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 LiNbO...

<ol>
	<li>Chin, C. D., Linder, V., Sia, S. 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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.

<table border="0" cellpadding="0" cellspacing="0" width="689">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">poly-dimethylsiloxane</td>
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
	</tbody>
</table>

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 ...

This method can help address key issues in the biomedical field, such as, manipulating of microparticles and assist in the sorting in a microfluidic channel for labs-on-a-chip. The main advantage of this technique is it can enhance the tunability of the commission of standing surface acoustic wave. Demonstrating the procedure will be Yannapol, a graduate student from my group.Begin with a mold for the microfluidic channel. This one is a negative tone photoresist pattern on a silicon wafer. More detail is provided by the photolithography pattern used to create it.Mix 10 parts PDMS with one part of elastomer base volume ratio. Degas the mixture in a vacuum oven for about 15 minutes. After degassing, take the mixture to the mold.There, pour it over the photoresist on the silicon wafer. Now, degas the covered silicon wafer for another 15 minutes. After that, heat the wafer in an incubator to solidify the PDSM.When the PDMS is cooled, remove it from the wafer. The microfluidic channel is ready for the next steps in the protocol. Prepare a substrate for fabricating the transducers.In this case, use a lithium niobate wafer four inches in diameter. At a spin coater, spin coat the wafer with positive photoresist. Then, use photolithography to pattern the photoresist with 20 150 nanometer strips in an aperture of two centimeters.After photolithography, this is the schematic depiction of the change in cross-section of the substrate. Once the photoresist is developed, sputter 20 nanometers of chromium onto the substrate followed by 400 nanometers of aluminum. Use acetone to remove the chromium and aluminum layer on the non-exposed areas.Take the substrate for surface treatment in oxygen plasma. Use a nitrogen to oxygen ratio of two to one with 30 watts of power for 60 seconds. When done, work with both the substrate and the microchannel.Align the PDMS microchannel and bond it with the lithium niobate substrate. Press the two together for a few seconds to bond them. Place the integrated device in the heating chamber at 60 degrees for three hours.After heating the chamber, the device is ready for studies using dual-frequency excitations of standing surface acoustical waves. For observations, use an inverted microscope with the device on its stage. Have the wafer with the interdigital transducers in contact with the stage and have the microchannel on top.Connect the transducers to the amplified signal of a function generator. Arrange for fluids from a syringe pump to pass over a magnetic stirrer. Prepare the solution for the experiments.Mix four micrometer polystyrene beads in deionized water. Spin the mixture in a vortex for two to three minutes. Follow this by placing the mixture in an ultrasound sonicator for 10 minutes.Transfer the mixture to a three milliliter syringe. Also add a stirrer bar to the syringe. Next, place the syringe on the syringe pump.Ensure the syringe is over the magnetic stirrer and is connected to the device inlet. Set the syringe pump flow rate to a few microliters per minute. Now, drive the device with the fundamental and third harmonic of the interdigital transducers.Observe the stabilized microparticles under the microscope and record images with the digital camera. Begin with no excitation and vary the phase difference between the two frequencies. Use the recorded images to determine the microparticle concentration at each pressure node.This is a plot of the pressure wave forms of a standing surface acoustic wave at the excitation frequencies of 6.2 and 18.6 megahertz. The vertical axis is the position along a 300 micrometer-wide microchannel. The curves represent different power ratios, the percentage of power in the fundamental mode versus the total power of 146 milliwatts.Here is the acoustic radiation force applied to four micrometer microspheres in the same channel under the same power conditions. At a power ratio above 90%the force is always in phase and produces a single pressure node at the 150 micrometer position in the channel. Additional nodes appear for a power ratio below 90%When four micrometer polystyrene microspheres are initially placed at a wall of the channel, their motion is determined by the power ratio.Note:For power ratios above 90%the particles go to the central node. For power ratios of 90%and below, they go to the side nodes. Comparison of the experimental data, plotted using symbols, with simulations plotted with dashed lines, for particle position as a function of the power ratio yields good agreement.Note this plot shows both the upper and lower side nodes. Similar agreement is evident in a plot of the particle concentration as a function of the power ratio. While attempting this procedure, it is important to remember to tune the standing acoustic wave in the microfluidic channel by adjusting the power ratio of the dual-frequency excitation.After its development, this technique paved the way for researchers in the field of biomedical engineering to explore rapid, effective and flexible manipulation of microparticles assist in labs-on-a-chip. Don't forget that working with electric instruments can be extremely hazardous and that precaution to prevent electric shock should always be taken while performing this procedure.

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6.9K visualizações.  Nanyang Technological University. This method can help address key issues in the biomedical field, such as, manipulating of microparticles and assist in the sorting in a microfluidic channel for labs-on-a-chip. The main advantage of this technique is it can enhance the tunability of the commission of standing surface acoustic wave. Demonstrating the procedure will be Yannapol, a graduate student from my group.Begin with a mold for the microfluidic channel. This one is a negative tone photoresist pattern on a silicon wa...