석사 학위논문 Master's Thesis

집중적인 표면 진행 탄성파를 이용한 입자

분리 및 화학적 구배 제어

Particle Separation and Chemical Gradient Control via

Focused Travelling Surface Acoustic Waves

굴람 데스트기르 (Ghulam Destgeer) 기계항공시스템학부 기계공학전공

School of Mechanical Aerospace & Systems Engineering, Division of Mechanical Engineering

KAIST

2013

집중적인 표면 진행 탄성파를 이용한 입자

분리 및 화학적 구배 제어

Particle Separation and Chemical Gradient Control via

Focused Travelling Surface Acoustic Waves

Particle Separation and Chemical Gradient Control via Focused Travelling Surface Acoustic Waves

Advisor: Professor Sung, Hyung Jin by

Ghulam Destgeer

School of Mechanical Aerospace & Systems Engineering, Division of Mechanical Engineering

KAIST

A thesis submitted to the faculty of KAIST in partial fulfillment of the re-

quirements for the degree of Master of Science and Engineering in the School of

Mechanical Aerospace & Systems Engineering, Division of Mechanical Engi-

neering. The study was conducted in accordance with Code of Research Ethics1

2013. 06. 10

Approved by

Professor Sung, Hyung Jin

Advisor

1 Declaration of Ethical Conduct in Research: I, as a graduate student of KAIST, hereby declare that I have not committed any acts that may damage the credibility of my research. These include, but are not limited to: falsifi-cation, thesis written by someone else, distortion of research findings or plagiarism. I affirm that my thesis con-tains honest conclusions based on my own careful research under the guidance of my thesis advisor.

집중적인 표면 진행 탄성파를 이용한 입자

분리 및 화학적 구배 제어

굴람 데스트기르 (Ghulam Destgeer)

위 논문은 한국과학기술원 석사학위논문으로

학위논문심사위원회에서 심사 통과하였음.

2013 년 6 월 10 일

심사위원장

심사위원

심사위원

성형진 (인)

김상수 (인)

이봉재 (인)

MME 20114526

굴람 데스트기르. Ghulam Destgeer. Particle Separation and Chemical Gradient Control via Focused Travelling Surface Acoustic Waves (F-TSAW). 적응적 임계치와 영역 확장을

이용한 맘모그램 영상의 종괴 검출 방법. School of Mechanical Aerospace & Systems Engineering, Division of Mechanical Engineering. 2013. 93 p. Advisor Prof. Sung, Hyung Jin.

Abstract A novel focused travelling surface acoustic waves (F-TSAW) based micro-chip is demonstrated here to

continuously separate microparticles, generate chemical gradient, and uniformly mix fluids inside a PDMS mi-

crofluidic channel. A pair of curved interdigitated electrodes deposited on a piezoelectric substrate (LiNbO3)

produced unidirectional F-TSAW when high frequency AC signal is applied across the terminals of the trans-

ducer. F-TSAW, when interacted with the fluid inside the microfluidic channel, imparted acoustic radiation force

(ARF) to the suspended microparticles and induced acoustic streaming flow (ASF). ARF is used to separate the

particles whereas ASF generated chemical gradient and uniformly mixed fluids. Previously reported acousto-

fluidic based particle separators use standing surface acoustic waves (SSAW) whereas chemical gradient gener-

ator and micro-mixer depends on acoustic streaming induced by oscillating bubbles. F-TSAW based micro-chip

did not require a trapped bubble and a cumbersome microchannel alignment step, which is essential for the work-

ing of SSAW particle separator, is also eliminated. All three functions – separation, gradient generation and mix-

ing – are performed on a single micro-chip with performance comparable with already reported devices. After

selecting appropriate microchannel (w×h: 200µm×40µm) and flow rate (100µL/h–1200µL/h or 3.5mm/s–

41.67mm/s), variable size polystyrene particles suspended in DI water are successfully separated with power

input in excess of 235mW (23.7dBm) and separation efficiency of 100%. ARF separated microparticles with

diameter 10µm from 3µm by inducing an acoustophoretic separation distance whereas effect of ASF is negligible

with the aforementioned experimental conditions. The trajectory of separated microparticles is slightly effected

by the ASF but it does not affect the separation efficiency. F-TSAW micro-chip harnessed ASF to generate chem-

ical gradient and mix fluids inside a microchannel. The dimensions of microchannel (w×h: 500µm×90µm) are

critical to generate high velocity ASF. At flow rate of 100µL/h (0.6mm/s) (fluid 1, rhodamine: 50µL/h, fluid 2,

DI water: 50 µL/h) and power input of 60–200mW (18–23dBm), chemical gradient profile is effectively con-

trolled. To uniformly mix two fluids flowing in the microchannel, acoustic device is actuated with a high power

input of 800mW (29dBm) while maintaining a total flow rate of 100µL/h. Oscillating-bubbles based devices

induce stronger ASF but require fragile trapped bubbles for functionality, whereas F-TSAW micro-chip operated

without any trapped bubbles.

Keywords: Microfluidics, Acoustofluidics, Particle separation, Micromixing, Gradient generation

i

Table of Contents

Abstract ......................................................................................................................................... i

Table of Contents .......................................................................................................................... ii

List of tables .................................................................................................................................iii

List of figures ............................................................................................................................... iv

1. Introduction........................................................................................................................... 1

1.1. Particle separation ........................................................................................................... 1

1.2. Chemical gradient generation ......................................................................................... 3

2. Theory .................................................................................................................................... 5

2.1. Surface acoustic waves (SAW) ....................................................................................... 5

2.2. Focusing unidirectional transducer (FUT) ...................................................................... 9

3. Device fabrication and experimental setup ...................................................................... 11

3.1. Device fabrication ......................................................................................................... 11

3.2. Experimental setup ....................................................................................................... 12

4. Results .................................................................................................................................. 14

4.1. Particle separation by CAPS 1-3 ................................................................................... 14

4.1.1. Microchannel Alignment ................................................................................... 17

4.1.2. Deflection of particles ........................................................................................ 18

4.2. Chemical gradient control ............................................................................................. 19

5. Summary and discussion .................................................................................................... 21

References .................................................................................................................................... 40

ii

List of tables

Table 1: Experimental parameters of F-TSAW micro-chip ...................................................................... 22

iii

List of figures

Figure 1: Tree diagram summarizes the commonly used separation techniques in distinct categories. The

current research route is shown in red. ....................................................................................... 23

Figure 2: Schematic of particle separation by F-TSAW is shown. ............................................................ 24

Figure 3: (a-c) Schematic of F-TSAW micro-chip – particle separation, chemical gradient generation and

uniform mixing inside a PDMS microchannel is shown (d) F-TSAW amplitude produced by

focusing unidirectional transducer (UDT) (e) Fabricated F-TSAW micro-chip......................... 24

Figure 4: Interdigitated transducer (IDT): metal electrodes deposited on a piezoelectric substrate when

given an AC signal produces surface acoustic waves (SAW) .................................................... 25

Figure 5: When AC signal is applied across the IDT terminals, particles of solid piezoelectric substrate

revolve in an elliptical trajectory as the SAW propagates on the surface of substrate (Courtesy

Dan Russell, 2011) ..................................................................................................................... 25

Figure 6: Two travelling waves propagating in opposite directions when interfere forms a standing wave

(Courtesy Dan Russell, 2011) .................................................................................................... 25

Figure 7: Comparison of acoustic radiation force by SSAW and TSAW on polystyrene particles dispersed

in water: (a) For constant wavelength of 30µm (b) For particle radius of 5µm while wavelength

is increased from 30 to 100µm. .................................................................................................. 26

Figure 8: Schematic of focusing Interdigitated Transducer (IDT) ............................................................. 26

Figure 9: Estimated F-TSAW profile: waves are focused in a narrow region by the circular electrodes.

Black color shows high wave amplitude region. ........................................................................ 27

Figure 10: F-TSAW amplitude vs. z plotted at a plane passing through the center of focusing IDT and

perpendicular to substrate surface and microchannel. ................................................................ 27

Figure 11: (a) Interdigitated Transducer (IDT): Metal electrodes are uniformly spaced with electrode width

and gap equal to /4 as is the SAW wavelength. Acoustic energy is equally transmitted in

either direction. (b) Single Phase Uni-Directional Transducer (SPUDT): Metal electrodes width

iv

and gap is adjusted in such a way that maximum acoustic energy is transmitted in the forward

(right) direction whereas very little energy is transferred in the backward direction. ................ 28

Figure 12: (a) Fabrication of PDMS microchannel via soft lithography process (b) Deposition of Ti/Au

metal electrode on a LiNbO3 substrate by e-beam evaporation method (c) Oxygen plasma

bonding of PDMS microchannel with LiNbO3 substrate. .......................................................... 28

Figure 13: F-TSAW based micro-chip after PDMS microchannel (green) is bonded to LiNbO3 substrate

(blue) while Au electrodes (yellow) are also shown. ................................................................. 29

Figure 14: Microchannel drawings for particle separation: (a) Device#1 and 2 (b) Device#3. (c) Device#4:

Microchannel drawing for micromixing and gradient control. .................................................. 29

Figure 15: Metal electrode drawings of focused unidirectional transducer with operating frequemcy: (a)

40MHz for Device #1 (b) 133.3MHz for Device#2-4. ............................................................... 30

Figure 16: Experimental setup for acoustofluidics. Signal generator and power supply (Agilent), amplifier

(miniCircuits), camera and microscope (Olympus), micro syringe pump (neMESYS) ............. 30

Figure 17: CAPS-2: (a) Schematic diagram of a PDMS microchannel. (b-c) The separation of 3 µm particles

and 10 µm particles was achieved under a flow rate of 150 µL/h and an input power of 275mW.

Particles are indicated by the dotted circles. When the TSAW was OFF, no significant separation

distance was observed between the particles of the two sizes. Once the TSAW was turned ON, a

distinct separation distance could be observed. (d) Trajectory followed by a 10 µm particle

influenced by acoustic streaming. .............................................................................................. 31

Figure 18: CAPS-2: Separation efficiency. (a) TSAW OFF: nearly all of the particles flowed through outlet

1. (b) TSAW ON: 3µm particles were collected at outlet 1, whereas 97.2% of the 10µm particles

passed through outlet 3. (Sample size: 2,000 3µm particles per 150 10µm particles.) .............. 32

Figure 19: F-TSAW absolute amplitude estimation for two separate actuation frequencies i.e. 133.3MHz

and 40MHz. Line plots depict acoustic wave amplitude at locatinos z=1.0R, 1.5R, 2.0R and

2.5R. ........................................................................................................................................... 32

Figure 20: CAPS-1: F-TSAW absolute amplitude profile is shown along the trajectory followed by a 30µm

diameter particle under the influence of acoustic radiation force theoretically (left) and observed

v

experimentally (center). The separation of 10µm diameter particle from 30µm diameter particle

is shown on right. ....................................................................................................................... 33

Figure 21: CAPS-3: Particle separation is captured by dark field imaging and several images are stacked

together to obtain the path line followed by a 10µm diameter particle. Polystyrene microparticles

with diameter 10µm are shown to be separated from 3µm by F-TSAW. ................................... 34

Figure 22: CAPS-3: When the TSAW is OFF (0mW), all of the particles flowed through outlet 1. When the

TSAW is turned ON (151mW), 3µm particles were collected at outlet 1, whereas 100% of the

10µm particles passed through outlet 3. ..................................................................................... 35

Figure 23: Microchannel alignment with FUT: L-shape markers are aligned with the side walls of acoustic

window ....................................................................................................................................... 35

Figure 24: TSAW alignment with the separation zone. A: TSAW amplitude profile. B: Separation zone

along with time steps is shown. C: Trajectory followed by 3µm (left) and 10µm (right) particles.

D: Acoustic streaming flow traced by 1µm particle. The arrows show the vortices formed by the

flow. E: Particle Image Velocimetry. Image C, D and E are obtained through ImageJ

Software. .................................................................................................................................... 36

Figure 25: Particle deflection is plotted against input power for CAPS-3: (a) Flow rate is kept constant at

100µL/h [Sheath + Sample = 80µL/h + 20µL/h]. (b) For particles with diameter 10µm while

flow rate is increased from 100µL/h up to 300µL/h. The inset shows deflection vs. flow rate for

constant input power. ................................................................................................................. 36

Figure 26: Particle deflection is plotted for variable input power. (Top) For 10µm particle. (Bottom) For

7µm particle. For a comparison, dashed rectangles are sketched for input power of 217mW and

variable flow rate. It is evident that 10µm particles experience significantly more deflection

distance than 7µm particles. This ensures the separation of these particles. .............................. 37

Figure 27: Acoustic streaming flow induced via F-TSAW is traced by 1µm polymer microspheres dispersed

in DI water. F-TSAW are propagating from right to left. (a) Left four images are captured at

consecutive time intervals where the microchannel is 50µm wide. (b) Right three images are for

microchannels 150µm x 45µm, 200µm x 40µm and 500µm x 90µm from left to right,

vi

respectively................................................................................................................................. 38

Figure 28: CAGG: Chemical gradient generation and uniform mixing of fluids. (a) Two fluids flowing

parallel in a microchannel being interrupted by F-TSAW. (b) Upstream mixing by diffusion. (c-

d) Concentration profile (normalized light intensity) varies downstream as the input power is

increased. ................................................................................................................................... 39

vii

1. Introduction

Over the last two decades, microfluidics has obtained significant popularity in the fields of diag-

nostics, biochemistry, pharmaceuticals, bioengineering, analytical chemistry and medicine because of its po-

tential in developing Micro Total Analysis Systems (µTAS), Lab on a Chip technology, Point of Care (POC)

devices and rapid diagnostics tools. In the race to develop novel microfluidic tools, Acoustofluidics is compet-

ing with other technologies like Optofluidics,1–11 Magnetofluidics,12 Dielectrophoresis,13 and Inertial Microflu-

idics.14 A summary of most commonly used particle or cell separation techniques along with their classification

is shown in a tree diagram (see Figure 1). Acoustofluidics based systems make use of ultrasonic waves for its

functionality and are broadly categorized as bulk acoustic waves (BAW)15–18 and surface acoustic waves

(SAW)19–26 based systems. Acoustofluidics based micro-object manipulation, rapid micromixing27 and efficient

chemical gradient control28 have certain advantages over other methodologies viz. biocompatibility, easy func-

tionality, amenability to miniaturization, integration with other microfluidics components, well developed fab-

rication techniques and cost effectiveness. Since the 2nd half of last decade, Surface Acoustic Waves (SAW)

based micro-object manipulation has gained due attention of acoustofluidics research community.29

In the present work, we have demonstrated an acoustofluidics based micro-chip to continuously

separate particles and generate chemical gradient profiles in a PDMS microfluidic channel. In the following

subsections, we have briefly summarized previous literature related to particle separation and chemical gradient

control.

1.1. Particle separation

Microparticulate separation techniques are of fundamental importance in many research fields,

including cell biology, diagnostics, and therapeutics.30 Many separation techniques have been developed to

date, including techniques based on pinched flow fractionation,31 hydrodynamic filtration,32 inertial microflu-

idics,33 deterministic lateral displacement,34 fluorescent activated cell sorting,35 magnetophoresis,36 optofluid-

- 1 -

ics,8,10,37 dielectrophoresis,13,38 or acoustofluidics.29,39,40 Acoustofluidics particle separation techniques are la-

bel-free and biocompatible, permitting the manipulation of living organisms. The low power requirements of

acoustofluidics devices facilitate their integration into micro total analysis systems and point of care devices.41

Pioneering theoretical studies and recent reports on acoustofluidic techniques suggest that virtually any type of

particle or cell can be separated based on their shape, size, density, or compressibility differences.42–44 Micro-

particulate acoustofluidics separation devices are generally based on either bulk acoustic waves (BAW) or

surface acoustic waves (SAW).45

In BAW devices, a piezoelectric ceramic bonded to a microchannel is actuated by an AC signal to

form ultrasonic standing waves inside the microchannel. The ultrasonic standing waves can concentrate the

particles laterally in a continuous flow to separate out a certain cell population during washing or medium

exchange.46 Augustsson et al.47 recovered prostate cancer cells from white blood cells using a BAW device

with an efficiency of 97.9%. Petersson et el.48 showed that the acoustofluidic properties of lipid particles could

be used to separate the particles from red blood cells with a separation efficiency close to 100%. The same

group concentrated a sample containing dilute cells by a factor of two-hundred using a multistage acousto-

fluidic device.18

The principle of SAW generation via interdigitated transducers (IDT) is not new, but the use of

SAWs in microfluidics devices is quite recent.49,50 SAW devices operate on the same principle as BAW devices

except that the waves travel on the surface of a piezoelectric substrate instead of propagating in the bulk of the

material. SAW devices can be further classified into standing surface acoustic waves (SSAW) and travelling

surface acoustic waves (TSAW) devices. Recently, very efficient SSAW devices have been developed for fo-

cusing,20,50 sorting25, separation,40 and the handling of single cells or particles.24 Size- and density-dependent

particle separation has also been reported.51,52 Acoustic radiation forces on particles dispersed in a fluid by

SSAW are much higher than the radiation forces exerted by TSAW at a given frequency and power input;

hence, most researchers have focused on SSAW for particle or cell manipulation. The effectiveness of TSAW

can be increased by using higher actuation frequencies, although the concomitant high acoustic streaming ve-

locity can disturb the laminar fluid flow.53,54 Several useful acoustic streaming applications were recently re-

ported.53–57 Acoustic streaming induced by oscillating bubbles has shown tremendous potential for applications

such as pumping,55 switching,54 and microorganism processing.58 TSAW-induced acoustic streaming is useful

- 2 -

for cell/droplet sorting,59,60 droplet size control,61 and pumping62 inside a polydimethylsiloxane (PDMS) chan-

nel. TSAW techniques have proven to be very useful for droplet translocation,63 the formation of a unique

thin liquid film,64 particle concentration, and mixing inside a droplet placed on a piezoelectric substrate sur-

face.65 Tan et al.66 used a double aperture focusing unidirectional transducer to manipulate microparticles using

a TSAW inside a trapezoidal microchannel etched into a LiNbO3 surface. A significant acoustical radiation

force was applied to the microparticles using a TSAW with a 30 MHz frequency and an input power of 3 W.

The TSAW propagated parallel to the trapezoidal microchannel, which was important for preventing the for-

mation of a SSAW.

The present work demonstrates a cross-type acoustic particle separator (CAPS) for continuous

microparticles separation inside a PDMS microchannel using F-TSAW as can be seen from Figure 2. A focused

unidirectional interdigitated transducer (FUT) with curved electrodes, as shown in Figure 3(a), generated a

low power and high frequency TSAW that propagated inside a rectangular microchannel without forming a

SSAW. The power requirement and operating flow rate of the CAPS are comparable with previously reported

SSAW based particle separator40 and it does not need a specialized microchannel.66 A sheath flow pushed the

sample flow carrying the variable size particles dispersed in deionized water towards a side wall, and then the

TSAW, which propagated perpendicular to the flow direction, differentiated between the particles and forced

them away from the wall, as shown in Figure 3(b).

1.2. Chemical gradient generation

It has been reported by several researchers that chemical gradient generation in microfluidic de-

vices is of fundamental importance for many chemical and biological processes especially pulsatile gradient

generation is critically significant for dynamic biochemical characterization.28 It has also been learned that

pulsatile chemical gradient can expose biological samples with time variable chemical concentration which is

useful for high throughput characterization of cells. Most of the previously reported gradient generation tech-

niques are capable to for linear gradient but very few techniques can generate dynamic chemical gradient.67

Recently reported acoustofluidics based chemical gradient generator formed linear as well as pulsatile gradient

- 3 -

inside a microchannel but required a series of trapped bubbles and an off-chip piezoelectric transducer to actu-

ate micro-bubbles.28 Acoustic streaming induced via oscillating bubbles then generated a chemical gradient

profile. Temporal actuation of transducer resulted in pulsatile gradient profile.

In this study, we generated a linear as well as pulsatile chemical gradient using F-TSAW while

eliminating the need of any trapped micro-bubble or off-chip transducer. Our device is simple and easy to

operate and can also uniformly mix two fluids when actuated at high power. A schematic of chemical gradient

generation and uniform mixing of fluids is shown in Figure 3(c).

Focusing unidirectional transducer, reported in this study, produced SAW that traveled in a narrow

beam like fashion and reached a farther distance. F-TSAW have a frequency dependent wave amplitude profile.

Figure 3(d) shows absolute wave amplitude estimated for 133.3MHz F-TSAW. After travelling certain dis-

tance away from the FUT, F-TSAW have maximum amplitude at the center which decreased gradually towards

the sides. At this frequency (133.3MHz), the maxima is located between 1.5R and 2.0R region in Figure 3(d).

A lower frequency will move this point towards left whereas a higher frequency will push it towards right. A

fabricated F-TSAW micro-chip is also shown in Figure 3(e) placed alongside a US one dime coin for size

comparison. In the inset interdigitated metal (Au/Ti) electrodes deposited on top of LiNbO3 substrate are

shown. The PDMS microchannel O2 plasma bonded to substrate has two inlets – one for sample fluid and other

for sheath fluid – and three outlets. Plastic tubing are attached to the inlets and outlets while black dye is

injected inside for better visualization.

- 4 -

2. Theory

The F-TSAW based device has two important functionalities, first, curved microelectrodes gave

focused surface acoustic waves and second, specialized design of electrodes viz. SPUDT directed maximum

of the acoustic energy in the forward direction. In the following sub-sections, we have discussed the focusing

unidirectional travelling surface acoustic waves in more detail.

2.1. Surface acoustic waves (SAW)

In section 1.1, a brief classification of acoustofluidics based microfluidics systems is already been

described where SAW are categorized as SSAW and TSAW. Nearly all of the BAW and most of the SAW

based micro systems make use of standing ultrasonic waves to manipulate micro-objects inside a microfluidic

channel as well as induce acoustic streaming flows for numerous applications. It is pertinent to mention that

the basic principle of SSAW and standing BAW is similar i.e. the formation of standing nodes and anti-nodes.

In the following few paragraphs we have explained the basic theory of TSAW and SSAW.

When the interdigitated metal electrodes are biased by an AC signal, SAW originate from the

electrodes and propagated away on the surface of LiNbO3 substrate. A single IDT as shown in Figure 4, gen-

erated TSAW on either sides of it which can be denoted as SAW for simplicity. Alternating voltage potential

at the electrodes, deformed piezoelectric substrate underneath it, in a cyclic way, such that each particle of the

solid substrate revolve in an elliptical trajectory as can be seen from Figure 5. While the solid substrate particles

revolved in a defined pattern, SAW propagated on the surface of the substrate. The intensity of SAW decayed

exponentially with the depth. It can be inferred that SAW energy is concentrated within few wavelengths of

depth from the surface. Solid particles movement is strongest at the surface but it decreased sharply as we move

inside the substrate.

For the generation of SSAW, it is important that two counter propagating SAW or TSAW interfere.

To accomplish this, a pair of IDTs is deposited on a piezoelectric substrate parallel to each other. TSAW orig-

inating from both IDTs interfere to form a SSAW. Figure 6 depicts the same scenario where TSAW moving in

- 5 -

opposite direction interfere and form a SSAW. Some fixed points on SSAW with zero amplitude are called

nodes which gives an impression of stationary wave. The points opposite to nodes are called anti-nodes whose

amplitude fluctuates between two opposite maxima.

Particles suspended in a fluid can be separated by acoustic radiation force (ARF). ARF is depend-

ent on the mode of acoustic waves i.e. TSAW or SSAW, density and compressibility differences between

particle and fluidic media and most importantly size of the suspended particles. It has be reported that ARF

exerted by TSAW on compressible microspheres suspended in a fluid is defined as:43

𝐹𝐹𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 = 2𝜋𝜋𝜌𝜌𝑓𝑓|𝐴𝐴|2(𝑘𝑘𝑘𝑘)6φ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 (1)

where,

φ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 =��1 − 𝛼𝛼(2 + 𝛼𝛼)𝛽𝛽2

3 �2

+ 2(1 − 𝛼𝛼)29 �

(2 + 𝛼𝛼)2

(2)

Similarly, ARF by SSAW is defined as:

𝐹𝐹𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 =13𝜋𝜋𝜌𝜌𝑓𝑓|𝐴𝐴|2 sin(2𝑘𝑘𝑘𝑘) (𝑘𝑘𝑘𝑘)3φ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 (3)

where,

φ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 = (5 − 2𝛼𝛼2 + 𝛼𝛼

− 𝛼𝛼𝛽𝛽2) (4)

Here, R is the radius of μ-particles and A is complex amplitude of the velocity potential function.

𝛼𝛼 and 𝛽𝛽 are dimensionless density and compressibility ratios, respectively, defined as:

𝛼𝛼 =𝜌𝜌𝑓𝑓𝜌𝜌𝑝𝑝

, 𝛽𝛽 =𝑐𝑐𝑓𝑓𝑐𝑐𝑝𝑝

(5)

The subscripts, f and p denotes fluid and particle, respectively.

The complex amplitude of velocity potential function is related to acoustic energy density (𝐸𝐸𝑎𝑎𝑎𝑎) as

follows:

- 6 -

|𝐴𝐴|2 =2 𝐸𝐸𝑎𝑎𝑎𝑎𝑘𝑘2𝜌𝜌𝑓𝑓

(6)

Acoustic energy density (𝐸𝐸𝑎𝑎𝑎𝑎) is a related to fluid density, SAW amplitude (𝑢𝑢) and frequency (𝑓𝑓)

as follows:

𝐸𝐸𝑎𝑎𝑎𝑎 = (4𝜋𝜋𝑓𝑓𝑢𝑢)2𝜌𝜌𝑓𝑓 (7)

Combining eq. (6) and (7) yields:

|𝐴𝐴|2 =2(4𝜋𝜋𝑓𝑓𝑢𝑢)2

𝑘𝑘2= 8(𝑐𝑐𝑢𝑢)2 (8)

The parameter 𝑘𝑘 = 2𝜋𝜋𝜆𝜆� is the wavenumber, 𝑐𝑐 is speed of sound on wafer surface and 𝜆𝜆 is the

wavelength of SAW. After substituting eq. (8) into (1), we obtain that 𝐹𝐹𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 ≈ 𝑢𝑢2𝑓𝑓6𝑘𝑘6 which shows ARF

is proportional to sixth power of frequency and particle radius, and square of input voltage (V) as SAW ampli-

tude (𝑢𝑢) is directly related to V.

For a comparison of ARF by TSAW and SSAW, a sample example is solved here with the follow-

ing parameters assumed: 𝜌𝜌𝑓𝑓 ≅ 1𝑔𝑔/𝑐𝑐𝑐𝑐3 , 𝜌𝜌𝑝𝑝 ≅ 1.05𝑔𝑔/𝑐𝑐𝑐𝑐3 , 𝑐𝑐𝑓𝑓 ≅ 1.5𝑘𝑘𝑐𝑐/𝑠𝑠 , 𝑐𝑐𝑝𝑝 ≅ 2.35𝑘𝑘𝑐𝑐/

𝑠𝑠 , avg (sin(2𝑘𝑘𝑘𝑘)) = 0.5 . This resulted in 𝛼𝛼 = 0.95, 𝛽𝛽 = 0.65 , φ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 = 0.042 and φ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 = 0.65 . The

square of absolute amplitude of velocity potential function is calculated as:

|𝐴𝐴|2 = 1.28 ∗ 10−10 (𝑐𝑐2/𝑠𝑠)2 (9)

Simplifying eq. (1) and (3) by substituting above calculated parameters and eq. (9) gave the fol-

lowing relations:

𝐹𝐹𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 = 7.94 ∗ 10−6(𝑘𝑘/𝜆𝜆)6 (10)

𝐹𝐹𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 = 1.08 ∗ 10−5(𝑘𝑘/𝜆𝜆)3 (11)

The above two equations, relate ARF with the particle radius and SAW wavelength only. For a

better comparison of ARFs eq. (10) and (11) are plotted in Figure 7 (a) and (b). In Figure 7 (a), ARF is plotted

- 7 -

against a variable particle size and fixed SAW wavelength of 30µm, whereas, in Figure 7 (b), ARF is plotted

against variable wavelength and fixed particle radius of 5µm. It is obvious from these plots that ARF by SSAW

is always greater in magnitude than ARF by TSAW. This is one of the reasons that most of the researchers

prefer SSAW over TSAW for microparticles’ manipulation. In this study, the focus is to harness advantages

offered by TSAW e.g. higher order dependency on particle radius and SAW frequency. That is why we have

preferred to use higher frequencies (40 and 133.3MHz) compared to frequencies (10-15MHz) incorporated by

SSAW and (1-10MHz) used by BAW. It is essential to use high frequency of 133.3MHz to manipulate smaller

particles of diameter in the range of 5-10µm.

Acoustic streaming flow (ASF) driven via the absorption of TSAW in the fluid, is a steady current

in a fluid. It has being learned from the literature that acoustic waves or sound is absorbed in to the fluid media

which comes into contact with it. There are two possible conditions where sound absorption occurs. First,

acoustic waves are absorbed in its medium of propagation. The Stokes’ law of sound attenuation defines the

sound attenuation coefficient (𝛼𝛼) inside medium of density and viscosity as:

𝛼𝛼 =2 2

3 c3 (12)

where, c is the speed of sound in the media and is the acoustic wave angular frequency de-

fined as = 2 𝑓𝑓. This attenuation effect is prominent at elevated frequencies and is much greater in media

of low density like air. For an acoustic wave with frequency of 1MHz, the attenuation in air and water occurs

over a distance (𝛼𝛼−1) of 10cm and 100m, respectively.

The second possibility of ASF formation is near the fluid solid interface. It is possible that the

sound waves propagate through the fluid and reach a boundary or the interface wall is vibrating in a still me-

dium. Shear waves are generated inside the fluid when the adjoining wall vibrates parallel to itself. The atten-

uated amplitude of the shear wave lies within the Stokes oscillating boundary layer. This effect is localized on

an attenuation length of characteristic size (�) whose order of magnitude is a few micrometers in both air and

water at 1MHz. The characteristic length (�) defined in eq. (13) is calculated to be 60, 40 and 35nm in water at

frequencies 40, 100 and 133.3MHz, respectively.

= �

(13)

- 8 -

Although, the second reason of ASF generation is more befitting to this work but it is not the sole

contributing factor. In reality, small perturbation in the form of strong vortices close to the wall is transmitted

deep inside the microchannel. It will be seen in section 4.2 that the formation of small but strong vortices by

F-TSAW adjacent to the microchannel side wall resulted in bigger vortices which are responsible for chemical

gradient generation and micromixing without the need of any trapped bubble.

2.2. Focusing unidirectional transducer (FUT)

A FUT has two special feature viz. it focuses TSAW in a narrow region and its specialized elec-

trode design helps in discharging maximum acoustic energy in one direction. Focusing effect is achieved by

introducing curved interdigitated electrodes. Figure 8 shows a schematic of focusing IDT i.e. curved electrodes

with radius R and angular aperture θ. Microfluidic channel is attached at an appropriate distance (z) away from

the IDT. The F-TSAW amplitude is estimated using a method proposed by Fang et al.68

𝑢𝑢(𝑥𝑥, 𝑧𝑧) ≈1

𝑍𝑍1/4 � 𝐺𝐺(𝑡𝑡) 𝑒𝑒𝑥𝑥𝑒𝑒[𝑗𝑗(𝑡𝑡4+𝑍𝑍′𝑡𝑡2 + 𝑋𝑋′𝑡𝑡)]𝑘𝑘𝑡𝑡+∞

−∞

(14)

where,

𝑋𝑋′ =−𝑋𝑋

√4.98𝑍𝑍4 (15)

𝑍𝑍′ =(0.145𝑍𝑍 − 𝑘𝑘𝑘𝑘0/2)

√4.98𝑍𝑍 (16)

𝑡𝑡 = √4.98𝑍𝑍4 𝐾𝐾1 (17)

and,

𝑍𝑍 = 𝑘𝑘0𝑧𝑧, 𝑋𝑋 = 𝑘𝑘0𝑥𝑥, 𝐾𝐾1 = 𝑘𝑘1/𝑘𝑘0 (18)

- 9 -

where, x and z are vertical and horizontal distances from the origin point placed next to the focus-

ing IDT as shown in Figure 9, whereas, 𝑘𝑘0 = 2𝜋𝜋𝜆𝜆� is wavenumber and 𝑘𝑘1 is its vertical component.

The distance (z) is carefully estimated as the focusing electrodes give a unique wave amplitude

profile as shown in Figure 9. F-TSAW amplitude profile is very unpredictable close to the electrodes but

stabilizes as distance z is increased. For a device actuated at 40MHz, the microfluidic channel can be attached

somewhere between 1.25R – 1.50R. Figure 10 provided a comparison between nine different cases calculated

using eq. set (14) - (18). In the top three plots, angular aperture θ of IDT is varied; in the middle, radius R of

the IDT is increased and bottom plots show that how frequency variation effects the amplitude profile. Im-

portant point which can be inferred from these plots are: when the angular aperture θ is increased from 20� to

40�, maximum wave amplitude also increased but a further increment in θ to 60� has decreased the maximum

amplitude; an increment in electrode radius results in higher wave amplitude which can travel to a farther

distance; as the frequency is increased from 20MHz to 60MHz, no significant change is observed but we will

see in later sections that an increment in frequency up to 133.3MHz can shift the maximum F-TSAW amplitude

peak towards right at a distance z close to 2.5R.

In a simple Interdigitated Transducer (IDT), metal electrodes are uniformly spaced with electrode

width and gap equal to �/4 as � is the SAW wavelength. Acoustic energy is equally transmitted in either direc-

tion as shown in Figure 11 (a). In most of the cases, half of the acoustic energy is wasted while only one half

of it can be brought to proper use. To reduce the energy losses, a specialized Single Phase Uni-Directional

Transducer (SPUDT) is incorporated to direct maximum energy in one direction. In SPUDT as shown in Figure

11 (b), metal electrodes width and gap is adjusted in such a way that maximum acoustic energy is transmitted

in the forward (rightward) direction whereas very little energy is transferred in the backward direction. A com-

bination of focusing IDT and SPUDT yields an efficient FUT.

- 10 -

3. Device fabrication and experimental setup

The fabrication of F-TSAW based device is carried out in two stages. At first stage metal electrodes

are being deposited on a piezoelectric substrate while in the second stage PDMS microfluidic channel is fabri-

cated using soft lithography process. The both of these fabrication processes are further discussed in detail in

the following sub sections. To conduct the experiments, an acoustofluidic experimental setup is established in

Flow Control Laboratory, KAIST. In the following subheadings, device fabrication and experimental setup are

described thoroughly.

3.1. Device fabrication

The F-TSAW based CAPS was fabricated in two parts. First, a bimetallic Au/Ti layer with a thick-

ness of 800 Å/50 Å was deposited onto a 128° y-cut LiNbO3 substrate (MTI Korea) via e-beam evaporation50

to form 30 pairs (or 20 pairs in another design) of curved interlocking electrodes with an inner radius of 4 mm

(in another design 6 mm) and an aperture angle of 40°. The whole process is explained in Figure 12 (a) and

(b). After cleaning the LiNbO3 substrate, a thin layer of photoresist (PR) is spin coated on the polished surface

of substrate. The photoresist is exposed to UV light through a mask and then developed in appropriate PR

developer to obtain the required pattern. This will expose the selective surface of the substrate for metal layer

deposition. A bi-metallic layer of Au and Ti is deposited using e-beam evaporation process. The excess PR and

metal is removed from the surface using a lift-off process. The end result is a neatly patterned interdigitated

metal electrodes on top of LiNbO3 substrate.

Second, a bunch of PDMS microchannels were fabricated using soft lithography process with dif-

ferent designs and heights. The whole process is delineated in Figure 12 (a). A layer of negative photoresist

(SU-8) was spin-coated onto a Si substrate. The height (40-110µm) of the PR is controlled by adjusting the

spin coater’s RPM as it will determine the height of the final PDMS microfluidic channel. After soft baking at

65°C and hard bake at 95°C for specific minutes (detailed run sheets are attached in appendix), the PR is

exposed to UV light for few seconds depending upon the height of PR through a chrome mask. After the UV

- 11 -

exposure, post baking at 65°C and 95°C is done for the specific time. The photoresist is developed in the special

SU-8 developer for a couple of minutes until the SU-8 pattern becomes visible and can be seen with the naked

eye. Developing step will leave a solid SU-8 mold on the surface of the Si substrate. A silicone elastomer base

is mixed in a 10:1 weight ratio with the curing agent and then mixed to obtain a homogeneous mixture. The

PDMS mixture is poured onto the solid SU-8 mold. The bubbles formed during the mixing step are removed

by placing the PDMS in vacuum for an hour. After degassing under vacuum, the PDMS is cured at 65°C in an

oven for 1 hour and then peeled off from the Si wafer. To make inlet and outlet ports in to the microchannel, a

manual hole-puncher is used. The PDMS is then bonded to the LiNbO3 chip by O2 plasma bonding (Femto

Science, Korea) to form a microchannel as shown in Figure 12 (c). The dissipation of acoustic energy in the

PDMS is minimized by introducing an acoustic window along the microchannel during the fabrication process.

In Figure 13, a schematic of the end product is shown with a curved metal electrodes and PDMS microchannel

having an acoustic window as well.

Experiments are conducted on four types of F-TSAW micro-chips, whose detail is enlisted in Ta-

ble 1. In the fabrication of these chips, three different microchannel designs are incorporated as shown in Figure

14 (a-c). Two types of metal electrodes designs used in the micro-chips with actuating frequency of 40MHz

and 133.3MHz are shown in Figure 15 (a) and (b).

3.2. Experimental setup

The overall experiment layout schematic is shown in Figure 16. A radio frequency (RF) signal

generator (N5181A, Agilent Technologies) can produce high frequency sinusoidal signals in the range of

100kHz to 3GHz and maximum voltage of 4.5Vpp (power 50mW or 17dBm). F-TSAW based micro-chip is

operated at frequency of 40MHz and 133.3MHz. The high frequency and low power signal is then fed into a

power amplifier (ZHL-1-2W, miniCircuits) that can increase the amplitude of the signal up to 28Vpp (power

2W or 33dBm). The output from the power amplifier can be adjusted by controlling the input signal generated

at the RF signal generator. The power amplifier is connected to a DC power supply (E3634A, Agilent Tech-

nologies) for its functionality. A DC voltage of 24V and current of 0.9A is given to the power amplifier. The

amplified signal is then given to the micro-electrodes at the F-TSAW micro-chip.

- 12 -

The RF signal generator have an output terminal of N-Type which is then connected to N to BNC

type convertor as most of the cables and power amplifier are equipped with BNC type connectors. To make

the connection easy, an extension wire with appropriate length (approx. 30cm but can be adjusted as per user)

is soldered to the micro-electrodes by using a solder gun. The connections can also be made by using copper

tape and (or) silver paste. After applying a small amount of silver paste to the micro-electrodes terminals,

copper tape is attached on top of it. Silver tape is useful in reducing the contact resistance. A connection made

with copper tape only will have a higher contact resistance. In the present work, mostly solder connections are

used for its low contact resistance.

A syringe pump (neMESYS, Cetoni GmbH) is used to inject the sample and sheath fluids in two

separate inlets. The syringe pump is remotely controlled through a graphical user interface from a personal

computer to adjust the flow rate at the inlets independent of each other. Plastic tubing is used to inject the fluid

inside the microchannel through the inlets and to collect it at the outlets. To observe the microfluidic separation

and chemical gradient generation inside the channel, a microscope (BX51, Olympus) is used. F-TSAW micro-

chip is attached to a glass slide with the help of transparent epoxy so that it can be easily fixed on microscope

stage. A digital camera (DP26, Olympus) is mounted on the microscope to record the experimental results. The

camera is also connected to the computer to save all the snapshots taken and movies recorded during the ex-

periment. A specially designed imaging software (cellSens, Olympus) is used to control the camera, record

results and later analyze them. Sometimes, when the flow rate in the microchannel is too high, DP26 camera

cannot record the results efficiently with the frame rate of 6-7fps. In that case, a high speed camera (pco.

1200hs) with a frame rate of around 1000fps can be used to record the results.

- 13 -

4. Results

In this study we have tested four F-TSAW devices listed in Table 1; three for particle separation

and one for chemical gradient control. The distinguishing parameters are the actuating frequency, FUT radius,

particles to be separates, microchannel dimensions and function to be performed by the device i.e. separation

or gradient generation. The working of these micro-chips and the results obtained from the experiments con-

ducted on them are briefly discussed in the subheadings to come. The devices are named as “Cross-type Acous-

tic Particle Separator (CAPS)” and “Cross-type Acoustic Gradient Generator (CAGG)”.

4.1. Particle separation by CAPS 1-3

The CAPS chip was placed on the stage of a microscope (Olympus BX53) outfitted with a camera

(Olympus DP26). A micro-syringe pump (Cetoni GmbH neMESYS) was used to inject a mixture of polysty-

rene particles (Thermo Scientific) dispersed in DI water into the microfluidic channel. For the testing of CAPS-

2, a radio frequency (RF) signal generator (Agilent A5181) was used to produce a 133.3 MHz and -3.4 dBm

sinusoidal signal that was then amplified by a power amplifier (miniCircuits ZHL-1-2W). The amplified signal

of 24.2 dBm (275 mW) was fed into FUT. F-TSAW originating from the transducer passed through the acoustic

window and interacted with the fluid inside the microchannel, as shown in Figure 17 (a). The sample fluid

carrying the particles of diameter 3 µm and 10 µm was then injected through the sample inlet at a flow rate of

25 µL/h while a sheath flow was injected at 125 µL/h to force the sample fluid closer to the side wall. A total

flow rate of 150 µL/h through the microchannel (45 µm x 150 µm) produced an average flow velocity of 6.2

mm/s. For better visualization and to achieve a larger spatial separation, the width of the microchannel was

broadened to 350 µm. Figure 17 (b) shows that when the TSAW were off, no significant separation distance

was observed between the particles. Later, when the TSAW were turned on, the larger particles were pushed

toward the opposite wall of the channel under the acoustic radiation force, thereby inducing a significant sep-

aration distance between the particles, as shown in Figure 17 (c). The trajectory followed by a 10 µm particle

that was diverted under the acoustic radiation force and slightly influenced by acoustic streaming induced by

- 14 -

the TSAW is shown in Figure 17 (d) (left). The acoustic streaming flow pattern developed in a stationary fluid

by TSAW is shown in Figure 17 (d) (right). Smaller particles (1 µm in diameter) dispersed inside the fluid

experienced a negligible acoustic radiation force and moved with the bulk fluid under the effect of the Stokes

drag force. In the central region, the fluid was pushed away from the left wall as the acoustic energy from the

TSAW dissipated into the fluid. Two symmetric strong vortices formed as a result of this dissipation, and the

vortices rotated in opposite directions. Considering the fact that narrow microchannels can reduce the effects

of acoustic streaming, 48 the CAPS was equipped with a 150 µm-wide microchannel. The acoustic streaming

velocity was on the order of a few microns per second, significantly lower than the average fluid flow velocity

(6.2 mm/s) in the microchannel; hence, the acoustic streaming did not significantly affect the particle trajectory,

as can be seen from Figure 17 (d) where the slow particle motions captured using a high-speed CMOS camera

(pco. 1200 hs).

The separation efficiency of the CAPS-2 chip was calculated to be 97.2% for a sample size of 150

10 µm particles per 2,000 3 µm particles. The separated particles could be collected at separate outlets after

traveling a certain retention distance. Figure 18 clearly shows that most of the particles were collected at outlet

1 in the absence of an applied TSAW. After the AC signal had been turned on, nearly all of the 10 µm particles

could be collected at outlet 3. The average particle dwell time in the active acoustic region varied because the

Poiseuille flow inside the rectangular cross sectional microchannel ensured that not all of the particles flowed

with the same velocity. Particles near the walls moved with a lower speed than the particles at the center of the

microchannel. Hence, few of the 10 µm particles experienced less acoustic radiation force that was not suffi-

ciently strong to divert the particles to outlet 3.

To investigate the effect of frequency on F-TSAW based acoustic field on the piezoelectric sub-

strate, absolute wave amplitude estimated by method discussed in section 2.2 is plotted in Figure 19. As the F-

TSAW based micro-chips are actuated at 40MHz and 133.3MHz, both of these cases are explored. The line

plots shows wave amplitude profile at different locations away from the circular FUT. For 133.3MHz and

40MHz cases maximum wave amplitude is obtained at location 2-2.5R and 1-1.5R, respectively. Based on

these findings, the microfluidic channel is attached at location 1.25R for 40MHz case and 2.5R for 133.3MHz

case. These locations are considered most appropriate for better acoustic energy transmission into fluid as the

wave amplitude profile compared to locations very close to the FUT becomes smooth with maximum at the

center.

- 15 -

As CAPS-1 is operated at 40MHz frequency, smaller particles, with diameter of 10µm or so, do

not experience enough force by the F-TSAW. To test the working of CAPS-1, a mixture of 10µm and 30µm

polymer particles dispersed in DI water is injected in the microfluidic channel at flow rate of 50µL/h by a

syringe pump. A FUT with the curvature of 6mm is actuated by a -7.5dBm AC signal which generates F-

TSAW to separate the particles flowing at an average speed of 0.84mm/s. The 30µm particle trajectory is

calculated theoretically as well as captured experimentally as shown in Figure 20. A set of 15 images are

stacked together in Figure 20 (center) to obtain the particle trail whereas Figure 20 (right) shows the actual

separation of 10µm particle from 30µm particle. It is pertinent to note here that 10µm particles do not experi-

ence any significant force by 40MHz F-TSAW and continue to follow laminar flow. From this figure, it can be

inferred that a higher frequency of F-TSAW must be used to manipulate particles with diameter in the range

of 10µm. This was one the reason behind redesigning CAPS-2&3 with high frequency of 133.3MHz.

In CAPS-3, the design of FUT microelectrodes is essentially the same but the microfluidic channel

is slightly modified as you can see in Figure 21. The microfluidic channel’s width is narrowed down to 200µm

at the active separation zone, so that the effect of acoustic streaming flow can be reduced. The downstream

microchannel width is designed as 500µm for separation enhancement. Dark field imaging is used for better

visualization; polymer microspheres are captured as white dots whose size varies with the actual diameter of

the particles. The mixture of 3µm and 10µm particles dispersed in DI water is injected through the particles

inlet and DI water as sheath fluid is injected through sheath inlet at flow rate of 25µL/h and 75µL/h, respec-

tively, as shown in Figure 21. It can be seen from the separation window that all the particles follow the laminar

flow when the F-TSAW is turned off but the particles get separated after F-TSAW is turned on. The outlet

window show particles flowing through the same outlet in the first case but in the latter case they are being

collected at separate outlets.

The separation efficiency of the CAPS-3 chip was calculated to be 100% for a power input of

151mW. The separated particles could be collected at separate outlets after traveling a certain retention dis-

tance. Figure 22 clearly shows that all of the particles were collected at outlet 1 in the absence of applied

TSAW. After the AC signal had been turned on with input power of 55mW, all of the 10 µm particles could

be collected at outlet 2. Further increment in power up to 151mW pushed all the 10 µm particles towards outlet

3.

- 16 -

4.1.1. Microchannel Alignment

To align the microfluidic channel with the focusing unidirectional transducer (FUT), L-shape

alignment markers are fabricated along with FUT electrode deposition on LiNbO3 by e-beam evaporation. The

L-shape markers as shown in Figure 23 should be aligned closely with the walls of the acoustic window. The

width and breadth of the marker is 1 mm, respectively, which could be seen easily without mounting the micro-

chip on microscope stage. After O2 plasma treatment of LiNbO3 chip and PDMS microfluidic channel, the

alignment is done manually with hand. Although the microchannel is not tightly aligned with the FUT, a mis-

alignment of approx. 200 µm can be tolerated. It has been shown experimentally that a misalignment of up to

200 µm in horizontal or vertical direction did not affect the separation significantly. In Figure 23, it can be seen

that the targeted centerline is at the center of microchannel but the misalignment shifts the centerline 200 µm

upward.

Figure 24 shows the alignment of TSAW beam with the microchannel (A), trajectory followed by

the 10 µm particle (B, C) and formation of acoustic streaming flow (D, E). The acoustic streaming flow pattern

developed in a stationary fluid by the high frequency (133.3 MHz) TSAW is traced by smaller particles (1 µm

in diameter) dispersed inside the fluid. Figure 24 (D and E) are obtained by a free image process software

Image J. The smaller particles experienced a negligible acoustic radiation force and moved with the bulk fluid

under the effect of the Stokes drag force. In the central region of TSAW beam, the fluid was pushed away from

the left wall as the acoustic energy from the TSAW dissipated into the fluid. Two pairs of symmetric strong

vortices formed as a result of this dissipation, and the vortices rotated in opposite directions.

Four vortices, named as V1, V2, V3 and V4, effect the trajectory of 10 µm particle. As the acoustic

radiation force FTSAW constantly pushed the particle from left towards right, the vortices assisted and opposed

the rightward motion of the particle at different locations. At the juncture of V1 and V2, particle got some

support from the vortices and pushed in the rightward direction. The same applied for the joining point of V3

and V4. In the central region, V2 and V3, respectively, opposed the rightward motion of particle as the acoustic

radiation force on particle is balanced by the Stokes drag force produced by the back flow of the vortices. The

streaming flow in the central region denoted by two U-turns is not as strong as flow by vortices V1-4. The

effect of these low velocity U-turn flows is so negligible that it can be ignored.

- 17 -

4.1.2. Deflection of particles

Deflection distance travelled by the particles under the influence of TSAW is measured down-

stream of the separation zone where the channel width is 500 µm. Figure 25 (a) shows the relationship between

deflection distance, power input and flow rate. Figure 25 (b) shows that the particles of size 3 µm can be easily

differentiated from 10 µm particles for a reasonable constant input power greater than 50mW. The 10 µm

particles can also be sorted into different outlets by regulating the input power as the deflection distance is

power dependent. For a constant deflection distance, the relationship between power-input against flow rate is

found to be directly proportional. As the flow rate is increased from 100µL/h to 100µL/h, the required power

to produce maximum deflection is increased from 200mW to 600mW.

A bar chart of particle deflection is plotted for variable input power in Figure 26 - for 10µm particle

(top) and. for 7µm particle (bottom). For a comparison, dashed rectangles are sketched for input power of

217mW and variable flow rate. It is evident that 10µm particles experience significantly more deflection dis-

tance than 7µm particles. This ensured the separation of larger particles from the smaller particles. All the

particles of diameter 10µm and 7µm flowed within a region of approximately 100µm width at all flow rates

with zero input power. The deflection distance distribution varied when the input power is increased. For a

10µm particles with flow rate of 100µL/h, the deflection distance gradually increased from below 100µm to

above 400µm as the input power is increased from 0mW to 217mW. Whereas, for the flow rate of 300µL/h,

the maximum deflection is only achieved when power input is increased at a level of 600mW. The overall

patterns that can be deduced from these bar charts is: (i) the deflection distance is increased with power input

for both types of particles but 7µm always lagged behind 10µm particles which is essential for their separation,

(ii) for higher flow rates, the power requirement is also higher to generate a similar deflection distance.

All the microfluidic separation chips, CAPS-1, 2, 3 successfully separatde the microparticles in a

continuous flow without any prior labelling. In the following subsection, a micro-chip is described to generate

chemical gradient and uniform mixing; have same electrode design but different microfluidic channel dimen-

sions.

- 18 -

4.2. Chemical gradient control

It has been observed during the particle separation experiments performed with the CAPS micro-

chips that high frequency F-TSAW generated considerable acoustic streaming flow (ASF). In the separation

experiment, the effect of ASF is tried to be suppressed so that the particle separation is not influenced. A

microfluidic channel with wider dimensions can enhance the ASF which is useful for generating chemical

gradient and micromixing applications.

To get an idea about ASF dependence on microchannel dimensions, four types of microchannels

with different cross-sections are injected with 1µm diameter polymer microspheres dispersed in DI water. F-

TSAW are applied to the fluid to see how ASF is formed inside the microchannel. After the acoustic waves

dissipate their energy inside the liquid, symmetric vortices are observed as shown in Figure 27. In the figure,

F-TSAW are propagating from right to left. Left four images in Figure 27 (a) are captured at four consecutive

time steps where the microchannel is 50µm wide and 40µm high. It can be seen that SSAW are formed as the

F-TSAW are reflected from the PDMS wall. The microparticles are trapped at three prominent pressure nodes.

This narrow microfluidics channel is not quite helpful in generating strong ASF instead the formation of SSAW

leads to undesirable results. Right three images in Figure 27 (b) are for microchannels 150µm x 45µm, 200µm

x 40µm and 500µm x 90µm from left to right, respectively. It can be inferred from these images that as the

microchannel width and height is increased, ASF is also enhanced. A 500µm x 90µm microchannel can be a

suitable choice for harnessing ASF for chemical gradient control and uniform micromixing of fluids flowing

continuously inside the PDMS microfluidic channel.

To investigate chemical gradient generation and micromixing by F-TSAW, rhodamine and DI

water are injected at separate inlets with flow rate of 50µL/h each. Laminar flow in microchannel does not

allow two fluids to mix easily even after travelling a long distance. Mixing of fluids is only limited to diffusion.

To rapidly mix two fluids inside a microchannel, some external impetus or special structural design is used to

disturb the laminar flow and induce some agitation in the form of turbulence or vortices. In this study, we

introduce this agitation by ASF based symmetrical vortices that not only uniformly mix two fluids but can also

generate precisely controlled chemical gradient. In Figure 28 (a) a cross-type acoustic gradient generator

(CAGG) is shown where two fluids flowing parallel in a microchannel being interrupted by F-TSAW. An

- 19 -

absence of F-TSAW does not affect the laminar flow and two fluids are smoothly flowing without any signif-

icant mixing (left) but F-TSAW with 133.3MHz and 200mW generate ASF with appropriate vortex strength

which is sufficient to form a required gradient profile as shown in central image. The uniform mixing of fluids

(right) is obtained when the input power is increased up to 800mW. Figure 28 (b-d) show chemical concentra-

tion profile corresponding to normalized-light-intensity. Figure 28 (b) depicts upstream mixing by diffusion

only whereas (c) and (d) provide a comparison of gradient profiles as the input power is increased from 0 to

800mW.

- 20 -

5. Summary and discussion

It can be concluded here that a TSAW are as effective as an SSAW for microparticulate separation

when actuated at a high frequency. Unlike SSAW-based devices which require a pair of IDTs for functionality,

the CAPS and CAGG devices described here operated on a single focusing transducer that eliminated the critical

microchannel alignment process. For most of SSAW based particle/cell separation and manipulation techniques,

the microfluidic channel/chamber should be aligned parallel to the pair of IDT. The microchannel alignment is

performed under the microscope which is a tiresome process carried out by placing a droplet of methanol/etha-

nol on the substrate before permanently bonding it with the PDMS via oxygen plasma bonding. The use of

TSAW eliminate the need of this tiresome process.

In a previously reported study, during the course of particle separation by TSAW, a single focusing

IDT also formed SSAW inside a microchannel etched in the substrate because of the reflection of waves from

the side walls. This problem was countered by using trapezoidal cross-sectional microchannel and high input

power was used to manipulate the particles. Our low power F-TSAW based devices did not require a trapezoidal

microchannel as PDMS do not reflect acoustic waves with low amplitude. The CAPS devices have successfully

separated smaller particles (3 or 10µm) from larger particles (10 or 30µm), respectively, with low input power

and a separation efficiency close to 100%. As the acoustic radiation force by TSAW is proportional to diameter

of particle power six, a higher separation resolution could be achieved; this means, particles with small size

differences can easily be separated, provided that they are tightly focused before entering the separation zone.

The CAGG is also another efficient F-TSAW device which does not require any complicated mi-

crochannel design or oscillating bubbles to form chemical gradient inside the channel. The CAGG device simply

makes use of the focused acoustic waves with maximum intensity at the center. The bell shape acoustic wave

amplitude profile produces the required ASF which forms the symmetrical vortices to generate chemical gradi-

ent and uniform mixing of fluids.

- 21 -

Tables

Table 1: Experimental parameters of F-TSAW micro-chip

- 22 -

Figures

Figure 1: Tree diagram summarizes the commonly used separation techniques in distinct categories. The

current research route is shown in red.

- 23 -

Figure 2: Schematic of particle separation by F-TSAW is shown.

Figure 3: (a-c) Schematic of F-TSAW micro-chip – particle separation, chemical gradient generation and

uniform mixing inside a PDMS microchannel is shown (d) F-TSAW amplitude produced by focusing

unidirectional transducer (UDT) (e) Fabricated F-TSAW micro-chip.

(a)

(d)

Chemical gradient Mixing

(c) Concentration

profile

- 24 -

Figure 4: Interdigitated transducer (IDT): metal electrodes deposited on a piezoelectric substrate when given

an AC signal produces surface acoustic waves (SAW)

Figure 5: When AC signal is applied across the IDT terminals, particles of solid piezoelectric substrate revolve

in an elliptical trajectory as the SAW propagates on the surface of substrate (Courtesy Dan Russell, 2011)

Figure 6: Two travelling waves propagating in opposite directions when interfere forms a standing wave

(Courtesy Dan Russell, 2011)

- 25 -

Figure 7: Comparison of acoustic radiation force by SSAW and TSAW on polystyrene particles dispersed in

water: (a) For constant wavelength of 30µm (b) For particle radius of 5µm while wavelength is increased from

30 to 100µm.

Figure 8: Schematic of focusing Interdigitated Transducer (IDT)

- 26 -

Figure 9: Estimated F-TSAW profile: waves are focused in a narrow region by the circular electrodes. Black

color shows high wave amplitude region.

Figure 10: F-TSAW amplitude vs. z plotted at a plane passing through the center of focusing IDT and

perpendicular to substrate surface and microchannel.

x+

x-

z

- 27 -

Figure 11: (a) Interdigitated Transducer (IDT): Metal electrodes are uniformly spaced with electrode width and

gap equal to /4 as is the SAW wavelength. Acoustic energy is equally transmitted in either direction. (b)

Single Phase Uni-Directional Transducer (SPUDT): Metal electrodes width and gap is adjusted in such a way

that maximum acoustic energy is transmitted in the forward (right) direction whereas very little energy is

transferred in the backward direction.

Figure 12: (a) Fabrication of PDMS microchannel via soft lithography process (b) Deposition of Ti/Au metal

electrode on a LiNbO3 substrate by e-beam evaporation method (c) Oxygen plasma bonding of PDMS

microchannel with LiNbO3 substrate.

- 28 -

Figure 13: F-TSAW based micro-chip after PDMS microchannel (green) is bonded to LiNbO3 substrate (blue)

while Au electrodes (yellow) are also shown.

Figure 14: Microchannel drawings for particle separation: (a) Device#1 and 2 (b) Device#3. (c) Device#4:

Microchannel drawing for micromixing and gradient control.

- 29 -

Figure 15: Metal electrode drawings of focused unidirectional transducer with operating frequemcy: (a) 40MHz

for Device #1 (b) 133.3MHz for Device#2-4.

Figure 16: Experimental setup for acoustofluidics. Signal generator and power supply (Agilent), amplifier

(miniCircuits), camera and microscope (Olympus), micro syringe pump (neMESYS)

- 30 -

Figure 17: CAPS-2: (a) Schematic diagram of a PDMS microchannel. (b-c) The separation of 3 µm particles

and 10 µm particles was achieved under a flow rate of 150 µL/h and an input power of 275mW. Particles are

indicated by the dotted circles. When the TSAW was OFF, no significant separation distance was observed

between the particles of the two sizes. Once the TSAW was turned ON, a distinct separation distance could be

observed. (d) Trajectory followed by a 10 µm particle influenced by acoustic streaming.

- 31 -

Figure 18: CAPS-2: Separation efficiency. (a) TSAW OFF: nearly all of the particles flowed through outlet 1.

(b) TSAW ON: 3µm particles were collected at outlet 1, whereas 97.2% of the 10µm particles passed through

outlet 3. (Sample size: 2,000 3µm particles per 150 10µm particles.)

Figure 19: F-TSAW absolute amplitude estimation for two separate actuation frequencies i.e. 133.3MHz and

40MHz. Line plots depict acoustic wave amplitude at locatinos z=1.0R, 1.5R, 2.0R and 2.5R.

- 32 -

Figure 20: CAPS-1: F-TSAW absolute amplitude profile is shown along the trajectory followed by a 30µm

diameter particle under the influence of acoustic radiation force theoretically (left) and observed experimentally

(center). The separation of 10µm diameter particle from 30µm diameter particle is shown on right.

- 33 -

Figure 21: CAPS-3: Particle separation is captured by dark field imaging and several images are stacked

together to obtain the path line followed by a 10µm diameter particle. Polystyrene microparticles with diameter

10µm are shown to be separated from 3µm by F-TSAW.

- 34 -

Figure 22: CAPS-3: When the TSAW is OFF (0mW), all of the particles flowed through outlet 1. When the

TSAW is turned ON (151mW), 3µm particles were collected at outlet 1, whereas 100% of the 10µm particles

passed through outlet 3.

Figure 23: Microchannel alignment with FUT: L-shape markers are aligned with the side walls of acoustic

window

- 35 -

Figure 24: TSAW alignment with the separation zone. A: TSAW amplitude profile. B: Separation zone along

with time steps is shown. C: Trajectory followed by 3µm (left) and 10µm (right) particles. D: Acoustic streaming

flow traced by 1µm particle. The arrows show the vortices formed by the flow. E: Particle Image Velocimetry.

Image C, D and E are obtained through ImageJ Software.

Figure 25: Particle deflection is plotted against input power for CAPS-3: (a) Flow rate is kept constant at

100µL/h [Sheath + Sample = 80µL/h + 20µL/h]. (b) For particles with diameter 10µm while flow rate is

increased from 100µL/h up to 300µL/h. The inset shows deflection vs. flow rate for constant input power.

- 36 -

Figure 26: Particle deflection is plotted for variable input power. (Top) For 10µm particle. (Bottom) For 7µm

particle. For a comparison, dashed rectangles are sketched for input power of 217mW and variable flow rate. It

is evident that 10µm particles experience significantly more deflection distance than 7µm particles. This ensures

the separation of these particles.

- 37 -

Figure 27: Acoustic streaming flow induced via F-TSAW is traced by 1µm polymer microspheres dispersed in

DI water. F-TSAW are propagating from right to left. (a) Left four images are captured at consecutive time

intervals where the microchannel is 50µm wide. (b) Right three images are for microchannels 150µm x 45µm,

200µm x 40µm and 500µm x 90µm from left to right, respectively.

- 38 -

Figure 28: CAGG: Chemical gradient generation and uniform mixing of fluids. (a) Two fluids flowing parallel

in a microchannel being interrupted by F-TSAW. (b) Upstream mixing by diffusion. (c-d) Concentration profile

(normalized light intensity) varies downstream as the input power is increased.

- 39 -

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Acknowledgment

First of all, it has been a great blessing of Almighty Allah that I am writing these lines today.

Secondly, I would like to express my deepest gratitude towards my parents and sisters; whose

utmost love and support always provided me the necessary motivation to set high goals and

pursue them. A bundle of appreciations to all those who braced me in carrying out this study

throughout the span of my master’s degree. I am thankful to my supervisor Prof. Hyung Jin Sung

for his sincere guidance during my research work. It has been a wonderful experience working

with all the lab members who were always willing to discuss any problem at hand and also pro-

posed possible solutions. I convey my regards to Dr. Sang Youl Yoon, Dr. Kyungheon Lee, Dr.

Mubashshir Ansari, Dr. Khalid Mahmood, Hyun Wook Kang, Kang Soo Lee, Emad Uddin,

Yong Suk Oh, Seung-hwan Kim, Muhammad Nadeem, Sunghyuk Im, Byunghang Ha, Jin Ho

Jung and Juyoung Leem. I would also acknowledge Dr. Tae Gyu Park (from SNU) for helping

in the fabrication process. In the end, special thanks to all of my friends in whose company time

really flies.

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Curriculum Vitae

Name : Ghulam Destgeer

Date of Birth : March 31, 1990

Place of birth : Pakistan

Email : destgeer@kaist.ac.kr

Education

2011.9-2013.9 M.S.

Department of Mechanical Engineering

KAIST

Daejeon, Korea

2006.8-2010.6 B.S.

Department of Mechanical Engineering

Ghulam Ishaq Khan Institute of Engineering Science and

Technology

Topi, Pakistan

2011.9-2013.9 High School

Army Public School and College

Gujranwala, Pakistan

Academic activities

1. Ghulam Destgeer and Hyung Jin Sung, “µ-particulate separation by travelling surface acoustic waves”,

7th Korea-China-Japan Student Symposium 2013, KAIST, Daejeon, Korea, March 21-23, 2013.

2. Ghulam Destgeer and Hyung Jin Sung, “Micro particulate separation by travelling surface acoustic

waves (TSAW)”, The Symposium of the Korean Society of Visualization 2013, KAERI, Daejeon,

Korea, April 26, 2013.

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