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Abstract
Terahertz chemical microscopy has been developed for measuring the pH of a solution using only a small volume. The microsolution wells were fabricated on the surface of the sensing plate using a conventional photolithograph technique. Because the pH value can be calculated from the amplitude of a terahertz wave directly radiated from a sensing plate by a femtosecond laser irradiation, this method does not require any reference electrode in the solution. Thus, pH measurement can be achieved with a volume as small as 16 nL.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Measuring ion concentrations in small volumes of solutions is an attractive challenge with particular applications in several fields such as clinical and environmental analysis. Among various types of ion sensors and/or ion measurements systems, ion-sensitive field-effect sensors (ISFETs) are good candidates for measuring ion concentrations [1–3]. An ISFET generally comprises an insulated gate field-effect transistor with an ion-selective material on the gate electrode. Therefore, the sensing area corresponds to the area of the gate electrode. Using well-developed semiconductor fabrication processes, a gate electrode with an area less than 100 μm2 can be achieved. However, this type of sensors requires a reference electrode to be injected in the solution in order to stabilize the electric potential of the solution. Thus, this reference electrode reduces the solution volume even if it is integrated in the ISFET. Light-addressable potentiometric sensors (LAPSs) enable ion-concentration measurements using a small sensing area [4–9]. LAPSs utilize a comprising a semiconductor thin film coated with an insulator layer on a substrate. Then, ion-selective materials are fabricated to change the electric potential on the surface of the chip and therefore the electric field inside the semi-conductor. By applying modulated-light irradiation to the backside of the semiconductor thin films, modulated photocurrents are produced. These, in turn, can be measured by forming a closed AC circuit including a reference electrode, which limits the minimum required volume of the sample solution.
Our research group has developed a terahertz (THz) chemical microscope (TCM) for measuring ion concentrations in solutions [10–16]. The TCM utilizes a “sensing plate,” which comprises semiconductor thin films similar to those used in the LAPS systems on a sapphire substrate. However, although the LAPS system utilizes modulated light to produce the photocurrents, the TCM utilizes an ultrafast laser pulse to produce the photocurrents with ultrafast modulation. Because the photocurrents produced by the ultrafast laser pulse emit a THz wave radiated directly into free space, a closed electric circuit, electrodes on the TCM sensing plate, and a reference electrode in the solution are not required. Consequently, the minimum required volume of the target solution for the TCM is potentially smaller than that for conventional measurement systems [17–19].
2. Experimental
Figure 1 shows a schematic of the sensing pate. The epitaxial Si thin film was fabricated on a sapphire substrate with a thickness of ~1 μm. The surface of the Si thin film was naturally oxidized; therefore, the SiO2 thin layer was formed on the surface. This SiO2 thin layer works as the insulator layer between the Si film and the sample solution on the sensing plate. The size of the sensing plate was 100 mm × 100 mm. No passivation layers were present against the solution on the surface of the sensing plate at this moment. The microsolution wells made from an ultraviolet (UV) curable resin were patterned on the sensing plate using a conventional photolithography technique. The diameters of the solution wells were in the range of 50–500 μm. The depth of the solution wells measured using white light interferometry was ~3 μm. In this experiment, we used wells with a diameter of 250 μm, which could store 16 nL of the water solution. When the water solutions were on the sensing plate, the silanol (Si–OH) groups are formed on the surface of the SiO2 thin layer. The Si–OH groups in the thermal equivalent state are expressed as follows:
Fig. 1 Schematic of the sensing plate when it is illuminated by the femtosecond laser. Inset is the microphotograph of the microsolution wells.
The Si–OH groups titrate with the protons from the solution and exist as either negatively charged SiO− or positively charged SiOH+ functions, and the electric double layer is formed at the surface of SiO2. The selectivity to the other ions attribute to the chemical reactions between the SiO2 films and the ions, and the selectivity coefficient of SiO2 for protons to other ions are generally more than 1000 [20]. In accordance with Nernst’s equation, the chemical potential at the surface is determined by the proton concentration. Because chemical potential can be considered as the voltage applied between the solution and the SiO2 surface, it changes the local electric field of the Si thin film. When the ultrafast laser pulse is incident onto the Si thin film from the substrate side the sensing plate, photocurrents with ultrafast modulation are produced. According to the classical electromagnetic theory, an electromagnetic wave is emitted from the modulated currents, and the far electric field E(t) of the emitted electromagnetic wave can be given by
where J(t) is the current density. Because J(t) is given by the product of the carrier density n(t), speed of the carrier v(t), and elementary charge e, the equation yieldsThe time derivative of v(t) is proportional to the electric field affecting the carriers. Thus, the amplitude of the electric field of the electromagnetic wave can be related to the chemical potential on the surface of the sensing plate [10,11]. In our experiments, a femtosecond laser with a pulse width and a full width at half maximum were used to excite the carriers. The center wavelength was ~790 nm, which has much higher photon energy than the energy gap of the Si thin film. The repetition rate of the laser pulses was 82 MHz. Because of the ultrafast modulation of the carriers by the femtosecond laser, the radiated electromagnetic wave had frequency components in the THz range. This radiated THz wave was detected by a bow-tie-type photoconductive antenna. We applied a pump-probe measurements system with a bow-tie-type photoconductive antenna to detect the THz wave. The time-delay line consisted with the retro-reflector mounted on the linear stage and the position of the time-delay was fixed at where the peak amplitude of THz wave was observed.
3. Results and discussion
The electric field distribution on the surface of the sensing plate was simulated by a finite-difference time-domain (FD-TD) method when a water droplet with a diameter of 250 μm was placed at the center of the sensing plate. The schematic of the simulated model is shown in Fig. 2(a). The dimensions and components of the model are the same as those of the actual sensing plate. The surface charges of the droplets were set to 1, 10, and 100 C for Figs. 2(b), 2(c), and 2(d), respectively. The electric fields in the Si thin films beneath the droplets were 5 × 1010, 5 × 1011, and 5 × 1012 V/m, respectively. This showed the linear relationship between the surface potential and the electric field on the Si film. In addition, in this simulation, the reference electrodes were not used because the volume of the water solution was extremely small and its potential might be increased due to electrical charge. Thus, the pH values could be stably measured without reference electrodes.
Fig. 2 (a) Schematic of the simulated model of the sensing plate. The water droplet with a diameter of 250 μm was placed at the center of the sensing plate. The model was the same as the actual sensing plate in terms of its dimensions and components. (b), (c), and (d) are the simulated distributions of the electric field in the Si films of the sensing plates, where the surface charges of the water droplets were set to be 1, 10, and 100 C, respectively.
Figures 3(a) and 3(b) shows the typical time-domain THz waveforms radiated from the sensing plate when the water solution was on the surface of the sensing plate and their frequency spectra, respectively. The microsolution wells were not fabricated for these experiments, therefore, the volume of the water solution was hundreds of microliters. Both of the waveforms and the spectra were independent of the pH values of the water solutions on the sensing plate, because changes in the pH values only result in changes in the electric fields in the Si thin films and the radiated THz waves do not interact with the water solutions. Therefore, the position of the time-delay was fixed at where the peak amplitude of THz wave was observed when the pH values were measured.
Fig. 3 (a) The typical time domain THz waveforms radiated from the sensing plate when the water solution was on the surface of the sensing plate and (b) their frequency spectra.
Figure 4 shows the THz peak amplitudes of the THz waveforms in Fig. 3(a) as a function of pH values of the buffer solutions. We confirmed that the amplitude was increased by increasing the pH values of the buffer solutions.
Fig. 4 THz peak amplitudes of the THz waveforms in Fig. 3(a) as a function of pH values of the buffer solutions.
As the next step, the microsolution wells were fabricated on the sensing plate to measure the small amount of the water solutions. Figure 5 shows the map of the change in the amplitude of radiated THz waves before and after injecting the buffer solutions into the wells. The buffer solutions were injected using a micro-dispenser whose discharge volumes are controlled by air pressure. The images were obtained by scanning the ultrafast laser pulses across the sensing plate surface. The dashed circles represent the microsolution wells fabricated on the sensing plate. Figures 5(a) and 5(b) show the results for the buffer solutions with the pH values of 6.86 and 10.01, respectively. The amplitude of the THz waves was enhanced beneath the wells filled with the pH solutions. The change in the amplitude of the THz waves for the solution with the pH value of 10.01 was much larger than that with the pH value of 6.86. These results suggest that the concentration of protons can be visualized using TCM. Figure 5(c) shows the map of the change in the amplitude superimposed on a microphotograph of the microsolution wells. The spatial resolution of the map was determined by the spot size of the laser focused on the backside of the sensing plate using an objective lens; thus, the spatial resolution was estimated to be ~300 μm [3–5].
Fig. 5 The map of the change in the amplitude of the THz wave radiated from the sensing plate before and after injecting the buffer solutions into the microsolution wells. (a) and (b) represent the maps for the buffer solutions with the pH values of 6.86 and 10.01, respectively, wherein the dashed circles indicate the position of the microsolution wells. (c) represents the map superimposed with the microphotograph of the microsolution wells.
The average THz amplitudes corresponding to pH 1.68, 6.86, and 10.01 on the microsolution wells of the buffer solution are plotted in Fig. 6. The accuracy of the pH measurement was estimated from the error bar and found to be approximately ± 0.22 at pH 6.86 for our experiments. Note that the accuracy is generally affected by a lot of factors such as impurity, temperature, and laser stability. The amplitude of the THz waves increased with increasing pH values, indicating that it depends on the concentration of protons in the sample solution, even though the volume is only 16 nL. The signs of the THz waves were crossing zero because the direction of the electric field changed. The relation between the sign of THz and the electric potential on the sensing plate was reported in Ref [16]. However, this change in the sign of the THz waves does not affect the measurement accuracy because the amplitude increases monotonically. The amplitude offset may change slightly owing to the alignment of the optical setup. Moreover, the quantitative measurement of the proton concentration could be accomplished by normalizing the measured THz amplitude. In addition, we developed a double-laser pulse method to stabilize the signals obtained by TCM [6]. This method can be integrated into the TCM as the next step. Furthermore, by fabricating the selective membranes for various types of ions on the bottom of the microsolution wells, this system can be integrated into multi-ion sensing systems, increasing the usefulness of the sensor for nano-volume applications.
Fig. 6 Average THz amplitudes of the microsolution wells as a function of pH values of the buffer solutions. Error bars indicate spatial deviations.
4. Conclusion
In conclusion, TCM has been proposed and developed for measuring ion concentration and pH values in water solutions. The microsolution wells were fabricated on the surface of the sensing plate using a conventional photolithograph technique. As a result, the pH value of a solution with a volume of only 16 nL could be measured. In a future work, we will attempt the integration for multi-ion sensing and reducing the laser spot size to improve the accuracy of TCM. We believe that the applications of TCM can be increased by fabricating the various micro-order membranes with various types of ionophore on the microsolution wells.
Funding
Japan Society for the Promotion of Science (JSPS) (KAKENHI B 16H03887).
Acknowledgments
The authors thank Masakai Kobayashi and Akinori Hashimoto, Kyowa fin-tech co. ltd. for helpful discussion on the optical design of the TCM.
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