Anal. Chem. 1997, 69, 977-981
Real-Time Imaging of Microscopic pH Distribution with a Two-Dimensional pH-Imaging Apparatus Satoshi Nomura,* Motoi Nakao, Tsuyoshi Nakanishi, Shuji Takamatsu, and Katsuhiko Tomita
Horiba Ltd., Kisshoinmiyanohigashimachi, Minami-ku, Kyoto 601, Japan
Real-time two-dimensional pH imaging has become possible with the development of a new type of potentiometry, by which the pH distribution can be visualized as pH images with submillimeter spatial resolution. The measurement time for one image is short enough for practical use. The transient change of pH distribution generated by the protons released from a single cation-exchange resin was observed and visualized as the pH images. From the pH images, the evaluation of the resin performance was also carried out.
As one of the most important properties of a solution, the pH is measured widely in various fields of science. Potentiometry is one of the most convenient and most common methods of pH measurement, and many types of pH electrodes and pH meters are now commercially available. Together with the improvement of the reliability of measurement, efforts have also been made to expand the applicability of potentiometry by, for example, reducing the electrode size.1 However, the applications of this measurement are still mainly limited to uniform pH distribution. Recently, the uniform pH distribution limit was surpassed following the development of the pH imaging sensor2-5 based on a new type of potentiometry employing semiconductor silicon.6 This sensor was applied to the measurement of pH distribution with submillimeter resolution, and the extracellular pH distribution of a microbiological colony could be observed as the pH images.4 In the present work, an attempt was made to improve the temporal resolution of the pH imaging to enhance its utility as a practical analytical method, and toward this end, a new twodimensional pH-imaging apparatus was developed. This apparatus enabled the real-time imaging of the pH distribution with submillimeter spatial resolution. By the continuous real-time imaging of pH distribution, the behavior of the protons released from a single cation exchange resin was visualized. The resin performance was also quantitatively evaluated based on the pH images. (1) Midgley, D.; Torrance, K. Potentiometric Water Analysis; John Wiley & Sons: Chichester, England, 1991. (2) Nakao, M.; Yoshinobu, T.; Iwasaki, H. Sens. Actuators B 1994, 20, 119123. (3) Nakao, M.; Yoshinobu, T.; Iwasaki, H. Jpn. J. Appl. Phys. 1994, 33, L394L397. (4) Nakao, M.; Inoue, S.; Oishi, R.; Yoshinobu, T.; Iwasaki, H. J. Ferment. Bioeng. 1995, 2, 163-1662. (5) Nakao, M.; Inoue, S.; Oishi, R.; Yoshinobu, T.; Iwasaki, H. Tech. Digest, 8th. Conf. Solid-State Sensors & Actuators (Transducers ‘95), 1995; p 451. (6) Hafeman, D. G.; Parce, J. W.; McConnell, H. M. Science 1988, 240, 11821185. S0003-2700(96)00909-2 CCC: $14.00
© 1997 American Chemical Society
EXPERIMENTAL SECTION Measurement Principle. The apparatus developed in this work is based on a scanning laser beam semiconductor pHimaging sensor studied by one of the authors.2-5 The measurement principles are explained in this section. A Si3N4 film/SiO2/Si structure was used as the pH sensor, and the electrolyte sample is in contact with the Si3N4 surface. The ac photocurrent induced by the modulated illumination of the silicon side of the sensor is measured with the bias voltage applied between the electrolyte and silicon. This ac photocurrent is affected by the proton concentration at the electrolyte/Si3N4 interface on the illuminated spot. Consequently, only the illuminated spot is the measurement point in the sensor. Therefore, the microscopic two-dimensional distribution of proton density, i.e., the pH value, can be observed by focusing and then scanning the illumination. The measured photocurrent at each illuminated spot is transformed to a pH value and displayed as a twodimensional pH image in synchronization with the X, Y position of the illuminated spot. The spatial resolution and pH resolution of this sensor is 0.1 mm and 0.1 pH, respectively.3 The measurement time is somewhat too long for practical use. For example, it takes almost 4 min to obtain 6.4 mm × 6.4 mm images with 64 × 64 measurement points. Instrumentation. The measurement principle and the sensor structure have been described elsewhere.2-5 However, in this study, the illumination scan method, which determines the measuring time, was mainly modified to improve the pH imaging as a practical analytical method. The measurement was controlled by a personal computer (PC1:NEC, PC9821Xa). The bias voltage was applied by a customdesigned potentiostat. A semiconductor Laser (Sakai Glass, Tokyo, Japan, λ ) 780 nm of 5 mW maximum power) was used as the light source. During the measurement, the power was set at 10 µW, and the frequency of the illumination was 5 kHz. Figure 1 shows the design of the electrochemical cell. This electrochemical cell was placed on the X, Y stage during the measurement. The sensor (A) is fixed in a plastic holder (B) so that the holder forms the cell wall. The bias voltage is applied using the Pt electrode (C) and the ohmic contact (D) from the potentiostat. A 27 mm × 27 mm area of the sensor is in contact with the electrolyte sample (E). In the illumination scanning, the sensor (electrochemical cell) is placed on the two-dimensional X, Y stage with the laser beam fixed. The stage (Sigmakoki MYST-55, Hidaka, Japan) has the highest speed available. The stage is controlled by PC1 using the driver supplied by the stage manufacturer. This stage control allows scanning of the stage at a speed of 9.5 mm/s. Analytical Chemistry, Vol. 69, No. 5, March 1, 1997 977
Figure 1. Electrochemical cell and measurement setup: (A) sensor, (B) holder, (C) Pt electrode, (D) ohmic contact, (E) sample electrolyte (agar film), (F) cation-exchange resin, and (G) proton release. Table 1. Measurement Times for One Image under Various Image Conditions measured area, mm
measuring point
measuring time, s
14.0 × 14.0 6.4 × 6.4 12.8 × 12.8 12.8 × 12.8 25.6 × 25.6
35 × 35 64 × 64 128 × 128 128 × 128 256 × 256
35 45 (225)a 90 180 (900)a 360
a
From ref 5.
The ac photocurrent is sampled in synchronization with the light modulation using the clock circuit at a time interval t given in eq 1, where d is the distance of each pixel of the pH image,
t ) d/9.5
(1)
that is the resolution of the pH image (mm), and 9.5 is the X, Y stage scan speed (mm/s). The ac photocurrent at each sampling time was regarded as that observed in the d mm × d mm region inside of which the light was illuminated. The sampled photocurrent was converted to the voltage output and digitized as a 12-bit digital signal and then sent to and stored in PC1. For continuous pH imaging, when all of the sampling for one pH image was complete, the data set was sent to PC2, making PC1 available for the successive imaging. The data set in PC2 was converted to pH values and processed to the pH image. This process by PC2 was possible even when the PC1 started controlling the successive imaging. The image processing at PC2 was carried out using commercially available software (IPP Win, Media Cybernetics, Silver Spring, MD). Image processing was also carried out, when required, using a Power Macintosh 7500/100 and image public domain NIH imaging program. The spatial resolution and pH resolution were evaluated in a manner reported previously.3 The measurement times for one image under various imaging condition are shown in Table 1. Reagents. Ion-exchanged water was used for preparing the electrolyte sample. Potassium chloride, sodium hydrate, and agar (Nakalai Tesque, Kyoto, Japan) were of analytical grade. Ionexchange resin (Amberlite IR-120B, sulfonated type, Organo, Tokyo, Japan) was purified by hydrochloride7 unless otherwise indicated. The purified resins were stored in ion-exchanged water. The diameter of the resin was 0.4 mm when they were stored in ion-exchanged water. Measurement Procedure. The measurement procedure is also shown in Figure 1. Agar film was used as the electrolyte, 978
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
and the cation-exchange resin (F) was placed on it. The gel film was prepared from solution consisting of 1.5% agar and 0.1 M potassium chloride. The solution pH was adjusted to 7.4 by adding sodium hydroxide. The agar film was prepared by pouring heated agar solution directly on the sensor followed by cooling, leaving an agar film 0.5 mm thick on the sensor surface. The cation-exchange resin was placed on the agar film. The transient pH change was imaged, which was generated by the protons released from the resin after penetrating the agar film to the sensor. RESULTS AND DISCUSSION Two-dimensional pH imaging was repeated using a single cation-exchange resin as the source for transient microscopic pH distribution. Under the first set of conditions, the first image was obtained 2 min after the resin was placed on the agar film and the successive measurements were carried out at a 2-min intervals. The measured area and the measuring points were 12.8 mm × 12.8 mm and 64 × 64, respectively. As shown in Figure 2, the pH distribution generated by the released protons was clearly observed in the pH images as the round acidified region. The temporal resolution of this apparatus was adequate, and the diffusion of released protons was visualized as a expansion of the acidified region. Inspection of the pH images revealed that the diameter of the acidified region (5 mm) was much larger than the diameter of the resin (0.4 mm) even on the first image obtained 2 min after the resin was placed on the agar film. This indicates that the release and diffusion of the protons occurred rapidly. While the acidified region expanded, the pH value remained almost the same at the center of the region, indicating a fairly constant supply of protons inside the resin and their constant release during the measurement. In the second experiment, the measurement was carried out by a different sequence with shorter time resolution. The first image was obtained soon after the resin was placed on the agar film, and successive images were obtained at a 1-min intervals. The measured area and the measuring points were 14.0 mm × 14.0 mm and 35 × 35, respectively. As shown in Figure 3, the diameter of the acidified region in the first image was smaller than that under the first set of conditions, but it was still 3-4 times greater than the actual resin diameter. This shows that the release and diffusion of the proton occurred soon after the resin was placed on the agar film. To examine in more detail the differences between each image in Figure 2, every image in the figure, except the last one, was subtracted from the following one, that is, the one obtained 2 min later. The images obtained by subtraction are shown in Figure 4. From the ring in each image, it was easily confirmed that the acidified region had continued to expand and that the pH value had remained constant at the center of the region. The ring may also be used for the evaluation of the proton diffusion in agar, because it shows the leading edge of the proton diffusion. Since the diffusion is a transient phenomenon, evaluation by real-time pH imaging is advantageous. Precise quantitative analysis of the proton diffusion characteristics is now underway. The release of protons from an unpurified resin was visualized, as shown in Figure 5, and compared with the release from a purified resin in the second experiment described above. The
Figure 2. Proton release from the cation-exchange resin: measured area, 12.8 mm × 12.8 mm; measuring points, 64 × 64; measurement interval, 2 min.
Figure 3. Proton release from the cation-exchange resin with shorter temporal resolution: measured area, 14.0 mm × 14.0 mm; measuring points 35 × 35; measurement interval, 1 min.
acidified region diameter was much smaller when the unpurified resin was used. Moreover, while the acidified region continued to expand until 9-10 min when the purified resin was used, it reached maximum size at around 6-7 min when the unpurified resin was used. From these results, we concluded that the resin performance was reflected in the pH image. The resin performance was quantitatively evaluated using all the images in Figures 3 and 5. The total released proton amount (N(t)) at each measurement time was calculated for both the purified and unpurified resins. They were obtained using eq 2,
∑(10
N(t) ) Ah
-pHxi,yi
- 10-pHback)
(2)
where pHxi,yi, pHback, A, and h are the pH values at each pixel (Xi, Yi), background pH value of the agar film (pH 7.4), pixel area (0.16 mm2), and agar film thickness (0.5 mm), respectively. The pH distribution perpendicular to the sensor was assumed to be zero. As shown in Figure 6, the total amount of the released protons was confirmed to be 100 times larger with the purified resin than with the unpurified resin. Moreover, the released proton amount became saturated at around 9 and 5 min with the purified and the unpurified resins, respectively. These data correspond to the pH images; that is, the diameter of the acidified region became constant at around 9 and 5 min, respectively. Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
979
Figure 4. Proton release from the cation-exchange resin (obtained by subtraction): measured area, 12.8 mm × 12.8 mm; measuring points, 64 × 64; measurement interval, 2 min.
Figure 5. Proton release from the unpurified cation-exchange resin with smaller time resolution: measured area, 14.0 mm × 14.0 mm; measuring points, 35 × 35; measurement interval, 1 min.
Figure 6. Amount of total proton release.
The ion-exchange capacitance (C) of the purified resin was calculated using eq 3,
C ) N11/NA/W
(3)
where N11, N4, and W are the total released proton amount at 11 min obtained by the above calculation, Avogadro’s number, and the resin weight, respectively.7 980
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
The calculated capacitance was 4.0 mg‚equiv/g, which is comparable to reference value 4.4 mg‚equiv.8 This indicates that a new method of resin characterization can potentially be developed by using this pH-imaging technique. It is particularly noteworthy that the resin performance can be evaluated using only a single resin. In the conventional method, acid/base titration must be carried out using a substantial amount of resins. A study is also underway for the evaluation of resin performance by pH imaging. In this study, real-time pH imaging was examined by potentiometry for the first time. Although pH imaging is a new concept in potentiometric measurement, the advantage of this real-time pH imaging was confirmed. In addition, new approaches may also come to be developed for the study of proton diffusion and resin performance may also come to be developed using the temporally resolved pH images. It is nevertheless clear that further improvement is required in the performance of the pH-imaging system in terms of pH resolution, spatial resolution, and temporal resolution. We are presently continuing our efforts toward this objective in order to expand the applications of this pH-imaging technique. (7) Nagashima, K.; Tomita, I. Analytical Chemistry; Shokabo: Tokyo, Japan, 1985. (8) Senoo, M.; Suzuki, T.; Abe, M. Ion Exchange Resin; Kodansha: Japan, 1991.
ACKNOWLEDGMENT
“Equipment for Monitoring Microorganism Activity” using twodimensional pH imaging.
We are grateful for Prof. Hiroshi Iwasaki in Osaka University for stimulating discussions. This work was financially supported by RITE (Research Institute of Innovative Technology for the Earth, Japan) in conjunction with which we are developing
Received for review September 10, 1996. December 11, 1996.X
Accepted
AC960909T X
Abstract published in Advance ACS Abstracts, February 1, 1997.
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
981
