This work was sponsored by the Academic Research Fund (AcRF) Tier 1 (RG171/15), Ministry of Education, Singapore.

1. Preparation of the Microfluidic Channel
<ol>
	<li>Mix poly-dimethylsiloxane (PDMS) with an elastomer base in a ratio of 10:1.</li>
	<li>Degas the mixture in a vacuum oven and pour it on a silicon wafer with a negative tone photoresist pattern on the top.</li>
	<li>Degas the patterned silicon wafer again and heat it at 70 &#176;C for 3 h in an incubator for solidification.</li>
</ol>
2. Fabrication of the Interdigital Transducers
<ol>
	<li>Deposit 20 nm of Cr and 400 nm of Al on a LiNbO3 wafer; pattern 20 strips with a width of 150 &#181;m and an aperture of 2 cm on a plastic mask for photolithography by depositing the positive photoresist on the substrate.</li>
	<li>Remove the Cr-Al layer on the non-exposed area with acetone.</li>
	<li>Treat the surface of them with oxygen plasma (with a nitrogen and oxygen ratio of 2:1) at the power of 30 W for 60 s.</li>
	<li>Align the PDMS microchannel and bond it to the LiNbO3 substrate by pressing it with a thumb for a few seconds.</li>
	<li>Place the integrated device in the heating chamber at 60 &#176;C for 3 h.</li>
</ol>
3. Dual-frequency Excitation
<ol>
	<li>Simultaneously apply two frequency components (f1 and f3, the fundamental frequency and its third harmonic of the fabricated IDT, respectively) with the phase difference &#966; between them to the pair of IDTs, so that the produced SAW may be expressed as follows. 
	<img alt="Equation 1" src="/files/ftp_upload/58085/58085eq1.jpg" /> 
	Here, 
	<img alt="Equation 2" src="/files/ftp_upload/58085/58085eq2.jpg" /> and <img alt="Equation 3" src="/files/ftp_upload/58085/58085eq3.jpg" /> = the acoustic pressures.</li>
	<li>Synthesize the dual-frequency waveform using the Equation Editor of the ArbExpress Application software at the sampling frequency of 100 MS/s and then store it to the function generator as arbitrary input for the SAW excitation in the experiment via a USB cable.</li>
	<li>Vary the power of the fundamental frequency to the total emitted power&#160;<img alt="Equation 4" src="/files/ftp_upload/58085/58085eq4.jpg" /> from 100% (excitation at the purely fundamental frequency) to 0% (excitation at the purely third harmonic); for a good comparison, change but keep the total power the same.</li>
	<li>Vary the phase difference of the dual-frequency excitation from 0&#176; to 360&#176;.</li>
</ol>
4. Numerical Simulation
<ol>
	<li>Describe the motion of the incompressible laminar flow with low Reynolds (i.e., Re = 0.55) and Mach numbers as follows35. 
	<img alt="Equation 5" src="/files/ftp_upload/58085/58085eq5.jpg" /> 
	<img alt="Equation 6" src="/files/ftp_upload/58085/58085eq6.jpg" /> 
	Here, 
	<img alt="Equation 7" src="/files/ftp_upload/58085/58085eq7.jpg" /> = the fluid velocity, 
	<img alt="Equation 8" src="/files/ftp_upload/58085/58085eq8.jpg" /> = the dynamic viscosity, 
	<img alt="Equation 9" src="/files/ftp_upload/58085/58085eq9.jpg" /> = the fluid density, 
	<img alt="Equation 10" src="/files/ftp_upload/58085/58085eq10.jpg" /> = the pressure on the fluid, 
	<img alt="Equation 11" src="/files/ftp_upload/58085/58085eq11.jpg" /> = the identity matrix, and 
	<img alt="Equation 12" src="/files/ftp_upload/58085/58085eq12.jpg" /> = an external force.</li>
	<li>Describe the produced Stoke drag force on the object as follows36. 
	<img alt="Equation 13" src="/files/ftp_upload/58085/58085eq13.jpg" /> 
	Here, 
	<img alt="Equation 14" src="/files/ftp_upload/58085/58085eq14.jpg" />&#160;= the radius of the microparticle, 
	<img alt="Equation 15" src="/files/ftp_upload/58085/58085eq15.jpg" />&#160;= the velocity of the fluid, and 
	<img alt="Equation 16" src="/files/ftp_upload/58085/58085eq16.jpg" />&#160;= the velocity of the microparticle.</li>
	<li>Derive the acoustic radiation force applied to the microparticle in the microchannel along the x-axis (across the microchannel width) at a single frequency as follows16 . 
	<img alt="Equation 17" src="/files/ftp_upload/58085/58085eq17.jpg" /> 
	Here, 
	<img alt="Equation 18" src="/files/ftp_upload/58085/58085eq18.jpg" /> = the volume of the microparticle, 
	<img alt="Equation 19" src="/files/ftp_upload/58085/58085eq19.jpg" /> = the density of the microparticle, 
	<img alt="Equation 20" src="/files/ftp_upload/58085/58085eq20.jpg" /> = the density of the medium, 
	<img alt="Equation 21" src="/files/ftp_upload/58085/58085eq21.jpg" /> = the compressibility of the microparticle, and 
	<img alt="Equation 22" src="/files/ftp_upload/58085/58085eq22.jpg" /> = the compressibility of the medium.</li>
	<li>Derive the resultant acoustic radiation force of the dual-frequency excitation as follows. 
	<img alt="Equation 23" src="/files/ftp_upload/58085/58085eq23.jpg" /></li>
	<li>Express the transverse motion across the channel width (along the y-axis) under both the acoustic radiation force and the Stokes drag force governed by Newton&#39;s second law as follows. 
	<img alt="Equation 24" src="/files/ftp_upload/58085/58085eq24.jpg" /></li>
	<li>Solve the ordinary differential equations (ODEs) above using the fourth order Runge-Kutta method on a personal computer.Set the time step and total duration as 1 &#181;s and 20 s, respectively.</li>
</ol>
5. Experimental Observation
<ol>
	<li>Spin the solution in the concentration of 5.9 x 107 with 4 &#181;m green fluorescent polystyrene beads per 1 mL by vortex for about 2 - 3 min and then immerse it in an ultrasound sonicator for 10 min to disrupt any agglomeration before each testing.</li>
	<li>Fill the mixture into a 3 mL syringe and then drive it with a syringe pump at a flow rate of 3 - 5 &#181;L/min.</li>
	<li>Drive the IDT with the dual-frequency signal from a function generator followed by a power amplifier.</li>
	<li>Observe the stabilized microparticles in the downstream microchannel under a light microscope at a 40X magnification and record the image with a digital camera.</li>
	<li>Measure the location of the accumulated microparticles in the captured digital images using ImageJ and the established scale, and then quantitatively determine the concentration of the accumulated microparticle with the normalized fluorescence brightness at each pressure node.</li>
</ol>

<ol>
	<li>Chin, C. D., Linder, V., Sia, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commercialization+of+microfluidic+point-of-care+diagnostic+devices.">Commercialization of microfluidic point-of-care diagnostic devices.</a> Lab on a Chip. 12 (12), 2118-2134 (2012).</li><li>Figeys, D., Pinto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-a-chip:+a+revolution+in+biological+and+medical+sciences.">Lab-on-a-chip: a revolution in biological and medical sciences.</a> Analytical Chemistry. 72 (9), 330 A-335 A (2000).</li><li>den Toonder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+the+Grand+Challenge.">Circulating tumor cells: the Grand Challenge.</a> Lab on a Chip. 11 (3), 375-377 (2011).</li><li>Yu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+of+lab-on-a-chip+in+isolation,+detection+and+post-processing+of+circulating+tumour+cells.">Advances of lab-on-a-chip in isolation, detection and post-processing of circulating tumour cells.</a> Lab on a Chip. 13 (16), 3163-3182 (2013).</li><li>Jorgenson, J. W., Lukacs, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+separations+based+on+electrophoresis+and+electroosmosis.">High-resolution separations based on electrophoresis and electroosmosis.</a> Journal of Chromatography A. 218, 209-216 (1981).</li><li>Gascoyne, P. R., Vykoukal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+separation+by+dielectrophoresis.">Particle separation by dielectrophoresis.</a> Electrophoresis. 23 (13), 1973 (1973).</li><li>Xia, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+microfluidic-micromagnetic+separation+of+living+cells+in+continuous+flow.">Combined microfluidic-micromagnetic separation of living cells in continuous flow.</a> Biomedical Microdevices. 8 (4), 299-308 (2006).</li><li>Garc&#233;s-Ch&#225;vez, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+organization+of+colloidal+microparticles+by+surface+plasmon+polariton+excitation.">Extended organization of colloidal microparticles by surface plasmon polariton excitation.</a> Physical Review B. 73 (8), 085417 (2006).</li><li>Zhu, T., Ye, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+Knudsen+forces+on+heated+microbeams.">Origin of Knudsen forces on heated microbeams.</a> Physical Review E. 82 (3), 036308 (2010).</li><li>Ashkin, A., Dziedzic, J., Yamane, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping+and+manipulation+of+single+cells+using+infrared+laser+beams.">Optical trapping and manipulation of single cells using infrared laser beams.</a> Nature. 330 (6150), 769-771 (1987).</li><li>Chiou, P. Y., Ohta, A. T., Wu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+manipulation+of+single+cells+and+microparticles+using+optical+images.">Massively parallel manipulation of single cells and microparticles using optical images.</a> Nature. 436 (7049), 370-372 (2005).</li><li>Yamada, M., Seki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+filtration+for+on-chip+particle+concentration+and+classification+utilizing+microfluidics.">Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics.</a> Lab on a Chip. 5 (11), 1233-1239 (2005).</li><li>Burguillos, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microchannel+acoustophoresis+does+not+impact+survival+or+function+of+microglia,+leukocytes+or+tumor+cells.">Microchannel acoustophoresis does not impact survival or function of microglia, leukocytes or tumor cells.</a> PLoS One. 8 (5), e64233 (2013).</li><li>Lin, S. -. C. S., Mao, X., Huang, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+acoustic+wave+(SAW)+acoustophoresis:+now+and+beyond.">Surface acoustic wave (SAW) acoustophoresis: now and beyond.</a> Lab on a Chip. 12 (16), 2766-2770 (2012).</li><li>Petersson, F., &#197;berg, L., Sw&#228;rd-Nilsson, A. -. M., Laurell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+flow+acoustophoresis:+microfluidic-based+mode+of+particle+and+cell+separation.">Free flow acoustophoresis: microfluidic-based mode of particle and cell separation.</a> Analytical Chemistry. 79 (14), 5117-5123 (2007).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+manipulation+of+single+microparticles,+cells,+and+organisms+using+surface+acoustic+waves.">On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves.</a> Proceedings of the National Academy of Sciences. 109 (28), 11105-11109 (2012).</li><li>Friend, J., Yeo, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+acoustofluidics:+Microfluidics+driven+via+acoustics+and+ultrasonics.">Microscale acoustofluidics: Microfluidics driven via acoustics and ultrasonics.</a> Reviews of Modern Physics. 83 (2), 647 (2011).</li><li>Destgeer, G., Lee, K. H., Jung, J. H., Alazzam, A., Sung, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+separation+of+particles+in+a+PDMS+microfluidic+channel+via+travelling+surface+acoustic+waves+(TSAW).">Continuous separation of particles in a PDMS microfluidic channel via travelling surface acoustic waves (TSAW).</a> Lab on a Chip. 13 (21), 4210-4216 (2013).</li><li>Guo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell-cell+interactions+using+surface+acoustic+waves.">Controlling cell-cell interactions using surface acoustic waves.</a> Proceedings of the National Academy of Sciences. 112 (1), 43-48 (2015).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+using+tilted-angle+standing+surface+acoustic+waves.">Cell separation using tilted-angle standing surface acoustic waves.</a> Proceedings of the National Academy of Sciences. 111 (36), 12992-12997 (2014).</li><li>Roshchupkin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+diffraction+by+standing+surface+acoustic+waves.">X-ray diffraction by standing surface acoustic waves.</a> Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms. 142 (3), 432-436 (1998).</li><li>Shi, J., Mao, X., Ahmed, D., Colletti, A., Huang, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focusing+microparticles+in+a+microfluidic+channel+with+standing+surface+acoustic+waves+(SSAW).">Focusing microparticles in a microfluidic channel with standing surface acoustic waves (SSAW).</a> Lab on a Chip. 8 (2), 221-223 (2008).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+acoustic+wave+microfluidics.">Surface acoustic wave microfluidics.</a> Lab on a Chip. 13 (18), 3626-3649 (2013).</li><li>King, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+acoustic+radiation+pressure+on+spheres.">On the acoustic radiation pressure on spheres.</a> Proceedings of the Royal Society of London A: Mathematical, Physical and Engineering Sciences. , 212-240 (1934).</li><li>Yosioka, K., Kawasima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+radiation+pressure+on+a+compressible+sphere.">Acoustic radiation pressure on a compressible sphere.</a> Acta Acustica United with Acustica. 5 (3), 167-173 (1955).</li><li>Ratier, C., Hoyos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+programming+in+step-split-flow+lateral-transport+thin+fractionation.">Acoustic programming in step-split-flow lateral-transport thin fractionation.</a> Analytical Chemistry. 82 (4), 1318-1325 (2010).</li><li>Mandralis, Z., Feke, D., Bolek, W., Burger, W., Benes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+synchronized+ultrasonic+and+flow-field+fractionation+of+suspensions.">Enhanced synchronized ultrasonic and flow-field fractionation of suspensions.</a> Ultrasonics. 32 (2), 113-122 (1994).</li><li>Laurell, T., Petersson, F., Nilsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chip+integrated+strategies+for+acoustic+separation+and+manipulation+of+cells+and+particles.">Chip integrated strategies for acoustic separation and manipulation of cells and particles.</a> Chemical Society Reviews. 36 (3), 492-506 (2007).</li><li>Liu, Y., Lim, K. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+separation+in+microfluidics+using+a+switching+ultrasonic+field.">Particle separation in microfluidics using a switching ultrasonic field.</a> Lab on a Chip. 11 (18), 3167-3173 (2011).</li><li>Brissaud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+piezoceramics.">Characterization of piezoceramics.</a> IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control. 38 (6), 603-617 (1991).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+patterning+of+microparticles+and+cells+using+standing+surface+acoustic+waves.">Tunable patterning of microparticles and cells using standing surface acoustic waves.</a> Lab on a Chip. 12 (14), 2491-2497 (2012).</li><li>Jo, M. C., Guldiken, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+manipulation+by+phase-shifting+of+surface+acoustic+waves.">Particle manipulation by phase-shifting of surface acoustic waves.</a> Sensors and Actuators A: Physical. 207, 39-42 (2014).</li><li>Meng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transportation+of+single+cell+and+microbubbles+by+phase-shift+introduced+to+standing+leaky+surface+acoustic+waves.">Transportation of single cell and microbubbles by phase-shift introduced to standing leaky surface acoustic waves.</a> Biomicrofluidics. 5 (4), 044104 (2011).</li><li>Sriphutkiat, Y., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+manipulation+using+standing+surface+acoustic+waves+(SSAW)+at+dual+frequency+excitation:+effect+of+power+ratio.">Particle manipulation using standing surface acoustic waves (SSAW) at dual frequency excitation: effect of power ratio.</a> Sensors and Actuators A: Physical. 263, 521-529 (2017).</li><li>Batchelor, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Fluid Dynamics. , (2000).</li><li>Glynne-Jones, P., Hill, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustofluidics+23:+acoustic+manipulation+combined+with+other+force+fields.">Acoustofluidics 23: acoustic manipulation combined with other force fields.</a> Lab on a Chip. 13 (6), 1003-1010 (2013).</li><li>Winkler, A., Br&#252;nig, R., Faust, C., Weser, R., Schmidt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+efficient+surface+acoustic+wave+(SAW)-based+microfluidic+actuators.">Towards efficient surface acoustic wave (SAW)-based microfluidic actuators.</a> Sensors and Actuators A: Physical. 247, 259-268 (2016).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standing+surface+acoustic+wave+(SSAW)-based+microfluidic+cytometer.">Standing surface acoustic wave (SSAW)-based microfluidic cytometer.</a> Lab on a Chip. 14, 916-923 (2014).</li><li>Devendran, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+travelling+wave+components+in+standing+surface+acoustic+wave+(SSAW)+systems.">The importance of travelling wave components in standing surface acoustic wave (SSAW) systems.</a> Lab on a Chip. 16, 3756-3766 (2016).</li><li>Destgeer, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicron+separation+of+microspheres+via+travelling+surface+acoustic+waves.">Submicron separation of microspheres via travelling surface acoustic waves.</a> Lab on a Chip. 14, 4665-4672 (2014).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standing+surface+acoustic+wave+(SSAW)+based+multichannel+cell+sorting.">Standing surface acoustic wave (SSAW) based multichannel cell sorting.</a> Lab on a Chip. 12, 4228-4231 (2012).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+enrichment+of+low-abundance+cell+samples+using+standing+surface+acoustic+waves+(SSAW).">Continuous enrichment of low-abundance cell samples using standing surface acoustic waves (SSAW).</a> Lab on a Chip. 14, 924-930 (2014).</li></ol>

The authors have nothing to disclose.

The microparticle motion in the microchannel by an SSAW at the dual-frequency excitation was extensively investigated in this study, and an effectively tunable patterning technique by varying the dual-frequency excitation signals was developed and tested. The production of such a waveform is easily realized by most function generators, and the adjusting approach is very convenient. Both the S12- and S11-frequency responses of the fabricated IDTs illustrate several resonant modes34<...

LOC technology integrates one or several functions on a microchip for biology, chemistry, biophysics, and biomedical processes. LOC allows a laboratory set-up on a scale smaller than sub-millimeters, fast reaction rates, a short response time, a high process control, a low volume consumption (less waste, lower reagents cost, and less required sample volume), a high throughput due to parallelization, a low cost in the future mass production and cost-effective disposables, a high safety for chemical, radioactive, or biological studies, and the advantages of a compact and portable device1,2. Precise cell manipulation (i.e., accumulation and separation) is critical in an LOC-based analysis and diagnosis3,4. However, the accuracy and reproducibility of microparticle manipulation have a variety of challenges. Many techniques, such as electro-osmosis5, dielectrophoresis (DEP)6, magnetophoresis7, thermophoresis8,9, an optical approach10, an optoelectronic approach11, a hydrodynamic approach12, and acoustophoresis13,14,15, have been developed. In comparison, acoustic approaches are appropriate for a LOC application because, theoretically, many types of microparticles/cells can be manipulated effectively and noninvasively with a sufficiently high contrast (density and compressibility) compared with the surrounding fluid. Therefore, compared to their counterparts, acoustic approaches are inherently eligible for most microparticles and biological objects, no matter their optical, electrical, and magnetic properties16.
Surface acoustic waves (SAWs) from the IDTs propagate mostly on the surface of a piezoelectric substrate at the thickness of several wavelengths and then leak at the Rayleigh angle into the fluid in the microchannel, according to the Snell&#39;s law17,18,19,20,21,22. They have the technical advantages of a high energy efficiency along the surface due to their localization of the energy, a great design flexibility at high frequency, a good system integration with the microfluidic channel and miniaturization using micro-electronic-mechanical system (MEMS) technology, and a high potential of mass production23. In this protocol, SAWs are generated from a pair of identical IDTs and propagated in the opposite direction to generate a standing wave, or SSAW, in the microchannel, where the suspended microparticles are pushed to pressure nodes, mostly by the applied acoustic radiation force24. The amplitude of such resultant force is determined by the excitation frequency, microparticle size, and its acoustic contrast factor22,25.
Such acoustophoresis has the limitation of predetermined manipulating patterns that are not easily adjustable. The excitation frequency of the IDTs is determined by their periodic distance, so the bandwidth is quite limited. Several strategies have been developed to enhance the tunability and manipulation capability. The first and second modes of acoustic standing waves applied in different parts of the microchannel could separate microparticles more effectively according to different motion speeds toward the nodal lines26. These two modes could also be applied to the whole part of the microchannel and switched alternatively27,28,29. However, for this, a large number of equipment (i.e., three function generators, two impedance matching units, and an electromagnetic relay) is required, with the increased cost and control complexity of the experimental set-up owing to the different electrical impedances at the fundamental frequency and third harmonic of the piezoceramic plate30. Furthermore, slanted-finger interdigital transducers (SFITs) could be applied to adjust the cells and the microparticles patterning by exciting a period of the slanted fingers for a certain resonance20,31. However, then, the bandwidth is inversely proportional to the number of slanted fingers. Multiple pressure nodal lines have a higher separation efficiency and sensitivity in comparison to the single nodal line in the conventional SSAW-based microparticle separator. Alternatively, the location of the pressure nodes could also be changed simply by adjusting the phase difference applied to the two IDTs in the design32,33.
The fundamental frequency and the third harmonic of IDTs have similar frequency responses so that they can be excited simultaneously, which provides more tunability for the microparticles manipulation34. In comparison to the conventional IDT excitation at a single frequency, adjusting the acoustic pressures of the dual-frequency excitation and the phase between them provides technical uniqueness, such as the up to ~2-fold reduced motion time to the pressure nodal line or the center of the microchannel, the varied number and location of the pressure nodal lines, and the microparticle concentrations.

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			<td style="width:147px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">poly-dimethylsiloxane elastomer base</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
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			<td height="21" style="height:21px;">silicon wafer</td>
			<td>Bonda Technology</td>
			<td>SI8PSPD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">negative tone photoresist</td>
			<td>Microchem</td>
			<td>SU-8</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">double-side polished LiNbO3 wafer</td>
			<td>University Wafer</td>
			<td style="width:119px;">Y-128&#176;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">positive photoresist</td>
			<td>Nicolaus-Otto-Stra&#223;e</td>
			<td>AZ 9260</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">oxygen plasma</td>
			<td>Harrick Plasma</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic mask</td>
			<td>Infinite Graphics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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microparticle manipulation by standing surface acoustic waves with dual-frequency excitations

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip (LOC) system. The simultaneous excitation of the fundamental frequency and its third harmonic, which is termed as dual-frequency excitation, to a pair of interdigital transducers (IDTs) could generate a new type of standing acoustic waves in a microfluidic channel. Varying the power and the phase in the dual-frequency excitation signals results in a reconfigurable field of the acoustic radiation force applied to the microparticles across the microchannel (e.g., the number and location of the pressure nodes and the microparticle concentrations at the corresponding pressure nodes). This article demonstrates that the motion time of the microparticle to only one pressure node can be reduced ~2-fold at the power ratio of the fundamental frequency greater than ~90%. In contrast, there are three pressure nodes in the microchannel if less than this threshold. Furthermore, adjusting the initial phase between the fundamental frequency and the third harmonic results in different motion rates of the three SSAW pressure nodes, as well as in the percentage of microparticles at each pressure node in the microchannel. There is a good agreement between the experimental observation and the numerical predictions. This novel excitation method can easily and non-invasively integrate into the LOC system, with a wide tenability and only a few changes to the experimental set-up.

The distributions of the acoustic pressure and the acoustic radiation force of an SSAW at the dual-frequency excitation (6.2 and 18.6 MHz) are shown in Figure 1. Here, the dual-frequency excitation occurs on polystyrene microparticles (4 &#181;m in diameter) in a microchannel with a width of 300 &#181;m at an acoustic power of 146 mW. The resultant acoustic pressure is always in phase when P1 &#62; 90% so that only one pressure node is pre...

Watch this Scientific Journal Video about Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations at JoVE.com

Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations

A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip ...

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6.9K Views.  Nanyang Technological University. A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

Video: Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations

Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.

Acoustic cavitation in liquids submitted to power ultrasound creates transient extreme conditions inside the collapsing bubbles, which are the origin of unusual chemical reactivity and light emission, known as sonoluminescence. In the presence of noble gases, nonequilibrium plasma is formed. The "hot" particles and the photons generated by collapsing bubbles are able to excite species in solution.

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic ...

Chemistry

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device.

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we ...

Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must be smaller than the incident wavelength of sound and the width of the fluidic channels are typically tens to hundreds of micrometers across, acoustofluidic devices typically use ultrasonic waves generated from a piezoelectric transducer pulsating at high frequencies (in the megahertz range). At characteristic frequencies that depend on the geometry of the device, it is possible to induce the formation of standing waves that can focus particles along desired fluidic streamlines within a bulk flow. Here, we describe a method for the fabrication of acoustophoretic devices from common materials and clean room equipment. We show representative results for the focusing of particles with positive or negative acoustic contrast factors, which move towards the pressure nodes or antinodes of the standing waves, respectively. These devices offer enormous practical utility for precisely positioning large numbers of microscopic entities (e.g., cells) in stationary or flowing fluids for applications ranging from cytometry to assembly.

Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Bioengineering

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Fabrication of Nanoheight Channels Incorporating Surface Acoustic Wave Actuation via Lithium Niobate for Acoustic Nanofluidics

Controlled nanoscale manipulation of fluids is known to be exceptionally difficult due to the dominance of surface and viscous forces. Megahertz-order surface acoustic wave (SAW) devices generate tremendous acceleration on their surface, up to 108 m/s2, in turn responsible for many of the observed effects that have come to define acoustofluidics: acoustic streaming and acoustic radiation forces. These effects have been used for particle, cell, and fluid manipulation at the microscale, although more recently SAW has been used to produce similar phenomena at the nanoscale through an entirely different set of mechanisms. Controllable nanoscale fluid manipulation offers a broad range of opportunities in ultrafast fluid pumping and biomacromolecule dynamics useful for physical and biological applications. Here, we demonstrate nanoscale-height channel fabrication via room-temperature lithium niobate (LN) bonding integrated with a SAW device. We describe the entire experimental process including nano-height channel fabrication via dry etching, plasma-activated bonding on lithium niobate, the appropriate optical setup for subsequent imaging, and SAW actuation. We show representative results for fluid capillary filling and fluid draining in a nanoscale channel induced by SAW. This procedure offers a practical protocol for nanoscale channel fabrication and integration with SAW devices useful to build upon for future nanofluidics applications.

We demonstrate fabrication of nanoheight channels with the integration of surface acoustic wave actuation devices upon lithium niobate for acoustic nanofluidics via liftoff photolithography, nano-depth reactive ion etching, and room-temperature plasma surface-activated multilayer bonding of single-crystal lithium niobate, a process similarly useful for bonding lithium niobate to oxides.

Controlled nanoscale manipulation of fluids is known to be exceptionally difficult due to the dominance of surface and viscous forces. Megahertz-order ...

Fabrication of Surface Acoustic Wave Devices on Lithium Niobate

Manipulation of fluids and particles by acoustic actuation at small scale is aiding the rapid growth of lab-on-a-chip applications. Megahertz-order surface acoustic wave (SAW) devices generate enormous accelerations on their surface, up to 108 m/s2, in turn responsible for many of the observed effects that have come to define acoustofluidics: acoustic streaming and acoustic radiation forces. These effects have been used for particle, cell, and fluid handling at the microscale&#8212;and even at the nanoscale. In this paper we explicitly demonstrate two major fabrication methods of SAW devices on lithium niobate: the details of lift-off and wet etching techniques are described step-by-step. Representative results for the electrode pattern deposited on the substrate as well as the performance of SAW generated on the surface are displayed in detail. Fabrication tricks and troubleshooting are covered as well. This procedure offers a practical protocol for high frequency SAW device fabrication and integration for future microfluidics applications.

Two fabrication techniques, lift-off and wet etching, are described in producing interdigital electrode transducers upon a piezoelectric substrate, lithium niobate, widely used to generate surface acoustic waves now finding broad utility in micro to nanoscale fluidics. The as-produced electrodes are shown to efficiently induce megahertz order Rayleigh surface acoustic waves.&#160;

Manipulation of fluids and particles by acoustic actuation at small scale is aiding the rapid growth of lab-on-a-chip applications. Megahertz-order ...

Fabrication and Characterization of Thickness Mode Piezoelectric Devices for Atomization and Acoustofluidics

We present a technique to fabricate simple thickness mode piezoelectric devices using lithium niobate (LN). Such devices have been shown to atomize liquid more efficiently, in terms of &#64258;ow rate per power input, than those that rely on Rayleigh waves and other modes of vibration in LN or lead zirconate titanate (PZT). The complete device is composed of a transducer, a transducer holder, and a &#64258;uid supply system. The fundamentals of acoustic liquid atomization are not well known, so techniques to characterize the devices and to study the phenomena are also described. Laser Doppler vibrometry (LDV) provides vibration information essential in comparing acoustic transducers and, in this case, indicates whether a device will perform well in thickness vibration. It can also be used to &#64257;nd the resonance frequency of the device, though this information is obtained more quickly via impedance analysis. Continuous &#64258;uid atomization, as an example application, requires careful &#64258;uid flow control, and we present such a method with high-speed imaging and droplet size distribution measurements via laser scattering.

Fabrication of piezoelectric thickness mode transducers via direct current sputtering of plate electrodes on lithium niobate is described. Additionally, reliable operation is achieved with a transducer holder and &#64258;uid supply system and characterization is demonstrated via impedance analysis, laser doppler vibrometry, high-speed imaging, and droplet size distribution using laser scattering.

We present a technique to fabricate simple thickness mode piezoelectric devices using lithium niobate (LN). Such devices have been shown to atomize liquid ...

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. Experimental observations suggest that the temporary permeabilization of these barriers may be due to shear stress that is exerted on cell tissues by cavitation microstreaming. Cavitation microstreaming is the generation of vortex flows which arise around oscillating ultrasound microbubbles. To produce such liquid flows, bubble oscillations must deviate from purely spherical oscillations and include either a translational instability or shape modes. Experimental studies of bubble-induced flows and shear stress on nearby surfaces are often restricted in their scope due to the difficulty of capturing shape deformations of microbubbles in a stable and controllable manner. We describe the design of an acoustic levitation chamber for the study of symmetry-controlled nonspherical oscillations. Such control is performed by using a coalescence technique between two approaching bubbles in a sufficiently intense ultrasound field. The control of nonspherical oscillations opens the way to a controlled cavitation microstreaming of a free surface-oscillating microbubble. High-frame rate cameras allow investigating quasi-simultaneously the nonspherical bubble dynamics at the acoustic timescale and the liquid flow at a lower timescale. It is shown that a large variety of fluid patterns may be obtained and that they are correlated to the modal content of the bubble interface. We demonstrate that even the high-order shape modes can create large-distance fluid patterns if the interface dynamics contain several modes, highlighting the potential of nonspherical oscillations for targeted and localized drug delivery.

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. ...

Assembly and Characterization of an External Driver for the Generation of Sub-Kilohertz Oscillatory Flow in Microchannels

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid samples, such as chemical reagents and cell cultures, is predominantly accomplished through steady flows that are typically driven by syringe pumps, gravity, or capillary forces. The use of complementary oscillatory flows is seldom considered in applications despite its numerous advantages as recently demonstrated in the literature. The significant technical barrier to the implementation of oscillatory flows in microchannels is likely responsible for the lack of its widespread adoption. Advanced commercial syringe pumps that can produce oscillatory flow, are often more expensive and only work for frequencies less than 1 Hz. Here, the assembly and operation of a low-cost, plug-and-play type speaker-based apparatus that generates oscillatory flow in microchannels is demonstrated. High-fidelity harmonic oscillatory flows with frequencies ranging from 10-1000 Hz can be achieved along with independent amplitude control. Amplitudes ranging from 10-600 &#181;m can be achieved throughout the entire range of operation, including amplitudes &#62; 1 mm at the resonant frequency, in a typical microchannel. Although the oscillation frequency is determined by the speaker, we illustrate that the oscillation amplitude is sensitive to fluid properties and channel geometry. Specifically, the oscillation amplitude decreases with increasing channel circuit length and liquid viscosity, and in contrast, the amplitude increases with increasing speaker tube thickness and length. Additionally, the apparatus requires no prior features to be designed on the microchannel and is easily detachable. It can be used simultaneously with a steady flow created by a syringe pump to generate pulsatile flows.

The protocol demonstrates a convenient method to produce harmonic oscillatory flow from 10-1000 Hz in microchannels. This is performed by interfacing a computer-controlled speaker diaphragm to the microchannel in a modular manner.

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid ...