Drewer was born in Havixbeck, Germany, in 1965 and received the Dipl.-Ing. in electrical engineering from the Fachhochschule of Munster in 1991. He then joined the Institut fUr Chemo- und Biosensorik e.V. in MUnster, where he heads a group for electronic and software development.

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J6rg Krause was born in Munster, Germany, in 1957. (c)

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He received the diploma in chemistry in 1986 and the Dr. rer. nat. in 1989 at the Westfalische WilhelmsUniversitat of Munster. Since 1990 he has led a group

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there at the Lehrstuhl fur Analytische Chemie working in the field of biosensors. KJJrl Cammann was born in Dusseldorf, Germany, in 1939. He received the Dipl.-Ing. in electrical en· gineering at the Staatl. Ingenieurschule fur Maschi· nenwesen in Essen in 1963. Then, he joined the Beckman Instruments Company for two years, where he led an electrochemistry group. He received the diploma in chemistry at the Technische Universitat in Munchen in 1970 and the Dr. rer. nat. at the Ludwig-Maximilian. Universitat in Munchen in 1975. After postdoctoral positions with GA Rechnitz (State University of New York at Buffalo) and S. Mazur (University of Chicago), he accepted an assistant professorship for analytical chemistry at the Universitat of Ulm. In 1986 he became associate professor of analytical chemistry at the Technische Universitiit in Miinchen and in 1987 full professor of analytical chemistry at the Westfalische WilhelmsUniversitat of Munster. Since 1991 he has additionally headed the Institut fur Chemo- und Biosensorik e. V., in which a team of 60 scientists works mainly in the field of chemical and biochemical sensors. Ralf KJJkerow was born in Dusseldorf, Germany, in 1965 and received the Dip!. Ing. degree in electrical engineering from the University of Duisburg in 1990. He then joined the Fraunhofer Institute of Micro·

electronic Circuits and Systems in Duisburg, where he is currently working on monolithic circuit design for sensing applications. Yiannos Manoli was born in Cyprus in 1954 and received the BA degree in physics and mathematics from Lawrence University in 1978, the M.S. degree in electrical engineering and computer science from the University of California, Berkeley, in 1980 and the Dr. Ing. degree from the University of Duisburg in 1987. Since 1985 he has been with the Fraunhofer Institute of Microelectronic Circuits and Systems in Duisburg, where he heads an integrated circuit design group. Wl1fried Mokwa was born in Doveren, Germany, in 1951. He received the diploma in physics in 1977 and the Dr. rer. nat. in 1981, both at the RWTH Aachen. From 1981 he worked at the 2. Phys. Institute RWTH Aachen and joined the Fraunhofer Institute of Microelectronic Circuits and Systems in 1985, where he leads a group working on silicon sensor technology. Matthias Rospert was born in Saarlouis, Germany, in 1962 and received the Dip!. Phys. degree from the RWTH Aachen in 1989. Then, he joined the Fraunhofer Institute of Microelectronic Circuits and Systems, where he is currently working in the field of integrated chemical sensors.