Measurement of the Thermal Effect of Standing Surface Acoustic Waves in Microchannel by Fluoresence Intensity

Temperature is an important parameter for many medical and biological applications. It is key to measuring the temperature of acoustofluidics devices for controlling the device’s temperature. In this paper, Rhodamine B was used to measure the temperature change of the microchannel induced by the SSAWs’ thermal effect in microfluidics. A thermocouple was integrated into the microfluidics device to calibrate the relationship between the fluorescent intensity ratios of Rhodamine B and the temperature. Then, the fluid temperature in the microchannel heated by the SSAWs was measured by the fluorescent signal intensity ratio in the acoustofluidics device. The fluid temperature with different input voltages and different flow rates was measured. The results show that SSAWs can heat the still fluid rapidly to 80 °c, and the flow rates will influence the temperature of the fluid. The results will be useful for precisely controlling the temperature of acoustofluidics devices.


Introduction
Acoustofluidics, which integrates acoustic waves with microfluidics to manipulate particles and fluids in microscale fluid, has been widely used in material engineering [1][2][3] and biotechnology [4,5], due to the advantages of noncontact, better biocompatibility, and compactness [6]. Surface acoustic waves (SAWs), which are generated and propagated on the surface, have attracted attention for the following reasons. First, SAWs have a high vibration velocity and acceleration and concentrate most of the energy on the surface, resulting in remarkably effective and efficient fluid-structural coupling [7]. Second, SAW devices are easy to fabricate and integrate with microfluidics [8]. Third, SAW devices are inexpensive and simple and can be made portable and flexible. As a result, SAWs have been widely used in acoustofluidics [9].
In the propagation of waves, SAWs will induce acoustic radiation force, acoustic streaming, and a thermal effect in microfluidics [10]. The three physical phenomena interact with each other in acoustofluidics [11]. It is important to study the mechanism of the three physical phenomena for better controlling the particle, droplet, and fluid in acoustofluidics [12]. The mechanisms of acoustic radiation force and acoustic streaming have been deeply studied and widely used in microfluidic systems [13]. The study of SAWs' thermal effect is still in its infancy. Most researchers have paid attention to the thermal effect induced by travelling surface acoustic waves (TSAWs) in open microfluidics [14]. The droplet thermal effect induced by TSAWs in open microfluidics was observed by the thermal infrared imager and provided a condition for controlling the droplet temperature and heating the micro system [14][15][16][17]. The TSAWs' thermal effect was also used as energy for thermal drug release from temperature-sensitive liposomes [18], assisting closed-vessel Suzuki coupling reactions [19], lysis cells, and the isothermal amplification of DNA [20,21]. Sung et al. observed the thermal effect of polydimethylsiloxane and developed an acoustothermal tweezer for sorting droplets [22][23][24]. The results demonstrate that the SAWs' thermal effect plays an important role in microfluidics. It is necessary to measure the thermal effect induced by SAWs.
Standing surface acoustic waves (SSAWs) have been widely used in manipulating particles [25], cells [26], and droplets [27]. Researchers found that the SSAWs' thermal effect is key in the application of acoustofluidics because of the destruction of bio-particles [28]. To overcome the high temperature in acoustofluidics, cooling equipment had to be integrated into microfluidics [6,29]. However, few have measured the temperature induced by the SSAWs' thermal effect in the microchannel, which limited the precise controlling and application of particles in the biological detection of SSAW-based devices. In acoustofluidics, there are two difficulties in measuring the temperature in the microchannel. First, the channel size is on the micro-scale, which makes it difficult to integrate contact sensors. Second, the vibration of the substrate and the acoustic streaming will both influence the detection precision of contact sensors. Rhodamine B (RhB) dye, with a high sensitivity to temperature, negligible pressure sensitivity, and independent absorption of PH, has been widely used in the measurement of temperature in microfluidics [30][31][32][33].
In this paper, the fluorescent signal intensity ratios of Rhodamine B were used to measure the temperature changes induced by SSAWs' thermal effect in microfluidics. The fluorescent signal intensity ratios were calibrated by a thermocouple in the microchannel. Then, the Rhodamine B was used to measure the temperature in acoustofluidics. The temperature of different input voltages and different fluid velocities was measured. The results show that SSAWs can rapidly heat microfluids, and fluid velocity can influence the temperature. This paper will promote the precise control of particles, cells, and droplets in acoustofluidics.

Fabrication of Calibration Microfluidic
The PDMS microchannel was fabricated by a standard soft-lithography. The mold was made by patterning SU-8 photoresist on silicon. Then, PDMS channel (width: 100 µm, height: 100 µm) was made by replica process. Two holes of the inlet and outlet were punched with a Harris Uni-core punch in PMDS channel. Then the PDMS channel and silicon were treated by a plasma cleaner with oxygen plasma for 60 s. The PDMS channel and thermocouple (F52-II, Fluke) were aligned and bonded to the silicon (as shown in Figure 1a). The microfluidic was cured at 95 • C for 2 h. induced by SAWs.

39
Standing surface acoustic waves (SSAWs) have been widely used in manipulating 40 particles [25], cells [26] and droplet [27]. The researchers found that the SSAWs thermal 41 effect is key in the application of acoustofluidics, because of the destructive to bio-42 particle [28]. To overcome the high temperature in acoustofluidics, cooling equipment 43 had to be integrated into microfluidic [6,29]. However, there are few people to measure 44 the temperature induced by SSAWs thermal effect in the microchannel, which lim-45 ited the particle precisely controlling and application in biological detection of SSAWs 46 based devices. In acoustofluidics, there are two difficulties to measure the tempera-47 ture in microchannel. First, the channel size is micro-scale, which makes it difficult 48 to integrate contact sensors. Second, the vibration of the substrate and the acoustic 49 streaming will both influence the detection precision of contact sensors. Rhodamine 50 B (RhB) dye, with high sensitivity to temperature, negligible pressure sensitivity and 51 independent absorption of PH, has been widely used in the measurement of temperature 52 in microfluidic [30][31][32][33].

Fabrication of Acoustofluidic
First, the patterning of two interdigital transducers (IDTs) was designed and patterned on the mask. The IDTs comprise 20 pairs of straight fingers with a period of 400 µm. Then the patterning was transferred to a 1 mm thick piezoelectric (Lithium niobite, LiNbO3) substrate by the process of lithography. Finally, the SSAW device was fabricated by the process of lift off. The IDTs were made by depositing dual layer metal (Au 200 nm/Cr 50 nm) on the LiNbO3 surface. The PDMS microchannel (width: 100 µm, height: 100 µm) was fabricated by a standard soft-lithography. The PDMS channel and SSAW device were treated by a plasma cleaner with oxygen plasma for 30 s. The PDMS channel was aligned and bonded to the LiNbO3 surface to form acoustofluidic device (As shown in Figure 1b). The acoustofluidic device was cured 3 h in 150 • C.

Calibration Experiment
The experimental system consists of a water bath kettle (HH.S 11-'1, Shanghai Boxun), a peristaltic pump (BT100L, Baoding Lead Fluid), and inverted fluorescence microscope (Ti-S Nikon). RhB solution was heated in the water bath kettle. Then, the solution was pump into the inlet of the microchannel through a silicone tube. The thermocouple was used to record the RhB solution temperature. The microchannel was put on the microscope. The microscope recorded the pictures to analyze the temperature of the RhB solution. The temperature measured by thermocouple and RhB was used to calibrate the relationship between the fluorescent signal intensity ratios and the temperature.
The concentration of RhB solution was 0.006 g/L. The flow velocity of the RhB solution was 1000 µL/min. The power of the laser was 100 w when the voltage was 12 V. The environmental temperature was 20 • C.

Temperature Measurement of Acoustofluidic
The experiment platform consisted of two systems. The first system was used to generate SSAWs and heat RhB solution, which consisted of a function generator (Tektronix AFG 3251C, Beaverton, OR, USA), amplifier (NF BA 4850, Kohoku-ku, Japan), and acoustofluidic device. The second system was used to measure the temperature of RhB solution, which consisted of a peristaltic pump and an inverted fluorescence microscope. The acoustofluidic device was put on the microscope. RhB was pumped by the peristaltic into the microchannel. Then the microscope was opened to record the fluorescent signal intensity. An AC signal with the frequency of 15.59 MHz, which was generated by the generator and amplified by the amplifier, was applied on the IDTs. The applied signal simulated the SSAWs to heat the RhB solution. The fluorescent signal intensity of the solution was recorded by the microscope.

The Relationship of the Fluorescent Intensity and Temperature
The relationship between the fluorescent signal intensity I and temperature T can be expressed by the equation: where I 0 is the intensity of the laser, C is the concentration of RhB in the solution, ϕ(T) is the luminescent intensity of RhB, and is absorption coefficient. Because of the linear relationship between the luminescent intensity and the temperature, the luminescent intensity can be fitted with a linear approximation in three regions: temperature region of 150 K to 200 K, 240 K to 360 K, and 380 K to 500 K [33]. The luminescent intensity of RhB ϕ(T) can be expressed as where a and b are the fitting coefficients.
To eliminate the experimental error, the relationship of relative luminescent intensity I and relative temperature difference ∆T is used to measure the fluid temperature, where T 0 is the environment temperature, and I(T 0 ) is the luminescent intensity of RhB when the temperature is T 0 . Substitute Equations (2)-(4) into Equation (1): where c and d are the fitting coefficients. Equation (5) represents the linear relationship between the luminescent intensity and the relative temperature. The linear relationship can be used to measure the temperature by luminescence after the coefficients are made sure.
To obtain the coefficients, five experiments were performed using the SSAW device. Three pictures were taken at the same temperature in each experiment to obtain the exact temperature. The temperature was measured by the thermocouple that was embedded in the microchannel. Because the distance between the thermocouple and the fluid is less than 50 µm, and the heated solution passes continuously for 5 min, the thermocouple temperature can represent the fluid temperature. Figure 2 shows the schematic of the thermocouple relative to the microchannel, and Figure 3 shows the RhB luminescent intensity of one time. It is obvious that the picture brightness decreases with the increase of the temperature. I(∆T) = c + d∆T (5 here c and d are the fitting coefficients. Eq. (5) represents the linear relationship betwee he luminescent intensity and the relatively temperature. The linear relationship can b sed to measure the temperature by luminescent, after the coefficients are made sure.
To obtain the coefficients, five experiments were performed using the SSAWs devic hree pictures were taken at the same temperature in each experiment to obtain the exa emperature. The temperature was measured by the thermocouple that was embedde n the microchannel. Because the distance between the thermocouple and the fluid is les han 50 µm, and the heated solution passes continuously for 5 minutes, the thermocoup emperature can represent the fluid temperature. Fig. 2 shows the schematic of th hermocouple relative to the microchannel, Fig. 3 shows the RhB luminescent intensit f one time. It is obvious that the picture brightness decreases with the increase of th emperature. The grayscale of the image is proportional to the luminescent intensity [32]. Th inescent intensity and the relatively temperature. The linear relationship can be measure the temperature by luminescent, after the coefficients are made sure. obtain the coefficients, five experiments were performed using the SSAWs device. ictures were taken at the same temperature in each experiment to obtain the exact rature. The temperature was measured by the thermocouple that was embedded icrochannel. Because the distance between the thermocouple and the fluid is less µm, and the heated solution passes continuously for 5 minutes, the thermocouple rature can represent the fluid temperature. Fig. 2 shows the schematic of the couple relative to the microchannel, Fig. 3 shows the RhB luminescent intensity time. It is obvious that the picture brightness decreases with the increase of the rature. he grayscale of the image is proportional to the luminescent intensity [32]. The s of luminescent intensity were imported MATLAB and translated into intensity s. Selected the luminescent intensity area and took the average grayscale. A line ted by the relatively average grayscale and the temperature changing, as shown The grayscale of the image is proportional to the luminescent intensity [32]. The images of luminescent intensity were imported into MATLAB and translated into intensity images. We selected the luminescent intensity area and took the average grayscale. A line was fitted by the relative average grayscale and the temperature change, as shown in

Fluid Temperature Measurement
The pump pumped the RhB solution into the channel. Then, the pump was closed. SSAWs were generated to heat the fluid. The frequency was 15.59 MHz. The input voltages of the generator were 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, and 0.8 V. Figure 5 shows the changing of the luminescent intensity with time when the input voltage was 0.6 V. The images were imported into MATLAB to obtain gray values. The temperature can be obtained by putting the gray value onto the line in Figure 4. The fluid temperature with different times can be used to describe the temperature rise process. Figure 6 shows the temperature rise process when the input voltage were 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, and 0.8 V. The results show that SSAWs can heat the fluid to a high temperature when the fluid is still. Many acoustofluidics devices are used in biological and chemical reactions, in which the temperature is an important parameter. It is necessary to integrate the temperature controlling system in the acoustofluidics when the fluid is still.
The flow rate is an important parameter in microfluidics. It is important to measure the temperature with different flow rates. Figure 7 shows the temperature rise process when the input voltage is 0.5 V and the flow rates are 0 µL· min −1 , 50 µL· min −1 , 100 µL· min −1 , 200 µL· min −1 , 300 µL· min −1 , 400 µL· min −1 , and 1000 µL· min −1 . Figure 8 shows the change of the highest temperature in Figure 7 with the flow rates. The temperature decreases from 55 • C to 32 • C when the flow rate increases from 0 µL· min −1 to 400 µL· min −1 . The temperature decreases rapidly with the increase of the flow rate. The temperature decreases from 32 • C to 31 • C when the flow rate increases from 400 µL· min −1 to 1000 µL· min −1 . The role of fluid flow in the cooling fluid can be neglected. The temperature is 11 • C higher than the environmental temperature. The results demonstrate that SSAWs can heat the fluid to a high temperature rapidly, which will limit the application of SSAWs in biological and chemical reactions. The flow rate plays an important role in cooling fluid. It is necessary to take into account the role of flow rate in the controlling system. different times can be used to describe the temperature rise process. Fig. 6 shows the 135 temperature rise process when the input voltage were 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V. 136 The results show that SSAWs can heat the fluid to a high temperature when the fluid is 137 still. Many acoustofludics devices are used in biological and chemical reactions, in which 138 the temperature is an important parameter. It is necessary to integrate the temperature 139 controlling system in the acoustofluidics when the fluid is still. The flow rate is an important parameter in microfluidics. It is important to mea-141 sure the temperature with different flow rates. Fig. 7 shows the temperature rise 142 process when the input voltage is 0.5 V and the flow rates are 0µL · min −1 , 50µL · min −1 , 143 100µL · min −1 , 200µL · min −1 , 300µL · min −1 , 400µL · min −1 , and 1000µL · min −1 . Fig.   144 8 shows the changing of the highest temperature in Fig. 7 with the flow rates. The    . Fluid temperature changing with time when the input voltage was 0.5 V, the flow rates were 0µL · min −1 , 50µL · min −1 , 100µL · min −1 , 200µL · min −1 , 300µL · min −1 , 400µL · min −1 , and 1000µL · min −1 .  Figure 7. Fluid temperature changing with time when the input voltage was 0.5 V, the flow rates were 0µL · min −1 , 50µL · min −1 , 100µL · min −1 , 200µL · min −1 , 300µL · min −1 , 400µL · min −1 , and 1000µL · min −1 .

Conclusions
In this paper, the fluid temperature in microchannel heated by SSAWs was measured by the fluorescent signal intensity ratios of Rhodamine B. First, a thermocouple was integrated into the microfluidics to calibrate the relationship between the fluorescent signal intensity ratios of Rhodamine B and the fluid temperature. Then, fluorescent signal intensity ratios of Rhodamine B were used to measure the fluid temperature heated by SSAWs. Finally, the fluid temperature with different input voltages and different flow rates was measured by the fluorescent signal intensity ratios of Rhodamine B. The

Conclusions
In this paper, the fluid temperature in the microchannel heated by SSAWs was measured by the fluorescent signal intensity ratios of Rhodamine B. First, a thermocouple was integrated into the microfluidics to calibrate the relationship between the fluorescent signal intensity ratios of Rhodamine B and the fluid temperature. Then, the fluorescent signal intensity ratios of Rhodamine B were used to measure the fluid temperature heated by the SSAWs. Finally, the fluid temperature with different input voltages and different flow rates was measured by the fluorescent signal intensity ratios of Rhodamine B. The results show that SSAWs can heat the still fluid rapidly to 80 • C, and the flow rates will influence the temperature of the fluid. The results will be useful for precisely controlling the temperature of acoustofluidics devices.