PAB: a newly designed potentiometric alternating biosensor system

Biosensors & Bioelectronics 10 (1995) 155-167

PAB: a newly designed potentiometric alternating biosensor system

M. Adami, M. Sartore & C. Nicolini*

Technobiochip, Consorzio di Ricerca Industriale Avanzata, Via Roma 28, 57030 Marciana (LI), Italy and Institute of Biophysics of the University of Genova, Via Giotto 2, 16153 Genova Sestri Ponente (GE), Italy

Abstract: A complete potentiometric alternating biosensor system (PAB) is described which utilizes a newly developed transducer based on the light addressable chemical sensor (LAPS) technology, following detailed theoretical optimizations of the relevant physical parameters. Our device, entirely designed and realized 'in house', combines measuring chambers with specific electronic cards and the related software programs; the system is completely automated and driven by a personal computer. The electronics was designed in order to obtain a good general signal-to-noise ratio, hence ensuring reproducible results. The software package consists of low-level programs (written both in C and in Assembler) to interact with the data acquisition and controlling cards, and of high-level programs (written in C) to get, display, save and print data files, and to present a suitable user interface. A very important peculiarity of our system is the possibility of connecting different measuring chambers to the same device. This allows the user to perform a wide variety of experiments, utilizing different biological elements, by simply changing the reaction chamber and selecting the appropriate acquisition software. The system has been shown to operate with living cells as well as with enzymes and antibodies. In addition, a particular chamber containing a gold-evaporated transducer can be connected to the main system, allowing redox potential measurements. The system utilizes a fast information recovery from the transducer output signal, and can acquire quantitative data every few fractions of a second. This feature can be utilized to monitor fast acidification or redox processes.

Keywords: PAB, LAPS, potentiometric sensors, cell biosensor, enzyme- and immuno-sensor

I N T R O D U C T I O N

It is now evident that FET-based transducers are not suitable for the construction of biosensors, mainly because of their extremely low lifetime

* Author to whom addressed. 22 April 1994.

all correspondence should be

and also because of the difficulty in managing some critical parameters such as drift and stability and the interfacing problems with biomolecules. This is confirmed by the fact that only a few ISFET-based devices (as simple pH-sensitive systems) are available on the market , e.g. the Sentron pH-meter (USA), although ISFET was invented more than 20 years ago, in 1970, by P. Bergveld of Twente University (Bergveld, 1970).

On the other hand, the recent introduction of

0956-5663/95/$07.00 © 1995 Elsevier Science Ltd. 155

M. Adami, M. Sartore & C. Nicolini Biosensors & Bioelectronics

the LAPS (Hafeman et al., 1988; Bousse et al., 1993) seemed to be promising for the construction of real biosensor devices. Hence, efforts were aimed at the development of a biosensor based on this type of transducer.

The acronym 'LAPS' (light addressable potentiometric sensor) is now as commonly used as 'ISFET', and indicates a heterostructure of silicon/ silica/silicon nitride, excited by a modulated light source to obtain a photocurrent, which results in the detection of surface potential variations after an electrochemical event (Massobrio et al., 1992; Sartore et al., 1992a, b). This transducer is now experimentally utilized by several research groups (Adami et al., 1992a, b, 1993; Winquist et al., 1992; Gavazzo et al., 1993) and is preferred to ISFET for the following reasons:

(i) The fabrication process is easier and compatible with standard microelectronics facilities. The usual first steps in a standard CMOS process consist of the formation of the aforementioned wafer structure; this means that no particular and specific technology need be designed for the production of chips.

(ii) The sensing surface is free of metal contacts. Hence encapsulation of the device into measuring chambers is simple and less critical. One could observe that back-contact ISFETs are available which also do not have contacts at the sensing surface. However, such components must be designed and realized taking into account the need for a precise back-to- front alignment that is critical and difficult to obtain. What happens in practice is that ISFETs are physically damaged after insertion into the measuring chamber, mainly because of their extreme thinness in the proximity of the rear contacts.

(iii) The sensitive area is extremely flat and allows satisfactory incorporation into very small (micro) volume measuring chambers.

All the above points are already presented in the references mentioned, but one of the most convincing arguments is offered by the presence on the US market of at least two systems based on LAPS transducers: the Cytosensor T M

Microphysiometer and the Threshold T M Machine, by Molecular Devices Corporation (brochure on the Cytosensor, 1990), the former device being

dedicated to the study of extracellular acidification, while the latter is used for immunoassays.

Our system, still under development and also based on LAPS transducers as previously stated, differs in many respects from commercially available devices based on the same principle. In fact we have already patented the PAB system described here (Nicolini et al., 1993) which is going to be introduced into the European and world markets by Technobiochip (Marciana (LI), Italy). The main differences are listed below:

• The PAB is intended as a general-purpose system and can be used in many applications in the same standard configuration. One original feature is the possibility of changing the measuring chamber (of our own design and fluid dynamics optimization) easily by merely substituting the connections of the tubing without any modifications to the measuring system.

• The monitoring of cell acidification processes is performed in a unique chamber in which cells are grown on glass cover slips. This facilitates the biologists' work, as the substrate is normally used for similar purposes, is compatible with cells and is disposable.

• Data acquisition has been optimized to be as fast as possible. Typical methods involve the acquisition of many data points and subsequent computation to retrieve the information on the surface potential. We use this procedure occasionally, but prefer (when possible) to acquire the photocurrent signal directly and to correlate the data quantitat- ively with the current potential at the sensitive layer. The complete approach will be given in detail in following sections. By biasing the device in the linear region, we have developed a new method for measuring the photocurrent from the transducer (Adami et al., 1992b), thereby allowing the monitoring of very of fast pH transients which would be otherwise impossible.

• We have previously demonstrated the possibility of connecting different ion-sensitive electrodes (Adami et al., 1992a; Della Ciana, 1991), in order to allow multiparameter detection.

• We also demonstrated the possibility of utilizing a standard MOS transistor instead of the LAPS transducer by inducing an

156

Biosensors & Bioelectronics

alternating current through a time-varying signal.

* Thermal regulation is achieved by a new non-linear temperature control system with a Peltier-based precision thermostat (Sartore et al., 1993).

• New chambers (still being designed) can in principle be connected to the system without additional modifications. For instance, we are going to design new chambers for measuring the acidification effects on either non-adherent cells or in tumour biopsies, and we intend to connect them to the existing system.

• PAB can combine the pH-sensitive and the redox-sensitive regions on a single chip, thus enlarging the field of applications and enhancing the reliability of the acquired data.

Moreover, the transducers used in our PAB are the subject of numerous investigations and analysis of LAPS parameters. The chips, which are made in Italy, are used in LAPS-based devices to continuously update physical parameters with respect to their quality and performance (shown to be wavelength dependent).

A description of the potentiometric alternating biosensor (PAB) system follows. After a general block diagram, a more detailed discussion on electronic, software and mechanical aspects is presented.

BLOCK DIAGRAM

The PAB system integrates mechanics, hardware and software aspects, and consists of a complete stand-alone device for biosensing purposes. A block diagram is shown in Fig. 1. The system is driven by a computer, which allows continuous measurements in time, and the automatic rec- ording of data. As the interfacing electronics is standard and based on a 16-bit AT-BUS, a normal personal computer can be utilized without any particular hardware modification.

Appropriate software programs have been written both for signal acquisition and processing, and for controlling the device. The computer 'talks' to the electronics through a digital interface, suitably programmed to guarantee high speed and fast acquisitions. In particular, an analog-to-digital board converts the measuring

Potentiometric alternating biosensor system

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~ Driving computer Coriol Unit

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Fig. 1. Schematic representation of the PAB system. A central control unit drives and controls the solution .flowing system and the measuring chambers containing the transducer. The control unit also dialogs with a personal computer, utilized to bias the device and acquire and process the experimental data via appropri-

ate AD/DA interfaces.

signal, while a digital-to-analog board gives the correct supply to bias the transducers. In addition, a digital output card provides eight digital signals to be used as switches for external controls. The main block is the central control unit, consisting of several cards devoted to precise signal amplification and conditioning, to the rectification and finally to the control of several parts of the system. This unit interfaces directly with the solution pumping system and with the measuring chambers. The latter always contains one or more transducers, directly biased and interfaced to the control unit.

The system was designed only after detailed study of the working principles of the transducer jointly carried out with our American colleagues and competitors (Sartore et al., 1992a, b; Bousse et al., 1993). We designed, realized and tested every block of the device in order to obtain good reliability. In addition, we also designed the transducers, which are very important for a continuous updating of the chips, after an accurate test of their performance by means of the device.

A description of the single blocks is now presented in more detail.

Hardware

A schematic block diagram of the electronics is shown in Fig. 2. Several circuits are necessary to drive the LAPS transducer and to measure the

157


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Fig. 2. Block diagram of the main electronics. The LAPS is biased via a potentiostat and the current signal is converted to a voltage, filtered and synchronously demodulated, before being digitized and computer- acquired. The figure also shows a digital output interface suitable for driving the flow circuit through an interface. In addition, a temperature control scheme is provided.

output signal. In this respect, the amount of circuitry is larger than in the case of ISFETs, where a few operational amplifiers can drive the transducer completely.

The figure shows the main blocks used to drive the transducer, and also some additional circuitry devoted to control a pump and for the thermal regulation of the measuring environment. The transducer is connected to a current-to-voltage converter. In fact, the measuring signal is an alternating current of the order of several micro- amperes, usually generated by means of modulated light impinging on the silicon surface. The voltage-converted signal is then filtered and measured as RMS values. As the driving signal is available, our system utilizes a synchronous demodulation technique to recover information.

A schematic diagram of this circuit is shown in Fig. 3. Here both the filtered input and the reference signals are fed in two multipliers. The first output consists of the product of the input signal with the reference component in phase with it, while the second output is derived by the 90 ° shifted reference component. These signals, low-pass filtered to cut out undesirable high-frequency components, are fed into a circuit for the calculation of the modulus after proper amplification. The output of this block is then a DC signal corresponding to the RMS of the actual (alternating) current signal, in a suitable form to be computer-acquired after digitalization.

Acquisition of this signal versus the bias potential yields to characteristic sigmoidal curves, as already reported (Hafeman et al., 1988; Sartore et al., 1992a). The surface potential detection method is always related to the shift of these curves on the V-bias axis after a modification in the surface charge. To detect variations in pH, it is possible to define the sensitivity of the transducer in terms of the shift amplitude per pH unit. On the other hand, for redox potential measurements, a similar definition can be expressed in terms of the redox pair components.

The normal procedure to detect these fluctu- ations of the signal with respect to the applied potential involves determination of the bias voltage corresponding to the inflection point of that potential. This value can then successfully be used as an indicator of the actual surface potential. When applying this technique, suitable electronics must be provided in order to achieve fast voltage scans and corresponding signal acquisitions. Then the inflection point can be computed either by hardware circuitry or by software. In either case, the information can be retrieved only after a complete voltage scan, global signal acquisition and relative signal processing (usually involving second-derivative computation). The computation time needed to perform all these steps is the limiting factor in the speed of acquisition.

Our system, as shown in Fig. 2, does not present any special circuit for this signal acquisition and processing. In fact, we only partially utilize the method described, as will be explained in the software section. The method implemented allows faster information detection and, at the same time, greatly simplifies the hardware.

To return to Fig. 2, we can now focus on the remaining blocks: the potentiostat, the thermal control and the interface for the flowing mechanism. The first circuit allows a proper biasing and control of the electrolyte-transducer system. The input consists of the digitally converted bias potential coming from the computer settings; a controlling electrode (Pt wire) and a reference electrode (SCE) are connected to this block. The functional feature of the potentiostat is to maintain a controlled potential between the two electrodes and the transducer, which is considered as the working electrode, regardless any impedance fluctuation.

Thermal control is a must for biosensing purposes, and is particularly necessary when a

158

Biosensors & Bioelectronics Potentiometric alternating biosensor system

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of these blocks is then filtered and amplified before being fed into the circuit for calculation of the modulus.

biological sample is utilized, as in the case of cells (to maintain the population in suitable living conditions) or enzymes (to guarantee the correct working temperature). In addition to the preser- vation of optimal biological conditions, temperature control is also necessary because of the use of a silicon transducer, which is extremely sensitive to thermal variations. Finally, a good thermostating system is necessary to obtain true reproducible results.

Our system is equipped with a temperature control system consisting of a circuit implementing a non-linear control algorithm whose heating/ cooling element is a Peltier cell. Of course, the set-up depends upon the application, and varies according to the actual measuring chamber. In fact, the Peltier cell is always fixed inside a chamber, together with a temperature sensor utilized in the feedback circuit. The thermostating system has been presented previously and is subject of a patent (Sartore et al., 1993).

The last block is the interface with the flowing circuit; a flow of a measuring solution, of a given compound (e.g. enzyme substrates), or of a cell medium is usually utilized in standard experiments. Our system makes use of a peristaltic pump which allows the solutions to flow from a beaker to the measuring chamber. The pump is completely automated in the sense that start and stop commands come from the computer. This allows a very precise flow-control in time, which is, for example, very useful in acidification experiments. In order to minimize the noise in the signal conditioning circuits, the interface with the motor pump has been designed by means of opto-couplers, which allows a good controlling

technique and avoids interference problems, as the motor and driving circuit grounds are physically separated.

The complete hardware system described above consists of an interface board to plug into a PC bus, a connection cable, and an external circuit connected to the pump, to the electrodes and to the measuring chambers.

Software

Another important part of the system is the set of programs to drive the circuitry, to acquire the data and to perform the necessary processing. A block diagram of the software is shown in Fig. 4; it represents the main procedures for a typical data acquisition session.

A shell provides a user-friendly interface, and allows both parameter input and complete data acquisition control. Of course, the acquisition procedure depends upon the particular experiment, but in general a surface potential measurement is requested versus time, either pH or redox. One can even acquire simple characteristic curves, for example to investigate some surface property or to test a particular sensitive layer. In this case the user connects a standard (static) measuring chamber, biases the transducer and records the corresponding current values. This procedure can be performed easily and is the basis for further and more complicate acquisitions.

If a measurement versus time is requested, as in enzymatic or cellular applications, then a particular algorithm runs. Referring to the preced- ing section, the program in this case constitutes

159


Fig. 4. Software organization of the PAB system. A shell offers a suitable user interface, and allows parameter input, data display, saving and printing; the usual procedure consists of the acquisition of a complete characteristic curve (current versus bias potential) and in the consequent determination of the inflection point. Our software then biases the device and continuously

acquires the digitized signals.

the main portion of the system, as it performs the work usually done by specific electronics.

The pH or redox detection method implies the acquisition of a characteristic curve (current signal versus bias voltage); then the program automatically computes the bias potential corresponding to the inflection point, which is a good indication of the actual surface potential. Once the V-bias is known, the system is biased at the inflection point corresponding to the middle of a characteristic region, which can be approxi- mated by a straight line, as we have already shown (Adami et al., 1992b). Because of this fact, if a small surface potential variation occurs, the characteristic curve shift can be regarded as a line shift; then the current value corresponding to the previous bias voltage can be linearly related to the surface potential change, as it can, first of all, be linearly related to transducer sensitivity. This argument shows that quantitative measurements can be performed by biasing the system at a given voltage and by simply reading the current values during time; each value can be converted into the corresponding pH or redox units by a linear formula depending on the chip sensitivity and the characteristic curve slope at the inflection point. The program essentially implements the procedure described. In particular, a complete characteristic curve is acquired

160

and data are stored in the memory; then a digital filtering procedure, based on the Savitsky-Golay algorithm (Press et al., 1990) is applied, and finally, data are fitted with a particular theoretical formula, and the bias potential at which the inflection point occurs is computed. The procedure is time-consuming, but in most cases it is applied only once. In fact, the system is biased at the value just found, and the current signal is then acquired continuously, and related to the surface potential, as previously stated. This procedure allows an information detection every few fractions of a second, and hence data acquisition can be performed to monitor fast pH or redox changes. The method described can only be applied when no impedance variation among the electrodes arises during the measurement. This can happen, for example, if a membrane is pressed against the transducer.

The remaining blocks of Fig. 4 show the program procedures described: inflection point calculation, consequent system biasing, and finally acquisition in time.

For the dialogue with the electronics, we utilize an interrupt-based structure; this guarantees faster communication from/to the circuits, and allows fast data acquisition. Basically, an interrupt occurs every time a data conversion is performed; control then passes to a specific routine which acquires the data and resets the program.

When communication with the flow circuit (pump on/off) is requested during acquisition, proper procedures interact with the hardware. Actual temperature monitoring is also possible with an additional AD converter and related software.

MEASURING CHAMBERS AND RESULTS

The system can be equipped with various measuring chambers whose purpose is to allow different experiments with the same device. Among the chambers already realized, is a standard static cell able to contain a solution in contact with the transducer. This system is depicted in Fig. 5. The solution is confined by an O-ring pressed against the chip, and the two electrodes are dipped from the top. This chamber is the simplest example, and it is useful for standard measurements. In addition, it can be used for experiments on the transducer, as it can be thermostated and generally guarantees that con-


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Fig. 5. Schematic of a very simple measuring chamber. A well in the upper part confines the measuring solution in a region delimited by an O-ring directly pressed against the LAPS surface. Counter and reference electrodes are dipped in the measuring solution from the top, while a LED confined in the lower part just near the back of the chip provides the light excitation.

ditions are maintained constant in time. For example, our test on the chips has been performed by this chamber.

Figure 6 shows a flow cell suitable for enzymatic

Reaction Chamber (with the possibility

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Fig. 6. Measuring flow chamber suitable for enzymatic experiments. The lower side is similar to that of Fig. 5; the upper part contains an inlet channel towards a well which is able to contain a membrane. Usually, some biological specimen is entrapped in the membrane at this level. The reaction products then flow in a small volume region near the sensitive surface, through a channel which is as small as possible, in order to reduce the delay time of the sensor response. The solution flows away through an outlet channel, contacting the

reference electrode.

applications. The solution flows through a reaction chamber where a membrane can easily be located; for example, the membrane could be a PALL Immunodyne T M for the immobilization of proteins (enzymes or antibodies). The figure also shows the transducer and the related light- emitting diode. Again the measuring volume is delimited by a small O-ring, and a Pt wire is placed in the proximity of the sensitive surface; the reference electrode can be located in the outlet channel. The membrane can also be placed in the small volume near the chip, but in this case the eventual remotion implies opening the entire chamber.

The chamber described above has been used for measurements of an enzymatic reaction cata- lyzed by alcohol dehydrogenase from yeast (YADH) (Adami et al., 1993). Figure 7 shows a typical acquisition for the determination of enzyme concentrations. The signal decay is due to H + production after the reaction took place, yielding to an acidification of the solution in contact with the transducer. Such exponential curves, from their initial slope as a function of ADH concentration, allow the calculation of calibration curves, such as the one shown in Fig. 8, which demonstrates the fine sensitivity of the system, since it was possible to discriminate enzyme concentrations of up to 10 -1° M.

Encouraged by these results, we are trying to use enzymes such as YADH or a redox enzyme such as horse radish peroxidase (HRP) for immuno-enzymatic assays: the enzyme is regarded

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Fig. 7. Experiment for the determination of enzyme concentration in a measuring solution. The acquired signal presents a flat region and a consequent decay due to the insertion of the substrates in the reaction chamber. The enzyme utilized here is ADH (from yeast). Taking the initial slope of the exponential-like portion of the curve gives an indication of the enzyme

concentration.

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Fig. 8. By means o f curves such as the one shown in Fig. 7, it is possible to draw calibration curves such as the one presented here. In this case, the concentration o f Y A D H was varied between 7 × 1 0 - 9 M and

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as a label to detect the reaction between antibodies and antigens.

By using a redox enzyme, the great advantage in these potentiometric measurements is overcoming buffering capacity problems, as the surface potential is affected by electrons instead of protons. In order to create a surface charge layer only on the transducer, we evaporated a metal spot on the silicon nitride. In effect, we are using a competitive method and a configuration based on (monoclonal) antibodies entrapped in a membrane; the competition appears between the free antigene and the antigene bound to the HRP molecules. As soon as the enzyme substrates are injected into the reaction chamber, we obtain a signal variation responsive to the immunocomplex formation. A sketch of the method described is shown in Fig. 9A. A typical signal variation after the immunocomplex formation is shown in Fig. 9B.

Another exciting application available in this system concerns measurements of extracellular acidification. Hence, a specific micro-volume flow chamber able to contain a cell population has been designed and realized, and is shown in Fig. 10. One important feature of the chamber is the possibility of inserting glass coverslips, routinely utilized by biologists. Other methods entail fixing the cells inside a porous membrane, but this method is not as standardized as the use of coverslips. Another flexibility offered by our measuring chamber is the possibility of varying the microvolume, i.e. the distance between the transducer and the cells. This is accomplished by using different teflon spacers. The set-up mechanism is very easy: the user should simply

take the coverslip containing living cells and insert it into the appropriate guides, as shown in the figure. Then the desired spacer (usually ranging between 50 and 400/zm) should be inserted and the chamber closed. It is necessary to thermostat the system a few minutes before starting this set-up procedure in order to offer good thermal conditions to the cell population, and to start the medium flow immediately after the set-up is complete. In usual applications the entire procedure takes only a few seconds to carry out. Hence no particular perturbation is caused to the cells, and quantitative measurements are possible.

The usual experiment consists of several flow- on/flow-off steps, and in the consequent signal acquisition, when the flow is stopped, acidification effects are detectable. A typical result is shown in Fig. 11. Data are relative to a CHO confluent cell population, maintained at 37°C and utilizing a standard F12 culture medium without hydrogen carbonate, in order to minimize the buffering capacity effects.

In a recent study carried out on 3T6 cells (Gavazzo et al., 1993) we were able to estimate the number of protons derived from the acidification effects, quantized in 6.5 x 108 protons per cell per second. In addition, a detailed evaluation of the extracellular acidification as related to cell viability has been performed.

A more recent application, currently under test and development, makes use of micro- organisms. In this case, and in many other applications, it is preferable to have a quantitative method to take into account eventual noise or possible artefacts deriving from the particular biological set-up. This can be accomplished using a differential scheme. The solution must use the electronics described above for both signals in order to ensure the same electrical characteristics and the same gains for the two measurements. A good solution consists of a digital-driven switch to select the two signals alternately. A related chamber has been designed and is shown in Fig. 12. Two chips are fixed in the lower side of the chamber and the flow circuit sequentially contacts both transducers. A reaction chamber (which can be a commercial membrane-containing teflon system) can easily be connected and removed in the area between the two measuring areas; in this case, the first chip gives a signal related to the solution, while the second one measures the effects of the reaction. As the chips are very

162


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Fig. 9. (A) The immunoreaction scheme, where the antibody is fixed onto a substrate and the antigene with an enzyme is injected into the solution to compete with the free antigene. The binding of enzyme-linked molecules is detectable after the production of electrons. (B) A typical acquisition curve (I/V characteristic) obtained utilizing horse radish peroxidase as the labeling enzyme, 2,4-dichlorophenossyacetic acid (2,4-D) as antigene and a monoclonal antibody against the pesticide 2,4-D. The right-hand curve is before binding: the left-hand side is after

binding.

close to each other and fixed in the same environment and with the same connections, statistically the noise should affect both chips in the same way, and hence a differential measurement appears capable of eliminating noise effects. The two chips used in this configuration must be carefully chosen, and must present electrical properties as similar as possible (mainly drift and sensitivity) in order to have the same response in time. However, even by utilizing a geometrically modified chip, a similar chamber can be designed using a single LAPS and so reducing the problems associated with different features.

Several studies have been performed in order to optimize the operating features of the transducers. It has been shown that the frequency and wavelength of the light emanating from the light emitting diode (LED) which is used for the illumination of the LAPS chip influences the

sensitivity and stability of the response (Bousse et al., 1993).

The optimization of the operational parameters of the PAB system lead to a significant increase in the signal stability, yielding a drift which is always lower than 0.01 pH per min. The sensitivity of a system with such a drift was approxi- mately 52 mV per unit pH.

Some modifications of the native device are possible, and will be investigated further. In particular, as we have already shown (Adami et al., 1992a), the same signal generation technique as LAPS can be utilized in conjunction with different ion-sensitive electrodes, yielding multiparameter detection within the same device. In addition, we proved that in principle a standard MOS transistor can be utilized instead of the LAPS chip, by inducing an alternating current through a time-varying small signal, and connecting some ion-selective electrodes. In addition

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Fig. 10. Measuring flow chamber for cellular acidification experiments. The counter electrode is inserted inside the inlet channel, while the reference electrode is in the outlet. The LAPS is separated from the cells immobilized onto a glass coverslip by a teflon spacer, whose thickness defines the micro-volume of the measuring chamber. Thermal control is achieved by a Peltier cell connected to appropriate circuitry and fixed on the top of the chamber. Heating is provided near the cells by a metal cylinder (usually aluminum). The right-hand side of the figure shows the top view of the gasket used to prevent medium leakage, the coverslip where cells are grown, and the teflon spacer,

giving an indication of the flow circuit inside the 'sandwich'.

to the advantage mentioned previously, it is also possible to consider some integration of the biosensor as standard processes are being utilized.

CONCLUSIONS

This work describes a novel potentiometric alternating biosensor (PAB) capable of functioning as an integrated system under different conditions and of being utilized with a variety of biological sensing elements for the analysis of a wide range of biological samples. This integration represents the major novelty, while the transducing element is an optimization of the well-known LAPS, or of various modifications of it depending on the application, which is one of the most reliable transducers in use in biosensor engineering.

The data acquisition and control systems of the PAB were completely re-designed to adapt the electronics to the general requirements of the biosensor and the interfacing. These sub- systems consist of several blocks interfaced by

means of AD/DA converters to an IBM (or compatible) PC. The set-up of the electronic cards has been specifically studied in order to minimize the noise and maximize the operation speed. Another important feature is the utilization of a separate 'measuring box' that incorporates all the necessary components and can easily be interfaced with the available PC. This implies that, instead of purchasing a dedicated processing unit, a connection with a conventional PC is sufficient.

A completely original software package has been written for proper system control, for signal measuring, and for data archivation and visualization. The software runs on a DOS operating system and allows the user to manage different types of signal acquisition. The main feature of the low-level software is the interrupt- based structure, which has proved to be most reliable and to allow good computer interfacing independently of the system clock.

The fact that the flow circuit of the PAB system is controlled by the same computer unit

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Fig. 11. Typical acidification experiment performed with a confluent CHO living cell population grown onto a glass coverslip. The figure shows the unfiltered acquired signal. Several 2 min 'flow ONT'flow OFF' steps allow measurement of the acidification effects; during the flow OFF condition, the signal decreases linearly, while during flow ON, a steady state corresponding to the medium pH is maintained. The medium used here was standard F12

without hydrogen carbonate.

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Cover

Fig. 12. A two-chip measuring chamber suitable for differential measurements. Here the flowing solution appears at the first chip, then passes through a removable reaction chamber before reaching a second chip. Of course, only the second LAPS senses the effects of the reaction that took place in the chamber; both chip should be affected by the same noise, as the particular design ensures the same environmental conditions for both. Hence, a differential

acquisition scheme detects only the true reaction signal.

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permits complete automation of the operations with flow-through chambers, especially the start/ stop operations, by means of the relevant electronic interfaces.

Different measuring chambers can be connected to the same device, rendering the system suitable for numerous applications: some chambers for standard solution measurements; a mini- volume chamber for enzymatic applications; a micro-volume flow-through chamber for cellular experiments. As an example, the last use has a completely new flow circuit which allows the realization of controlled and variable micro- volumes, simply by inserting different Teflon spacers.

The PAB system is thermostatted by means of an ad hoc temperature controlling system, based on a Peltier cell, which has high precision and reliability.

With respect to future development, even though our silicon technology already offers an optimized transducer, we are considering the possibility of designing a gallium arsenide-based transducer. In addition to the GaAs property of a very high charge mobility, it is possible to build extremely sensitive layers by LB technology. This method should provide the possibility of having a surface sensitive to many ions and not only to hydrogen, as in the case of silicon nitride transducers.

As stated earlier, one of the advantages of the PAB lies in the possibility of uniting the pH- sensitive and redox-sensitive elements on a single sensor chip. Moreover, the potential of utilizing several LEDs at different wavelengths addressing regions covered by biological sensing elements with various specificities (e.g. different enzymes) renders the PAB system extremely useful for combined assays of complex samples.

It should be noted that even for conventional silicon-based transducers, new procedures will be elaborated for the development of sensitive layers by means of surface modifications of silicon nitride.

ACKNOWLEDGEMENTS

We are very grateful to the people of the research laboratories of Technobiochip and of the Institute of Biophysics and the University of Genova. In particular we would like to thank M. Martini, L. Piras, A. Sardi, D. Nardelli, A. Rossi, L.

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Vergani, P. Gavazzo, S. Paddeu, M. Lanzi, A. Fanigliulo and M. Zunino for their constant assistance during the development of the system and in the present work. A particular acknowl- edgement goes to Dr Sergey Vakula for his generous help in the preparation of this manu- script. This work was supported by a research grant from Technobiochip.

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PAB: a newly designed potentiometric alternating biosensor system

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