Two-dimensional acoustic particle focusing device and acoustic concentration device using it

Abstract

Provided are a particle two-dimensional acoustic focusing device that can efficiently focus and concentrate particles present in liquid flowing in a channel by using the acoustic effect of ultrasonic waves while having a simple configuration, and an acoustic concentration device using the particle two-dimensional acoustic focusing device. This particle two-dimensional acoustic focusing device is configured so as to focus particles contained in liquid flowing in a channel to the center of the cross-section of the channel by using ultrasonic waves and comprises: a rectangular channel 10 having a substantially rectangular cross-sectional shape when broken orthogonally to the extension direction of the channel; and a single ultrasonic wave generator 20 that simultaneously irradiates the interior of the rectangular channel 10 with a first ultrasonic wave and a second ultrasonic wave in a composite state, the first ultrasonic wave being generated so that the length of a long side a of the rectangle of the rectangular channel 10 is substantially equivalent to the length of a half wavelength, the second ultrasonic wave being generated so that the length of a short side b of the rectangle of the rectangular channel 10 is substantially equivalent to the length of the half wavelength.

Description

[Technical Field]

[0001] The present invention relates to a two-dimensional acoustic particle focusing device configured to use ultrasonic waves to focus particles contained in a liquid flowing within a flow channel to the center of the cross section of the flow channel, and an acoustic concentration device using the same. [Background technology]

[0002] Previously, research into the acoustic focusing of particles contained in liquids has been reported, for example, as a biological particle manipulation technique using ultrasonic radiation pressure, which uses ultrasound to align, orient, and concentrate particles suspended in a solution (see Non-Patent Document 1). The principle of ultrasonic radiation pressure exerted on particles in standing waves is that when a standing ultrasonic wave is introduced into a fluid, the pressure amplitude of the standing wave's sound pressure is minimized at the nodes and maximized at the antinodes. Meanwhile, the displacement of the medium is maximized at the nodes and minimized at the antinodes. When particles with a density and compressibility different from that of the medium are introduced into the sound field, the particles attempt to move so as to minimize the work done on the particles by the ultrasonic pressure vibrations and medium vibrations as a whole. This is ultrasonic radiation pressure (Non-Patent Document 1, legend to Figure 2).

[0003] Furthermore, the present inventors have previously disclosed the results of their research into the applicability of acoustic focusing technology for particles contained in liquids as a means of enabling the continuous recovery and analysis of microplastics in the context of the plastic problem in the environment (see Non-Patent Document 2).

[0004] Regarding the problem of plastics in the environment, plankton nets with a mesh size of approximately 0.3 mm (300 μm) have traditionally been widely used to sample microplastics. As a result, anything smaller than this mesh size is not collected, and their actual status remains unknown. To collect smaller microplastics, a finer mesh is required, but the finer the mesh, the more likely it is to become clogged. Microplastics collected with the mesh are then manually picked up one by one with tweezers for analysis, but this task is difficult for the tiny pieces. Therefore, there was a need for technology that could continuously collect and analyze microplastics.

[0005] In conventional particle focusing devices using an acoustic method for particles contained in a liquid flowing through a channel, horizontal focusing using a one-dimensional (1D) standing wave is known as one that utilizes one-dimensional focusing (see Figure 9). Note that Figure 9 shows the state in which acoustic focusing is turned off and the change from that state to the state in which acoustic focusing is turned on. In this system, a half-wavelength standing wave is generated in the horizontal direction within a microchannel (microchannel), forming a node on the vertical centerline, and the particles are focused there. Note that the research results disclosed in Non-Patent Document 2 utilize this one-dimensional focusing.

[0006] Furthermore, in conventional acoustic particle focusing devices, the simplest method for utilizing two-dimensional focusing is to use a square channel. This allows the horizontal and vertical resonance frequencies to match, and two-dimensional focusing can be achieved with a single ultrasonic generator without the need for additional equipment or changes to the device design. Two-dimensional focusing using a circular channel has also been reported.

[0007] In contrast, when using two-dimensional focusing, two-dimensional focusing in a rectangular channel (flow path) is more common, but conventionally, two vibration systems with different frequencies are required (see Patent Document 1). One frequency is for horizontal focusing, selected so that half the wavelength corresponds to the width of the microchannel (microchannel). The other frequency is for vertical focusing, selected so that half the wavelength corresponds to the depth of the microchannel (microchannel). These two vibration systems require two piezoelectric vibrators (PZTs), two signal generators, and two high-frequency power amplifiers (amplifiers), making the entire system complex and nearly twice as costly as a single vibration system for one-dimensional focusing. Furthermore, two PZTs with different resonant frequencies corresponding to the width and depth must be attached to the microfluidic device, which places constraints on the design of the microfluidic device. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 6-241977 (

[0023] ) [Non-Patent Document 1] Kenji Yasuda, "Bio-particle manipulation technology using ultrasonic radiation pressure," Applied Physics, Vol. 67, No. 3 (1998), pp. 323-326 [Non-patent document 2] Yoshitake Akiyama, Takatoshi Egawa, Kiyoshi Koyano, Hiroshi Moriwaki, “Acoustic focusing of microplastics in microchannels: A promising continuous collection approach,” Sensors and Actuators B: Chemical, published online on November 10, 2019 Summary of the Invention [Problem to be solved by the invention]

[0009] The issues to be resolved with regard to a two-dimensional acoustic particle focusing device and an acoustic concentration device using it are, first, that while acoustic focusing is widely used in the collection of fine particles in a channel, with conventional one-dimensional focusing it is significantly affected by disturbances in the acoustic field at the branching section, limiting the concentration factor to around 10. To improve this, two-dimensional focusing has been proposed, and it has been reported that this can increase the concentration factor to around 60. However, this conventional two-dimensional focusing, particularly in the case of a typical rectangular channel, requires the preparation of two independent vibration systems, which makes the system complicated and costly, and so it has not been used in many cases.

[0010] Therefore, the object of the present invention is to provide a two-dimensional acoustic particle focusing device that utilizes the acoustic effect of ultrasound to efficiently focus and concentrate particles present in a liquid flowing through a flow path with a simple configuration, and an acoustic concentration device using the same. [Means for solving the problem]

[0011] In order to achieve the above object, the present invention has the following configuration. According to one example of a two-dimensional acoustic particle focusing device of the present invention, the device is configured to use ultrasonic waves to focus particles contained in a liquid flowing through a flow channel to the center of the cross section of the flow channel, and includes a rectangular flow channel whose cross section when broken perpendicular to the extension direction of the flow channel is substantially rectangular, and an ultrasonic generator that simultaneously irradiates into the rectangular flow channel a first ultrasonic wave generated so that the length of the long side of the rectangle of the rectangular flow channel is substantially equal to a half wavelength, and a second ultrasonic wave generated so that the length of the short side of the rectangle of the rectangular flow channel is substantially equal to a half wavelength, in a synthesized state.

[0012] Furthermore, according to one example of the two-dimensional acoustic particle focusing device of the present invention, the ultrasonic generator can be characterized by including a signal adjustment means for adjusting the mixing ratio and magnitude of two signals of different frequencies that generate the first ultrasonic wave and the second ultrasonic wave.

[0013] Furthermore, according to one example of the two-dimensional acoustic particle focusing device of the present invention, the ultrasonic generator can be characterized by comprising, as components, a signal generator that generates signals of two different frequencies, and a piezoelectric vibrator that receives signals from the signal generator and generates ultrasonic vibrations.

[0014] Furthermore, according to one example of the two-dimensional acoustic particle focusing device of the present invention, the portion of the rectangular flow path to which at least the first ultrasonic wave and the second ultrasonic wave are irradiated can be characterized in that it is installed in an upright position so that the liquid flows vertically.

[0015] Furthermore, according to one example of the two-dimensional acoustic particle focusing device of the present invention, an optical measurement device whose measurement capability is affected by the depth of field is disposed at a position facing the rectangular flow path.

[0016] Furthermore, an example of an acoustic concentration device using the two-dimensional acoustic particle focusing device of the present invention can be characterized in that it is equipped with the two-dimensional acoustic particle focusing device, and also has a three-branch flow path formed in a downstream flow path of the rectangular flow path so as to branch into three flow paths: a branch flow path on one side, a branch flow path on the other side, and a central branch flow path provided to flow particles focused to the center of the rectangular flow path by the two-dimensional acoustic particle focusing device.

[0017] Furthermore, according to one example of an acoustic concentration device using the two-dimensional acoustic particle focusing device of the present invention, the three-branch flow path can be formed by dividing the long side of the rectangle into three in the longitudinal direction.

[0018] Furthermore, according to one example of an acoustic concentration device using the two-dimensional acoustic particle focusing device of the present invention, the acoustic concentration device using a plurality of the two-dimensional acoustic particle focusing devices is configured to be connected in series with respect to the flow of liquid containing the particles.

[0019] Furthermore, according to one example of an acoustic concentration device using the two-dimensional acoustic particle focusing device of the present invention, the acoustic concentration device using a plurality of the two-dimensional acoustic particle focusing devices is configured to be connected in parallel with respect to the flow of liquid containing the particles. [Effects of the Invention]

[0020] The two-dimensional acoustic particle focusing device and the acoustic concentration device used therein according to the present invention have the particularly advantageous effect of utilizing the acoustic effect of ultrasound to efficiently focus and concentrate particles present in a liquid flowing through a flow channel with a simple configuration. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a cross-sectional view taken perpendicular to the extension direction of a flow channel (flow direction of a liquid) and schematically showing an example of a two-dimensional acoustic particle focusing device according to the present invention. [Figure 2] 1A and 1B are explanatory diagrams of an example of an acoustic concentrating device using a two-dimensional acoustic particle focusing device according to the present invention, in which multiple three-branched channels are connected in series. (a) A perspective view showing an example of the channel configuration and a schematic explanatory diagram of the three-branched channel. (b) A perspective view of a microfluidic chip. (c) A perspective view showing the state in which the microfluidic chip is placed on a piezoelectric vibrator. [Figure 3] 1 is a graph showing an example of the relationship between "Sample output flow rate" and "Concentration factor" according to the present invention. [Figure 4] FIG. 1 is an explanatory perspective view of an example of an acoustic concentrating device using a two-dimensional acoustic particle concentrating device according to the present invention, in which a plurality of three branched flow paths are connected in parallel. [Figure 5]Figure 1 illustrates the experimental setup for acoustic focusing of microparticles in an embodiment of the present invention. (a) Schematic diagram of the fully set up microfluidic chip. The inset shows a typical acoustic pressure field with standing waves. (b) Design of the microfluidic chip with a rectangular microchannel. Note that acoustic focusing was evaluated by observing the area before the three branches with a confocal fluorescence microscope from the opposite side of the PZT. [Figure 6] This graph shows the frequency dependence of electrical measurements of a PZT according to an example of the present invention. Several peaks are observed around 500 kHz, but no peaks are observed above 800 kHz, and the admittance increases slightly. The dashed line indicates 515 kHz. [Figure 7] Reconstructed cross-sectional images of fluorescent microparticles excited at single and dual frequencies in an embodiment of the present invention. The microparticles are observed in the green fluorescence image, and when overlaid with the red fluorescence image of the microchannel, the microparticles appear yellow (the bright areas in this grayscale image). The horizontal focusing amplitude is shown on the left side of the image, and the vertical focusing amplitude is shown above it. The image with the clearest 2D focusing is outlined in thick gray. The scale bar indicates 500 μm. [Figure 8] Figures 1A and 1B illustrate numerical simulations for acoustic focusing in an embodiment of the present invention. (a) Schematic of a 2D numerical model. (b) Average acoustic energy density in the fluid domain as a function of frequency. The white and black arrows indicate the frequencies used in the horizontal and vertical focusing experiments, respectively. (c) Pressure and acoustic radiation force (ARF) distributions for 1D focusing. The length of the arrows is proportional to the magnitude of the ARF. (d) Superimposed ARF distributions for 2D focusing, similar to (c). (e) Sequential images of particle movement over a 10-second period. Within 5 seconds, most particles move toward the vertical centerline at speeds up to 0.25 mm / s. [Figure 9] This is a schematic diagram illustrating conventional one-dimensional acoustic focusing, showing the change from a state where acoustic focusing is turned off to a state where acoustic focusing is turned on. DETAILED DESCRIPTION OF THE INVENTION

[0022] An embodiment of the two-dimensional acoustic particle focusing device according to the present invention will be described in detail with reference to the accompanying drawings (Figs. 1 to 8). The two-dimensional acoustic particle focusing device according to the present invention is configured to focus particles contained in a liquid flowing through a flow channel to the center of the cross section of the flow channel using ultrasonic waves.

[0023] As shown in FIG. 1, the two-dimensional acoustic particle focusing device of the present invention includes a rectangular flow channel 10 having a substantially rectangular cross-sectional shape when broken perpendicular to the extension direction of the flow channel (the direction of liquid flow), and an ultrasonic generator 20 that simultaneously irradiates into the rectangular flow channel 10 a first ultrasonic wave generated so that the length of the long side a of the rectangle of the rectangular flow channel 10 is substantially equal to half the wavelength, and a second ultrasonic wave generated so that the length of the short side b of the rectangle of the rectangular flow channel 10 is substantially equal to half the wavelength, in a synthesized state.

[0024] 1, in the two-dimensional acoustic particle focusing device according to the present invention, a horizontal half-wavelength standing wave S1 is generated by a first ultrasonic wave, and a vertical half-wavelength standing wave S2 is generated by a second ultrasonic wave, so that two-dimensional acoustic focusing can be obtained with a simple configuration, and particles contained in the liquid flowing through the channel can be efficiently focused at the center of the cross section of the rectangular channel 10. That is, by simultaneously driving a single piezoelectric element (piezoelectric vibrator 23) at two different frequencies, two-dimensional focusing can be achieved with a single vibration system, which prevents the system from becoming complicated and reduces manufacturing costs.

[0025] Furthermore, by providing the ultrasonic generator 20 with a signal adjustment means for adjusting the mixing ratio and magnitude of the two different frequency signals that generate the first ultrasonic wave and the second ultrasonic wave, it is possible to respond to various conditions such as sampling conditions and rationally adjust the signals to optimize two-dimensional acoustic focusing. As this signal adjustment means, a signal generator that generates signals of two different frequencies or a high-frequency power amplifier 22 (amplifier) ​​that can separately adjust the amplification of each of the two different frequency electrical signals can be used.

[0026] Furthermore, since the ultrasonic generator 20 comprises, as its components, a signal generator 21 that generates signals of two different frequencies and a piezoelectric vibrator 23 that receives a signal from the signal generator 21 and generates ultrasonic vibrations, the ultrasonic generator 20 can be constructed simply and rationally, thereby reducing costs.

[0027] Furthermore, in the two-dimensional acoustic particle focusing device according to the present invention, the rectangular channel 10 portion of the channel to which at least the first ultrasonic wave and the second ultrasonic wave are irradiated is installed in an upright position so that the liquid flows vertically. This makes it possible to reduce the influence of gravity on particles contained in the liquid during two-dimensional acoustic focusing within the rectangular channel 10, and to more appropriately focus the particles toward the center of the rectangular channel 10.

[0028] In this embodiment, as shown in FIG. 1, an optical system measuring device 30 whose measurement capability is affected by the depth of field is disposed at a position facing the rectangular flow channel 10. This allows the particles flowing through the rectangular flow channel 10 to be controlled so as to flow within the allowable range of the depth of field within which the optical measurement device can be focused, thereby improving the accuracy of measurements of the particles by the optical measurement device. For example, size measurements involving particle recognition and tracking by image processing, and material identification by microscopic Raman spectroscopy can be performed more accurately.

[0029] In addition, as shown in FIG. 2, this embodiment is equipped with the two-dimensional acoustic particle focusing device, and also equipped with a three-branch flow path 40 formed in the downstream flow path of the rectangular flow path 10 so as to branch into three flow paths: a branch flow path 41 on one side, a branch flow path 42 on the other side, and a central branch flow path 43 provided to allow particles focused to the center of the rectangular flow path 10 by the two-dimensional acoustic particle focusing device to flow.

[0030] Furthermore, since the three-branch flow path 40 is formed by dividing the long side of the rectangle into three in the longitudinal direction, it has the advantage of being able to appropriately utilize the shape of the flat rectangular flow path 10 and easily divide and branch into three flow paths (41, 42, 43).

[0031] According to the three-branch flow path 40 of this embodiment, for example, as shown in Fig. 2, the flow rates in the left branch flow path (branch flow path 41 on one side), the central branch flow path 43, and the right branch flow path (branch flow path 42 on the other side) are set to be 1.1:1:1.1. As a result, the concentration of microparticles (MP) in the liquid can be concentrated 3.2 times by this one-stage three-branch flow path 40. By arranging this in four stages in series as shown in Fig. 2 and repeating concentration, it becomes possible to concentrate the MP by about 100 times.

[0032] Here, a method for determining the "Concentration factor" will be described with reference to Fig. 3. Fig. 3 is a graph showing an example of the relationship between the "Sample output flow rate" and the "Concentration factor" according to the present invention. The relationship is concentration rate = output flow rate / input flow rate, and "Concentration factor" = recovery rate × concentration rate. In this example, the input flow rate is 300 μL / min, and when one-dimensional acoustic focusing is used with only 516.2 kHz ultrasound, and when two-dimensional acoustic focusing is used with both 516.2 kHz and 1051 kHz ultrasound, it is shown that one-dimensional acoustic focusing has a concentration rate of up to 10 times (output flow rate of 30 μL / min), while two-dimensional acoustic focusing has a practical concentration rate of up to 30 times (output flow rate of 10 μL / min). Furthermore, when the concentration rate is 30 times (300 μL / min / 10 μL / min) with two-dimensional acoustic focusing, the recovery rate is 90%, and the "Concentration factor" is calculated to be 27.

[0033] Furthermore, as shown in Figure 2, an acoustic concentration device using multiple two-dimensional acoustic particle focusing devices is configured by connecting them in series with respect to the flow of liquid containing the particles, thereby enabling the concentration rate to be exponentially increased.

[0034] Furthermore, as shown in Figure 4, an acoustic concentration device using multiple two-dimensional acoustic particle focusing devices is configured in parallel with the flow of liquid containing the particles, thereby increasing the processing flow rate.

[0035] Next, specific research results (examples) regarding two-dimensional acoustic focusing of particles in a rectangular microchannel (microchannel) using a single transducer (piezoelectric vibrator) excited at two different frequencies will be described in detail below.

[0036] (overview) Two-dimensional focusing can dramatically improve the efficiency of particle concentration devices, but it has not been widely used due to the complexity of the two-dimensional focusing system. Conventional two-dimensional focusing devices require two independent vibration systems operating at different frequencies. In contrast, the proposed method enables two-dimensional focusing by simply simultaneously vibrating a piezoelectric transducer (PZT) at two frequencies. In the proposed method, two-dimensional focusing is achieved simply by simultaneously vibrating a PZT at two frequencies using a single vibration system and inputting a pre-combined signal into a high-frequency power amplifier. This method can be easily applied to most conventional acoustic systems and can improve their efficiency.

[0037] In this example, we propose a two-dimensional (2D) focusing method that achieves horizontal and vertical focusing in a rectangular microchannel (flow path) using a single piezoelectric transducer (PZT) excited at two frequencies. In this proposed method, a single PZT is excited by combining signals of different frequencies in an appropriate ratio. This proposed method can be achieved using almost the same system as 1D focusing, making 2D focusing easy and low-cost.

[0038] Conventional two-dimensional focusing in rectangular microchannels requires a complex system because two types of PZT are excited separately at their respective resonant frequencies. In this experiment, two-dimensional focusing was achieved using a single PZT by adjusting the amplitude ratio between horizontal focusing at the resonant frequency and vertical focusing at the non-resonant frequency. Numerical simulations also demonstrated that two-dimensional focusing is possible by superimposing excitations of two different frequencies at the same ratio as in the experiment.

[0039] Various techniques have been developed to continuously separate small particles using acoustic, electrical, magnetic, optical, and inertial forces. 1) Among them, the acoustic method, which can achieve high efficiency by increasing the input energy, 2) is in the bio field 3),4) Biomedical field 5),6),7) Chemistry 8) Widely applied in the environmental field 9),10) Acoustic methods primarily rely on focusing of particles by acoustic radiation force (ARF), which moves acoustic contrast agents such as cells and plastic particles to nodes of an acoustic standing wave. 11) .

[0040] Conventional acoustic particle concentration devices use horizontal focusing by one-dimensional (1D) standing waves. 12) This system generates a horizontal half-wave standing wave in the microchannel, forming a node on the vertical centerline, where the particles are converged. By adjusting the flow rate and branching ratio of the three-way branch, the converged particles flow only into the central branch, from which concentrated particles can be recovered. Ideally, the concentration ratio should depend on the ratio between the width of the microchannel and the convergence width of the particles, and can be set to 50 or more. 13) The practical concentration ratio is generally 3 to 10 9),14),15) This limitation is mainly due to the instability of acoustic focusing at the bifurcation, which is caused by the disturbance of the acoustic pressure field due to the change in the bifurcation geometry from a straight channel to a three-way branch. 16)In particular, this instability affects small particles that flow slowly along the upper and lower walls of the bifurcation, following the parabolic profile of the pressure-driven flow. To suppress the effects of this instability, two-dimensional focusing (simultaneous horizontal and vertical focusing) to a single central spot has been reported. This two-dimensional focusing improves the limit of the concentration factor, and a 67-fold concentration was achieved for polystyrene particles with a diameter of 5 μm. 13) .

[0041] The simplest way to achieve two-dimensional focusing is to use a square channel, which matches the horizontal and vertical resonant frequencies and achieves two-dimensional focusing without any additional equipment or changes to the equipment design. 17),18),19) Two-dimensional focusing in circular channels has also been reported. Two-dimensional focusing in rectangular channels is more common, but requires two oscillation systems at different frequencies. 13),21),22) One frequency is for horizontal focusing, selected so that half the wavelength corresponds to the width of the microchannel. The other frequency is for vertical focusing, selected so that half the wavelength corresponds to the depth of the microchannel. These two vibration systems require two piezoelectric vibrators (PZTs), two signal generators, and two high-frequency power amplifiers, making the entire system complex and nearly twice as costly as a single vibration system for one-dimensional focusing. Furthermore, two PZTs with different resonant frequencies corresponding to the width and depth must be attached to the microfluidic device, which may impose constraints on the design of the microfluidic device and may affect the sound pressure field.

[0042] In this study, we report two-dimensional focusing of particles in a rectangular channel using a single PZT excited at two frequencies: horizontal and vertical focusing. Specifically, horizontal focusing is efficient at the PZT's resonant frequency, while the inefficient vertical focusing at non-resonant frequencies is compensated for by increasing the input power to the PZT. These signals are first combined at an appropriate ratio and amplified to excite the PZT. Therefore, two-dimensional focusing is achieved using the same vibration system as one-dimensional focusing, except that two excitation signals are required. First, horizontal and vertical focusing are confirmed by exciting the PZT at each frequency. Next, two-dimensional focusing is demonstrated by simultaneously exciting the PZT at two frequencies. Finally, numerical simulations of the experimental setup were performed to elucidate the mechanism of two-dimensional focusing.

[0043] Figure 5a shows the experimental setup for acoustic focusing. 3 The microchannel was fabricated by thermally bonding three 0.7 mm thick borosilicate glass plates (Tempax Float, Schott). A rectangular microchannel (1.42 mm wide, 0.7 mm deep) was obtained by machining a trifurcated through-groove in the central plate (Fig. 5b). The thickness-mode resonant frequency was 500 kHz. The dimensions of the microchannel were 40 × 34.5 × 4.2 mm. 3 A PZT (C-213, Fuji Ceramics Co., Ltd.) was attached to the microfluidic chip with epoxy adhesive. Positioning pins were used to accurately position the microfluidic chip and the microfluidic connector. A sine wave of the required frequency generated by a signal generator with two-channel output was amplified by a high-frequency power amplifier (HSA4011, NF Co., Ltd.) to operate the PZT. Two-dimensional focusing was achieved by adding and amplifying signals of different frequencies generated by the signal generator using an amplifier. Green fluorescent polyethylene particles (diameter 50 μm, density 1.025 g mL) were used. -1 , manufactured by Cospheric) was suspended in distilled water containing 0.2 wt% Tween 20, and the resulting suspension (10 4 pieces mL -1Finally, 1 ppm of Nile Red solution was added to the microchannel so that regions of the microchannel could be identified by fluorescence imaging. The microfluidic chip was placed vertically to prevent the particles from settling due to gravity. The suspension was introduced into the microfluidic chip by aspirating it from all outlets with a syringe pump (flow rate: 300 μL min -1 The alignment of the microparticles in the microchannel was observed by green and red fluorescence imaging using a macro confocal microscope (AZ-C2+, Nikon).

[0044] The horizontal and vertical excitation frequencies depend on the microchannel geometry. To generate a standing wave with one node in the horizontal direction, half the wavelength must be close to the microchannel width. Using a frequency of 1480 m / s (the speed of sound in water at room temperature), the excitation frequency was predicted to be approximately 521 kHz. Similarly, the vertical excitation frequency should be approximately 1060 kHz. Distilled water was flowed through the fully assembled PZT microchannel, and the impedance was measured using an impedance analyzer (IM3570, Hioki Corporation) to evaluate the resonance characteristics. As shown in Figure 6, several admittance peaks were observed around 500 kHz, with the phase approaching zero. If these peaks correspond to horizontal focusing, efficient focusing would be achieved at low amplitudes. On the other hand, no admittance peaks were observed above 800 kHz, and the phase was nearly constant at -85°, suggesting that vertical focusing is inefficient and requires a higher power input than focusing at the admittance peak around 500 kHz.

[0045] Next, one-dimensional focusing was performed in both directions, and cross-sectional images were constructed using a confocal microscope. The alignment of the particles was observed just before the three-way intersection, and excitation frequencies were searched for around 500 kHz and 800 kHz. Consequently, the excitation frequencies for horizontal and vertical focusing were determined to be 515 kHz and 1051 kHz, respectively, and remained fixed throughout this study. For horizontal focusing, the PZT was excited with an amplitude of 5 V or 10 V. As shown in the leftmost image in Figure 7, even at the lowest amplitude of 5 V (middle image), the particles were confirmed to be aligned along the central vertical line. For vertical focusing, voltages of 5, 10, 15, 20, 25, and 30 V were applied. As predicted by the impedance measurements, vertical focusing required higher amplitudes than horizontal focusing. Vertical focusing at 10 V did not align the particles in as narrow an area as horizontal focusing at 5 V. Therefore, vertical focusing was examined with amplitudes up to 30 V. Furthermore, for vertical focusing at high amplitudes, the particles did not line up in a single line but instead focused into two spots. This is thought to be because vibrations at frequencies that cause vertical focusing can form not only one horizontal nodal line but also two vertical nodal lines. Furthermore, because the aspect ratio (width / depth) of the rectangular channel used was approximately 2, it also functioned as the second harmonic of the horizontal focusing, which likely resulted in the two-spot focusing resulting from the two nodal lines.

[0046] We experimentally confirmed the proposed 2D focusing by simultaneously exciting a single PZT with two excitation frequencies. Signals at two different frequencies were added together, amplified, and applied to the PZT. Figure 7 shows cross-sectional images of 2D focusing with various amplitude combinations. In the experiment, 2D focusing was clearly observed when vertical focusing was performed at 20, 25, and 30 V and horizontal focusing at 10 V. When the vertical focusing amplitude was below 15 V, horizontal focusing predominated, and the vertical focusing had little effect on the particles. On the other hand, when the vertical focusing was too strong compared to the horizontal focusing, the particles were split into two spots, as seen in the images obtained with vertical focusing at 25 and 30 V and horizontal focusing at 5 V. These results indicate that 2D focusing in a rectangular microchannel using a single PZT is possible, and that the balance between the horizontal and vertical focusing forces needs to be adjusted.

[0047] To investigate the mechanism of two-dimensional focusing due to excitation at two different frequencies, we performed frequency-domain numerical simulations using COMSOL Multiphysics (ver. 5.6, COMSOL Inc.). Due to computational resource limitations, a cross-sectional two-dimensional model was used (Figure 8a). The model consisted of four domains: fluid, glass microfluidic chip, epoxy adhesive, and PZT. The center of the PZT was positioned 1.25 mm off-center from the center of the microfluidic chip, as in the experiment. In the fluid domain, the Helmholtz equation was solved using the "Pressure Acoustics Module," ignoring the viscous boundary layer. Because the particles used in this study were small enough that the acoustic hydrodynamic force could be ignored, only the ARF was considered. In all solid domains, including the glass, epoxy, and PZT, the Cauchy equation of motion was solved using the "Solid Mechanics Module," and the piezoelectricity of the PZT domain was considered as a stress-charge regime by coupling with the "Electrostatics Module." The epoxy adhesive layer was represented as a "Thin Elastic Layer," and its thickness was measured in micrometers. The boundary conditions for the fluid and glass were defined by setting the “Acoustic-Structure Boundary.” The material property parameters, including damping, were taken from the literature. 23),24) As in the experiment, the amplitude of excitation of the PZT was applied between the upper and lower boundaries. The amplitudes of horizontal and vertical focusing were 10 V and 30 V, respectively.

[0048] The excitation frequencies for horizontal and vertical convergence were determined by referring to the acoustic energy density Eac, which is calculated by the following equation:

[0049]

number

[0050] where 〈.〉 is the time average, ρ f is the fluid density, c f is the sound velocity in the fluid, p is the sound pressure, and v is the sound velocity. Figure 8b shows the E acThe average values ​​of are plotted around both frequencies. Around 515 kHz, the resonant frequency shifted slightly lower than the experimental results, and a fairly large peak was observed at 499 kHz, which was therefore adopted for horizontal focusing. In the region above 1000 kHz for vertical focusing, a large peak was observed at 1020 kHz, along with several smaller peaks. However, at this large peak, there was no horizontal pressure nodal line, and two vertical sound pressure nodal lines corresponding to the second harmonic frequency of the horizontal frequency were observed. Therefore, a search was conducted for a frequency in a higher region for vertical focusing, and 1083 kHz was selected for vertical focusing based on the distribution of sound pressure and ARF.

[0051] The acoustic pressure and ARF distribution in the fluid domain at each excitation frequency are shown in Figure 8c. The ARF F acting on the polyethylene particle generated from the acoustic pressure field is given by Equation (2): 24) was calculated using

[0052]

number

[0053] where f0=1-ρ f c f 2 / ρ p c p 2 is the monopole scattering coefficient of the particle, f1=2(ρ p -ρ f ) / (2ρ p +ρ f ) is the dipole scattering coefficient of the particle, V is the particle volume, ρ f is the particle density, c p is the particle speed of sound. In the simulation of horizontal focusing, a pressure nodal line formed almost vertically at the center. The ARF was horizontal and pointed everywhere to the vertical centerline. The maximum ARF value was 94 pN. In the simulation of vertical focusing, the pressure nodal line formed horizontally but was curved overall. Due to this behavior, the ARF distribution was partially weak, particularly in the central region below the center and near the upper wall. Despite the applied amplitude being three times that of horizontal focusing, the maximum ARF in vertical focusing was 46 pN, about half that of horizontal focusing.

[0054] To reproduce the two-dimensional focusing, the ARF distributions excited at two frequencies were estimated according to the principle of superposition. That is, the ARF distributions calculated separately for horizontal and vertical focusing were summed (Figure 8d). As expected, the ARFs pointed to the center of the microchannel at almost all locations, which means that the particles essentially focused two-dimensionally to a central spot. Here, the ARF in the region near the center of the top wall pointed toward the center horizontally but toward the top vertically. Thus, the particles in this region ultimately adhered to the center of the top wall.

[0055] Two-dimensional convergence was confirmed by particle tracking simulations using the "Particle Tracing Module" in COMSOL Multiphysics. First, 14 × 7 particles were placed on grid points with 0.1 mm spacing and zero velocity. For each particle, the ARF and inertial drag forces were solved using Newtonian formalism. Figure 8e and Movie S1 show particle positions and trajectories over a 10-second period. Overall, the microparticles initially moved horizontally and then moved vertically toward the center. This microparticle movement can be explained by the dominance of horizontal forces. For the first 5 seconds, the microparticles converged to the vertical centerline, after which most of the microparticles converged to a central spot. As predicted from Figure 8d, the microparticles near the center of the upper wall did not converge at the center but instead moved toward the upper wall. Six microparticles adhered to the wall, accounting for 6.1% of the total. Considering the parabolic velocity distribution of Poiseuille flow, it seems that the adhesion of microparticles to the wall in the simulations is negligible. In fact, it was confirmed that no microparticles adhered to the upper wall.

[0056] In the two-dimensional focusing experiment, almost all of the particles were collected from the central outlet. The average flow velocity under these experimental conditions was 5 mm / s. Assuming the Hagen-Poiseuille law, a maximum flow velocity of 10 mm / s was obtained at the center line of the microchannel. Particles introduced to the center at the inlet reach the three branches in 3.5 seconds. Since almost all of the particles converge in 3 seconds, it is thought that if the flow is divided equally among the three outlets, the particles will be collected from the central microchannel. The results of this simulation are consistent with the experimental results.

[0057] In this study, we demonstrated two-dimensional focusing in a rectangular microchannel using a single PZT. By exciting the PZT at two frequencies, we were able to focus microparticles to a single spot in the center of the microchannel. Conventional two-dimensional focusing requires two vibration systems, including a PZT, a signal generator, and a high-frequency power amplifier. However, our proposed method achieves two-dimensional focusing with a single vibration system, including a signal generator with two output channels. Furthermore, the simple configuration of a single PZT increases the flexibility of device design. Furthermore, numerical simulations demonstrated that two-dimensional focusing is possible by superimposing excitations at two different frequencies. This method is expected to be widely applicable because it achieves stable particle focusing with a higher concentration factor than conventional two-frequency focusing.

[0058] (References) 1) A. Lenshof and T. Laurell, Chem Soc Rev 39, 1203 (2010). 2)P. Sajeesh and AK Sen, Microfluid Nanofluid 17, 1 (2014). 3) H. Boehm, LG Briarty, KC Lowe, JB Power, E. Benes, and MR Davey, Biotech Bioeng 82, 74 (2003). 4)B. Hammarstroem, M. Vassalli, and P. Glynne-Jones, J Appl Phycol 32, 339 (2020). 5)F. Trampler, S. A. Sonderhoff, P. W. S. Pui, D. G. Kilburn, and J. M. Piret, Nat Biotechnol 12, 281 (1994). 6)F. Petersson, A. Nilsson, C. Holm, H. Joensson, and T. Laurell, Analyst 129, 938 (2004). 7)P. Augustsson, C. Magnusson, M. Nordin, H. Lilja, and T. Laurell, Anal Chem 84, 7954 (2012). 8)I. Leibacher, P. Reichert, and J. Dual, Lab Chip 15, 2896 (2015). 9)Y. Akiyama, T. Egawa, K. Koyano, and H. Moriwaki, Sens Actuators B: Chem 127328 (2019). 10)LN Perera and ME Piyasena, Sep Purif Technol 288, 120649 (2022). 11)H. Bruus, Lab Chip 12, 1014 (2012). 12)T. Laurell, F. Petersson, and A. Nilsson, Chem. Soc. Rev. 36, 492 (2007). 13)M. Nordin and T. Laurell, Lab Chip 12, 4610 (2012). 14)A. Nilsson, F. Petersson, H. Joensson, and T. Laurell, Lab Chip 4, 131 (2004). 15)O. Jakobsson, S. S. Oh, M. Antfolk, M. Eisenstein, T. Laurell, and H. T. Soh, Anal Chem 87, 8497 (2015). 16) P. Augustsson, J. Persson, S. Ekstrom, M. Ohlin, and T. Laurell, Lab Chip 9, 810 (2009). 17)M. Antfolk, P. B. Muller, P. Augustsson, H. Bruus, and T. Laurell, Lab Chip 14, 2791 (2014). 18)C. Perfetti and C.S. Iorio, Acoust Sci Technol 37, 221 (2016). 19)J. Lei, F. Cheng, K. Li, and Z. Guo, Appl Phys Lett 116, 033104 (2020). 20)G. Goddard and G. Kaduchak, J Acoust Soc Am 117, 8 (2005). 21)O. Jakobsson, C. Grenvall, M. Nordin, M. Evander, and T. Laurell, Lab Chip 14, 1943 (2014). 22)C. Grenvall, C. Antfolk, C. Z. Bisgaard, and T. Laurell, Lab Chip 14, 4629 (2014). 23)JS Bach and H. Bruus, Phys Rev Lett 124, 214501 (2020).). 24)A. Tahmasebipour, L. Friedrich, M. Begley, H. Bruus, and C. Meinhart, J Acoust Soc Am 148, 359 (2020).

[0059] The present invention has been described above in various ways using preferred embodiments, but the present invention is not limited to these embodiments, and it goes without saying that many modifications can be made within the scope of the invention without departing from the spirit of the invention. [Explanation of symbols]

[0060] a Long side b Short side S1 Horizontal half-wave standing wave S2 Vertical half-wave standing wave 10 Rectangular channel 20 Ultrasonic generator 21 Signal Generator 22 High frequency power amplifier 23 Piezoelectric vibrator 30 Optical measurement equipment 40 3-branch flow path 41 Branch flow path on one side 42 Branch flow path on the other side 43 Central branch channel

Claims

1. A two-dimensional acoustic particle focusing device configured to focus particles contained in a liquid flowing through a flow path to a center of a cross section of the flow path using ultrasonic waves, a rectangular flow channel having a substantially rectangular cross-sectional shape when cut perpendicular to the extension direction of the flow channel; A two-dimensional acoustic particle focusing device comprising an ultrasonic generator that simultaneously irradiates into the rectangular flow channel a first ultrasonic wave generated so that the length of the long side of the rectangle of the rectangular flow channel is substantially equal to the length of half the wavelength, and a second ultrasonic wave generated so that the length of the short side of the rectangle of the rectangular flow channel is substantially equal to the length of half the wavelength, in a synthesized state.

2. 2. The particle two-dimensional acoustic focusing device according to claim 1, wherein the ultrasonic generator comprises a signal adjusting means for adjusting the mixing ratio and magnitude of two signals of different frequencies that generate the first ultrasonic wave and the second ultrasonic wave.

3. 3. The particle two-dimensional acoustic focusing device according to claim 2, wherein the ultrasonic generator comprises, as components, a signal generator that generates signals of two different frequencies, and a piezoelectric vibrator that receives signals from the signal generator and generates ultrasonic vibrations.

4. 4. The particle two-dimensional acoustic focusing device according to claim 3, wherein the portion of the rectangular flow path to which at least the first ultrasonic wave and the second ultrasonic wave are irradiated is installed in an upright position so that the liquid flows vertically.

5. 5. The particle two-dimensional acoustic focusing device according to claim 1, wherein an optical measurement device whose measurement capability is affected by the depth of field is disposed at a position facing the rectangular flow channel.

6. The particle two-dimensional acoustic focusing device according to any one of claims 1 to 4 is provided, An acoustic concentration device using a two-dimensional particle acoustic focusing device, characterized in that the downstream flow path of the rectangular flow path is provided with a three-branch flow path formed to branch into three flow paths: a branch flow path on one side, a branch flow path on the other side, and a central branch flow path provided to flow particles focused to the center of the rectangular flow path by the two-dimensional particle acoustic focusing device.

7. 7. The acoustic concentrating device using a particle two-dimensional acoustic focusing device according to claim 6, wherein the three-branched flow path is formed by dividing the rectangular shape into three in the longitudinal direction of the long side of the rectangle.

8. 7. The acoustic concentration device using a two-dimensional particle acoustic focusing device according to claim 6, wherein a plurality of the acoustic concentration devices using the two-dimensional particle acoustic focusing devices are connected in series with respect to the flow of the liquid containing the particles.

9. 7. The acoustic concentration device using a two-dimensional particle acoustic focusing device according to claim 6, wherein a plurality of the acoustic concentration devices using the two-dimensional particle acoustic focusing devices are connected in parallel with respect to the flow of the liquid containing the particles.

Images