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Submicron separation of microspheres via travelling surface acoustic waves Ghulam Destgeer, Byung Hang Ha, Jin Ho Jung and Hyung Jin Sung*
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
Submicron separation is the segregation of the particles having diameter difference of less than one micrometre. We present an acoustofluidic particle separator with submicron separation resolution to study the continuous, label-free, and contactless separation of polystyrene (PS) particles based on their acoustofluidic parameters such as size, density, compressibility and shape. In this work, the submicron separation of PS microspheres, having marginal size difference, is achieved inside a polydimethylsiloxane (PDMS) microfluidic channel via travelling surface acoustic waves (TSAW). The TSAW of different frequencies (200, 192, 155, and 129 MHz), propagating normal to the fluid flow direction inside the PDMS microchannel, realized continuous separation of particles with diameter difference as low as 200 nm. A theoretical framework, composed of the rigid and elastic theories, is presented to support the experimental results.
Introduction The precise separation and dexterous manipulation of microscale objects is critical to many bio-chemical processes in cell biology, analytical chemistry, drug development, cancer diagnostics and therapeutics.1–3 Since the advent of microfluidics, a range of micro-fluid actuation and micro-object (particle, droplet, cell) manipulation techniques have been developed.4–8 Surface acoustic waves (SAW) based acoustofluidic techniques have been a popular choice for carrying out this job due to the following associated advantages. Owning to its widespread use in telecommunication industry, the micro-fabrication process for fabricating miniaturized, cheaper and versatile SAW devices is well developed. A SAW based microfluidic system is biocompatible; permits contact-free micro-object manipulation and fast fluid actuation. A series of interlocking comb-shaped metallic electrodes, i.e. an interdigitated transducer (IDT), have a strong electro-mechanical coupling with the piezoelectric substrate which make an energy efficient SAW device.9,10 The SAW devices are broadly categorized based on standing surface acoustic wave (SSAW) and travelling surface acoustic wave (TSAW) modes. A SSAW is formed when two TSAWs originating from two distinct IDTs, propagating in the opposite directions, interfere. A SSAW consists of regions of low and high acoustic pressure, known as pressure node (PN) and antinode, respectively. It has been reported that the SSAW can separate particles,11 manipulate cells,12–14 sort droplets15 and perform density dependent separation16 of micro-objects providing that the positions of the PNs are effectively controlled inside the microfluidic channel. The suspended particles move towards or away from the PNs if the carrying fluid is lighter or heavier than the particles, respectively. A precise control of the PNs requires tight alignment of microchannels with the IDTs. The use of chirped17 and
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slanted18 IDTs resolved this issue to some extent as the position of PN can be manipulated easily, however, the microchannel is still needed to be parallel with the IDTs. For an operation of most of the SSAW devices, the width () of the microchannel has to be in reasonable agreement with the sound wavelength (λ ) in fluid. Usually ≤ (1/2) ∙ λ , if a single PN is needed inside the microchannel, however, multiple PNs could be formed by increasing the width of the microchannel. Recently, Ding et al.19 reported a tilted-angle SSAW device, which incorporates a wider microchannel encompassing multiple PNs for separation of particles and cells, and does not require to be tightly aligned with the microchannel. The TSAW based acoustofluidic devices have been equally effective for a variety of microfluidic operations such as concentrating particles in micro sessile droplets,20 cell enrichment,1 droplet generation21 and size control,22,23 particle deflection and separation,24–27 sorting droplets and cells,28–30 mixing in nano-scale droplets,31 droplet coalescence in a microchannel,32 driving flow in a thin film,33 rotating a miniaturized disc,34 cell detachment cum label-free separation35 and generating microfluidic concentration gradient.36 A single IDT is employed to generate the TSAW while making sure there is no significant reflection of waves from micro-chip edges or polydimethylsiloxane (PDMS) walls that could form SSAW. The TSAW based devices have the advantage of easy integration of microchannel with the IDT on the piezoelectric substrate and the flexibility in the microchannel design. Particle separation in a continuous flow is desirable for increasing throughputs, enabling real-time monitoring of samples, and facilitating integration of other upstream/downstream microfluidic gadgets. To give an overview of the SAW based separation devices, some notable research works are summarized here. Shi et al.11 demonstrated the continuous separation of polystyrene (PS) microparticles having diameters ( ) of 0.87 µm from particles of diameter
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4.16 µm by using 12.6 MHz frequency SSAW in a PDMS microchannel under continuous flow conditions. Later on, Collins et al.37 used a single interdigitated transducer (IDT) to generate a standing wave acoustic pressure field inside a microfluidic channel to continuously separate 5.0 and 6.6 µm PS particles from 6.6 and 7.0 µm particles using 39 MHz frequency waves. Ding et al.19 used 19.4 MHz tilted-angle SSAW to separate 7.3 and 9.9 µm PS particles. On the other hand, the separation of microparticles was also realized by TSAW. Skowronek et al.24 successfully separated 4.5 µm particles from 3.0 µm particle with the TSAW frequency as high as 160 MHz within a bi-layer PDMS microfluidic channel. We previously demonstrated a cross-type acoustic particle separator (CAPS) comprising a single layer PDMS microchannel equipped with an acoustic window to separate 3 µm particles from 10 µm particles with a 100% efficiency.25 It can be deduced from the above mentioned studies that the minimum diameter difference between the separated particles is 0.4 µm and 1.5 µm, segregated by standing37 and travelling24 waves, respectively. The present study demonstrated the submicron separation of PS particles, having diameter difference as low as 0.2 µm, inside a PDMS microfluidic channel by harnessing the potential of TSAW. For the proposed TSAW based device, the width of the
Journal Name DOI: 10.1039/C4LC00868E microchannel and its precise location against the IDT are not critical as compared to other SSAW based devices, however, the PDMS microchannels were designed with reasonable dimensions; not too wide that the TSAW would damp inside the fluid and fail to reach the targeted particles effectively on the farther end, nor too small than the reflection of waves from microchannel walls becomes significant. High frequency TSAWs were used for contactless, label-free and continuous separation of nanoparticles (0.71 µm) from microparticles (3.0 µm) as well as microparticles from microparticles with size difference as low as 200 nm. The TSAWs of frequencies, 200, 192, 155, and 129 MHz, realized continuous separation of particles with diameter 3.0, 3.0, 3.4 and 4.2 µm from those of diameters 3.2, 3.4, 4.2 and 5.0 µm, respectively. The separation of 3.4, 4.2, and 4.5 µm PS particles from 4.2, 4.5, and 5.0 µm particles, respectively, was also demonstrated using a 129 MHz TSAW with power inputs of 104.3, 34.1, and 13.3 mW, respectively. Furthermore, the 155 MHz TSAW continuously separated 3.2 µm fluorescent particles from 4.2 µm particles at a flow rate as high as 1,250 µL/h. In the main body of this paper the focus is on the experimental results, whereas a detailed theoretical description is provided in the ESI.
Fig. 1 (a) Schematic diagram showing the cross type acoustic particle separator (CAPS). The TSAW generated by the IDT are coupled with the fluid inside the PDMS microchannel. The sample – particle carrying fluid, is sandwiched between two sheath flows to focus the particles prior to entering the separation zone. The particles separated by TSAW are collected at separate outlets. (b) (Bottom) Side view of the device. The TSAW (Rayleigh waves) coupled to the fluid form longitudinal leaky TSAW that interact with the suspended particles. The leaky TSAW propagate in the fluid at the Rayleigh angle ( ), which governs the trajectory of the particle. (Top) The plane wave scattered from the surface of the solid particle produces an ARF that is always directed along the direction of the wave propagation. (c) The 200 MHz TSAW separated 0.71 µm particles from 3.0 µm particles, whereas, the plot is showing the scattering light intensity from the particles across the width of the microchannel. (See ESI Movie I) (d) Separation of 3.0 µm particles from 3.2 µm particles. (See ESI Movies II and III) The dark field microscopy images are stacked to obtain the particle trajectories. The scale bars in (c) and (d) indicate 200 µm.
Experimental setup, materials, and methods Separation mechanism In order to characterize and compare various separation devices based on SAW, an acoustofluidic dimensionless parameter ( ) is defined, such that ( 2 / ) is the
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wavenumber, is the frequency of waves, is the speed of sound in the fluid and ( /2) is the radius of the particle. For acoustic waves interacting with a sphere, the factor gives a measure of the particle radius ( ) relative to the sound wavelength (λ /), and is also an important parameter to characterize particle separation via TSAW. Acoustic radiation force factor (ARFF) – another dimensionless parameter is defined as the acoustic radiation force (ARF) per unit acoustic
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Journal Name energy density per unit cross sectional area of the microsphere. The ARFF, a function of , is estimated using two theoretical models, the rigid and elastic theories, based on the rigid and elastic particle assumptions, respectively. The dependency of ARFF on and its usage in predicting particles separation via TSAW is also presented (for details, see ESI). Skowronek et al.24 reported that the net ARF applied by the TSAW on a small particle vanished for < 1 , due to spherically isotropic scattering, however, for ≳ 1 (i.e. λ ≲ 2 ), the scattering was no more isotropic and a net momentum transfer to the particle occurred as the backscattering dominated. The high frequency TSAW could be used to deflect or separate particles having radii comparable to or larger than the TSAW wavelength. The separation of microparticles by TSAW and SSAW can be attributed to the anisotropic scattering of waves and the presence of PNs (and anti-PNs) inside the fluid, respectively. The values of for various frequencies () and particle diameters ( ) are listed in Tab. S1, which highlights that a small difference in is enough to separate particles marginally different in diameter. A schematic of the CAPS device used to demonstrate the submicron separation of particles is shown in Fig. 1a. The TSAW, produced at the focused IDT, propagates in a beam-like fashion before interacting with the fluid inside the microchannel. Fig. 1b (top) describes the interaction of a plane TSAW propagating through a fluid with a suspended spherical particle. If λ ≲ 2 , the wave imparts a radiation force of sufficient magnitude to the particle to induce a lateral migration (from left to right in the frame of the page). The plane wave is then scattered from the surface of sphere in an anisotropic manner and the particle experiences the ARF in the forward direction. The magnitude of the ARF is determined by the size of the particle relative to the wavelength of TSAW and the density difference between the fluid ( ) and the spherical particle ( ). A side view of the device in Fig. 1b (bottom), shows a TSAW (Rayleigh wave) coupling with the fluid inside the microchannel to form a leaky TSAW. Given the speed of sound on the surface of a lithium niobate (LiNbO3) substrate, ≈ 3,850 m/s, and an incident angle = 90°, the angle of transmitted leaky TSAW (i.e. Rayleigh angle) may be found by employing Snell’s law, sin ! ( ⁄ ) ≈ 22.8°. Therefore, each deflected particle follows a two-step trajectory. First, the particle is pushed along an inclined line at angle . Second, the particle approaches the ceiling of microchannel and travel horizontally. Recently, Collins et al.27 reported a TSAW based particle valve by taking advantage of such a motion of the particles. Device fabrication The microfluidic particle separation device CAPS was composed of a single layered PDMS microchannel mounted on top of a piezoelectric substrate lithium niobate - LiNbO3 (128° Y-X cut, 4" dia. x 0.5 mm, 2sp, MTI Korea ). The IDT was deposited on the substrate to generate high frequency acoustic waves. The fabrication process for the micro device was similar to that used previously.25 The focused IDT comprised of a bimetallic Au/Cr layer 1000 Å/300 Å thick, deposited via an ebeam evaporation process. Three different designs of IDTs were fabricated to produce TSAW with nominal wavelengths of 30 µm (device 1), 25 µm (device 2) and 20 µm (device 3) and frequencies 129, 155 and 192 MHz, respectively. All the devices had 30 electrode pairs arranged such that the innermost electrode followed an arc radius of 4 mm and an aperture angle
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ARTICLE DOI: 10.1039/C4LC00868E of 40°. The device with 30 µm nominal wavelength had a unidirectional transducer (IDT) that could focus a maximal amount of energy in the forward direction (for detail, see the ESI in Destgeer et al.36). The devices 1-3 were found, experimentally, to produce the maximum electrical energy transfer into mechanical vibration (i.e. TSAW) when actuated at frequencies 129, 155 and 192 MHz, respectively. The devices could also be operated at slightly different frequencies, but the input power required to produce similar effects would have been higher. The particle separation shown in Fig. 1c,d was obtained by device 3 when actuated at 200 MHz. The power input was as high as 3.8 W, as the optimum frequency would have been 192 MHz for the same device (for details, see ESI Movie I-III). The PDMS microchannel was fabricated using standard soft lithography process. The width of the microchannel at the separation zone was 200 µm in some experiments and 500 µm in others. The separation zone was the central region of the microchannel within which the particles of variable sizes were separated by TSAW as they deviated from the primary flow streamline, as shown in Fig. 1a. The microchannel was fabricated by spin-coating a 40 µm thick layer of the SU-8 photoresist on top of a Si substrate and forming the desired channel shape by exposing the photoresist to UV light through a patterned chrome mask. The SU-8 pattern acted as a mould that could be transferred to the PDMS. The PDMS base and curing agents were mixed in a 10:1 ratio, and the mixture was poured on top of the SU-8 mould. The bubbles formed in the liquid PDMS during mixing were removed under vacuum. The PDMS was cured in a 65 °C oven for at least two hours and later peeled off the Si substrate. The inlet/outlet ports were punched through the PDMS with a punching tool (Harris Uni-Core). The LiNbO3 substrate and PDMS surfaces were treated in a multi-purpose oxygen plasma system (Covance, Femto Science, Korea) for 3 min (at 150 W and 750 mTorr)15 prior to bonding them together. The microchannel did not need to be tightly aligned with the IDT. The L-shaped alignment marks indicated the appropriate location to place the microchannel manually on the substrate (see Fig. 1a). After bonding, the device was cured in 95 °C oven for at least 15 min to strengthen the bond. Experimental method The microfluidic separation devices were tested using PS microspheres with mean diameter of 0.71, 3.0, 3.2, 3.4, 4.2, 4.5 and 5.0 µm. Red fluorescent particles (0.71 and 3.2 µm) were used to easily distinguish them from others and NIST (National Institute of Standards and Technology) standard particles (3.0, 3.4, 4.2 and 5.0 µm) were used to ensure particle size control. The sample fluid was prepared by mixing the selected particles (Particle Technology by Thermo Scientific, Inc. or PolyBeads by PolySciecnes, Inc) in DI water to a sufficient dilution level where the effect of acoustic scattering on the adjoining particles was negligible (e.g. 1 drop of particle suspension per ml of DI water). The sheath fluids (DI water mixed with surfactant, volumetric ratio 9:1) focused the particles in a narrow stream prior to separation. The surfactant prevented particle coagulation inside the microchannel and clinging to the PDMS walls. The CAPS device was operated at a high frequency alternating current signal generated by an RF signal generator (Agilent N5181A) and amplified by a power amplifier (miniCircuits LZT-22+). The amplified signal was then delivered to the IDT, which disseminated the TSAW for the particle separation. The sample fluid carrying the particles along with the sheath flows was injected by a syringe pump
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(Cetoni GmbH neMESYS) through three separate inlet ports, as shown in Fig. 1a. The sample fluid sandwiched between the sheath flows I and II passed through the separation zone. The particles were focused in a narrow stream. The flow rates used were 22.5, 2.5 and 100 µL/h for sheath flow I, sample flow and sheath flow II, respectively, unless otherwise mentioned. The hydrodynamically focused particles were separated according to their size difference and were later collected at separate outlets. The motion of separated particles was captured by mounting the CAPS device onto a microscopic stage (Olympus BX53) equipped with a camera (Olympus DP26). Dark field imaging technique was used to obtain good quality images with a black background and showing the particles as white bright spots. The microscopic images were processed with the help of an image processing software (imageJ, imagej.nih.gov) to remove background noise from the image and obtain particle trajectories by stacking images together.
Results The main focus of this study was to show the submicron separation of particles having diameters that differed by less than a micrometre. The TSAW based CAPS device was used to
Journal Name DOI: 10.1039/C4LC00868E segregate particles along the length of a continuous flow according to the diameter of the particles. The separation of particles was directly dependent on the factor that indicates the relative size of a particle compared to the wavelength of acoustic wave. The frequencies and the particles’ diameters used in this study and the corresponding values are listed in Tab. S1. The highlighted values correspond to the experimental separation of pairs of microparticles having different sizes using a single frequency. Fig. 1c,d show the separation of 0.71 and 3.0 µm particles from 3.0 and 3.2 µm particles, respectively, using a 200 MHz TSAW inside a microchannel 200 µm wide and 40 µm high. The narrow separation region of the microchannel widened downstream to 500 µm to enhance the separation of particles (Fig. 1d). The power inputs in these experiments (2.3 – 3.8 W) were on the high side, as 200 MHz frequency results in lower electrical to mechanical energy transfer for device 3 – having the optimum actuation frequency of 192 MHz; however, separation could still be achieved which proves the versatility of the CAPS device. These results showed that a single device could be actuated over a range of frequencies at the expense of the high input power while adjusting the factor to suit the needs of the experiment.
Fig. 2 The separation of particles having various sizes with diameter differences of less than 1 µm was achieved using a 129 MHz TSAW. (a) The microchannel used in this particular experiment was 200 µm wide in the separation zone and 500 µm wide downstream, close to the outlet zone. The scale bars indicate 1 mm. (b) The zone highlighted with the red dashed rectangle in (a) is shown in (b). PS particles were deflected by the 129 MHz TSAW with a variable power input. (c) The plot shows the positions of the particles across the microchannel width. The average light intensity measured within the red dashed rectangle in (b) is plotted in arbitrary units (a.u.) versus the microchannel width. (d) The separation of particles having various diameters was achieved by tuning the input power. (e) The corresponding positions of the microparticles are plotted. The scale bars in (b) and (d) indicate 200 µm.
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The deflection of microparticles and their submicron separation are further illustrated in Fig. 2. The microchannel design used in this particular experiment, as shown in Fig. 2a, was 200 µm wide at the separation zone and expanded to 500 µm downstream near the outlet, to increase the lateral separation. PS particles of diameter 5 µm were used with a 129 MHz TSAW. The power input was varied to characterize its effect on the particle deflection distance across the microchannel width (), as shown in Fig. 2b,c. The power input was switched on from 0 mW to 4.8 mW and was gradually increased to 19.2 mW, producing a shift in the particles’ streamlines from left to right. The outlet port for the particles was steadily changed with increasing the power. After deflection, the particles were no longer focused in a narrow stream due to the random initial vertical positions of the particle within the microchannel prior to entering the separation zone. As the particles were not aligned or focused along a single line upstream of the separation zone, small differences in their positions dispersed their streamlines downstream. Moreover, small variance in the diameter of particles, already marginally different in size, results in some particles moving out through wrong outlet ports during separation as seen in ESI Movie III. The ability to direct the microparticles into the desired outlet port demonstrates the potential of CAPS device for particle sorting and separation via modulation of the input power for a fixed flow rate. The locations of the particles’ streamlines across the 500 µm wide channel, outlined by a red dashed-line rectangle in Fig. 2b, are plotted as a function of the input power in Fig. 2c. The vertical
axis in Fig. 2c indicates the average light intensity corresponding to the particles’ position, measured from the stacked images in arbitrary units (a.u.). The spread of streamlines was smallest for 0 mW, when all particles were aligned along a vertical plane; however, the streamlines were not focused in the vertical direction. A power input of 4.8 - 19.2 mW yielded a wide streamline spread due to the differing initial positions of the particles. In Fig. 2d, the separation of 3.4, 4.2, and 4.5 µm diameter particles from 4.2, 4.5, and 5.0 µm diameter particles using power inputs of 104.3, 34.1, and 13.3 mW, respectively, was successfully achieved. In order to not to confuse the particles of different diameters, this set of experiments was conducted with one type of particles being deflected at a time. The trajectories of particles obtained separately were combined together to get the final figure presenting the separation. The differences between the diameters of the separated particle pairs were 0.8, 0.3, and 0.5 µm, respectively. The streamlines obtained from stacking a series of images confirmed the separation of particles flowing through distinct outlet ports for a separation device actuated at 129 MHz with a tuneable input power (Fig. 2d). The corresponding positions of particle streamlines are plotted in Fig. 2e and clearly indicate that the particles were separated from each other by a separation distance of no less than 100 µm in all experiments. Collection of different particles at separate outlets was achieved by adjusting the input power.
Fig. 3 (a) Schematic of the microfluidic channel. The scale bar is 1 mm long. (b-d) The separation of particles having various sizes via a TSAW actuated at different frequencies. The first three images from the left indicate that the separation distance gradually increased as the input power to the CAPS increased (left to right). The particles’ trajectories on the right corresponded to larger diameter particles (5.0, 4.2, and 3.4 µm) and those on the left corresponded to smaller diameter particles (4.2, 3.4, and 3.0
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Another microchannel design having width x height: 500 µm x 40 µm (Fig. 3a) was used with IDT actuation frequencies 129, 155 and 192 MHz to separate particles of diameter 5.0, 4.2 and 3.4 µm from 4.2, 3.4 and 3.0 µm, respectively, as shown in Fig. 3b-d. The first three images from the left show the trajectories of the separated particles for input powers that increased from left to right. The corresponding images on the right side in Fig. 3b-d show the acoustic streaming flow patterns captured by tracing 1 µm PS particles, which experienced negligible ARF under the influence of TSAW, as < 1 (0.27, 0.33, and 0.40, for 129, 155, and 192 MHz, respectively). The vortices produced by the acoustic streaming flow (ASF) evidently affected the trajectories of the particles to some extent; however, particle separation was still possible. The 129 MHz TSAW produced a pair of symmetric vortices, which assisted the lateral migration of 5 µm particles beyond migration due to the ARF (Fig. 3b). A sudden bend in the trajectory of the 5 µm particles resulted from a strong ASF between the two vortices directed to the right. The PS particles 4.2 µm in diameter were further split into two separate streamlines, as shown in Fig. 3b. The rightward stream of particles was governed by the ARF; however, the leftward stream of particles was dominated by the ASF, as they could not escape the vortex and pushed against the left wall. As the overall separation of particles was governed by the ARF, the steaming flow-induced drag force did not significantly affect the separation distance in general. The relative effects of the acoustic radiation force and acoustic streaming flow-induced drag force were also demonstrated previously.25 The variable actuation frequency of the TSAW produced slightly different ASF patterns in microchannels with similar dimensions. Alghane et al. described the frequency38 and scaling effects39 on the ASF produced in a droplet, which provide a reasonably good understanding of the current phenomena. The 155 and 192 MHz TSAW produced two symmetric pairs of ASF vortices (see Fig. 3c-d). A strong flow between the vortices at two positions further deflected the trajectories of the particles. The regions in which the streaming flows were strong corresponded to the bends in the particle trajectories. The separation distance between particles increased gradually for the 155 MHz TSAW (Fig. 3c); however, the separation distance did not change significantly after reaching a certain value for the 192 MHz TSAW (Fig. 3d), even though both types of particles (3.0 and 3.4 µm) migrated further to the right as the input power increased. The separation distance between particles, normalized by the width of the microchannel, is plotted against the input voltage normalized by the maximum voltage for a particular experiment, as shown in Fig. 3d. The particles’ behaviours under a high-frequency TSAW (192 MHz) could be explained using Stokes’ law of sound attenuation, which states that the amplitude of sound waves decreases exponentially in the medium of propagation at an attenuation rate $ ( 2%(2)& ((3 & ) ), which is proportional to the square of the frequency (where, % is the viscosity of the fluid). The higher absorption of high-frequency sound waves in a fluid leads to a stronger ASF and a weaker ARF deep inside the fluid. Particle separation in the presence of the 192 MHz TSAW was induced by the ARF, as the particles were closer to the left wall and the amplitude of the TSAW did not decrease significantly. Once the particles had moved away
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from the left wall of the microchannel into a region in which the ARF effects were weaker, the ASF induced a drag force (proportional to the particles’ radius) that affected both types of particles equally. No further separation between particles was induced. Fig. 3e shows that even though the particles migrated further toward the right, the separation distance between them was retained. The separation of 3.2 µm (red fluorescent) from 4.2 µm (nonfluorescent) particles in a stationary as well as continuous flow was also realized by 155 MHz frequency TSAW (see ESI Movie IV and V). For particles’ separation in the stationary flow, the particles were first flown in a focused stream and then the flow was suddenly stopped to bring the particles to an approximate stand still position. An input power of 13 mW was used to segregate the particles as the smaller 3.2 µm particles were dominated by the ASF, whereas the larger 4.2 µm particles overcome by the ARF were pushed towards the opposite microchannel wall. The continuous separation of particles was achieved at the net flow rate of 125 and 1,250 µL/h and the power input of 53 and 479 mW, respectively. The performance of the CAPS device was tested against time by continuously running the device at 479 mW to separate the particles. The device was found to work effectively without any loss in particle separation.
Discussion The interaction of sound waves with a spherical particle resulted in scattering of the waves which imparted an acoustic radiation force to the spherical body. The TSAW propagated through a liquid (DI water) and interacted with the suspended particles. It has been discussed in this section that how the theoretical models can help in predicting the submicron separation of the PS particles. A comprehensive estimation of ARFF on a solid rigid particle by King40 is referred to as the rigid theory in this text. The analytical expressions of ARFF (see Eq. S3 and S4 for travelling and standing waves, respectively) are valid for the spherical body that could be assumed as rigid. The ARFF estimation for compressible41 and elastic42,43 spheres was assessed based on a non-zero compressibility of the fluid and suspended body. The derivation of the ARFF for travelling waves (see Eq. S6) by Hasegawa et al.42 is referred to as the elastic theory in this text. (see ESI for detailed derivation of the theoretical models). To find out the relative strength of radiation force acting on a PS particle by TSAW or SSAW, the ARFF estimated by rigid theory for travelling and standing waves and their ratio is plotted against in Fig. S1. It is found that the dominating force of standing waves is overcome by the travelling waves at a critical value of ≈ 1.05. Yosioka et al.43 found a critical value of ≈ 1.3 for steel sphere. The theoretical models are tested by assuming fused silica (FS) particles suspended in water, so that a comparison could be made with the already reported results. The ARFF calculated by the rigid and elastic theories are comparable for the given range of (0 < < 1.5) (see Fig. S2). A good agreement with the previous study44 confirms the validity of the computational models. The ARFF was then calculated for PS particles dispersed in water; which is found to be rising steeply after
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µm) in each experiment, respectively. The right-most images show the streaming patterns formed by 1 µm particles in a stationary fluid subjected to the TSAW. (e) The separation distance (dn) between particles normalized by the microchannel width (500 µm) is plotted against the input voltage (Vn) normalized by the maximum voltage applied in a particular experiment. The scale bars in (bd) indicate 200 µm.
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Journal Name > 1 when using the elastic theory owing to the fact that the PS particles are more fitting to be elastic than rigid (see Fig. S2). A sudden rise in ARFF for a small increase in , opens up the possibility of separation of particles that are slightly larger than the rest. In light of the theoretical predictions, a guideline for the TSAW frequency selection can be presented for the separation of PS particles with particular sizes. The ARFF estimated by elastic theory is plotted against and particles’ diameter ( ) for different frequencies (see Fig. S3). The submicron separation of pairs of PS particles, whose diameters range within 4-5 µm, 3.2-4.2 µm and 2.4-3.4 µm, is predicted by travelling waves of frequency 129, 155 and 192 MHz, respectively. Future work: The separation of targeted malignant or diseased cells from a whole blood sample is critical to numerous biochemical studies. This study could be useful for isolation of target cells that are insignificantly different in size than others like diabetic fat cells from healthy fat cells.
Conclusions We successfully separated PS particles having size differences of less than one micrometre using high-frequency TSAW. The separation of particles with diameter differences as low as 0.3 µm was achieved using a 129 MHz TSAW applied to a continuous flow. The separation of 3.0 µm particles from 3.2 µm red fluorescent particles (diameter difference of 0.2 µm) was demonstrated using a 200 MHz TSAW. The versatility of the CAPS device was confirmed by applying a variety of other frequencies (155 and 192 MHz) to separate different combinations of particles. The separation of PS particles was explored theoretically using the rigid and elastic theories, which estimate the acoustic radiation force applied to particles of various sizes under various actuation frequencies. The force factors estimated for the PS particles under the rigid and elastic theories were significantly different, as the rigid particle assumptions were not applicable to the PS particles tested here. The ARF on the PS particles needed for deflection from its original streamline was effective for ≳ 1, which is true for a range of particle diameters and TSAW frequencies. Although this work focused on providing an experimental demonstration of particle separation, theoretical treatments are also summarized. King’s rigid theory is suitable for describing particles that can be modelled as rigid, given their physical properties, whereas elastic materials like PS must be modelled using the elastic theory of Hasegawa et al. for predicting the acoustic radiation force acting on an elastic particle.
ARTICLE DOI: 10.1039/C4LC00868E 1. 2.
3. 4. 5. 6. 7. 8. 9. 10.
11. 12.
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20. 21. 22. 23. 24. 25. 26. 27.
Acknowledgements
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This work was supported by the Creative Research Initiatives (No. 2014-001493) program of the National Research Foundation of Korea (MSIP).
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Notes and references
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Department of Mechanical Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea. Email: [email protected]. † Electronic Supplementary Information (ESI) available: [Detailed derivation of the theoretical models. Movie I: Separation of 0.71 µm and 3.0 µm particles. Movie II and III: Separation of 3.0 µm and 3.2 µm (red fluorescent) particles. Movie IV and V: Separation of 3.2 µm (red fluorescent) particles from 4.2 µm particles by 155 MHz TSAW.]. See DOI: 10.1039/b000000x/
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Cite this: DOI: 10.1039/x0xx00000x
Submicron separation of microspheres via travelling surface acoustic waves Ghulam Destgeer, Byung Hang Ha, Jin Ho Jung and Hyung Jin Sung*
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
Submicron separation is the segregation of the particles having diameter difference of less than one micrometre. We present an acoustofluidic particle separator with submicron separation resolution to study the continuous, label-free, and contactless separation of polystyrene (PS) particles based on their acoustofluidic parameters such as size, density, compressibility and shape. In this work, the submicron separation of PS microspheres, having marginal size difference, is achieved inside a polydimethylsiloxane (PDMS) microfluidic channel via travelling surface acoustic waves (TSAW). The TSAW of different frequencies (200, 192, 155, and 129 MHz), propagating normal to the fluid flow direction inside the PDMS microchannel, realized continuous separation of particles with diameter difference as low as 200 nm. A theoretical framework, composed of the rigid and elastic theories, is presented to support the experimental results.
Introduction The precise separation and dexterous manipulation of microscale objects is critical to many bio-chemical processes in cell biology, analytical chemistry, drug development, cancer diagnostics and therapeutics.1–3 Since the advent of microfluidics, a range of micro-fluid actuation and micro-object (particle, droplet, cell) manipulation techniques have been developed.4–8 Surface acoustic waves (SAW) based acoustofluidic techniques have been a popular choice for carrying out this job due to the following associated advantages. Owning to its widespread use in telecommunication industry, the micro-fabrication process for fabricating miniaturized, cheaper and versatile SAW devices is well developed. A SAW based microfluidic system is biocompatible; permits contact-free micro-object manipulation and fast fluid actuation. A series of interlocking comb-shaped metallic electrodes, i.e. an interdigitated transducer (IDT), have a strong electro-mechanical coupling with the piezoelectric substrate which make an energy efficient SAW device.9,10 The SAW devices are broadly categorized based on standing surface acoustic wave (SSAW) and travelling surface acoustic wave (TSAW) modes. A SSAW is formed when two TSAWs originating from two distinct IDTs, propagating in the opposite directions, interfere. A SSAW consists of regions of low and high acoustic pressure, known as pressure node (PN) and antinode, respectively. It has been reported that the SSAW can separate particles,11 manipulate cells,12–14 sort droplets15 and perform density dependent separation16 of micro-objects providing that the positions of the PNs are effectively controlled inside the microfluidic channel. The suspended particles move towards or away from the PNs if the carrying fluid is lighter or heavier than the particles, respectively. A precise control of the PNs requires tight alignment of microchannels with the IDTs. The use of chirped17 and
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slanted18 IDTs resolved this issue to some extent as the position of PN can be manipulated easily, however, the microchannel is still needed to be parallel with the IDTs. For an operation of most of the SSAW devices, the width () of the microchannel has to be in reasonable agreement with the sound wavelength (λ ) in fluid. Usually ≤ (1/2) ∙ λ , if a single PN is needed inside the microchannel, however, multiple PNs could be formed by increasing the width of the microchannel. Recently, Ding et al.19 reported a tilted-angle SSAW device, which incorporates a wider microchannel encompassing multiple PNs for separation of particles and cells, and does not require to be tightly aligned with the microchannel. The TSAW based acoustofluidic devices have been equally effective for a variety of microfluidic operations such as concentrating particles in micro sessile droplets,20 cell enrichment,1 droplet generation21 and size control,22,23 particle deflection and separation,24–27 sorting droplets and cells,28–30 mixing in nano-scale droplets,31 droplet coalescence in a microchannel,32 driving flow in a thin film,33 rotating a miniaturized disc,34 cell detachment cum label-free separation35 and generating microfluidic concentration gradient.36 A single IDT is employed to generate the TSAW while making sure there is no significant reflection of waves from micro-chip edges or polydimethylsiloxane (PDMS) walls that could form SSAW. The TSAW based devices have the advantage of easy integration of microchannel with the IDT on the piezoelectric substrate and the flexibility in the microchannel design. Particle separation in a continuous flow is desirable for increasing throughputs, enabling real-time monitoring of samples, and facilitating integration of other upstream/downstream microfluidic gadgets. To give an overview of the SAW based separation devices, some notable research works are summarized here. Shi et al.11 demonstrated the continuous separation of polystyrene (PS) microparticles having diameters ( ) of 0.87 µm from particles of diameter
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4.16 µm by using 12.6 MHz frequency SSAW in a PDMS microchannel under continuous flow conditions. Later on, Collins et al.37 used a single interdigitated transducer (IDT) to generate a standing wave acoustic pressure field inside a microfluidic channel to continuously separate 5.0 and 6.6 µm PS particles from 6.6 and 7.0 µm particles using 39 MHz frequency waves. Ding et al.19 used 19.4 MHz tilted-angle SSAW to separate 7.3 and 9.9 µm PS particles. On the other hand, the separation of microparticles was also realized by TSAW. Skowronek et al.24 successfully separated 4.5 µm particles from 3.0 µm particle with the TSAW frequency as high as 160 MHz within a bi-layer PDMS microfluidic channel. We previously demonstrated a cross-type acoustic particle separator (CAPS) comprising a single layer PDMS microchannel equipped with an acoustic window to separate 3 µm particles from 10 µm particles with a 100% efficiency.25 It can be deduced from the above mentioned studies that the minimum diameter difference between the separated particles is 0.4 µm and 1.5 µm, segregated by standing37 and travelling24 waves, respectively. The present study demonstrated the submicron separation of PS particles, having diameter difference as low as 0.2 µm, inside a PDMS microfluidic channel by harnessing the potential of TSAW. For the proposed TSAW based device, the width of the
Journal Name DOI: 10.1039/C4LC00868E microchannel and its precise location against the IDT are not critical as compared to other SSAW based devices, however, the PDMS microchannels were designed with reasonable dimensions; not too wide that the TSAW would damp inside the fluid and fail to reach the targeted particles effectively on the farther end, nor too small than the reflection of waves from microchannel walls becomes significant. High frequency TSAWs were used for contactless, label-free and continuous separation of nanoparticles (0.71 µm) from microparticles (3.0 µm) as well as microparticles from microparticles with size difference as low as 200 nm. The TSAWs of frequencies, 200, 192, 155, and 129 MHz, realized continuous separation of particles with diameter 3.0, 3.0, 3.4 and 4.2 µm from those of diameters 3.2, 3.4, 4.2 and 5.0 µm, respectively. The separation of 3.4, 4.2, and 4.5 µm PS particles from 4.2, 4.5, and 5.0 µm particles, respectively, was also demonstrated using a 129 MHz TSAW with power inputs of 104.3, 34.1, and 13.3 mW, respectively. Furthermore, the 155 MHz TSAW continuously separated 3.2 µm fluorescent particles from 4.2 µm particles at a flow rate as high as 1,250 µL/h. In the main body of this paper the focus is on the experimental results, whereas a detailed theoretical description is provided in the ESI.
Fig. 1 (a) Schematic diagram showing the cross type acoustic particle separator (CAPS). The TSAW generated by the IDT are coupled with the fluid inside the PDMS microchannel. The sample – particle carrying fluid, is sandwiched between two sheath flows to focus the particles prior to entering the separation zone. The particles separated by TSAW are collected at separate outlets. (b) (Bottom) Side view of the device. The TSAW (Rayleigh waves) coupled to the fluid form longitudinal leaky TSAW that interact with the suspended particles. The leaky TSAW propagate in the fluid at the Rayleigh angle ( ), which governs the trajectory of the particle. (Top) The plane wave scattered from the surface of the solid particle produces an ARF that is always directed along the direction of the wave propagation. (c) The 200 MHz TSAW separated 0.71 µm particles from 3.0 µm particles, whereas, the plot is showing the scattering light intensity from the particles across the width of the microchannel. (See ESI Movie I) (d) Separation of 3.0 µm particles from 3.2 µm particles. (See ESI Movies II and III) The dark field microscopy images are stacked to obtain the particle trajectories. The scale bars in (c) and (d) indicate 200 µm.
Experimental setup, materials, and methods Separation mechanism In order to characterize and compare various separation devices based on SAW, an acoustofluidic dimensionless parameter ( ) is defined, such that ( 2 / ) is the
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wavenumber, is the frequency of waves, is the speed of sound in the fluid and ( /2) is the radius of the particle. For acoustic waves interacting with a sphere, the factor gives a measure of the particle radius ( ) relative to the sound wavelength (λ /), and is also an important parameter to characterize particle separation via TSAW. Acoustic radiation force factor (ARFF) – another dimensionless parameter is defined as the acoustic radiation force (ARF) per unit acoustic
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Journal Name energy density per unit cross sectional area of the microsphere. The ARFF, a function of , is estimated using two theoretical models, the rigid and elastic theories, based on the rigid and elastic particle assumptions, respectively. The dependency of ARFF on and its usage in predicting particles separation via TSAW is also presented (for details, see ESI). Skowronek et al.24 reported that the net ARF applied by the TSAW on a small particle vanished for < 1 , due to spherically isotropic scattering, however, for ≳ 1 (i.e. λ ≲ 2 ), the scattering was no more isotropic and a net momentum transfer to the particle occurred as the backscattering dominated. The high frequency TSAW could be used to deflect or separate particles having radii comparable to or larger than the TSAW wavelength. The separation of microparticles by TSAW and SSAW can be attributed to the anisotropic scattering of waves and the presence of PNs (and anti-PNs) inside the fluid, respectively. The values of for various frequencies () and particle diameters ( ) are listed in Tab. S1, which highlights that a small difference in is enough to separate particles marginally different in diameter. A schematic of the CAPS device used to demonstrate the submicron separation of particles is shown in Fig. 1a. The TSAW, produced at the focused IDT, propagates in a beam-like fashion before interacting with the fluid inside the microchannel. Fig. 1b (top) describes the interaction of a plane TSAW propagating through a fluid with a suspended spherical particle. If λ ≲ 2 , the wave imparts a radiation force of sufficient magnitude to the particle to induce a lateral migration (from left to right in the frame of the page). The plane wave is then scattered from the surface of sphere in an anisotropic manner and the particle experiences the ARF in the forward direction. The magnitude of the ARF is determined by the size of the particle relative to the wavelength of TSAW and the density difference between the fluid ( ) and the spherical particle ( ). A side view of the device in Fig. 1b (bottom), shows a TSAW (Rayleigh wave) coupling with the fluid inside the microchannel to form a leaky TSAW. Given the speed of sound on the surface of a lithium niobate (LiNbO3) substrate, ≈ 3,850 m/s, and an incident angle = 90°, the angle of transmitted leaky TSAW (i.e. Rayleigh angle) may be found by employing Snell’s law, sin ! ( ⁄ ) ≈ 22.8°. Therefore, each deflected particle follows a two-step trajectory. First, the particle is pushed along an inclined line at angle . Second, the particle approaches the ceiling of microchannel and travel horizontally. Recently, Collins et al.27 reported a TSAW based particle valve by taking advantage of such a motion of the particles. Device fabrication The microfluidic particle separation device CAPS was composed of a single layered PDMS microchannel mounted on top of a piezoelectric substrate lithium niobate - LiNbO3 (128° Y-X cut, 4" dia. x 0.5 mm, 2sp, MTI Korea ). The IDT was deposited on the substrate to generate high frequency acoustic waves. The fabrication process for the micro device was similar to that used previously.25 The focused IDT comprised of a bimetallic Au/Cr layer 1000 Å/300 Å thick, deposited via an ebeam evaporation process. Three different designs of IDTs were fabricated to produce TSAW with nominal wavelengths of 30 µm (device 1), 25 µm (device 2) and 20 µm (device 3) and frequencies 129, 155 and 192 MHz, respectively. All the devices had 30 electrode pairs arranged such that the innermost electrode followed an arc radius of 4 mm and an aperture angle
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ARTICLE DOI: 10.1039/C4LC00868E of 40°. The device with 30 µm nominal wavelength had a unidirectional transducer (IDT) that could focus a maximal amount of energy in the forward direction (for detail, see the ESI in Destgeer et al.36). The devices 1-3 were found, experimentally, to produce the maximum electrical energy transfer into mechanical vibration (i.e. TSAW) when actuated at frequencies 129, 155 and 192 MHz, respectively. The devices could also be operated at slightly different frequencies, but the input power required to produce similar effects would have been higher. The particle separation shown in Fig. 1c,d was obtained by device 3 when actuated at 200 MHz. The power input was as high as 3.8 W, as the optimum frequency would have been 192 MHz for the same device (for details, see ESI Movie I-III). The PDMS microchannel was fabricated using standard soft lithography process. The width of the microchannel at the separation zone was 200 µm in some experiments and 500 µm in others. The separation zone was the central region of the microchannel within which the particles of variable sizes were separated by TSAW as they deviated from the primary flow streamline, as shown in Fig. 1a. The microchannel was fabricated by spin-coating a 40 µm thick layer of the SU-8 photoresist on top of a Si substrate and forming the desired channel shape by exposing the photoresist to UV light through a patterned chrome mask. The SU-8 pattern acted as a mould that could be transferred to the PDMS. The PDMS base and curing agents were mixed in a 10:1 ratio, and the mixture was poured on top of the SU-8 mould. The bubbles formed in the liquid PDMS during mixing were removed under vacuum. The PDMS was cured in a 65 °C oven for at least two hours and later peeled off the Si substrate. The inlet/outlet ports were punched through the PDMS with a punching tool (Harris Uni-Core). The LiNbO3 substrate and PDMS surfaces were treated in a multi-purpose oxygen plasma system (Covance, Femto Science, Korea) for 3 min (at 150 W and 750 mTorr)15 prior to bonding them together. The microchannel did not need to be tightly aligned with the IDT. The L-shaped alignment marks indicated the appropriate location to place the microchannel manually on the substrate (see Fig. 1a). After bonding, the device was cured in 95 °C oven for at least 15 min to strengthen the bond. Experimental method The microfluidic separation devices were tested using PS microspheres with mean diameter of 0.71, 3.0, 3.2, 3.4, 4.2, 4.5 and 5.0 µm. Red fluorescent particles (0.71 and 3.2 µm) were used to easily distinguish them from others and NIST (National Institute of Standards and Technology) standard particles (3.0, 3.4, 4.2 and 5.0 µm) were used to ensure particle size control. The sample fluid was prepared by mixing the selected particles (Particle Technology by Thermo Scientific, Inc. or PolyBeads by PolySciecnes, Inc) in DI water to a sufficient dilution level where the effect of acoustic scattering on the adjoining particles was negligible (e.g. 1 drop of particle suspension per ml of DI water). The sheath fluids (DI water mixed with surfactant, volumetric ratio 9:1) focused the particles in a narrow stream prior to separation. The surfactant prevented particle coagulation inside the microchannel and clinging to the PDMS walls. The CAPS device was operated at a high frequency alternating current signal generated by an RF signal generator (Agilent N5181A) and amplified by a power amplifier (miniCircuits LZT-22+). The amplified signal was then delivered to the IDT, which disseminated the TSAW for the particle separation. The sample fluid carrying the particles along with the sheath flows was injected by a syringe pump
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(Cetoni GmbH neMESYS) through three separate inlet ports, as shown in Fig. 1a. The sample fluid sandwiched between the sheath flows I and II passed through the separation zone. The particles were focused in a narrow stream. The flow rates used were 22.5, 2.5 and 100 µL/h for sheath flow I, sample flow and sheath flow II, respectively, unless otherwise mentioned. The hydrodynamically focused particles were separated according to their size difference and were later collected at separate outlets. The motion of separated particles was captured by mounting the CAPS device onto a microscopic stage (Olympus BX53) equipped with a camera (Olympus DP26). Dark field imaging technique was used to obtain good quality images with a black background and showing the particles as white bright spots. The microscopic images were processed with the help of an image processing software (imageJ, imagej.nih.gov) to remove background noise from the image and obtain particle trajectories by stacking images together.
Results The main focus of this study was to show the submicron separation of particles having diameters that differed by less than a micrometre. The TSAW based CAPS device was used to
Journal Name DOI: 10.1039/C4LC00868E segregate particles along the length of a continuous flow according to the diameter of the particles. The separation of particles was directly dependent on the factor that indicates the relative size of a particle compared to the wavelength of acoustic wave. The frequencies and the particles’ diameters used in this study and the corresponding values are listed in Tab. S1. The highlighted values correspond to the experimental separation of pairs of microparticles having different sizes using a single frequency. Fig. 1c,d show the separation of 0.71 and 3.0 µm particles from 3.0 and 3.2 µm particles, respectively, using a 200 MHz TSAW inside a microchannel 200 µm wide and 40 µm high. The narrow separation region of the microchannel widened downstream to 500 µm to enhance the separation of particles (Fig. 1d). The power inputs in these experiments (2.3 – 3.8 W) were on the high side, as 200 MHz frequency results in lower electrical to mechanical energy transfer for device 3 – having the optimum actuation frequency of 192 MHz; however, separation could still be achieved which proves the versatility of the CAPS device. These results showed that a single device could be actuated over a range of frequencies at the expense of the high input power while adjusting the factor to suit the needs of the experiment.
Fig. 2 The separation of particles having various sizes with diameter differences of less than 1 µm was achieved using a 129 MHz TSAW. (a) The microchannel used in this particular experiment was 200 µm wide in the separation zone and 500 µm wide downstream, close to the outlet zone. The scale bars indicate 1 mm. (b) The zone highlighted with the red dashed rectangle in (a) is shown in (b). PS particles were deflected by the 129 MHz TSAW with a variable power input. (c) The plot shows the positions of the particles across the microchannel width. The average light intensity measured within the red dashed rectangle in (b) is plotted in arbitrary units (a.u.) versus the microchannel width. (d) The separation of particles having various diameters was achieved by tuning the input power. (e) The corresponding positions of the microparticles are plotted. The scale bars in (b) and (d) indicate 200 µm.
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DOI: 10.1039/C4LC00868E
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The deflection of microparticles and their submicron separation are further illustrated in Fig. 2. The microchannel design used in this particular experiment, as shown in Fig. 2a, was 200 µm wide at the separation zone and expanded to 500 µm downstream near the outlet, to increase the lateral separation. PS particles of diameter 5 µm were used with a 129 MHz TSAW. The power input was varied to characterize its effect on the particle deflection distance across the microchannel width (), as shown in Fig. 2b,c. The power input was switched on from 0 mW to 4.8 mW and was gradually increased to 19.2 mW, producing a shift in the particles’ streamlines from left to right. The outlet port for the particles was steadily changed with increasing the power. After deflection, the particles were no longer focused in a narrow stream due to the random initial vertical positions of the particle within the microchannel prior to entering the separation zone. As the particles were not aligned or focused along a single line upstream of the separation zone, small differences in their positions dispersed their streamlines downstream. Moreover, small variance in the diameter of particles, already marginally different in size, results in some particles moving out through wrong outlet ports during separation as seen in ESI Movie III. The ability to direct the microparticles into the desired outlet port demonstrates the potential of CAPS device for particle sorting and separation via modulation of the input power for a fixed flow rate. The locations of the particles’ streamlines across the 500 µm wide channel, outlined by a red dashed-line rectangle in Fig. 2b, are plotted as a function of the input power in Fig. 2c. The vertical
axis in Fig. 2c indicates the average light intensity corresponding to the particles’ position, measured from the stacked images in arbitrary units (a.u.). The spread of streamlines was smallest for 0 mW, when all particles were aligned along a vertical plane; however, the streamlines were not focused in the vertical direction. A power input of 4.8 - 19.2 mW yielded a wide streamline spread due to the differing initial positions of the particles. In Fig. 2d, the separation of 3.4, 4.2, and 4.5 µm diameter particles from 4.2, 4.5, and 5.0 µm diameter particles using power inputs of 104.3, 34.1, and 13.3 mW, respectively, was successfully achieved. In order to not to confuse the particles of different diameters, this set of experiments was conducted with one type of particles being deflected at a time. The trajectories of particles obtained separately were combined together to get the final figure presenting the separation. The differences between the diameters of the separated particle pairs were 0.8, 0.3, and 0.5 µm, respectively. The streamlines obtained from stacking a series of images confirmed the separation of particles flowing through distinct outlet ports for a separation device actuated at 129 MHz with a tuneable input power (Fig. 2d). The corresponding positions of particle streamlines are plotted in Fig. 2e and clearly indicate that the particles were separated from each other by a separation distance of no less than 100 µm in all experiments. Collection of different particles at separate outlets was achieved by adjusting the input power.
Fig. 3 (a) Schematic of the microfluidic channel. The scale bar is 1 mm long. (b-d) The separation of particles having various sizes via a TSAW actuated at different frequencies. The first three images from the left indicate that the separation distance gradually increased as the input power to the CAPS increased (left to right). The particles’ trajectories on the right corresponded to larger diameter particles (5.0, 4.2, and 3.4 µm) and those on the left corresponded to smaller diameter particles (4.2, 3.4, and 3.0
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Journal Name DOI: 10.1039/C4LC00868E
Another microchannel design having width x height: 500 µm x 40 µm (Fig. 3a) was used with IDT actuation frequencies 129, 155 and 192 MHz to separate particles of diameter 5.0, 4.2 and 3.4 µm from 4.2, 3.4 and 3.0 µm, respectively, as shown in Fig. 3b-d. The first three images from the left show the trajectories of the separated particles for input powers that increased from left to right. The corresponding images on the right side in Fig. 3b-d show the acoustic streaming flow patterns captured by tracing 1 µm PS particles, which experienced negligible ARF under the influence of TSAW, as < 1 (0.27, 0.33, and 0.40, for 129, 155, and 192 MHz, respectively). The vortices produced by the acoustic streaming flow (ASF) evidently affected the trajectories of the particles to some extent; however, particle separation was still possible. The 129 MHz TSAW produced a pair of symmetric vortices, which assisted the lateral migration of 5 µm particles beyond migration due to the ARF (Fig. 3b). A sudden bend in the trajectory of the 5 µm particles resulted from a strong ASF between the two vortices directed to the right. The PS particles 4.2 µm in diameter were further split into two separate streamlines, as shown in Fig. 3b. The rightward stream of particles was governed by the ARF; however, the leftward stream of particles was dominated by the ASF, as they could not escape the vortex and pushed against the left wall. As the overall separation of particles was governed by the ARF, the steaming flow-induced drag force did not significantly affect the separation distance in general. The relative effects of the acoustic radiation force and acoustic streaming flow-induced drag force were also demonstrated previously.25 The variable actuation frequency of the TSAW produced slightly different ASF patterns in microchannels with similar dimensions. Alghane et al. described the frequency38 and scaling effects39 on the ASF produced in a droplet, which provide a reasonably good understanding of the current phenomena. The 155 and 192 MHz TSAW produced two symmetric pairs of ASF vortices (see Fig. 3c-d). A strong flow between the vortices at two positions further deflected the trajectories of the particles. The regions in which the streaming flows were strong corresponded to the bends in the particle trajectories. The separation distance between particles increased gradually for the 155 MHz TSAW (Fig. 3c); however, the separation distance did not change significantly after reaching a certain value for the 192 MHz TSAW (Fig. 3d), even though both types of particles (3.0 and 3.4 µm) migrated further to the right as the input power increased. The separation distance between particles, normalized by the width of the microchannel, is plotted against the input voltage normalized by the maximum voltage for a particular experiment, as shown in Fig. 3d. The particles’ behaviours under a high-frequency TSAW (192 MHz) could be explained using Stokes’ law of sound attenuation, which states that the amplitude of sound waves decreases exponentially in the medium of propagation at an attenuation rate $ ( 2%(2)& ((3 & ) ), which is proportional to the square of the frequency (where, % is the viscosity of the fluid). The higher absorption of high-frequency sound waves in a fluid leads to a stronger ASF and a weaker ARF deep inside the fluid. Particle separation in the presence of the 192 MHz TSAW was induced by the ARF, as the particles were closer to the left wall and the amplitude of the TSAW did not decrease significantly. Once the particles had moved away
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from the left wall of the microchannel into a region in which the ARF effects were weaker, the ASF induced a drag force (proportional to the particles’ radius) that affected both types of particles equally. No further separation between particles was induced. Fig. 3e shows that even though the particles migrated further toward the right, the separation distance between them was retained. The separation of 3.2 µm (red fluorescent) from 4.2 µm (nonfluorescent) particles in a stationary as well as continuous flow was also realized by 155 MHz frequency TSAW (see ESI Movie IV and V). For particles’ separation in the stationary flow, the particles were first flown in a focused stream and then the flow was suddenly stopped to bring the particles to an approximate stand still position. An input power of 13 mW was used to segregate the particles as the smaller 3.2 µm particles were dominated by the ASF, whereas the larger 4.2 µm particles overcome by the ARF were pushed towards the opposite microchannel wall. The continuous separation of particles was achieved at the net flow rate of 125 and 1,250 µL/h and the power input of 53 and 479 mW, respectively. The performance of the CAPS device was tested against time by continuously running the device at 479 mW to separate the particles. The device was found to work effectively without any loss in particle separation.
Discussion The interaction of sound waves with a spherical particle resulted in scattering of the waves which imparted an acoustic radiation force to the spherical body. The TSAW propagated through a liquid (DI water) and interacted with the suspended particles. It has been discussed in this section that how the theoretical models can help in predicting the submicron separation of the PS particles. A comprehensive estimation of ARFF on a solid rigid particle by King40 is referred to as the rigid theory in this text. The analytical expressions of ARFF (see Eq. S3 and S4 for travelling and standing waves, respectively) are valid for the spherical body that could be assumed as rigid. The ARFF estimation for compressible41 and elastic42,43 spheres was assessed based on a non-zero compressibility of the fluid and suspended body. The derivation of the ARFF for travelling waves (see Eq. S6) by Hasegawa et al.42 is referred to as the elastic theory in this text. (see ESI for detailed derivation of the theoretical models). To find out the relative strength of radiation force acting on a PS particle by TSAW or SSAW, the ARFF estimated by rigid theory for travelling and standing waves and their ratio is plotted against in Fig. S1. It is found that the dominating force of standing waves is overcome by the travelling waves at a critical value of ≈ 1.05. Yosioka et al.43 found a critical value of ≈ 1.3 for steel sphere. The theoretical models are tested by assuming fused silica (FS) particles suspended in water, so that a comparison could be made with the already reported results. The ARFF calculated by the rigid and elastic theories are comparable for the given range of (0 < < 1.5) (see Fig. S2). A good agreement with the previous study44 confirms the validity of the computational models. The ARFF was then calculated for PS particles dispersed in water; which is found to be rising steeply after
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µm) in each experiment, respectively. The right-most images show the streaming patterns formed by 1 µm particles in a stationary fluid subjected to the TSAW. (e) The separation distance (dn) between particles normalized by the microchannel width (500 µm) is plotted against the input voltage (Vn) normalized by the maximum voltage applied in a particular experiment. The scale bars in (bd) indicate 200 µm.
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Journal Name > 1 when using the elastic theory owing to the fact that the PS particles are more fitting to be elastic than rigid (see Fig. S2). A sudden rise in ARFF for a small increase in , opens up the possibility of separation of particles that are slightly larger than the rest. In light of the theoretical predictions, a guideline for the TSAW frequency selection can be presented for the separation of PS particles with particular sizes. The ARFF estimated by elastic theory is plotted against and particles’ diameter ( ) for different frequencies (see Fig. S3). The submicron separation of pairs of PS particles, whose diameters range within 4-5 µm, 3.2-4.2 µm and 2.4-3.4 µm, is predicted by travelling waves of frequency 129, 155 and 192 MHz, respectively. Future work: The separation of targeted malignant or diseased cells from a whole blood sample is critical to numerous biochemical studies. This study could be useful for isolation of target cells that are insignificantly different in size than others like diabetic fat cells from healthy fat cells.
Conclusions We successfully separated PS particles having size differences of less than one micrometre using high-frequency TSAW. The separation of particles with diameter differences as low as 0.3 µm was achieved using a 129 MHz TSAW applied to a continuous flow. The separation of 3.0 µm particles from 3.2 µm red fluorescent particles (diameter difference of 0.2 µm) was demonstrated using a 200 MHz TSAW. The versatility of the CAPS device was confirmed by applying a variety of other frequencies (155 and 192 MHz) to separate different combinations of particles. The separation of PS particles was explored theoretically using the rigid and elastic theories, which estimate the acoustic radiation force applied to particles of various sizes under various actuation frequencies. The force factors estimated for the PS particles under the rigid and elastic theories were significantly different, as the rigid particle assumptions were not applicable to the PS particles tested here. The ARF on the PS particles needed for deflection from its original streamline was effective for ≳ 1, which is true for a range of particle diameters and TSAW frequencies. Although this work focused on providing an experimental demonstration of particle separation, theoretical treatments are also summarized. King’s rigid theory is suitable for describing particles that can be modelled as rigid, given their physical properties, whereas elastic materials like PS must be modelled using the elastic theory of Hasegawa et al. for predicting the acoustic radiation force acting on an elastic particle.
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Acknowledgements
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This work was supported by the Creative Research Initiatives (No. 2014-001493) program of the National Research Foundation of Korea (MSIP).
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Notes and references
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Department of Mechanical Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea. Email: [email protected]. † Electronic Supplementary Information (ESI) available: [Detailed derivation of the theoretical models. Movie I: Separation of 0.71 µm and 3.0 µm particles. Movie II and III: Separation of 3.0 µm and 3.2 µm (red fluorescent) particles. Movie IV and V: Separation of 3.2 µm (red fluorescent) particles from 4.2 µm particles by 155 MHz TSAW.]. See DOI: 10.1039/b000000x/
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