- Email: [email protected]
A novel low-noise measurement principle for LAPS and its application to faster measurement of pH
Sensors and Actuators B 74 (2001) 112±116
A novel low-noise measurement principle for LAPS and its application to faster measurement of pH Abu Bakar Md. Ismaila,*, Hirokazu Sugiharab, Tatsuo Yoshinobua, Hiroshi Iwasakia a
Department of Quantum Molecular Devices, The Institute of Scienti®c and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan b Health and Medical Care Planning Of®ce, Corporate Research Division, Matsushita Electric Co., Ltd., Soraku, Kyoto, Japan
Abstract A novel measurement principle for the light-addressable potentiometric sensor (LAPS) system is proposed in this paper. With several features like low-noise and low-background, this measurement scheme extends the application of LAPS in faster high-sensitive direct monitoring of ionic concentration and small change in the surface potential without the need of costly instruments like potentiostat, lock-in ampli®er. Using two out-of-phase modulated light sources applied at two different points of the Si surface of the heterostructure, a kind of differential LAPS response is obtained. Due to the addition of the two out-of-phase photocurrents, low-frequency components associated with the photocurrents are canceled out and a low-noise and hence highly sensitive response is expected. As an application of the new principle, the pH measurement of an electrolyte solution is investigated and a new instrumentation for the direct, high-sensitive, and faster measurement of pH is proposed. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Light-addressable potentiometric sensor (LAPS); Differential measurement; pH sensor; Chemical sensor
1. Introduction Recently, biosensors based on light-addressable potentiometric sensors (LAPS) have become popular in different chemical and biological applications [1±3]. Generally, in these applications pH and/or other ion concentrations in the environment are measured. With the conventional LAPS measurement technique, the quantitative assessment of the pH change is a relatively slow process as it involves several operations to be performed on the photocurrent response characteristics of the LAPS. In applications like determination of dissociation/binding constant of anti-DNA-antibody/ antibody-antigen [4,5], sensing and measuring the electrical activity of the neuron, it is dif®cult to extract the very weak measured signal, or, extra-cellular potential transients of the neural action potential, those are modulated with the very strong photocurrent (background) signal. Despite the immense importance of improved measurement technique for the LAPS, very few works [6±8] have been done so far. Bousse et al. [6] investigated the combined measurement of surface potential and zeta potential with two sensors and two LEDs by using the difference of the measured potential * Corresponding author. Tel.: 81-6-6879-8404; fax: 81-6-6879-8404. E-mail address: [email protected] (A.B. Md. Ismail).
output between LED1 and LED2, as a function of time. Adami et al. [7] tried to obtain a differential LAPS by two sensors and two LEDs and with the help of a comparator. Sasaki et al. [8] also tried to obtain differential measurement with LAPS by a time sharing technique using a differential ampli®er. We propose a simple, low-noise, and low-cost measurement principle using a single sensor and two out-ofphase modulated light sources. In particular, this measurement principle is ef®cient in the applications where lowbackground is a precondition. As an application of the novel measurement principle, a new instrumentation of direct, faster, and highly sensitive pH measurement, is investigated in this article. 2. Measurement principle The LAPS consists of an electrolyte-insulator [Si3 N4 SiO2 ]-semiconductor [Si] (EIS) structure. If this structure is biased in depletion, the width of the depletion layer is a function of the local value of the surface potential, which depends on the pH value of the electrolyte. The local value of the depletion capacitance can be read out with ac photocurrent that is generated when an intensity-modulated light source is shone at the bulk Si. Instead of one light source, we
0925-4005/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 0 ) 0 0 7 1 9 - X
A.B. Md. Ismail et al. / Sensors and Actuators B 74 (2001) 112±116
113
Fig. 1. Schematic representation of the new sensing principle.
propose a LAPS measurement principle with two out-ofphase light sources that provides low-noise as being a sort of differential approach. The block diagram of the principle is shown in Fig. 1. In this scheme, the Si layer in the heterostructure (Si3N4/SiO2/ Si) is illuminated with two light sources (LASER1 and LASER2) at two different points. Measuring and reference cells are constructed on the Si3N4 surface just at the top of the illuminated points. The intensity of the two light sources is modulated with the same frequency, but out-of-phase. If the various parameters, such as intensity of light sources, light-beam diameter, area of the cell, volume and electrical conductivity of the electrolytes, etc. are kept identical, the corresponding photocurrent from the two cells will be identical, but opposite in phase. Fig. 2 shows the timing sequence of the LAPS signal for the new measurement approach. Due to the addition of the two out-of-phased currents, as they are collected by a common metallic back contact, their base components as well as common noises cancel
Fig. 2. Timing sequence (drawn on the same scale) of the new measurement principle.
each other and low-noise response is achieved, as can be seen in the plot (C) of Fig. 2. If there is a change in the pH value of the electrolyte or change in surface potential in one cell, the photocurrent will also change in that cell accordingly, and a difference-signal proportional to the change will appear at the output. Once the calibration curve of the sensor is obtained, the pH can be measured directly from the difference-signal and since it involves no operation like differentiation on the sampled data, the measurement is much faster than the conventional approach. 3. Application of the new principle to pH measurement Using the LAPS, the pH of an electrolytic solution is conventionally determined from the bias shift of the photocurrent response of the LAPS. The shift in the photocurrent response in the potential axis is determined from the difference of the in¯exion potentials of the measuring pH buffer from any reference pH buffer. Regarding LAPS, the in¯exion potential is the bias potential at which the second derivative of the photocurrent response crosses the zero value. Fig. 3 shows the photocurrent response of the LAPS and determination of the in¯exion point (inset). This operation to ®nd the in¯exion point is done on the sampled data, which are affected by noise, and a preliminary smoothing ®ltering of the curve is necessary. Moreover, the steeper the curve, the better the calculation, because the zero crossing point will consequently be more accurate and the pH measurement more precise [9]. The new measurement scheme can be applied in two modes, namely, ``difference-signal'' and ``bias-difference'' mode, for the direct measurement of pH. In both cases, identical solution is put into the two cells prior to the measurement and the bias voltage of the measurement cell Vm is adjusted to minimize the difference-signal DI for an appropriately chosen bias voltage of the reference cell. Let us suppose that a minimum difference-signal DI0 is obtained for a bias Vmi at the measurement cell.
114
A.B. Md. Ismail et al. / Sensors and Actuators B 74 (2001) 112±116
electro-deposition technique. A parallel combination of a 10 MO resistor and 100 mF capacitor is connected in series with the power supply to reduce the dc leakage current. The difference photocurrent is collected by the back metalcontact and is ampli®ed and converted to voltage by a pre-ampli®er with a conversion factor of 106 V/A. From now on, all the ac currents will be expressed as ac voltage measured with an oscilloscope. 3.2. Experimental performance and discussion
Fig. 3. Conventional way of determining the inflexion point.
In the difference-signal mode, the bias voltages of the two cells are ®xed, and the difference-signal DI is recorded for the new solution in the measurement cell. From the value of DI ÿ DI0 , the pH value can be determined if it is known whether the pH value increased or decreased, because the amplitude of the difference-signal for DpH from the reference pH is the same. In the bias-difference mode, Vmi is adjusted to VmpH to minimize the difference-signal for the new solution. The value V mpH ÿ V mi represents the pH sensitivity of the Si3N4 surface. From this value, the new pH value can be easily determined. Either or both of the differencesignal and bias-difference modes can be used for the quantitative assessment of pH. A PC can be used for the bias scan to get fully automated and faster measurement of pH.
Various buffers in the range of 3.55±5.65 pH were experimented with this system of measurement. These buffers were prepared in the laboratory and their pH values were always checked just before the experiment. Since the amplitude of the photocurrent is dependent on many parameters of the electrolytic solution, it is required that the non-pH characteristics of the reference and measuring solutions, e.g. electrical conductivity, concentration of interfering ions, temperature, etc. are identical. For this experimental purpose, buffer solutions mixed with 0.1 M/l NaCl [10] as an ionic strength adjuster, are experimented at room temperature. At ®rst, ``measuring'' and ``reference'' cells are ®lled with pH 4.55 buffer as reference solution. Turning on only LASER1, or, only LASER2, or LASER1 and LASER2 both together, the photocurrent responses are obtained. The operating point for the pH measurement is then carefully chosen from the characteristics as shown in Fig. 4, so that the photocurrent remains under saturation at the highest pH value to be measured. The photocurrents at the two cells are not exactly the same at the deep inversion. This is maybe due the non-uniformity on the sensing surfaces at the two cells, difference in intensities and beam-diameter of the lasers, etc. The dc biases at the two cells are adjusted around the operating point, for this experiment, adjusted to 1.390 V (Vri) and 1.445 V (Vmi) at the reference and measuring cells,
3.1. Experimental setup The sensor is a heterostructure consisting of Au/Si/SiO2/ Si3N4 layers from n-type Si (1±10 O cm, 180 mm thick) with h1 0 0i orientation. The backside of the Si is illuminated by two infrared lasers (l 830 nm), LASER1 and LASER2 at two different points. The lasers are modulated at a frequency of 30 kHz, but 1808 out-of-phase by a phase-shifter circuit. Adjustment of phase by the phase shifter is sometimes necessary to obtain minimum difference photocurrent signal. Two cells, 25 mm apart, are constructed on the top Si3N4 surface just above the two illuminated points. Only a small area of diameter 8 mm, in each cell, is exposed to the electrolytes by a silicon rubber sealing. One cell is used for the reference buffer and the other is for the measuring buffer. Two Ag/AgCl rod is used in the two cells as the electrode to de®ne the reference potential of the electrolyte through two dc power supplies. This Ag/AgCl rod was prepared with thick coating of AgCl on Ag rod by the
Fig. 4. Photocurrent response for various combination of laser illumination.
A.B. Md. Ismail et al. / Sensors and Actuators B 74 (2001) 112±116
115
Fig. 5. Difference-signal for the various pH.
Fig. 6. Bias-difference at various pH.
respectively, to obtain a minimum difference-signal (Vr), which is 13.3 mVp±p for this experiment. Ideally Vr should be zero, but as it depends on many parameters it is not always zero. Therefore, a minimum difference-signal is always obtained by adjusting biases at the two cells, intensity of the lasers, and phase-shift between the modulating signals to the lasers around 1808. The minimum differencesignal at the above mentioned operating biases was checked several times during the experiment, and a less than 1% change in the difference-signal was noticed. This drift in minimum difference-signal may be attributed to the difference in intensity of the lasers with time. Since in this experiment identical lasers were used, a nearly identical laser-intensity pro®le with time is expected. The change in photocurrents due to the change in intensities of the two lasers are nearly equal and opposite resulting a nearly unchanged minimum difference-signal. The base photocurrent signals for the measuring and reference cells were found to be 211 and 210 mVp±p, respectively. Now, for example, the solution at the measuring cell is changed to another buffer of pH 3.55 keeping the pH 4.55 buffer unaltered at the reference cell. As a result of change in pH value the surface potential and the photocurrent amplitude also changes accordingly at the measuring cell producing another difference-signal (VmpH) of 25.3 mVp±p. The change in photocurrent due to the change in pH is calculated from the difference between the two difference-signals, i.e. (V mpH ÿ V r ). Fig. 5 shows typical difference-signal calibration for difference-signal mode of operation. The amplitude of the difference-signal is assumed to be positive for the increase in pH value, and negative for the decrease in pH value, from the reference pH buffer. Conceptually, the change in photocurrent is due to the change in insulator surface potential that is sensitive to pH of the buffer. Now if the dc bias at the measuring cell is increased, the photocurrent at that cell increases and VmpH
decreases to Vr at a new bias of 1504.5 mV (VmpH). The difference in initial bias (Vmi) and the new bias (VmpH) at the measuring cell is exactly the pH sensitivity (mV/pH) of the sensor. When the pH value of the measuring buffer is higher than the reference buffer, the bias at the measuring cell is decreased to obtain the minimum difference-signal (Vr). As shown in Fig. 6, the difference in bias from the bias for reference pH buffer, are plotted for various pH value, and the pH sensitivity is obtained. An average pH sensitivity of 57.6 mV/pH, which is very close to the Nernstian value, is obtained in the experimented pH range. The proposed measurement system has limitation on pH measurement range. As we can see from the Fig. 4, the sensor switches from the accumulation to the inversion within 350 mV of the surface potential change. If the sensitivity is ÿ58 mV/pH as obtained from the experiment, pH range of 6 units (6 58 350), e.g. 4±10 pH, can be measured. With a photocurrent sensitivity of 22.4 mV/pH and a drift of 0.1 mV in the minimum difference-signal, a maximum error of 4 mpH in the measured pH is expected. The whole process of the pH measurement can be automated by employing a PC for the data collection, bias scan at the measuring buffer, and laser intensity control. 4. Conclusion In this article a generalized novel measurement scheme for the LAPS is presented. In principle, this measurement system offers a low-noise and low-background approach for the LAPS based instrumentation for the biosensing applications. Among the various expected applications, pH measurement is investigated in two modes, namely, differencesignal and bias-difference mode. The pH sensitivity of 57.6 mV/pH, which is very close to the Nernstian value, is obtained in the range of 3.55±5.65 pH. Without costly
116
A.B. Md. Ismail et al. / Sensors and Actuators B 74 (2001) 112±116
equipments like potentiostat, lock-in-ampli®er, etc., a direct, faster and highly sensitive instrumentation for the pH measurement using LAPS is proposed.
[9] M. Sartore, M. Adami, C. Nicolini, Computer simulation and optimization of a light-addressable potentiometric sensor, Biosens. Bioelectron. 7 (1992) 57±64. [10] Y. Murakami, et al., An organic pollution sensor based on surface photovoltage, Sens. Actuators B 53 (1998) 163±172.
Acknowledgements The authors would like to thank the New Energy and Industrial Technology Development Organization (NEDO), the Ministry of International Trade and Industry (MITI), the Government of Japan, for supporting this work (contract no. 98S18-001). References [1] T. Yoshinobu, H. Iwasaki, M. Nakao, S. Nomura, T. Nakanishi, S. Takamatsu, K. Tomita, Bioimages, Visualization of pH change of E. coli with a novel pH imaging microscope 5 (1997) 143±147. [2] G. Gehring, D.L. Patterson, S. Tu, Use of light addressable potentiometric sensor for the detection of Escherichia coli O157:H7, Anal. Biochem. 258 (2) (1998) 293±298. [3] K.A. Uithoven, J.C. Schmidt, M.E. Ballman, Rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system, Biosens. Bioelectron. 14 (2000) 761±770. [4] K. Dill, et al., Determination of dissociation constant and concentration of an anti-DNA antibody by using the light-addressable potentiometric sensor, J. Biochem. Biophys. Methods 31 (1996) 17±21. [5] V.T. Kung, et al., Picogram quantitation of total DNA using DNAbinding proteins in a silicon based system, Anal. Biochem. 187 (1990) 220±227. [6] L.J. Bousse, S. Mostarshed, D. Hafeman, A combined measurement of surface potential and zeta potential of an electrolyte/insulator interface of LAPS, Sens. Actuators B 10 (1992) 67±71. [7] M. Adami, M. Sartore, E. Baldini, A. Rossi, C. Nicolini, New measuring principle for LAPS, Sens. Actuators B 9 (1992) 25±31. [8] Y. Sasaki, Y. Kanai, H. Uchida, T. Katsube, Highly sensitive taste sensors with a new differential LAPS method, Sens. Actuators B 24/ 25 (1995) 819±822.
Biographies Abu Bakar Md. Ismail was born in Rajshahi, Bangladesh. He received his BSc (Hons) and MSc degrees in Applied Physics and Electronics from the University of Rajshahi, Bangladesh. In 1998 he received his PhD degree in Advanced Instrumentation from the Saga University, Japan. He has been serving as an Assistant Professor in the Department of Applied Physics and Electronics, Rajshahi University, Bangladesh. At present, he is engaged in imaging of electrical activity of living cells in the Department of Quantum Molecular Devices, The Institute of Scientific and Industrial Research, Osaka University, Japan. Hirokazu Sugihara received his BS degree in Bioengineering from the Osaka University, Japan, in 1985. He is currently working in Matsushita Electronic Industrial Co., Ltd., as a senior scientist. His research interests are developmental change and plasticity of biological neural networks, and design of new devices for multi-site neural response recording using micro-fabrication technique. Tatsuo Yoshinobu, born in Kyoto in 1964, received BE, ME, and PhD degrees in Electrical Engineering from Kyoto University in 1987, 1989, and 1992, respectively, for his study on gas source molecular beam epitaxy of silicon carbide. Since April 1992, he has been engaged in development of the scanning chemical microscope at The Institute of Scientific and Industrial Research, Osaka University. Hiroshi Iwasaki was born in 1945 and received the BS and MS degrees and the PhD degree in electronic engineering from Osaka University, in 1967, 1969, and 1975, respectively. From 1971 to 1986, he worked on Surface Physics including oxidation of silicon as a member of the faculty of Osaka University, and from 1983 to 1984, he worked at the University of Maryland as a Visiting Professor. From 1986 to 1991, he worked on ULSI's for Matsushita Electric Industrial Co., Ltd., Osaka. Since April 1991, he has been working at the Institute of Scientific and Industrial Research, Osaka University, as a Professor.
A novel low-noise measurement principle for LAPS and its application to faster measurement of pH Abu Bakar Md. Ismaila,*, Hirokazu Sugiharab, Tatsuo Yoshinobua, Hiroshi Iwasakia a
Department of Quantum Molecular Devices, The Institute of Scienti®c and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan b Health and Medical Care Planning Of®ce, Corporate Research Division, Matsushita Electric Co., Ltd., Soraku, Kyoto, Japan
Abstract A novel measurement principle for the light-addressable potentiometric sensor (LAPS) system is proposed in this paper. With several features like low-noise and low-background, this measurement scheme extends the application of LAPS in faster high-sensitive direct monitoring of ionic concentration and small change in the surface potential without the need of costly instruments like potentiostat, lock-in ampli®er. Using two out-of-phase modulated light sources applied at two different points of the Si surface of the heterostructure, a kind of differential LAPS response is obtained. Due to the addition of the two out-of-phase photocurrents, low-frequency components associated with the photocurrents are canceled out and a low-noise and hence highly sensitive response is expected. As an application of the new principle, the pH measurement of an electrolyte solution is investigated and a new instrumentation for the direct, high-sensitive, and faster measurement of pH is proposed. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Light-addressable potentiometric sensor (LAPS); Differential measurement; pH sensor; Chemical sensor
1. Introduction Recently, biosensors based on light-addressable potentiometric sensors (LAPS) have become popular in different chemical and biological applications [1±3]. Generally, in these applications pH and/or other ion concentrations in the environment are measured. With the conventional LAPS measurement technique, the quantitative assessment of the pH change is a relatively slow process as it involves several operations to be performed on the photocurrent response characteristics of the LAPS. In applications like determination of dissociation/binding constant of anti-DNA-antibody/ antibody-antigen [4,5], sensing and measuring the electrical activity of the neuron, it is dif®cult to extract the very weak measured signal, or, extra-cellular potential transients of the neural action potential, those are modulated with the very strong photocurrent (background) signal. Despite the immense importance of improved measurement technique for the LAPS, very few works [6±8] have been done so far. Bousse et al. [6] investigated the combined measurement of surface potential and zeta potential with two sensors and two LEDs by using the difference of the measured potential * Corresponding author. Tel.: 81-6-6879-8404; fax: 81-6-6879-8404. E-mail address: [email protected] (A.B. Md. Ismail).
output between LED1 and LED2, as a function of time. Adami et al. [7] tried to obtain a differential LAPS by two sensors and two LEDs and with the help of a comparator. Sasaki et al. [8] also tried to obtain differential measurement with LAPS by a time sharing technique using a differential ampli®er. We propose a simple, low-noise, and low-cost measurement principle using a single sensor and two out-ofphase modulated light sources. In particular, this measurement principle is ef®cient in the applications where lowbackground is a precondition. As an application of the novel measurement principle, a new instrumentation of direct, faster, and highly sensitive pH measurement, is investigated in this article. 2. Measurement principle The LAPS consists of an electrolyte-insulator [Si3 N4 SiO2 ]-semiconductor [Si] (EIS) structure. If this structure is biased in depletion, the width of the depletion layer is a function of the local value of the surface potential, which depends on the pH value of the electrolyte. The local value of the depletion capacitance can be read out with ac photocurrent that is generated when an intensity-modulated light source is shone at the bulk Si. Instead of one light source, we
0925-4005/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 0 ) 0 0 7 1 9 - X
A.B. Md. Ismail et al. / Sensors and Actuators B 74 (2001) 112±116
113
Fig. 1. Schematic representation of the new sensing principle.
propose a LAPS measurement principle with two out-ofphase light sources that provides low-noise as being a sort of differential approach. The block diagram of the principle is shown in Fig. 1. In this scheme, the Si layer in the heterostructure (Si3N4/SiO2/ Si) is illuminated with two light sources (LASER1 and LASER2) at two different points. Measuring and reference cells are constructed on the Si3N4 surface just at the top of the illuminated points. The intensity of the two light sources is modulated with the same frequency, but out-of-phase. If the various parameters, such as intensity of light sources, light-beam diameter, area of the cell, volume and electrical conductivity of the electrolytes, etc. are kept identical, the corresponding photocurrent from the two cells will be identical, but opposite in phase. Fig. 2 shows the timing sequence of the LAPS signal for the new measurement approach. Due to the addition of the two out-of-phased currents, as they are collected by a common metallic back contact, their base components as well as common noises cancel
Fig. 2. Timing sequence (drawn on the same scale) of the new measurement principle.
each other and low-noise response is achieved, as can be seen in the plot (C) of Fig. 2. If there is a change in the pH value of the electrolyte or change in surface potential in one cell, the photocurrent will also change in that cell accordingly, and a difference-signal proportional to the change will appear at the output. Once the calibration curve of the sensor is obtained, the pH can be measured directly from the difference-signal and since it involves no operation like differentiation on the sampled data, the measurement is much faster than the conventional approach. 3. Application of the new principle to pH measurement Using the LAPS, the pH of an electrolytic solution is conventionally determined from the bias shift of the photocurrent response of the LAPS. The shift in the photocurrent response in the potential axis is determined from the difference of the in¯exion potentials of the measuring pH buffer from any reference pH buffer. Regarding LAPS, the in¯exion potential is the bias potential at which the second derivative of the photocurrent response crosses the zero value. Fig. 3 shows the photocurrent response of the LAPS and determination of the in¯exion point (inset). This operation to ®nd the in¯exion point is done on the sampled data, which are affected by noise, and a preliminary smoothing ®ltering of the curve is necessary. Moreover, the steeper the curve, the better the calculation, because the zero crossing point will consequently be more accurate and the pH measurement more precise [9]. The new measurement scheme can be applied in two modes, namely, ``difference-signal'' and ``bias-difference'' mode, for the direct measurement of pH. In both cases, identical solution is put into the two cells prior to the measurement and the bias voltage of the measurement cell Vm is adjusted to minimize the difference-signal DI for an appropriately chosen bias voltage of the reference cell. Let us suppose that a minimum difference-signal DI0 is obtained for a bias Vmi at the measurement cell.
114
A.B. Md. Ismail et al. / Sensors and Actuators B 74 (2001) 112±116
electro-deposition technique. A parallel combination of a 10 MO resistor and 100 mF capacitor is connected in series with the power supply to reduce the dc leakage current. The difference photocurrent is collected by the back metalcontact and is ampli®ed and converted to voltage by a pre-ampli®er with a conversion factor of 106 V/A. From now on, all the ac currents will be expressed as ac voltage measured with an oscilloscope. 3.2. Experimental performance and discussion
Fig. 3. Conventional way of determining the inflexion point.
In the difference-signal mode, the bias voltages of the two cells are ®xed, and the difference-signal DI is recorded for the new solution in the measurement cell. From the value of DI ÿ DI0 , the pH value can be determined if it is known whether the pH value increased or decreased, because the amplitude of the difference-signal for DpH from the reference pH is the same. In the bias-difference mode, Vmi is adjusted to VmpH to minimize the difference-signal for the new solution. The value V mpH ÿ V mi represents the pH sensitivity of the Si3N4 surface. From this value, the new pH value can be easily determined. Either or both of the differencesignal and bias-difference modes can be used for the quantitative assessment of pH. A PC can be used for the bias scan to get fully automated and faster measurement of pH.
Various buffers in the range of 3.55±5.65 pH were experimented with this system of measurement. These buffers were prepared in the laboratory and their pH values were always checked just before the experiment. Since the amplitude of the photocurrent is dependent on many parameters of the electrolytic solution, it is required that the non-pH characteristics of the reference and measuring solutions, e.g. electrical conductivity, concentration of interfering ions, temperature, etc. are identical. For this experimental purpose, buffer solutions mixed with 0.1 M/l NaCl [10] as an ionic strength adjuster, are experimented at room temperature. At ®rst, ``measuring'' and ``reference'' cells are ®lled with pH 4.55 buffer as reference solution. Turning on only LASER1, or, only LASER2, or LASER1 and LASER2 both together, the photocurrent responses are obtained. The operating point for the pH measurement is then carefully chosen from the characteristics as shown in Fig. 4, so that the photocurrent remains under saturation at the highest pH value to be measured. The photocurrents at the two cells are not exactly the same at the deep inversion. This is maybe due the non-uniformity on the sensing surfaces at the two cells, difference in intensities and beam-diameter of the lasers, etc. The dc biases at the two cells are adjusted around the operating point, for this experiment, adjusted to 1.390 V (Vri) and 1.445 V (Vmi) at the reference and measuring cells,
3.1. Experimental setup The sensor is a heterostructure consisting of Au/Si/SiO2/ Si3N4 layers from n-type Si (1±10 O cm, 180 mm thick) with h1 0 0i orientation. The backside of the Si is illuminated by two infrared lasers (l 830 nm), LASER1 and LASER2 at two different points. The lasers are modulated at a frequency of 30 kHz, but 1808 out-of-phase by a phase-shifter circuit. Adjustment of phase by the phase shifter is sometimes necessary to obtain minimum difference photocurrent signal. Two cells, 25 mm apart, are constructed on the top Si3N4 surface just above the two illuminated points. Only a small area of diameter 8 mm, in each cell, is exposed to the electrolytes by a silicon rubber sealing. One cell is used for the reference buffer and the other is for the measuring buffer. Two Ag/AgCl rod is used in the two cells as the electrode to de®ne the reference potential of the electrolyte through two dc power supplies. This Ag/AgCl rod was prepared with thick coating of AgCl on Ag rod by the
Fig. 4. Photocurrent response for various combination of laser illumination.
A.B. Md. Ismail et al. / Sensors and Actuators B 74 (2001) 112±116
115
Fig. 5. Difference-signal for the various pH.
Fig. 6. Bias-difference at various pH.
respectively, to obtain a minimum difference-signal (Vr), which is 13.3 mVp±p for this experiment. Ideally Vr should be zero, but as it depends on many parameters it is not always zero. Therefore, a minimum difference-signal is always obtained by adjusting biases at the two cells, intensity of the lasers, and phase-shift between the modulating signals to the lasers around 1808. The minimum differencesignal at the above mentioned operating biases was checked several times during the experiment, and a less than 1% change in the difference-signal was noticed. This drift in minimum difference-signal may be attributed to the difference in intensity of the lasers with time. Since in this experiment identical lasers were used, a nearly identical laser-intensity pro®le with time is expected. The change in photocurrents due to the change in intensities of the two lasers are nearly equal and opposite resulting a nearly unchanged minimum difference-signal. The base photocurrent signals for the measuring and reference cells were found to be 211 and 210 mVp±p, respectively. Now, for example, the solution at the measuring cell is changed to another buffer of pH 3.55 keeping the pH 4.55 buffer unaltered at the reference cell. As a result of change in pH value the surface potential and the photocurrent amplitude also changes accordingly at the measuring cell producing another difference-signal (VmpH) of 25.3 mVp±p. The change in photocurrent due to the change in pH is calculated from the difference between the two difference-signals, i.e. (V mpH ÿ V r ). Fig. 5 shows typical difference-signal calibration for difference-signal mode of operation. The amplitude of the difference-signal is assumed to be positive for the increase in pH value, and negative for the decrease in pH value, from the reference pH buffer. Conceptually, the change in photocurrent is due to the change in insulator surface potential that is sensitive to pH of the buffer. Now if the dc bias at the measuring cell is increased, the photocurrent at that cell increases and VmpH
decreases to Vr at a new bias of 1504.5 mV (VmpH). The difference in initial bias (Vmi) and the new bias (VmpH) at the measuring cell is exactly the pH sensitivity (mV/pH) of the sensor. When the pH value of the measuring buffer is higher than the reference buffer, the bias at the measuring cell is decreased to obtain the minimum difference-signal (Vr). As shown in Fig. 6, the difference in bias from the bias for reference pH buffer, are plotted for various pH value, and the pH sensitivity is obtained. An average pH sensitivity of 57.6 mV/pH, which is very close to the Nernstian value, is obtained in the experimented pH range. The proposed measurement system has limitation on pH measurement range. As we can see from the Fig. 4, the sensor switches from the accumulation to the inversion within 350 mV of the surface potential change. If the sensitivity is ÿ58 mV/pH as obtained from the experiment, pH range of 6 units (6 58 350), e.g. 4±10 pH, can be measured. With a photocurrent sensitivity of 22.4 mV/pH and a drift of 0.1 mV in the minimum difference-signal, a maximum error of 4 mpH in the measured pH is expected. The whole process of the pH measurement can be automated by employing a PC for the data collection, bias scan at the measuring buffer, and laser intensity control. 4. Conclusion In this article a generalized novel measurement scheme for the LAPS is presented. In principle, this measurement system offers a low-noise and low-background approach for the LAPS based instrumentation for the biosensing applications. Among the various expected applications, pH measurement is investigated in two modes, namely, differencesignal and bias-difference mode. The pH sensitivity of 57.6 mV/pH, which is very close to the Nernstian value, is obtained in the range of 3.55±5.65 pH. Without costly
116
A.B. Md. Ismail et al. / Sensors and Actuators B 74 (2001) 112±116
equipments like potentiostat, lock-in-ampli®er, etc., a direct, faster and highly sensitive instrumentation for the pH measurement using LAPS is proposed.
[9] M. Sartore, M. Adami, C. Nicolini, Computer simulation and optimization of a light-addressable potentiometric sensor, Biosens. Bioelectron. 7 (1992) 57±64. [10] Y. Murakami, et al., An organic pollution sensor based on surface photovoltage, Sens. Actuators B 53 (1998) 163±172.
Acknowledgements The authors would like to thank the New Energy and Industrial Technology Development Organization (NEDO), the Ministry of International Trade and Industry (MITI), the Government of Japan, for supporting this work (contract no. 98S18-001). References [1] T. Yoshinobu, H. Iwasaki, M. Nakao, S. Nomura, T. Nakanishi, S. Takamatsu, K. Tomita, Bioimages, Visualization of pH change of E. coli with a novel pH imaging microscope 5 (1997) 143±147. [2] G. Gehring, D.L. Patterson, S. Tu, Use of light addressable potentiometric sensor for the detection of Escherichia coli O157:H7, Anal. Biochem. 258 (2) (1998) 293±298. [3] K.A. Uithoven, J.C. Schmidt, M.E. Ballman, Rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system, Biosens. Bioelectron. 14 (2000) 761±770. [4] K. Dill, et al., Determination of dissociation constant and concentration of an anti-DNA antibody by using the light-addressable potentiometric sensor, J. Biochem. Biophys. Methods 31 (1996) 17±21. [5] V.T. Kung, et al., Picogram quantitation of total DNA using DNAbinding proteins in a silicon based system, Anal. Biochem. 187 (1990) 220±227. [6] L.J. Bousse, S. Mostarshed, D. Hafeman, A combined measurement of surface potential and zeta potential of an electrolyte/insulator interface of LAPS, Sens. Actuators B 10 (1992) 67±71. [7] M. Adami, M. Sartore, E. Baldini, A. Rossi, C. Nicolini, New measuring principle for LAPS, Sens. Actuators B 9 (1992) 25±31. [8] Y. Sasaki, Y. Kanai, H. Uchida, T. Katsube, Highly sensitive taste sensors with a new differential LAPS method, Sens. Actuators B 24/ 25 (1995) 819±822.
Biographies Abu Bakar Md. Ismail was born in Rajshahi, Bangladesh. He received his BSc (Hons) and MSc degrees in Applied Physics and Electronics from the University of Rajshahi, Bangladesh. In 1998 he received his PhD degree in Advanced Instrumentation from the Saga University, Japan. He has been serving as an Assistant Professor in the Department of Applied Physics and Electronics, Rajshahi University, Bangladesh. At present, he is engaged in imaging of electrical activity of living cells in the Department of Quantum Molecular Devices, The Institute of Scientific and Industrial Research, Osaka University, Japan. Hirokazu Sugihara received his BS degree in Bioengineering from the Osaka University, Japan, in 1985. He is currently working in Matsushita Electronic Industrial Co., Ltd., as a senior scientist. His research interests are developmental change and plasticity of biological neural networks, and design of new devices for multi-site neural response recording using micro-fabrication technique. Tatsuo Yoshinobu, born in Kyoto in 1964, received BE, ME, and PhD degrees in Electrical Engineering from Kyoto University in 1987, 1989, and 1992, respectively, for his study on gas source molecular beam epitaxy of silicon carbide. Since April 1992, he has been engaged in development of the scanning chemical microscope at The Institute of Scientific and Industrial Research, Osaka University. Hiroshi Iwasaki was born in 1945 and received the BS and MS degrees and the PhD degree in electronic engineering from Osaka University, in 1967, 1969, and 1975, respectively. From 1971 to 1986, he worked on Surface Physics including oxidation of silicon as a member of the faculty of Osaka University, and from 1983 to 1984, he worked at the University of Maryland as a Visiting Professor. From 1986 to 1991, he worked on ULSI's for Matsushita Electric Industrial Co., Ltd., Osaka. Since April 1991, he has been working at the Institute of Scientific and Industrial Research, Osaka University, as a Professor.