Acoustic Radiation and Working Principle

Figure1 shows the schematic illustrationof the developedmicrofluidic device and the working principle of size-based particleseparation using a SSAW field. The SSAW generator is basically a pairof interdigital transducers (IDTs) patterned on a piezoelectric substrate.A microchannel is located between the two IDTs to form the microfluidicdevice. When an ac signal is applied to the IDTs, two series of identicalSAWs propagate toward the microchannel in opposite directions. Constructiveinterference of the two SAWs gives rise to a SSAW field across thechannel. Particles through the SSAW field are subjected to a time-averagedacoustic radiation force, given as³¹

1

In the above,V_p is the volume of the particle, ρ_f isthe densityof the fluid medium,c_f is the speed ofsound in the fluid medium, β = ρ_p/ρ_f is the density ratio, and γ =c_p/c_f is the speed of sound ratio.The subscripts “p” and “f” denote, respectively,the material parameters for solid particle and fluid medium. p isthe pressure generated by the acoustic wave,k =ω/c_f is the wavenumber with ωdenoting the angular frequency. Considering a one-dimensional modelwithx denoting the distance from the pressure node,the pressure can be expressed as

2

wherep₀ is thepressure magnitude. Substituting eq2 into eq1, we can get

3

The time average term, ⟨cos²(ωt)⟩ = 1/2, can further simplifyeq3 as³²

4

where

5

is the acoustic contrast factor. When φ(β,γ)> 0, the acoustic radiation force pushes particles to the pressurenode where the pressure change is always zero. On the contrary, particlesare attracted to the antipressure node when φ(β,γ)< 0. In general, most solid particles and biological cells suspendedin aqueous solutions have a positive acoustic contrast factor andare thus attracted to the pressure node.⁸ The resonant frequency of the generated SSAW mainly depends on thedistance between two adjacent electrode fingers of the IDTs. In thisdevice, the wavelength of the resonant SSAW is approximately twicethe channel width, and the pressure nodes are located at the two sidewallsof the channel, as shown in Figure1. The entranceof the device has three inlets with the particle mixture solutionin the middle and the sheath flow at the two sides. The faster sheathflow hydrodynamically focuses the particle mixture into a very narrowstream along the centerline of the channel, as shown in Figure1. When particles enter the SSAW field, the size-dependentacoustic radiation force starts to attract particles into the pressurenode. Larger particles laterally move to the pressure node at thesidewalls faster than smaller particles, resulting in the size-basedparticle separation. Therefore, larger particles are switched to theside outlets and are accordingly separated from smaller particlesflowing into the middle outlet, as shown in Figure1.

Numerical Modeling

A finite element method (FEM) basednumerical model (COMSOL Multiphysics 4.3,www.comsol.com) was developed to study the acoustic-piezoelectric interaction problem.As the SSAW is uniform in the longitudinal direction of the channel,we simply considered a 2D modeling of the device cross-section ina frequency analysis. Therefore, the acoustic-piezoelectric interactionmodule at frequency domain was selected to perform the modeling ofthe developed SSAW-based device. A PDMS layer was located on the topof a piezoelectric substrate, and a tiny fluid layer was sealed betweenthem. The propagation of SAW in a piezoelectric substrate is governedby the Maxwell’s equations for electric field and the stress–strainequations for mechanical motion. The linear piezoelectric constitutiveequations are given as

6

7

whereT is the mechanical stressvector,C_E is the elasticity matrix,S is the strain vector,e is the piezoelectricstress matrix,E is the electric field vector,D is the electric displacement vector, andε is the dielectric matrix. The superscript “tr” representsthe transpose of the matrix. The acoustic pressure field in the fluidand PDMS domains is governed by the well-known Helmholtz equation,

8

where

9

In the above, ρ_i,c_i, and α_i are the density, speedof sound, and acousticattenuation coefficient in the corresponding domain,j = (−1)^1/2 is the imaginary unit. The acousticvelocity is given by

10

For theelectric field in the piezoelectricsubstrate, a sinusoidal ac signal with a peak-to-peak magnitude of10 V was applied to the interdigital electrodes on the piezoelectricsubstrate. The other boundaries of the piezoelectric substrate wereassumed zero charge surfaces. For the elastic mechanical motion arisingfrom the piezoelectric effect, the surface with interdigital electrodes,excluding boundaries in contact with the fluid and PDMS domains, wasset to free, referring to no force loads or constraints. Because ofthe interaction between the elastic mechanical motion and acousticpressure field, a force load was applied on the boundaries in contactwith the fluid and PDMS domains,

11

wheren is the unite normal vectorof the applied boundaries. Zero normal displacement was applied onall the other boundaries. The harmonic vibration of the piezoelectricsubstrate propagated acoustic waves into the fluid and PDMS at theinterface, which is mathematically described by an acceleration boundarycondition

12

Strictly speaking,the standing acoustic wavefield in the fluid across the channel was actually generated by theSAWs radiated into the fluid from the piezoelectric substrate. Theouter surface of the PDMS domain was specified as a sound hard boundary

13

Acoustic pressure and velocitywere continuousacross the interface between the fluid and PDMS. Simulations withdifferent mesh sizes were implemented to ensure that numerical resultswere converged and mesh-independent. A coarse mesh with a size of20 μm was generated near the bottom of the piezoelectric substrate,and a fine mesh with a size of 3 μm was employed in the microchannelto accurately capture the pressure nodes of the SSAW field.

Chip Fabrication

The PDMS channel in the device wasfabricated using a standard soft lithography technique.¹¹ Briefly, a 25 μm thick negative photoresist(SU-8 25, MicroChem Corp., Newton, MA) was first spin-coated on aclean glass slide, followed by a two-step soft bake (65 °C for3 min and 95 °C for 7 min). The photoresist covered by a 20 000dpi mask with a channel pattern was then exposed to 365 nm ultravioletlight with an energy density of 150 mJ/cm² (Figure2a), followed by another two-step hard bake (65 °Cfor 1 min and 95 °C for 3 min). Subsequently, a master mold wasobtained by developing the photoresist in a commercial SU-8 developersolution for 4 min (Figure2b). Degassed PDMSmixture (Sylgard184 Silicone Elastomer Kit, Dow Corning Corp., Freeland,MI) of prepolymer and curing agent at a ratio of 10:1 by weight werepoured over the master and cured at 65 °C for 4 h (Figure2c). The fully cured PDMS was peeled off from themaster mold. Inlet and outlet holes were created using a small drillbit for external tubing interconnection. The IDTs for SSAW generationwere fabricated on a 128° rotated Y-cut X-propagating lithiumniobate (LiNbO₃) piezoelectric substrate using a lift-offtechnique. Basically, the LiNbO₃ substrate was first spin-coatedwith a 1.25 μm thick positive photoresist (AZ 5214E-IR, CapitolScientific, Dallas, TX), followed by a soft bake (100 °C for60 s). The mask-covered photoresist was patterned by exposing to theultraviolet light with an energy density of 70 mJ/cm²,followed by a 35 s development in AZ 300 MIF developer solution (Figure2d). A double metallic layer (Cr/Au, 5 nm/80 nm)was then deposited onto the developed LiNbO₃ substrateby an electron beam evaporator (Figure2e).Subsequently, the LiNbO₃ substrate was sonicated in acetonefor half an hour to remove photoresist and undesired Cr/Au layer onits top (Figure2f). The previously obtainedPDMS substrate and the patterned LiNbO₃ substrate wereloaded into an oxygen plasma cleaner (Harrick Plasma Inc., Ithaca,NY) for surface activation (Figure2g). Later,the two substrates were well aligned under a microscope with the assistanceof markers on both substrates and were eventually brought into contactto form permanent bonding (Figure2h).

Experimental Setup

Figure3 showsthe image of the final microfluidic device. The width and height ofthe main channel are 120 and 25 μm, respectively. The lengthsof the main channel and IDTs are 15 mm and 9 mm, respectively. EachIDT has 20 electrode finger pairs with 300 μm finger pitch and75 μm finger width, corresponding to a SAW wavelength of λ= 300 μm. The channel width is slightly smaller than the halfwavelength, which aims to provide a reasonable tolerance for the alignmentprior to the permanent bonding. Therefore, a tight alignment of IDTswith the microchannel is not critical to achieve an efficient separation.The speed of sound in LiNbO₃ substrate is approximately3900 m/s, leading to a resonant frequency around 13 MHz. In practice,the best resonant frequency of the fabricated IDTs was found to be13.0168 MHz by an impedance analysis. A sinusoidal ac signal at theresonant frequency was generated by a signal generator (Tektronix,Beaverton, OR) and then amplified by a power amplifier (OPHIR RF,Los Angeles, CA). The IDTs were excited by the amplified ac signalto generate a SSAW field across the channel. The device was loadedon the stage of a fluorescence microscope (Zeiss Axioskop Microscope)to conduct the separation experiments. Particle mixtures (cell mixtures)and DI water (1× phosphate buffered saline solution) were, respectively,injected into the device through the inlets labeled “particlemixture” and “sheath flow” using syringe pumps(New Era Pump Systems Inc., Farmingdale, NY). In order to avoid particleor cell adhesion to the channel wall, all the solutions were mixedwith 0.5% surfactant of Pluronic F68 (Invitrogen Corp., Carlsbad,CA). Particle motion and separation were captured and recorded at30 frames per second by a Sony camcorder installed on the microscope.Fluorescence filters were manually switched to visualize a specificexcitation and emission light of fluorescent particles or stainedcells. The recorded videos were later processed by a free image processingprogram, ImageJ (National Institutes of Health,http://rsbweb.nih.gov/ij/).

Sample Preparation

Two differentparticle mixtureswere used in the separation experiments. The first mixture includedtwo types of fluorescent synthetic microspheres (Particle I, 1.2 μmin diameter, green emission; Particle II, 5.86 μm in diameter,red emission, both from Polysciences Inc., Warrington, PA). Both particleswere diluted to a concentration of 2 × 10⁷ particles/mL.The second sample was a mixture of purified human PBMCs (BioreclamationLLC, Hicksville, NY) andE. coli bacteria. PBMCswere washed and then fixed for 15 min using 2% formaldehyde in phosphatebuffered saline (PBS) solution. Cells were stained with 1 μg/mLHoechst 33342 (Calbiochem, Billerica, MA) for 10 min, spun down andresuspended in PBS. The average size of PBMCs is approximately 7.23μm in diameter. Thermostable green protein (TGP, ECGP123 variant)expressing BL21E. coli bacteria were induced withisopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h at 30 °Cand then grown on kan agar overnight at 37 °C.³³ Later, the bacteria were resuspended in PBS and mixed withthe PBMC solution.E. coli bacteria are typicallyrod-shaped with a diameter of 0.5 μm and a length of 2 μm,which has a similar volume as a sphere with a diameter of 1.1 μm.The concentration of both cells is approximately 3 × 10⁶ particles/mL. Separation purity of the cell mixture was determinedby a flow cytometric analysis (LSR II, Becton Dickenson, San Jose,CA). Pure samples were used to calibrate the settings and also excludethe debris from the cytometric results. A combination of relativesize based on light scatter and whether the cells were positive forHoechst or TGP-10 was used to evaluate purity.

Simulationof SSAW Field

To theoretically verify thedesign of the SSAW-based particle separation, we first applied theFEM model to simulate the SAW propagation and acoustic pressure fieldgenerated on the cross-section of the fabricated device. A frequencyanalysis from 12.2 to 13.2 MHz was performed to find the resonantfrequency, which was around 12.98 MHz and agreed well with the practicalvalue. The attenuation coefficients of water and PDMS at 13 MHz are36.67 dB/m³⁴ and 8224 dB/m,³⁵ respectively. Figure4a shows the numerical result of the acoustic velocity field on thecross-section of the device, in which the fluid and PDMS are locatedin the midpoint between two IDTs. Two identical SAWs propagate fromboth sides toward the fluid and PDMS. When the SAW first encountersthe PDMS, it partially radiates into the PDMS at a Rayleigh angle,θ_R = arcsin (c_PDMS/c_L), wherec_PDMS andc_L are, respectively, the speed of sound in thePDMS and LiNbO₃. As the two individual SAWs travel furtherand meet each other, the constructive interference of the two SAWsforms a SSAW field in the LiNbO₃ and also across the fluidand PDMS layers, as shown in Figure4a. Thedistance between two adjacent peaks is half of the SSAW wavelength.It is calculated from the numerical modeling that the SSAW wavelengthin the LiNbO₃ is about 300 μm, equal to the designedIDT pitch. The SSAW in the PDMS layer along the vertical directionhas a shorter wavelength because the speed of sound in the PDMS islower than that in the LiNbO₃. Because of the attenuationeffect, the SSAW strength gradually decreases as it is located furtherfrom the interface between PDMS and LiNbO₃. Figure4b shows the resulting acoustic pressure field insidethe fluid. The pressure node with a zero magnitude is a single planeperpendicular to the LiNbO₃ substrate, located in the horizontalcenter of the channel. As a result, suspended particles with a positiveacoustic contrast factor are attracted to the middle of the channel,referring to the acoustic focusing effect.¹² In Figure4c, the fluid and PDMS are shifted75 μm toward the IDT on the right-hand side. Similarly, a SSAWfield is generated over the entire cross-section of the device. However,the pressure node is shifted to the two sidewalls of the channel,as shown in Figure4d. Therefore, the locationof the pressure node across the channel can be precisely controlledduring the alignment process prior to the permanent bonding. To verifythe location of the pressure node in the fabricated device, a highlyconcentrated 5.86 μm Particle II was injected into the channeland eventually became stationary with a random distribution. A SSAWfield was subsequently turned on with a power of 23.8 dBm (1 dBm =10 log(U), whereU is the inputpower applied on the IDTs in the unit of mW). Movie file 1 in theSupporting Information shows that all the particleswere quickly attracted and accumulated at the two sidewalls of thechannel once the SSAW field was turned on. This observation confirmedthat the pressure node in this device is located at the sidewalls.Some particles, initially located at the same distance from one ofthe sidewalls, moved toward the sidewall at different speeds. Thisphenomenon may be attributed to the nonuniform acoustic pressure fieldat different heights of the channel, which was verified by the numericalresults shown in Figure4b,d.

Separation of Synthetic Microspheres

Next, the separationof the first mixture (1.2 μm Particle I and 5.86 μm ParticleII) was demonstrated using the fabricated device. The flow rates ofthe particle mixture and the sheath flow were 0.2 μL/min and1.6 μL/min, respectively. The sheath flow was later evenly splitinto two side streams at the inlet junction (Figure5a); therefore, the flow rate of each individual side sheathflow was 0.8 μL/min. As the inlet junction is away from theSSAW field, a pure hydrodynamic focusing of both particles to thecenterline of the channel was observed (Figure5a,b). When the SSAW field was turned off, both particles kept flowingnear the centerline along the entire channel due to the nature ofa laminar flow. Therefore, both particles flowed into the middle outlet(Figure5c,d). The motion of both particlesat the outlet junction without a SSAW field can be seen from moviefile 2 in theSupporting Information. Later,a SSAW field with a power of 23.8 dBm was turned on. When the particlemixture entered the SSAW field, the acoustic radiation force actingon the 5.86 μm Particle II was much greater than that actingon the 1.2 μm Particle I. Therefore, the larger Particle IIwas pulled out of the particle mixture, laterally moving toward thepressure node at the sidewalls. The acoustic radiation force was maximizedat the antipressure node and gradually decreased to zero when approachingthe pressure node. As a result, the lateral movement stopped at thepressure node, and Particle II exactly followed the sheath flow thereafter.The acoustic radiation force acting on the smaller Particle I wasinsufficient to pull it out of the middle stream before leaving theSSAW field. Thus, Particle I remained near the centerline at the outletjunction and flowed into the middle outlet (Figure5e). Particle II, however, was switched to the side outlets(Figure5f), indicating a successful separation.The separation of the two particles using a SSAW field can be seenfrom movie file 3 in theSupporting Information.

Figure6 shows the particle trajectoriesin the middle region of the SSAW field with different input powers.When the SSAW field was turned off, the two particles both remainednear the centerline (Figure6a,b), exactlyas they were at the inlet junction. When a SSAW field with a powerof 19.3 dBm was turned on, Particle I stayed near the centerline dueto a weak acoustic radiation force and an insufficient SSAW exposuretime (Figure6c). In contrast, Particle IIstarted to laterally shift toward the two sidewalls (Figure6d). When the power of SSAW field was further increasedto 25.3 dBm, the stream width of Particle I became larger as a resultof a minor lateral movement (Figure6e). Meanwhile,Particle II was further laterally shifted near the sidewalls becauseof an increased acoustic radiation force (Figure6f). An efficient SSAW-based separation relies on a sufficientdifference in lateral movements of dissimilar particles. In orderto achieve the SSAW-based separation at high flow rates, one can increasethe input power or maintain a sufficient SSAW exposure time by extendingthe SSAW field.

Separation ofE. coli and PBMCs

Finally,we used this device to separateE. coli bacteriafrom PBMC samples. Both cells were found to move toward the pressurenode when exposed to a SSAW field, indicating a positive acousticcontrast. We increased the flow rates of both cell mixture and sheathflow to test the throughput of the developed device. Hence, the powerapplied on the IDTs was increased to maintain a sufficient lateralmovement for PBMCs. The optimum acoustic power was determined by observingthe cell separation at the outlet junction under the microscope. Thefluorescence of stainedE. coli bacteria was notvery bright, and when the flow rate of the cell mixture was too high,it was quite difficult to observe the trajectory ofE. coli bacteria. Therefore, the visibility of the cell separation processlimits the throughput of the developed device. To clearly visualizethe separation process, the maximum flow rate of the cell mixtureshould not exceed 0.5 μL/min, which was used in the separationofE. coli and PBMCs. Accordingly, the flow rateof the sheath flow was adjusted to 4 μL/min to maintain a highlyfocused middle stream before entering the SSAW field. A SSAW fieldwith a power of 26.7 dBm was turned on for about 4 h to separate thetwo cells in a continuous flow. The separatedE. coli bacteria and PBMCs were, respectively, collected from the outletslabeled “Outlet A” and “Outlet B”, asshown in Figure3. The premixture and collectedsamples were analyzed in a flow cytometer to quantify the respectivecell contents. The ratio of each cell type was defined as the numberof corresponding cells detected through the flow cytometer to thetotal number of counted cells. The cell populations were plotted interms of forward scatter (FSC) and side scatter (SSC) to show thecell content in each sample. Figure7a confirmsthat the premixture mainly consisted ofE. coli andPBMCs with very little debris. The ratios ofE. coli and PBMCs in the premixture were, respectively, 46.23% and 53.06%(Figure7d), as they were intended to mix ata similar cell concentration. After flowing through the SSAW field,E. coli bacteria were successfully extracted from the premixture,as shown in Figures7b and7c. The ratios ofE. coli and PBMCs in thesamples collected from the outlets labeled “Outlet A”and “Outlet B” were, respectively, 95.65% and 91.48%(Figure7d). The difference in density andcompressibility ofE. coli and PBMCs could resultin the different acoustic contrast factors and eventually affect theacoustic radiation forces acting on the cells. However, we found thatthe cell size remains the key factor influencing the separation ofE. coli from PBMCs. These results demonstrated that thedeveloped device could effectively separate synthetic particles orbiological cells based on their sizes using a SSAW field.

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Supplementary Materials