3.1. Turbidity Sensing
Before the turbidity sensing experiment, we performed calibration to clarify the correlation between turbidities and output photovoltages. In accordance with international standard ISO 7027 [
11], a formazine solution is recommended as the turbidity standard solution for measuring turbidity by the optical method. However, formazine is difficult to handle because a hydrazine sulfate, which is a carcinogenic agent, is necessary in the mixing process to obtain the formazine solution [
12]. On the other hand, kaolin has been prevalently utilized as a turbidity standard solution for a long time. Since kaolin is a clay mineral, it is completely free of anything harmful, is easy to handle, and is a low-cost material. However, a kaolin solution settles easily. It causes variations in turbidity during the measurement. In order to resolve the problem, a styrene-divinylbenzene (St-DVB) copolymer microbeads solution was proposed as the turbidity standard solution. However, a St-DVB copolymer microbeads turbidity standard solution is much more expensive than other turbidity standard solutions. In the calibration, we utilized both a kaolin solution and a St-DVB copolymer microbeads solution.
First, we prepared 7 different concentration calibration solutions of kaolin. The prepared kaolin concentrations are 0, 10, 20, 50, 100, 200, and 500 mg/L, as shown in
Figure 4. Then, we measured the photovoltages for each kaolin solution with the optical measurement setup shown in
Figure 3. In the measurement, the red LED was set to the LED1 position in
Figure 1b, and we measured the output photovoltages at three positions: PD1, PD2, and PD3, also shown in
Figure 1b. The measurement results are represented in
Figure 5. The photovoltages measured at the position of PD2 are not represented in
Figure 5 because the position of PD2 is aligned with LED1 and because the photovoltages were out of range. In other words, PD2 measured the light intensity of the light source LED1 rather than that of the scattered light caused from the suspended kaolin particles. However, if the sample is a dense suspension or if the LED and PD are far away from each other, the measurement with LED1 and PD2 may also be valid.
As a result, we were able to distinguish the degrees of turbidity according to the measured photovoltages, but we could not distinguish them with the naked eye, especially for low concentration samples shown in
Figure 4. Furthermore, we were also able to confirm the linear relationship between the concentration of the calibration solution and the scattered light intensity. The linearity was confirmed for the positions of both PD1 and PD3. We observed more accurate linearity in the measurement results from PD3. This linearity comes from the rapid precipitation effect of kaolin in the measurement, which means that the photovoltage measurement at the bottom of the sample yields a more accurate turbidity. Moreover, especially in low concentration regions of less than 50 mg/L, which is a region that is more meaningful for the sensors during detection, it shows better linearity. In turbidity sensing with environmental water samples, zero calibration is necessary only when the scattered light intensity is considered.
To convert the photovoltage value to the Nephelometric Turbidity Unit (NTU), we measured the same kaolin calibration solutions with a commercial turbidity sensor (Hydrolab DS5X, OTT Hydromet GmbH, Kempten, Germany) calibrated with a formazine calibration solution. The NTU is defined by the formazine calibration solution. The turbidity measurement results are shown in
Figure 6. Using the measurement results in
Figure 6, the measured photovoltages were converted to NTU turbidities.
We also performed turbidity measurements with environmental water samples from Lake Koyamaike located in the Tottori Prefecture of Japan. We collected eight measurement samples in Lake Koyamaike, and the places are indicated in
Figure 7. Sample Nos. 3 and 4, and Nos. 5 and 6 were collected at the same place, but Sample Nos. 4 and 6 were collected after a pause. In the measurement, we measured the photovoltage for each sample, and it was converted to NTU turbidity using the relationship shown in
Figure 6. The NTU turbidity measurement results are summarized in
Figure 8. The photovoltage measurements were performed by PD1 and PD3, and the results were in agreement. The turbidity of Sample Nos. 4 and 5 are relatively higher than the others due to the muddiness of the stream during sampling.
We also performed calibration with the St-DVB copolymer microbeads solutions for comparison. For calibration solutions of St-DVB copolymer microbeads, we prepared eight different concentration calibration solutions. The prepared St-DVB copolymer microbeads solution concentrations were 0, 1, 2, 5, 10, 20, 50, and 100 mg/L and are shown in
Figure 9. In the measurement, the measurement setup and procedure were the same as that used for the kaolin calibration solutions. The measurement results are shown in
Figure 10. Here, we only measured the photovoltages from PD1 and PD3.
With the St-DVB copolymer microbeads calibration solution, we were also able to distinguish the degrees of turbidity according to the measured photovoltages. Moreover, we were able to successfully confirm the linear relationship between the concentration of the calibration solution and the scattered light intensity. Although the measurement results between PD1 and PD3 have small differences, we could obtain linearity with almost identical slopes. This means that the St-DVB copolymer microbeads are uniformly distributed in the solution and the precipitation effect can be ignored. The zero calibration is also necessary only to consider the scattered light intensity when we measure the turbidity of the environmental water sample.
We also performed turbidity measurements with the same environmental water samples, which were used in previous turbidity measurements. The measurement results based on the St-DVB copolymer microbeads solution calibration are shown in
Figure 11. The NTU turbidity conversion was performed by using the relationship shown in
Figure 6 as well. The turbidity values measured by PD1 agreed with those measured by PD3. Although the obtained turbidity values based on the St-DVB copolymer microbeads solution calibration were slightly smaller than those based on kaolin solution calibration. Specifically, in most low turbidity areas, the turbidity showed almost the same values. According to the measurement results for each calibration solution shown in
Figure 5 and
Figure 10, the St-DVB copolymer microbeads solution calibration is considered as the more accurate method. However, in real applications, it is more important and meaningful to detect and clarify low turbidity water instead of high turbidity water. Therefore, the cheaper and easier method using the kaolin calibration solution is considered to be the effective one.
3.2. Chlorophyll a Concentration Sensing
We also performed a chlorophyll
a concentration sensing experiment with the same optical measurement setup shown in
Figure 3. Before the measurement with an environmental water sample, we calibrated to clarify the correlation between chlorophyll
a concentrations and output photovoltages. As calibration solutions of chlorophyll
a, we prepared eight different concentration calibration solutions. The prepared chlorophyll
a concentrations were 0, 5, 10, 20, 50, 100, 200, and 500 µg/L, which are shown in
Figure 12. With the naked eye, the concentration differences cannot be distinguished. In the measurement, the measurement setup and procedure are the same as what we used for the turbidity measurement, except for the LED. For chlorophyll
a measurements, we utilized a blue LED instead of a red LED for the excitation of fluorescence. The blue LED was set to the LED2 position in
Figure 1b. The measurement results are shown in
Figure 13. In the measurement, the photovoltages were detected by PD3 only because detection by PD3 yielded better results in turbidity measurement. Moreover, we set a red filter sheet on PD3 to consider only the fluorescence around 685 nm in the measurement. For convenience in the red filter setup, we chose PD3 for the detection. As a result, we were also able to obtain good linearity between chlorophyll
a concentrations and photovoltages. In other words, with the same simple optical measurement setup, we can easily detect both the turbidity and the chlorophyll
a concentration. Commonly used commercial chlorophyll
a sensors measure water soluble uranine (fluorescein disodium salt, C
20H
10Na
2O
5) fluorescence intensity for calibration, and measurement results are then converted to chlorophyll
a concentration. However, by using our measurement setup, we can directly calibrate with the chlorophyll
a that is dissolved in ethanol and diluted with water. In chlorophyll
a concentration sensing with an environmental water sample, zero calibration is necessary to consider fluorescence intensity only.
We performed chlorophyll
a concentration measurements with the same environmental water samples, which were used in previous turbidity measurements. The measured chlorophyll
a concentrations are summarized in
Figure 14. The chlorophyll
a concentration was measured successfully with the same optical measurement setup applied to turbidity sensing. The chlorophyll
a concentration is also one of the more important quantities in defining water quality. For instance, the turbidity of Sample No. 4 showed the highest value, but the chlorophyll
a concentration of it was not high. This means that the environmental water sample at the Sample No. 4 position was only muddy and not polluted by phytoplankton.
In the chlorophyll
a concentration measurement, we used a red filter in front of PD3 to effectively detect fluorescence. PD3 is also used for turbidity detection, and we performed the turbidity measurement again with a red filter on PD3 to investigate influence of the red filter installation on turbidity measurements. In the measurements, we measured photovoltages and NTU turbidities for kaolin calibration solutions shown in
Figure 4 by using our measurement setup and a commercial sensor, respectively. The measurement results with and without the red filter on PD3 are shown in
Figure 15. Although the output photovoltages are decreased due to scattered light intensity reduction for the high concentration sample, the linearity still exists; specifically, there are no remarkable variations for low concentration samples, which are samples that are more useful in real applications. As a result, it reveals that two LEDs and one PD are sufficient to simultaneously detect both the turbidity and chlorophyll
a concentration with our measurement setup.
In order to improve the strength of output photovoltages, we investigated the reflection effect of the socket. We put Al foil tape inside and around the socket to gather reflected scattered or fluorescent light to increase the output photovoltage. In the measurement, we used chlorophyll
a solutions as samples shown in
Figure 12. Moreover, a larger LED which has a 5 mm diameter was also tested. The measurement results are shown in
Figure 16. Of course, all measurement results showed good linearity. There were no remarkable changes in the strength of photovoltages between 3 mm and 5 mm diameter LEDs. However, the output photovoltage strength was much more improved when the reflective Al foil tape was applied. For instance, for the chlorophyll
a concentration of the 5 µg/L sample, which is sample with the lowest concentration, the output photovoltage strength was increased by 14.9%. It is obvious that using reflection in the measurement setup is effective and should be considered when turbidity and chlorophyll
a concentration sensors are integrated into a microfluidic sensor module, as shown in
Figure 17.
In environmental water measurement, it is important to guarantee constant LED intensity. To do so, the ageing effect of LED intensity must be clarified, which will be considered as a follow-up to this work. Moreover, the intensity of the LEDs and the sensitivity of the PD should be optimized to obtain an optimal device size.