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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 device^1,2. Precise cell manipulation (i.e., accumulation and separation) is critical in an LOC-based analysis and diagnosis^3,4. However, the accuracy and reproducibility of microparticle manipulation have a variety of challenges. Many techniques, such as electro-osmosis⁵, dielectrophoresis (DEP)⁶, magnetophoresis⁷, thermophoresis^8,9, an optical approach¹⁰, an optoelectronic approach¹¹, a hydrodynamic approach¹², and acoustophoresis^13,14,15, 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's law^{17,18,19,20,21,22}. 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 factor^22,25.

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 alternatively^27,28,29. 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 resonance^20,31. 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 design^32,33.

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.

Protokół

1. Preparation of the Microfluidic Channel

1. Mix poly-dimethylsiloxane (PDMS) with an elastomer base in a ratio of 10:1.
2. Degas the mixture in a vacuum oven and pour it on a silicon wafer with a negative tone photoresist pattern on the top.
3. Degas the patterned silicon wafer again and heat it at 70 °C for 3 h in an incubator for solidification.

2. Fabrication of the Interdigital Transducers

1. Deposit 20 nm of Cr and 400 nm of Al on a LiNbO₃ wafer; pattern 20 strips with a width of 150 µm and an aperture of 2 cm on a plastic mask for photolithography by depositing the positive photoresist on the substrate.
2. Remove the Cr-Al layer on the non-exposed area with acetone.
3. 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.
4. Align the PDMS microchannel and bond it to the LiNbO₃ substrate by pressing it with a thumb for a few seconds.
5. Place the integrated device in the heating chamber at 60 °C for 3 h.

3. Dual-frequency Excitation

1. Simultaneously apply two frequency components (f₁ and f₃, the fundamental frequency and its third harmonic of the fabricated IDT, respectively) with the phase difference φ between them to the pair of IDTs, so that the produced SAW may be expressed as follows.
   
   Here,
   and = the acoustic pressures.
2. 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.
3. Vary the power of the fundamental frequency to the total emitted power  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.
4. Vary the phase difference of the dual-frequency excitation from 0° to 360°.

4. Numerical Simulation

1. Describe the motion of the incompressible laminar flow with low Reynolds (i.e., Re = 0.55) and Mach numbers as follows³⁵.
   
   
   Here,
   = the fluid velocity,
   = the dynamic viscosity,
   = the fluid density,
   = the pressure on the fluid,
   = the identity matrix, and
   = an external force.
2. Describe the produced Stoke drag force on the object as follows³⁶.
   
   Here,
    = the radius of the microparticle,
    = the velocity of the fluid, and
    = the velocity of the microparticle.
3. 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 .
   
   Here,
   = the volume of the microparticle,
   = the density of the microparticle,
   = the density of the medium,
   = the compressibility of the microparticle, and
   = the compressibility of the medium.
4. Derive the resultant acoustic radiation force of the dual-frequency excitation as follows.
   
5. 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's second law as follows.
   
6. 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 µs and 20 s, respectively.

5. Experimental Observation

1. Spin the solution in the concentration of 5.9 x 10⁷ with 4 µm 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.
2. Fill the mixture into a 3 mL syringe and then drive it with a syringe pump at a flow rate of 3 - 5 µL/min.
3. Drive the IDT with the dual-frequency signal from a function generator followed by a power amplifier.
4. Observe the stabilized microparticles in the downstream microchannel under a light microscope at a 40X magnification and record the image with a digital camera.
5. 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.

Wyniki

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 µm in diameter) in a microchannel with a width of 300 µm at an acoustic power of 146 mW. The resultant acoustic pressure is always in phase when P₁ > 90% so that only one pressure node is pre...

Dyskusje

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 modes^34<...

Materiały

Name	Company	Catalog Number	Comments
poly-dimethylsiloxane	Dow Corning	Sylgard 184
poly-dimethylsiloxane elastomer base	Dow Corning	Sylgard 184
silicon wafer	Bonda Technology	SI8PSPD
negative tone photoresist	Microchem	SU-8
double-side polished LiNbO3 wafer	University Wafer	Y-128°
positive photoresist	Nicolaus-Otto-Straße	AZ 9260
oxygen plasma	Harrick Plasma
plastic mask	Infinite Graphics