acoustofluidic manipulation of microspheres via vertical...

Master’s Thesis

Acoustofluidic Manipulation of Microspheres via Vertical

Hydrodynamic Focusing and Upward Migration

2017

Husnain Ahmed

Korea Advanced Institute of Science and Technology

Acoustofluidic Manipulation of Microspheres via Vertical

Hydrodynamic Focusing and Upward Migration

Husnain Ahmed

Advisor: Hyung Jin Sung

A dissertation/thesis submitted to the faculty of

Korea Advanced Institute of Science and Technology in

partial fulfillment of the requirements for the degree of

Master of Science in Mechanical Engineering

Daejeon, Korea

June 12, 2017

Approved by

Hyung Jin Sung

Professor of Mechanical Engineering Department

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

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:

falsification, thesis written by someone else, and distortion of research findings or plagiarism. I affirm that my

thesis contains honest conclusions based on my own careful research under the guidance of my thesis advisor

MME

20154599

Abstract

Advancements in microfluidic manipulation devices are vital for the development of

future lab-on-a-chip technologies with applications in the biological, chemical, and materials

sciences. Surface acoustic wave (SAW)-based acoustofluidic separation devices, using low

power densities, offer non-contact and label-free sorting capabilities using differences in

mechanical properties (density, compressibility, etc.) or sizes of the micro-objects. A particle

suspended in a fluid within a microfluidic channel experiences a direct acoustic radiation force

(ARF) when travelling surface acoustic waves (TSAW) couple with the fluid at the Rayleigh

angle. Most SAW-based microfluidic devices rely on the horizontal component of the ARF to

migrate pre-focused particles laterally across a microchannel width. Although the magnitude

of the vertical component of the ARF is more than twice the magnitude of the horizontal

component, it has been long ignored due to polydimethylsiloxane (PDMS) microchannel

fabrication limitations and difficulties in particle focusing along the vertical direction. In the

present work, we have devised a single layered PDMS microfluidic chip for hydrodynamically

focusing particles in the vertical plane while explicitly taking advantage of the horizontal ARF

component to slow down the selected particles and the stronger vertical ARF component to

push the particles in the upward direction to realize continuous particle separation. An

acoustofluidic device with a straight PDMS microchannel placed directly on top of a straight

interdigitated transducer (IDT) was used to produce high frequency (140 MHz) TSAWs.

Contrary to the conventionally used two sheath flows for particle focusing, a single sheath flow

was used to pinch the particles close to the bottom of the microchannel. The TSAWs originating

from the IDT pushed the focused larger 4.8 µm diameter particles in the upward direction to

isolate them from smaller 2.0 µm or 3.2 µm diameter particles. The proposed particle

separation device offers high-throughput operation with purity > 97% and recovery rate > 99%.

It is simple in its fabrication and versatile due to the single layered microchannel design,

combined with vertical hydrodynamic focusing and the use of both the horizontal and vertical

components of the ARF. Using the concept of upward migration of particles, another

Husnain Ahmed. Acoustofluidic Manipulation of Microspheres via Vertical

Hydrodynamic Focusing and Upward Migration. School of Mechanical

Aerospace & Systems Engineering, Division of Mechanical Engineering.

2017. 38+vi pages. Advisor Prof. Sung, Hyung Jin.

acoustofluidic device was presented for the concentration and separation of four different sized

micro-objects inside a single-layered straight polydimethylsiloxane (PDMS) microchannel

without using external pumps. Two parallel placed interdigitated transducers (IDTs) were used

to produce high frequency (73 MHz & 140 MHz) traveling surface acoustic waves (tSAWs)

that trap and concentrate the 12 µm and 4.8 µm diameter particles at two different locations

inside the PDMS microchannel without the assistance of microfabricated PDMS membrane,

while allowing the 2.1 µm particles to filter through the chromatography of different size

microspheres.

Keywords: Acoustofluidics, particle separation, vertical component, acoustic radiation force,

trapping, concentration, chromatography.

i

Table of Contents

Table of Contents …………………………………………………………………………….……....i

List of Figures………………………………………………………………………………….……..ii

Chapter 1. Introduction

1.1. Acoustofluidic separation of vertically focused particles in a continuous flow………………….2

1.2. Acousto-chromatography based batchwise concentration and separation of particles…………...6

Chapter 2. Material and methods

2.1. Device fabrication………………………………………………………………………………...7

2.2. Tap based microchannel fabrication……………………………………………………………...9

2.3. Particles solution preparation……………………………………………………………………..9

2.4. Experimental setup………………………………………………………………………………..9

Chapter 3. Acoustofluidic separation of vertically focused particles in a continuous flow

3.1. Working Mechanism…………………………………………………….………………………11

3.2. Results and discussion……………………………………………….…………………………..15

3.2.1. Device operation at low flow rates…………………………………………………………….15

3.2.2. Characterization of inlets and outlets flow rate………………………………………………..17

3.2.3. Device operation at high flow rates……………………………………………………………19

3.2.4. Tape based microchannel for the separation of particles………………………………………23

3.3. Conclusions………………………………………………………………………………………23

Chapter 4. Acousto-chromatography based batchwise concentration and separation of particles

4.1. Working Mechanism………………………………………………………..……………………25

4.2. Results and discussion……………………………………………………….…………………..26

4.2.1. Concentration of particles by TSAWs…………………………………………………………26

4.2.2. Particles trapping from extremely low concentrated solution…………………………………30

4.2.3. A pumpless device for the concentration and separation of particles………………………….31

4.2.4. Naked eye view of device operation, sample collection and separation analysis……………...32

4.3. Conclusions………………………………………………………………………………………33

Bibliography…………………………………………………………………………………………35

Acknowledgement…………………………………………………………………………………...38

Curriculum Vitae…………………………………………………………………………………....39

ii

List of Figures

Figure 1.1. Tree diagram summarizes the most commonly used separation methods in

different categories. The current research route is shown in green……………………………..1

Figure 1.2. Uniformly spaced metal electrodes deposited on top of a piezoelectric substrate

(Lithium niobate, LiNbO3). Interdigitated transducer (IDT) was actuated by providing high

frequency AC signal. Only a certain AC frequency can actuate the IDT that depends on the

SAW speed 𝑐𝑆𝐴𝑊 , and inter-electrode gaps and widths

𝜆𝑆𝐴𝑊…………………………………..3

Figure 1.3. Calculation of the components of acoustic radiation force (ARF). The interaction

between TSAWs and the fluid results in leaky acoustic waves that radiate at an angle of ~22°

such that the vertical component Fv of ARF is more than twice its horizontal component

Fh…............................................................................................................................................4

Figure 1.4. Acoustofluidic particles separation using horizontal (a) and vertical (b)

components of acoustic radiation force (ARF)……………………………………………........5

Figure 2.1. (a) Deposition of Cr/Au metal electrodes on a LiNbO3 substrate by e-beam

evaporation method. (b) Fabrication of PDMS microchannel via soft lithography technique. (c)

Oxygen plasma bonding of PDMS microchannel with LiNbO3 substrate……………………...8

Figure 2.2. Experimental setup for acoustofluidics consisting of signal generator, DC power

supply, amplifier, micro syringe pump, microscope and camera, computer and acoustofluidic

device…………………………………………………………………………………………10

Figure 3.1. A schematic illustration showing the vertical migration of particles to realize size

based separation. The separation device is composed of a straight interdigitated transducer

(IDT) patterned on the lithium niobate (LiNbO3) substrate, a SiO2 layer and a straight PDMS

microchannel mounted on top. The top and side views of the device are illustrated as the

particles separation zone is enhanced when the power was turned off (a) and on (b),

respectively. Particles are slowed down by the horizontal component of the ARF Fh and pushed

in the upward direction depending on particle sizes due to the vertical component of ARF

Fh……………………………………………………………………………………………..13

iii

Figure 3.2. (a) A fabricated particle separation device. The PDMS microchannel was bonded

on top of the gold electrodes deposited onto a LiNbO3 substrate. Red dye was used to highlight

the microchannel and its ports. (b) The 3D solid geometry of the microchannel, with a top view.

(c) Side view of simulated streamlines within the straight microchannel……………………..14

Figure 3.3. (a) A 3D geometry and side view of the simulation of streamlines inside the straight

microchannel when the diameter of the punched hole is smaller than the width of the

microchannel. (b) The diameter of the punched holes is equal to the width of the

microchannel…………………………………………………………………………………15

Figure 3.4. (a) Simulation of the streamlines inside the microchannel at 𝑄1/𝑄2 = 𝑄4/𝑄3 with

zoom in side view of the streamlines after the 1st inlet. (b) Simulation of the streamlines inside

the microchannel at 𝑄1/𝑄2 < 𝑄4/𝑄3 with zoom in side view of the streamlines leaving

through 1st and 2nd outlet……………………………………………………………………...15

Figure 3.5. Photographic images of the particle separation experiment based on the upward

movement of particles under an applied ARF. (a) Power off: the particle mixture (green & red)

flowed together through the lower streamlines and resulted in no separation. (b) Power on:

green particles migrated upward toward the upper streamlines, resulting in separation………16

Figure 3.6. A side view graphic supporting the photographic images at the outlet pipes. (a)

Power off: a control experiment showing that particles of different sizes were collected through

the 2nd outlet. (b) Power on: Larger (green) and smaller (red) particles were collected through

1st and 2nd outlet, respectively.................................................................................................17

Figure 3.7. Particle separation results for 𝑄1/𝑄2 = 1/9 and 𝑄3/𝑄4 = 0.67/1. Flow cytometry

graphs showing the ratio of particles of different sizes collected at the collection (a) and waste

(b), outlets respectively……………………………………………………………………….18

Figure 3.8. Particle separation purity measures at different ratios of the inlet and outlet flow

rates. (a) Percentage of purity at various outlet flow ratios 𝑄3: 𝑄4 was varied from 9:1 to 0.67:1,

holding the inlet flow ratio fixed at 𝑄1: 𝑄2 = 1: 9. (b) Percentage of purities across inlet flow

ratios of 𝑄1: 𝑄2 = 1: 9 to 1: 1, holding the outlet flow ratio fixed at 𝑄3: 𝑄4 = 0.67: 1. (c), (d)

Hemocytometer images obtained showing the particle size compositions collected at different

outlet and inlet flow ratios.........................................................................................................19

iv

Figure 3.9. (a) A side view schematic diagram illustrating the height of the sheath flow and the

mixed particle flow within sections of the microchannel. Particles with 𝜅 > 1 were affected by

the ARF, whereas particles with 𝜅 < 1 were predominantly influenced by the streaming flow.

(b) Top view of the experimental images, illustrating the effects of SAWs on particles of

different sizes…………………………………………………………………………………20

Figure 3.10. Purity and recovery measures at different net flow rates QNET by holding the

conditions (𝑄1/𝑄2 = 1/9) and (𝑄3 < 𝑄4) for the isolation of green, 4.8 µm and red, 3.2 µm

particles………………………………………………………………………………………21

Figure 3.11. Hemocytometer images of sample collection at first outlet. Varied the net

flowrates (𝑄NET) by fixing the inlets and outlets flow ratio, 𝑄1 : 𝑄2 = 1 : 9 and 𝑄3 < 𝑄4 for

the separation of green, 4.8 µm and red, 3.2 µm particles………………………...…………...22

Figure 3.12. Hemocytometer images of sample collection at second outlet. Varied the net

flowrates (𝑄NET) by fixing the inlets and outlets flow ratio, 𝑄1 : 𝑄2 = 1 : 9 and 𝑄3 < 𝑄4 for

the separation of green, 4.8 µm and red, 3.2 µm particles……………………………………..22

Figure 3.13. Simulation of the streamlines inside 250 µm wide microchannel with 1mm

punched hole at 𝑄1/𝑄2 = 1/9 and 𝑄3/𝑄4 = 0.67/1 for different net flow rates…………..23

Figure 3.14. Simulation of the streamlines inside 250 µm wide microchannel with 500 µm

punched hole at 𝑄1/𝑄2 = 1/9 and 𝑄3/𝑄4 = 0.67/1 for different net flow rates……………23

Figure 3.15. (a) A top view and (b) side view of the schematic with experimental images of

the manually fabricated scotch tape based microchannel……………………………………..24

Figure 4.1. A schematic diagram showing the concentration and separation of three different

diameter particles. The device is composed of two parallel placed straight interdigitated

transducer (IDT) patterned on the lithium niobate (LiNbO3) substrate, a SiO2 layer and a

straight PDMS microchannel loosely positioned on top without plasma bonding. The top and

side views of the device are showed as the concentration and separation zone is enlarged during

SAW off (a) and on (b) respectively. Particles slowed down by the horizontal component of

v

ARF Fh and concentrated by the vertical force component Fv depending on particle

diameters………………………………………………………………………………..........27

Figure 4.2. A schematic diagram supported by a photographic image of particle concentration

inside the microchannel due to the combination of horizontal and vertical force components.

Hemocytometer images of inlet sample solution and outlet collected sample illustrated the

efficient concentration of microspheres inside the microfluidic channel……………………..28

Figure 4.3. Characterization of particle concentration inside the microchannel. The

concentration of the sample solution is 400 particles/µL. (a) Experimental images of top view

of the microchannel and graph (b) showing the linear increase in the particle concentration

from 1 sec to 30 sec. (c) Bar chart illustrate the decrease in the number of particles per microliter

in the sample solution results in the increase in required time to achieve fixed

concentration…………………………………………………………………………………28




from 6 sec to 60 sec…………………………………………………………………………...29




from 15 sec to 120 sec………………………………………………………………………...29




from 1 min to 10 min………………………………………………………………………….30




from 3min to 30 min…………………………………………………………………………..30

vi


concentration of the sample solution is 1 particle/µL. (a) Experimental images of top view of

the microchannel and graph (b) showing the linear increase in the particle concentration from

10 min to 120 min…………………………………………………………………………….31

Figure 4.9. Efficient particles trapping by the vertical component of ARF from extremely low

concentrated particles solution. The concentration of sample solution is 10 particles/ml. 99 out

of 100 targeted particles were captured in 2 hour……………………………………………..32

Figure 4.10. Photographic images of the particle concentration and separation experiment

based on the stronger force component of an applied ARF imposed by TSAWs. (a) Power on:

blue particles passed through the chromatography of red and green particles. (b) Power on:

after washing with ethanol red and green particles concentration is displayed. (c) Naked eye

view of the concentrated (different diameter) particles inside the microchannel. (d)

Hemocytometer images obtained displaying the particle size compositions of concentrated and

separated particles…………………………………………………………………………….33

Figure 4.11. Photographic image of the experimental setup for naked eye view of the particles

chromatography and sample collection from the outlet for separation analysis………………34

1

Chapter 1. Introduction

Microfluidic particle and cell separation takes over the conventional method of cell

separation. The later method usually employed membrane based filtering schemes which are

limited by the membrane pore size and are easily susceptible to clogging after a particular time

period. In contrast, microfluidics offer various advantages including: reduced sample volumes,

fast sample processing, high sensitivity, low cost, integrated systems minimized the chances of

sample contamination and increased flexibility in potential for point-of-care diagnostic in the

areas which lacks clinical labs and skilled staffs. In the competition to develop innovative

microfluidic tools, acoustofluidics is competing with other microfluidic technologies like,

opto-fluidics, dielectrphoresis, electrophoresis, hydrodynamic filtration and inertial

microfluidics. A summary of most commonly used particle or cell separation methods along

with their classification is shown in the tree diagram (see Figure 1.1).

Figure 1.1. Tree diagram summarizes the most commonly used separation methods in different categories. The

current research route is shown in green.

In the present work, we designed two acoustofluidic devices for the continuous (device

1) and batch-wise separation (device 2) of different diameter particles. In the following

Label based

Label free

Current research

route

Active methods

Cell/particle separation methods

Acoustofluidics

Dielectrphoresis

Electrophoresis

Optical sorting

FACS

MACS

Hydrodynamic filtration

Pinched flow fractionation

Inertial microfluidics

Pillar and weir structure

SAW

BAW

TSAW

SSAW

Horizontal force component

Vertical force component

Active techniques: rely on an external force field for functionality.

Passive techniques: rely entirely on the channel geometry and inherent hydrodynamic forces for functionality.

Label based techniques: the cell surface markers are identified via specific antibodies labeled with fluorescent molecules or magnetic beads. e.g. fluorescence-activated cell sorting (FACS) & magnetically actuated cell sorter (MACS)

Label free techniques: separation is often based on intrinsicproperties of the sample components, such as their size, charge or their polarizability.

SAW – Surface Acoustic WavesBAW – Bulk Acoustic WavesTSAW – Travelling Surface Acoustic WavesSSAW – Standing Surface Acoustic Waves

Passive methods

Separation markers:

shape, size, density, compressibility

2

subsections, we briefly summarized previous literature related to the separation and

concertation of particles and proposed our new findings.

1.1. Acoustofluidic separation of vertically focused particles in a

continuous flow

Over the past few decades, microfluidic particle and cell separation techniques have

played a vital role in cell biology, disease diagnostics, drug screening, drug discovery, and

biochemical analysis.1,2 For example, cancer treatments requires the isolation of circulating

tumor cells (CTCs) from peripheral blood samples.3,4 Likewise, the cure for malaria relies on

the separation of malaria-infected red blood cells from healthy cells.5–7 Sorting the different

types of stem cells, such as embryonic stem cells (hESCs),8,9 tissue-specific stem cells,10

mesenchymal stem cells (MSCs)11 and induced pluripotent stem cells (iPSCs)12,13 is essential

for the analysis of different diseases and organ-on-a-chip technology14 for the development of

new therapies and drugs.

To date, many researchers have explored various microfluidic methods for the

manipulation of particles inside minute volumes of fluids, such as inertial microfluidics,15

hydrodynamic filtration,16 magnetophoresis,17 dielectrophoresis,18,19 optofluidics,20,21 and

acoustophoresis.22–24 Acoustophoresis-based microfluidic separation techniques have been

preferred due to the contactless handling of the biological samples, low power requirement,

biocompatible nature of acoustic waves, etc. that ease their incorporation into micro total

analysis systems. Acoustophoresis based devices are classified based on their driving

mechanisms: bulk acoustic wave (BAW) and surface acoustic wave (SAW). BAW devices

consist of a piezoelectric transducer attached to microfluidic chip with acoustic waves

propagating in bulk of the material25 which generates ultrasonic standing waves to concentrate

or separate the particles inside the microchannel. However, in SAW based particle separation

devices a pair of interdigitated electrodes patterned on a piezoelectric substrate (see Figure

1.2) produces acoustic waves that travel along the surface of the substrate to manipulate the

particles.26 As most of the acoustic energy is concentrated on the surface of the substrate, SAW-

based devices are more energy efficient.

SAWs are classified into standing surface acoustic waves (SSAWs) and travelling

surface acoustic waves (TSAWs).27 A SSAW is a combination of two constructively interfering

3

TSAWs propagating in opposite directions. SSAWs form pressure nodes and anti-nodes inside

the fluid and are used to push particles toward regions of low pressure inside the microchannel,

achieving particle focusing and separation.28–30 On the other hand, TSAWs have been used in

Figure 1.2. Uniformly spaced metal electrodes deposited on top of a piezoelectric substrate (Lithium niobate,

LiNbO3). Interdigitated transducer (IDT) was actuated by providing high frequency AC signal. Only a certain AC

frequency can actuate the IDT that depends on the SAW speed 𝑐𝑆𝐴𝑊, and inter-electrode gaps and widths 𝜆𝑆𝐴𝑊.

cross-type acoustic particle separators to laterally migrate particles and realize separation

across the microchannel width or within a sessile droplet, because particles predominantly

migrate within the horizontal plane.31–34 Most SAW-based acoustofluidic separation techniques

utilize forces that act on micro-objects suspended in a horizontal plane while pushing them

laterally inside the microchannel.35–39 The interaction between TSAWs and the fluid results in

leaky acoustic waves that radiate at an angle of ~22° (in systems comprising water and a lithium

niobate substrate) inside the microfluidic channel, such that the vertical component (Fv) of the

acoustic radiation force (ARF) acting on the suspended particles is ~2.5 times greater than the

horizontal component of the force (Fh), i.e., Fv ≅ 2.5Fh (see Figure 1.3). The SAW-based

acoustofluidic devices that utilize the horizontal component of an ARF are composed of an

interdigitated transducer (IDT) integrated into the side of a single layered PDMS

microchannel37,40,41 (see Figure 1.4(a)). A multiple-layered PDMS microchannel with a post

beneath the micro-object manipulation zone was also used in a similar fashion to deflect

particles37 and sort cells or droplets.35 IDTs may also be positioned directly beneath a

microchannel to induce desired particle migration. Collins et al. used a single IDT to produce

standing acoustic waves within a single layered microchannel to achieve particle separation.42

However, TSAWs, produced by an IDT placed beneath a single layered microchannel, have

not yet been demonstrated to be capable of inducing vertical particle migration by imposing a

λλ/4

SAW SAW

Straight Interdigitated transducer (SIDT)

LiNbO3

~

𝑆𝐴𝑊 =𝑐𝑆𝐴𝑊𝜆𝑆𝐴𝑊

= 𝐴

4

direct ARF onto particles directly from the bottom of the microchannel (see Figure 1.4(b)).

Previously our research group developed a cross-type particle separation device that showed

vertical particle migration, although the effect was not harnessed for an application.24 Collins

et al.43 took advantage of the vertical component of the ARF and employed a focused IDT to

trap and concentrate selected particles behind a micro-fabricated PDMS membrane. However,

the particle trapping capacity of the membrane would reach a saturation value in a short period

of time; therefore, a continuous flow separation device offers improved particle separation

performance..22,29

Figure 1.3. Calculation of the components of acoustic radiation force (ARF). The interaction between TSAWs

and the fluid results in leaky acoustic waves that radiate at an angle of ~22° such that the vertical component Fv

of ARF is more than twice its horizontal component Fh.

In the present work, a straight IDT was deployed with a straight loosely aligned

microchannel positioned perpendicularly to the uniformly spaced metal electrodes. Compared

to most SSAW-based devices29,44,45 that usually require a pair of parallel IDTs tightly aligned

with the microchannel, the present device did not require tight alignment with the microchannel

similar to other TSAW-based devices.22,24 However, tilted angle SSAWs have been utilized to

circumvent such limitations.46 The proposed design provided an additional advantage of

utilizing both the horizontal and stronger vertical components of the ARF acting on the

particles. The height and width of the microchannel did not significantly alter the device

performance, unlike the SSAW-based devices, in which the microchannel aspect ratio

significantly affected the locations of the pressure nodes and anti-nodes.28,44,45,47 The present

SAW

x

Y+ + --

Side viewLiNbO3

Snell’s law:

= 90

= 𝑐

𝑐 ( )

TSAW

LeakyTSAW

𝑐

𝑐

𝑐 000

𝑐 1 00

=

;

= = .

5

device utilized a comparatively low input power because the energy loss to PDMS walls was

minimal as the IDT was directly exposed to the fluid and both the vertical and horizontal

components of the ARF were employed. A single layered PDMS microchannel with a simple

design was used to continuously isolate the selected particles based on their diameters by

inducing vertical migration. Furthermore, particle focusing based on two sheath flows, as used

in most SAW-based particle separation devices,4,23,29,38 was circumvented in the present work

by focusing the particles along the vertical direction using a single sheath flow. Note that some

SPLITT or H-filter based separation devices used a single sheath flow configuration. For

instance, Hawkes et al.48 used a single sheath flow to focus the yeast cells along the side of the

microchannel wall prior to washing them using SSAWs. However, the migration of the cells

was in horizontal direction and the device required a fabrication of multiple layered

microchannel. Recently Chen et al.49 demonstrated the separation of platelets from whole blood

by focusing the biological sample vertically and then pushing them upward using BAWs in a

multiple layered microchannel. In contrast, the present device strictly distinguishes the external

force components (horizontal from vertical), which is available with TSAW device only. The

mechanism for SSAW and BAW based devices would be different. Among the single layered

PDMS based devices, similar kind of focusing is not reported before. Due to the utilization of

the principal component of ARF, the present device successfully operated for particle

separation at net flow rate up to 1.3 mL/min which is approximately 100 times higher than used

in previously reported SAW devices.22–24,29,38,39,42,47,50,51 The throughput of the proposed device

can be further improved with ease by increasing the width of the microchannel since the

microchannel is placed directly on the IDT. This low power, high throughput tiny device will

be useful for point-of-care testing applications based on biological sample separation.

Figure 1.4. Acoustofluidic particles separation using horizontal (a) and vertical (b) components of acoustic

radiation force (ARF).

TSAW in

horizontal

direction

Top view

x

z

SAW

IDT(a)

SAW

TSAW in

vertical

direction

x

Y

Side view PDMS microchannel

(b)

6

1.2. Acousto-chromatography based batchwise concentration and

separation of particles

The separation and concentration of a specific analyte such as circulating tumor cells or

malaria-infected red blood cells are fundamental for its diagnostic or therapeutic efficacy. In

addition to the high throughput separation of particles in a continuous flow, a pumpless

microfluidic device was designed to trap and concentrate the extremely low concentrated

particles solution. As described earlier, Collins et al.43 employed the focused IDT to

concentrate the selected particles behind the microfabricated membrane inside the

microchannel, while allowing the smaller particles to separate. However, the fabrication of

complex geometry of microchannel and micro-membrane is difficult in comparison to the

fabrication of a single layered PDMS microchannel. Li et al concentrated the particles inside

the sessile droplet of water by using SAWs. Similarly Destgeer et al. concentrated and

separated the different sized particles inside the sessile droplet.31 However, this kind of

separation can only be useful for diagnostics which requires only visualization. It’s not possible

to collect the sample after separation of particles inside the sessile droplet; as, the distinct

regimes of different sized particles might get disturb while collecting the separated samples

with pipette.

In the present work, a SAW-based pump-free particle concentration and separation

device was proposed, which used a single-layer PDMS microchannel (without microfabricated

membrane) to concentrate the particles based on their diameter at various locations inside the

microchannel by taking advantage from the vertical component of ARF, while allowing the

smaller particles to separate through the concentration of different size micro-particles. After

concentration of particles inside the microchannel we successfully collected each separated

sample at the outlet for analysis.

7

Chapter 2. Materials and methods

The fabrication of acoustofluidic devices was carried out in two steps. At first step metal

electrodes were deposited on a piezoelectric substrate while in the second step PDMS

microfluidic channel was fabricated using soft lithography technique (see Figure 2.1). These

fabrication processes and particles solution preparation is further discussed in detail in the

following subsections.

2.1. Device Fabrication

The acoustofluidic device as shown in Figure 3.2(a) was composed of a piezoelectric

(Lithium Niobate, LiNbO3) substrate (500 µm thick, 128 Y-X cut, MTI Korea, Korea) with

an IDT deposited on top and a PDMS microfluidic channel. A bimetallic (Cr/Au, 300 Å/1000

Å) layer of interdigitated electrodes was deposited, using the e-beam evaporation method and

lift-off process, to form an IDT having a total aperture of 1 mm and 20 electrode finger pairs

with uniform widths and spacings in between (λ/4 = 6.5 μm). A thin layer of SiO2 (2000 Å)

was also deposited on top of the electrodes using plasma enhanced chemical vapor deposition

to keep them safe from mechanical damage and enhance bonding. The whole process is brifely

explained in Figure 2.1(a).

A commonly used soft photolithographic process was used to fabricate PDMS

microchannel; 80 µm thick layer of SU-8 2100 (Micro Chem, USA) was spin coated on top of

a Si substrate. Later on, after soft baking at 65 °C for 5 min and 95 °C for 20 min photoresist

was exposed to UV light through hard contact of patterned chrome mask to make the desired

shape of the microchannel. After that, the SU-8 pattern was post-baked at 65 °C for 5 min and

95 °C for 10 min. At the end, it was dipped in the SU-8 developer to remove the unexposed

area of negative photoresist. Finally, the desired SU-8 pattern was rinsed with isopropyl alcohol

and DI water. The SU-8 pattern on Si substrate acted as a mold that could be transmitted to the

PDMS. A PDMS base and a curing agent (Sylgard 184A & 185B, Dow corning, U.S.A) mixed

in a 10:1 ratio and poured on top of the SU-8 mold. The bubbles formed during mixing of the

PDMS base and the curing agent were removed by putting the mixture under vacuum. PDMS

was cured at 65 °C for at least two hours and later on peeled off the Si substrate. SU-8 mold

8

was exposed to trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane (Sigma-Aldrich, U.S.A) under

vacuum for the surface treatment prior to pouring PDMS mixture. It helps in the easy release

of PDMS from the SU-8 mold after hard baking. After the inlet and outlet ports were punched

through the PDMS using punching tool (Harris Uni-Core), the microchannel and LiNbO3

substrate were exposed to oxygen plasma52 in a multipurpose oxygen plasma system (Covance,

Femto Science, Korea) for 2 min at 150 W and 750 mTorr. The microchannel was aligned

perpendicular to the IDT and bonded to the substrate in such a way that IDT would be manually

placed between the second inlet and first outlet. As the PDMS microchannel was 250 µm in

width and 80 µm in height, it did not require a tight alignment against the IDT with 1 mm

aperture. This process of microchannel fabrication is brifely explained in Figure 2.1(b,c).

Figure 2.1 (a) Deposition of Cr/Au metal electrodes on a LiNbO3 substrate by e-beam evaporation method. (b)

Fabrication of PDMS microchannel via soft lithography technique. (c) Oxygen plasma bonding of PDMS

microchannel with LiNbO3 substrate.

LiNbO3 substrate

Positive photoresist

UV exposure through chrome mask

Develop photoresist

Deposit Cr/Au layer

Lift –off process

(a) Electrode deposition on LiNbO3

Si substrate

Negative (SU-8) photoresist

UV exposure

Develop photoresist

Pour PDMS and cure

Peel off PDMS

(b) Fabrication of PDMS microchannel

(c) Oxygen plasma bonding

SAW device PDMS microchannel

Final device

Deposition of SiO2 layer

9

2.2. Tape based microchannel fabrication

In addition to standard MEMS and soft lithography method of microchannel fabrication,

we also fabricated the microchannel using scotch tape manually; as height and width of the

microchannel does not play a critical role in our device for particle separation. In contrast to

spin coating a photoresist on top of a silicone substrate and then making a mold of a photoresist

using photomask and UV aligner, a piece of a polyimide silicone adhesive tape (thickness of

70 µm that defines microchannel height, Tianjin Fortune, China) was manually cut (width ~800

µm) and pasted on top of the Si substrate which acted as a mold for the PDMS microchannel.

After a mixture of PDMS and its curing agent (10:1) was poured on top of the scotch-tape mold,

it was vacuumed to remove unnecessary bubbles, and was baked at 65 °C for at least two hours.

Rest of the process is similar to what has already been explained in the previous section. This

process of making a microchannel could be a cost-effective alternative compared to the

standard soft lithography process.

2.3. Particles solution preparation

Three different sized polystyrene particles having the same density were used in two pairs

(4.8 µm + 2.0 µm, and 4.8 µm + 3.2 µm) to demonstrate the separation mechanism: 4.8 µm

green fluorescent (<5% uniformity, No.G1000, Thermo Scientific, CA, USA), 3.2 µm red

fluorescent (<5% uniformity, No.R0300, Thermo Scientific, CA, USA) and 2.0 µm red

fluorescent (<5% uniformity, No.R0200, Thermo Scientific, CA, USA). Each of the particle

solution consisted of 1% solid microspheres per microliter of liquid as the total number of

beads/µl were counted (green 4.8 µm: 1.72 × 105, red 3.2 µm: 5.81 × 105, red 2.0 µm: 2.39 ×

106) for each particle solution. To add an equal number of particles in a sample, 13.9 µL of

green (4.8 µm) and 1 µL or 3.4 µL of red (2.0 µm or 3.2 µm) particle solutions per 1 ml of DI

water were added. Later on, 21.8 wt. % glycerin and 1 wt. % tween 20 was added.53 The

addition of glycerin matched the density of the particles with water and prevented the particles

from settling down in water. Tween 20 was added to avoid the formation of doublets and

triplets of the particles by preventing particle-to-particle adhesion.

10

2.4. Experimental setup

A schematic of experiment layout is shown in Figure 2.2. The device was mounted on

top of a fluorescent microscope stage (BX53, Olympus, Japan) for visualization using an

appropriate lens (2x, 4x or 10x magnification). The mixture of fluorescent polystyrene particles

with diameters 4.8 µm (green) and 2.0 µm (red) was pumped (neMESYS, Cetoni GmbH,

Germany) into the microchannel through the first inlet. DI water was pumped as a sheath flow

through the second inlet to force the particles solution to flow in the lower streamlines. One of

the two outlets was connected with a pump to suck the fluid out by applying a negative pressure

and another outlet was opened to atmospheric pressure for the collection of sample for analysis.

The inlet flow rates used were 50 µL/hr and 450 µL/hr for sample and sheath flows,

respectively, unless otherwise mentioned.

Figure 2.2 Experimental setup for acoustofluidics consisting of signal generator, DC power supply, amplifier,

micro syringe pump, microscope and camera, computer and acoustofluidic device.

The flow rates at the outlets were readily adjusted to achieve a maximum separation. An RF

signal generator (N5181A, Agilent Technologies, U.S.A) was used to generate an AC signal at

140 MHz frequency and tunable amplitude 4.5-50 mV that was amplified (LZT-22+, Mini-

Circuits, U.S.A) up to 11-1330 mW before feeding into the device. The images of green and

red fluorescent particles were captured by CCD camera (DP72, Olympus, Japan) using separate

filters and later on stacked together to obtain a single image. The captured microscopic images

Signal generator, N5181A [3 GHz]

DC power supply, E3634A

μ-pump, neMESYS

Microscope, BX51

Camera, DP72

Power amplifier,

ZHL-1-2W

1st

Inlet

2nd

Inlet

1st

outlet

2nd

outlet

LiNbO3

Au electrodes

PDMS

11

were processed to remove the background noise using ImageJ (http://imagej.nih.gov/ij/)

software for better image quality. The collected sample particles at two outlets were analyzed

through ImageJ software and counter-analyzed with a c-chip disposable hemocytometer

(Digital Bio, Korea). At the end, the particles were counted using flow cytometry at the specific

flow ratios at the inlets and outlets to confirm the efficient separation of the particles.

12

Chapter 3. Acoustofluidic separation of vertically focused

particles in a continuous flow

3.1. Working Mechanism

A schematic diagram of the particle separation device displays a simple PDMS

microchannel attached to a piezoelectric substrate (LiNbO3) with an IDT deposited on top of it

(see Figure 3.1). A silicon dioxide (SiO2) layer on top of the IDT protected the electrodes from

mechanical damage and enhanced PDMS–substrate bonding. A straight PDMS microfluidic

channel consisting of two inlet ports and two outlet ports was mounted on top of the IDT in

such a way that the IDT was positioned between the second inlet and the first outlet. A mixture

of two different sized particles (larger green, smaller red) was injected through the first of the

two inlets at a flow rate of 𝑄1, and a sheath fluid in the form of deionized (DI) water was

introduced through the second inlet at a flow rate of 𝑄2. The purpose of the sheath flow was to

pinch the sample mixture in the lower streamlines using the DI water flowing in the upper

streamlines to create a vertical double-layered flow while hydrodynamically focusing the

particles close to the bottom of the microchannel.54,55 The first of the two outlets was used to

pump out the fluid at a flow rate of 𝑄3 by applying a negative pressure, while the remaining

fluid was collected through the second outlet, which was open to the atmosphere, with a flow

rate of 𝑄4. The sheath flow induced all particles to flow through the lower fluidic streamlines,

and particles could be collected through the second outlet, without separation when the power

was off (see Figure 3.1(a)). Previous studies demonstrated that the value of the 𝜅-factor,

defined as 𝜅 = 𝜋𝑑 /𝑐 , where 𝑑 is the particle diameter, is the TSAW frequency, and 𝑐 is

the speed of sound in the fluid, could be used to effectively characterize the particle motion

under a TSAW.27 The TSAW frequency and the particle diameters were chosen such that 𝜅 >

1 for larger (green) particles and 𝜅 < 1 for smaller (red) particles. Once the AC signal was

applied to the IDT, it generated an acoustic wave that applied a significant ARF to the larger

(green) particles in the horizontal and vertical directions. The horizontal (X-direction)

component of the ARF (Fh) slowed down the particles against the direction of the flow due to

the resultant drag force Fd on the particle, whereas the major (Y-direction) component of the

ARF (Fv) pushed the green particles vertically upward into the upper streamlines, which were

ultimately collected through the first outlet. On the other hand, smaller (red) particles continued

to flow in the lower streamlines, nearly unaffected by the ARF, and were ultimately collected

13

through the second outlet (see Figure 3.1(b)). As a result, the green particles and red particles

were continuously separated through the first and second outlets based on their size difference.

Figure 3.1. A schematic illustration showing the vertical migration of particles to realize size based separation.

The separation device is composed of a straight interdigitated transducer (IDT) patterned on the lithium niobate

(LiNbO3) substrate, a SiO2 layer and a straight PDMS microchannel mounted on top. The top and side views of

the device are illustrated as the particles separation zone is enhanced when the power was turned off (a) and on

(b), respectively. Particles are slowed down by the horizontal component of the ARF Fh and pushed in the upward

direction depending on particle sizes due to the vertical component of ARF Fh.

A fabricated particle separation device is shown in Figure 3.2(a). A red dye was used to

highlight the microchannel, along with the inlet and outlet ports. The vertical migration of

particles induced by the stronger Y-directional component of the ARF could be harnessed to

induce particle separation only in the presence of the double-layered flow within the

microchannel. The streamlines in the microchannel were simulated using various inlet and

outlet port sizes to optimize the geometry for stabilizing the double-layer flow. The 3D

geometry of the microchannel is shown in Figure 3.2(b). If the diameter of the inlets and outlets

holes were smaller than the width of the microchannel, the streamlines from the first inlet

followed a path around the streamlines coming from the second inlet. This flow resulted in a

horizontally triple-layered flow that was not suitable for separating particles, because they

weren’t focused prior to exposure to the TSAW (see Figure 3.3(a)). If the size of the punched

14

holes was equal to (see Figure 3.3(b)) or larger than the width of the microchannel, however,

the device favored the desired double-layered flow, as the streamlines (blue) emerging from

the second inlet readily pinched the streamlines (red) emerging from the first inlet (see Figure

3.2(c)). The streamlines originating from the first and second inlets were collected through the

second outlet and first outlet, respectively. In general, it was not possible to manually punch

inlet/outlet holes with a diameter equal to the width of the microchannel; therefore, a

microchannel design with inlet and outlet ports larger than the microchannel width was

preferred. A top view of the microchannel inlet port, shown in Figure 3.2(b), reveals that the

diameter D of the punched hole exceeded the width w of the microchannel to form a double-

decker flow inside the microchannel, as predicted by the simulation results shown in Figure

3.2(c). The breadth of the punched holes (with respect to the width of the microchannel) was

fixed, and the streamlines at different flow ratios of the inlets and outlets were simulated. The

purpose of this simulation was to observe the height ratio of the sheath flow streamlines and

particle mixture streamlines during steep focusing of the sample mixture streamlines in the

perpendicular plane (see Figure 3.4(a)), and to study the consequences of the streamline height

(at the outlets) on the separation efficiency (see Figure 3.4(b)).

Figure 3.2. (a) A fabricated particle separation device. The PDMS microchannel was bonded on top of the gold

electrodes deposited onto a LiNbO3 substrate. Red dye was used to highlight the microchannel and its ports. (b)

The 3D solid geometry of the microchannel, with a top view. (c) Side view of simulated streamlines within the

straight microchannel.

15

Figure 3.3. (a) A 3D geometry and side view of the simulation of streamlines inside the straight microchannel

when the diameter of the punched hole is smaller than the width of the microchannel. (b) The diameter of the

punched holes is equal to the width of the microchannel.

Figure 3.4. (a) Simulation of the streamlines inside the microchannel at 𝑄1/𝑄2 = 𝑄4/𝑄3 with zoom in side view

of the streamlines after the 1st inlet. (b) Simulation of the streamlines inside the microchannel at 𝑄1/𝑄2 < 𝑄4/𝑄3

with zoom in side view of the streamlines leaving through 1st and 2nd outlet.

16

Figure 3.5. Photographic images of the particle separation experiment based on the upward movement of particles

under an applied ARF. (a) Power off: the particle mixture (green & red) flowed together through the lower

streamlines and resulted in no separation. (b) Power on: green particles migrated upward toward the upper

streamlines, resulting in separation.

3.2. Results and discussion

3.2.1. Device operation at low flow rates

A mixture of microspheres was separated using a single IDT placed under a straight

microchannel, as shown in Figure 3.5. Experimental images revealed the extent of particle

separation under the applied vertical component of the ARF. The particle mixture (green, 4.8

µm & red, 2.0 µm) and DI water were injected through the first and second inlets with flow

rates of 50 µL/hr (𝑄1) and 450 µL/hr (𝑄2), respectively. The first and second outlets were used

to collect the particles at flow rates of 200 µL/hr (𝑄3) and 300 µL/hr (𝑄4), respectively. When

the device was turned off, the large (green) and small (red) particles flowed together over the

IDT along the lower streamlines below the sheath flow streamlines, as shown in Figure 3.5(a).

These conditions resulted in no separation of particles. The 𝜅-factor values for the 4.8 µm

particles (green) and 2.0 µm particles (red) were calculated to be 1.407 and 0.586 ,

respectively.23 Therefore, when the power was turned on, the green particles were pushed

17

upward due to the vertical component of the ARF, because 𝜅 > 1, whereas the smaller red

particles with 𝜅 < 1 flowed along the lower streamlines, as shown in Figure 3.5(b). The green

particles were collected at the first outlet whereas the red particles moved toward the second

outlet. These effects produced the successful separation of particles, as indicated in the

photographic images of the outlets, shown in Figure 3.6.

Figure 3.6. A side view graphic supporting the photographic images at the outlet pipes. (a) Power off: a control

experiment showing that particles of different sizes were collected through the 2nd outlet. (b) Power on: Larger

(green) and smaller (red) particles were collected through 1st and 2nd outlet, respectively.

A side view of the outlet pipes confirmed the separation of particles. When the power

was turned off, all of the particles (green & red) were collected at the second outlet. Not a

single particle flowed through the first outlet, as shown in Figure 3.6(a). When the power was

turned on, however, the particles were effectively separated as the larger particles (green)

passed through the first outlet and the smaller (red) particles passed through the second outlet,

as shown in Figure 3.6(b).

Flow cytometry was used to count the particles collected from the first outlet (collection)

and the second outlet (waste) under the experimental conditions 𝑄1/𝑄2 = 1/9 and 𝑄3/𝑄4 =

0.67/1, as shown in Figure 3.7. The size and location of the square boxes were adjusted by

using the flow cytometry results obtained from pure samples of green (large) and red (small)

particles. Our aim was to isolate the green particles from the mixture of green and red particles

by pushing the larger particles in the upward direction. The green particles were separated

through the collection outlet with a 99.1% purity, and the red particles were separated through

the waste outlet with a 99.9% recovery rate.

18

Figure 3.7. Particle separation results for 𝑄1/𝑄2 = 1/9 and 𝑄3/𝑄4 = 0.67/1. Flow cytometry graphs showing

the ratio of particles of different sizes collected at the collection (a) and waste (b), outlets respectively.

3.2.2. Characterization of inlets and outlets flow rate

In addition to the 𝜅-factor, we examined the effect of the flow ratio at the inlets (𝑄1, 𝑄2)

and outlets (𝑄3, 𝑄4). We first fixed the inlet flow ratio (𝑄1/𝑄2 = 1/9) so as to focus the

particles in the lower streamlines while varying the flow rate ratio at the outlets. We started

with an outlet flow ratio of 𝑄3/𝑄4 = 9/1. Under these flow conditions, all of the green (large)

particles were separated through the first outlet; however, a number of red (small) particles also

made their way into the green particle stream, resulting in only a 21.3% separation efficiency.

This low separation efficiency was attributed to the streaming flow produced by the TSAWs,

which disturbed the laminar flow streamlines inside the microchannel and resulted in mixed

particle sample collection at the outlets. The ratio of the sheath flow and sample flow heights

above the IDT was adjusted to achieve a high separation efficiency. We decreased the height

of the sheath flow and increased the height of the sample mixture flow after the IDT by

increasing the flow rate 𝑄4 and decreasing the flow rate 𝑄3 until the point(𝑄3/𝑄4 = 0.67/1).

Beyond this point, the green (large) particles passed through the second outlet along with the

red particles, reducing the recovery rate. As shown in Figure 3.8(a), the separation efficiency

increased as 𝑄3 decreased compared to 𝑄4. At 𝑄3/𝑄4 = 1. /1, the efficiency reached 90% and

exceeded 99% at 𝑄3/𝑄4 = 0.67/1.

We studied the effect of the inlet flow ratio by fixing the outlet flow ratio. Disruptions to

the flow separation by the streaming flow were avoided by selecting an outlet flow ratio at

19

which the separation reached a maximum (𝑄3/𝑄4 = 0.67/1). The flow rate at the inlet was

then varied to determine the effect of vertical particle focusing. As the inlets flow ratio 𝑄1/𝑄2

was increased from 1: 9 to 1: 1, as shown in Figure 3.8(b), the separation efficiency decreased

because the sample mixture flow height increased and the sheath flow decreased. The particles

were then dispersed under the sheath flow (not tightly focused). If the particles were dispersed

prior to reaching the separation zone, a non-uniform ARF effect on the particles would have

been observed.

Figure 3.8. Particle separation purity measures at different ratios of the inlet and outlet flow rates. (a) Percentage

of purity at various outlet flow ratios 𝑄3: 𝑄4 was varied from 9:1 to 0.67:1, holding the inlet flow ratio fixed

at 𝑄1: 𝑄2 = 1: 9. (b) Percentage of purities across inlet flow ratios of 𝑄1: 𝑄2 = 1: 9 to 1: 1, holding the outlet flow

ratio fixed at 𝑄3: 𝑄4 = 0.67: 1. (c), (d) Hemocytometer images obtained showing the particle size compositions

collected at different outlet and inlet flow ratios.

The flow ratios were adjusted accordingly to achieve a high separation efficiency. The

particle samples collected from the outlets at different inlet and outlet flow rate conditions were

quantitatively evaluated using the ImageJ software and a hemocytometer. Photographic images

showing a top view of the hemocytometer are presented in Figure 3.8(c, d) for different flow

ratios at the inlets and outlets.

The experimental results obtained at 𝑄1/𝑄2 = 1/9 and 𝑄3/𝑄4 = 0.67/1 are interpreted

as shown in the schematic diagram in Figure 3.9(a). As discussed earlier, the height of the

sheath flow 𝐻2 before the separation zone should be sufficiently greater than 𝐻1(𝐻1: 𝐻2 = 1: 9)

to pinch the green and red particles in the lower streamlines. By contrast, after the separation

zone, the height of the sheath flow streamlines 𝐻3 leaving through the first outlet should be

smaller than the height of the streamlines 𝐻4 exiting through the second outlet (𝐻3: 𝐻4 = : 3)

to avoid the effect of acoustic streaming on the separation efficiency.

20

Green particles with 𝜅 > 1 were affected by the ARF, whereas the red particles with 𝜅 <

1 were affected by the acoustic streaming. An experiment was performed to verify this

prediction. Particles were held stationary inside the microchannel. When the power was turned

on, the green particles with 𝜅 > 1 were pushed in the lateral and perpendicular directions due

to the horizontal and vertical components of the ARF;24 however, red particles with 𝜅 < 1 were

under the influence of the acoustic streaming flow,24 so they travlled along the circular

streamlines, displaying mixing behavior inside the microchannel, as shown in Figure 3.9(b).

Figure 3.9. (a) A side view schematic diagram illustrating the height of the sheath flow and the mixed particle

flow within sections of the microchannel. Particles with 𝜅 > 1 were affected by the ARF, whereas particles with

𝜅 < 1 were predominantly influenced by the streaming flow. (b) Top view of the experimental images, illustrating

the effects of SAWs on particles of different sizes.

3.2.3. Device operation at high flow rates

In addition to investigating the particles separation using the principal component of ARF

by uniquely focusing the particles in the vertical direction; we tested our device at higher flow

rates for the separation of 4.8 µm and 3.2 µm particles. We fixed the inlet flow ratio (𝑄1/𝑄2 =

1/9) and passively regulated the outlet flow ratio (𝑄3/𝑄4) by differential fluidic resistance,

and thus to reduce the number of external equipment. Flow rate at the first outlet 𝑄3 should be

greater than the flow rate at the second outlet 𝑄4, as we already characterized by giving a

negative pressure at one of the two outlets to avoid the effect of eckart streaming on the

separation efficiency. Figure 3.10 shows the purity and recovery measures at the first and

second outlets, respectively. Starting with the net flow rate, QNET = 5,000 µL/hr (cross sectional

velocity: V = 69.4 mm/s), we increased the QNET by doubling the prior one. As we increased

21

the QNET from 5,000 µL/hr to 80,000 µL/hr (V = 1110.4 mm/s) the purity gradually decreases

from 97.7% to 71.2%. In contrast, the recovery remains > 99% even at a much higher flow rate

(QNET = 80,000 µL/hr). Hemocytometer images of the sample collection at first and second

outlet are presented in Figure 3.11 and Figure 3.12 respectively for different net flow rates.

The gradual decrease in the purity with the increase of QNET was investigated. The effect of

streaming flow explained in Figure 3.9 might not play a major role in the decrease of purity at

higher flow rates; therefore we simulated the streamlines inside the microchannel to see the

behavior of streamlines at higher flow rates by varying the port size (inlets, outlets) and net

flow rate (QNET) .

Figure 3.10. Purity and recovery measures at different net flow rates QNET by holding the conditions (𝑄1/𝑄2 =

1/9) and (𝑄3 < 𝑄4) for the isolation of green, 4.8 µm and red, 3.2 µm particles.

Streamlines were simulated inside 250 µm wide microchannel with 1 mm inlets and

outlets port size, holding the inlets and outlets flow ratio constant (𝑄1/𝑄2 = 1/9 ; 𝑄3/𝑄4 =

0.67/1) as characterized before. As the 𝑄NET increased from 500µL/hr to 40,000µL/hr, there

was a formation of vortices at the first outlet due to the sudden change in the geometry.

Although 𝑄3 < 𝑄4 a few sample flow streamlines were still going with the sheath flow

streamlines resulting in a decrease of purity at the first outlet (see Figure 3.13). This effect of

vortices formation can be minimized by decreasing the port size. Simulation of streamlines

inside the microchannel with 500 µm diameter ports shows that there is a decrease of vortices

formation at the 1st outlet (See Figure 3.14). This problem can further be minimized by using

the hole size equal to the width of the microchannel in order to achieve high purity at both

outlets.

22

Figure 3.11. Hemocytometer images of sample collection at first outlet. Varied the net flowrates (𝑄NET) by fixing

the inlets and outlets flow ratio, 𝑄1 : 𝑄2 = 1 : 9 and 𝑄3 < 𝑄4 for the separation of green, 4.8 µm and red, 3.2 µm

particles.

Figure 3.12. Hemocytometer images of sample collection at second outlet. Varied the net flowrates (𝑄NET) by

fixing the inlets and outlets flow ratio, 𝑄1 : 𝑄2 = 1 : 9 and 𝑄3 < 𝑄4 for the separation of green, 4.8 µm and red,

3.2 µm particles.

23

Figure 3.13. Simulation of the streamlines inside 250 µm wide microchannel with 1mm punched hole at 𝑄1/𝑄2 =

1/9 and 𝑄3/𝑄4 = 0.67/1 for different net flowrates.

Figure 3.14. Simulation of the streamlines inside 250 µm wide microchannel with 500 µm punched hole at

𝑄1/𝑄2 = 1/9 and 𝑄3/𝑄4 = 0.67/1 for different net flowrates.

24

3.2.4. Tape based microchannel for the separation of particles

It should be noted that a simple, straight, manually fabricated tape-based microchannel

was used to successfully separate particles using the horizontal and vertical components of the

ARF. A mixture of 4.8 µm (green) and 2.0 µm (red) particles, at the characterized flow rate

conditions (𝑄1/𝑄2 = 1/9 ; 𝑄3/𝑄4 = 0.67/1) , was used to demonstrate the separation

process. Figure 3.15(a) shows the separation of particles inside the microchannel, supporting

with the experimental images of side view of the outlet pipes (see Figure 3.15(b)).

Figure 3.15. (a) A top view and (b) side view of the schematic with experimental images of the manually

fabricated scotch tape based microchannel.

3.3. Conclusions

We demonstrated the use of vertical hydrodynamic focusing to separate microparticles

in a straight microchannel using a single sheath flow. This technique is dissimilar from

previously reported methods of particle focusing using a pair of sheath flows that horizontally

focused particles in the center of a microchannel. The vertical focusing mechanism employed

here was implemented using a smaller device footprint compared to that used in horizontal

focusing techniques. Unlike previous acoustofluidic particles separation techniques, we used

the vertical component of the ARF to separate particles in a straight single layered

microchannel. The SAWs that leaked into water exerted a force along the vertical direction

(Fv) that far exceeded the force exerted in the horizontal direction (Fh), i.e. Fv ≅ 2.5 Fh. After

hydrodynamically pinching the particles in the downward streamlines (vertical direction), the

horizontal component of the ARF was used to slow down the motions of the larger particles

while at the same time pushing them upward using the vertical (major) component of the ARF.

We took advantage of the major component of the ARF to continuously separate 4.8 μm (green)

from 2.0 μm (red) and 3.2 μm (red) polystyrene microparticles. This method enables the highly

25

efficient (> 99% purity and recovery rates) separation of particles over a wide range of flow

rates using tens of milliwatts of power. Moreover, the device operates at higher flow rates (upto

80,000 µL/hr) with reasonable separation performance (purity 71.2%, recovery 99.4%).

Furthermore, we fabricated a straight microchannel using a strip of tape manually cut into the

desired shape. This method costs less than $1 for microchannel fabrication. This microchannel

fabrication procedure can replace standard MEMS and soft-lithography device fabrication

processes that cost more than $500 due to the use of expensive glass/chrome-based

photomasks. These characteristics render this particle separation technique a useful addition to

micro total analysis systems and point-of-care device.

26

Chapter 4. Acousto-chromatography based batchwise

concentration and separation of particles

4.1. Working Mechanism

A schematic of the pumpless microfluidic device shows a PDMS microchannel placed

on the SAW device which consists of a piezoelectric substrate (LiNbO3) with two IDTs

deposited on top of it (see Figure 4.1). A straight microfluidic channel with one inlet and one

outlet port was loosely aligned directly on top of the IDTs without plasma bonding. Firstly, a

few microliter of ethanol was injected through the inlet well with the help of pipette to make

the microchannel hydrophilic. Secondly, an inlet well was filled with the solution of different

diameter particles (red, green and blue fluorescent). The particles were flowed through the

microchannel due to the hydrodynamic pressure generated due to the height of the inlet well

and capillary action inside the microchannel. During SAW off, the particles mixture were

flowed through the microchannel and collected through the outlet which results in no separation

(see Figure 4.1(a)). Our study group and other researchers showed the interaction of TSAW

with water results in leaky SAW which radiate at an angle of 22o (with the microchannel side

wall based on Snell’s law) exerts force in the vertical direction (Fv) that far exceeded the force

in the horizontal direction (Fh), i.e. Fv ≅ 2.5Fh. Destgeer et al. illustrated the dependence of

ARF on particle diameter and TSAW in terms of a dimensionless number; 𝜅-factor, defined as

𝜅=𝜋𝑑 /𝑐 , where 𝑑 is the particle diameter, is the TSAW frequency, and 𝑐 is the speed of

sound in the fluid. This factor could be used to portray the particle motion under a TSAW. The

TSAW frequency of the IDT and the particle diameters were chosen such that the 𝜅>1 for red

particles and 𝜅<1 for green particles against first IDT. However, 𝜅>1 for green particles against

second IDT. The value of 𝜅-factor for smallest (blue) particles was less than one against both

the IDTs to ensure the least effect of ARF. During SAW on, two different sized particles with

𝜅>1 (red and green respectively) were trapped and concentrated beside first and second IDT

due to the vertical force component Fv generated from each IDT, while the smallest particles

with 𝜅<1 remained nearly unaffected by the ARF and filtered through the chromatography of

two different sized particles. Note that the horizontal (X-direction) component of the ARF Fh

was almost balanced with the drag force Fd on the particle; however, major (Y-direction)

component of the ARF Fv pushed the particles in the upward direction and seized its motion

27

through the microchannel based on the diameter of a particle and frequency of the particular

IDT. As a result, two different sized particles (red and green) were concentrated in specific

regions inside a single layered microchannel while the smallest (blue) particles were ultimately

collected through the outlet (see Figure 4.1(b)).

Figure 4.1. A schematic diagram showing the concentration and separation of three different diameter particles.

The device is composed of two parallel placed straight interdigitated transducer (IDT) patterned on the lithium

niobate (LiNbO3) substrate, a SiO2 layer and a straight PDMS microchannel loosely positioned on top without

plasma bonding. The top and side views of the device are showed as the concentration and separation zone is

enlarged during SAW off (a) and on (b) respectively. Particles slowed down by the horizontal component of ARF

Fh and concentrated by the vertical force component Fv depending on particle diameters.

4.2. Results and discussion

4.2.1. Concentration of particles by TSAWs

A sample solution containing similar sized particles was concentrated by using a single

IDT placed under a straight single-layered microchannel consisting of one inlet and one outlet.

A sample solution of 9.9 µm (green) diameter particles was pumped through the inlet with the

flowrate of 1000 µL/hr (Q1). When the device was turned off, all the green particles flowed

through the single-layered microchannel and collected through the outlet. It results in no

concentration of particles inside the microfluidic channel. The 𝜅-factor value for 9.9 µm

particles (green) was calculated to be 1.5. Therefore, when the device was actuated with AC

signal of 73 MHz frequency, the particles were pushed upward due to the vertical component

of the ARF and concentrated beside the IDT (without the assistance of microfabricated

membrane in a single-layered microchannel). However, the solution containing no particle was

h

h

LiNbO3

LiNbO3

PDMS

PDMS

IDT IDT

IDT IDT

Inlet well

Inlet well

PDMS

PDMS

LiNbO3

LiNbO3 Side view

Side view

Enlarged view

(a) SAW off

(b) SAW on

Top view

Top view Enlarged view

1st

IDT

> 1 < 1

> 1 < 1

2nd

IDT

28

collected at the outlet with flow rate of Q2. The hemocytometer images of inlet sample and

outlet sample can be seen in Figure 4.2.

Figure 4.2. A schematic diagram supported by a photographic image of particle concentration inside the

microchannel due to the combination of horizontal and vertical force components. Hemocytometer images of inlet

sample solution and outlet collected sample illustrated the efficient concentration of microspheres inside the

microfluidic channel.

In addition, the concentration of the polystyrene particles were characterized (w.r.t time) inside

the PDMS microchannel by changing the concentration of particles sample solution. With the

constant input power and flow rate as the sample concentration decreased the time required to

achieve the fixed concentration increases.

Figure 4.3. Characterization of particle concentration inside the microchannel. The concentration of the sample

solution is 400 particles/µL. (a) Experimental images of top view of the microchannel and graph (b) showing the

linear increase in the particle concentration from 1 sec to 30 sec. (c) Bar chart illustrate the decrease in the number

of particles per microliter in the sample solution results in the increase in required time to achieve fixed

concentration.

Outlet collected sample

Inlet Outlet

Inlet sample solutionTop view

Power on

SAWFrequency

73 MHz

Q1 Q2

Q1 (µL/hr)1000

0

10

20

30

40

50

0 10 20 30Time (s)

Inte

nsi

ty(a

.u)

0

5

10

15

400 200 100 20 10 1

110

115

120

Number of particles/µL

Tim

e (

s)

(b)

(c)

400 particles/µLInlet OutletTop view(a)

1 S

3 S

9 S

27 S

30 S

21 S

15 S

750 µm

29

We started with the sample concentration of 400 particles/µL. As the time increased from 1 sec

to 30 sec the intensity of the particles increased, which correspond to the increase in

concentration of particles inside the microchannel (see Figure 4.3(a,b)). Similarly, the

experiments were performed with the sample concentration of 200 particles/µL, 100

particles/µL, 20 particles/µL, 10 particles/µL and 1 particle/µL as shown in Figure 4.4 to

Figure 4.8 respectively. As the number of particles per microliter decreased from 400 to 1, the

time required to achieve the fixed intensity (concentration) increased from 0.38 min to 118 min

(see Figure 4.3(c)).



linear increase in the particle concentration from 6 sec to 60 sec.



linear increase in the particle concentration from 15 sec to 120 sec.

0

10

20

30

40

50

0 20 40 60Time (s)

Inte

nsi

ty(a

.u)

(b)

200 particles/µL

(a)6 S

12 S

18 S

24 S

30 S

36 S

42 S

48 S

54 S

60 S

0

10

20

30

40

50

0 20 40 60 80 100 120Time (s)

Inte

nsi

ty(a

.u)

100 particles/µL

(b)15 S

30 S

45 S

60 S

75 S

90 S

105 S

120 S

(a)

30



linear increase in the particle concentration from 1 min to 10 min.



linear increase in the particle concentration from 3min to 30 min.

(a)1 m

2 m

3 m

4 m

5 m

6 m

7 m

8 m

9 m

10 m

Time (m)

Inte

nsi

ty(a

.u)

(b)

200 particles/µL

0

10

20

30

40

50

0 5 10

20 particles/µL

(a)3 m

6 m

9 m

12 m

15 m

18 m

21 m

24 m

27 m

30 m

Time (m)

Inte

nsi

ty(a

.u)

(b)

200 particles/µL

0

10

20

30

40

50

60

70

0 10 20 30

10 particles/µL

31


solution is 1 particle/µL. (a) Experimental images of top view of the microchannel and graph (b) showing the

linear increase in the particle concentration from 10 min to 120 min.

4.2.2. Particles trapping from extremely low concentrated solution

We demonstrated a highly sensitive SAW device for the capturing of particles from

extremely low concentrated particles solution. A device consisting of an inlet and outlet

mounted on the IDT, which was actuated by providing an AC signal of 73 MHz frequency. A

sample solution containing 10 particles/mL (9.9 µm, green) was injected through the inlet hole.

The experiment was run for two hours with the flow rate of 5 ml/hr; so, the target was to trap

100 particles. The images captured with time lap of 10 min revealed that almost all the particles

were successfully trapped and concentrated beside an IDT due to the principal component of

ARF, which seized its motion through the microchannel because of 𝜅>1 (see Figure 4.9). After

1 min of the process, 1 particle was captured. The number of captured particles was increased

to nearly half of the target after 1 hour. It kept on increasing with the passage of time and after

completion of 2 hours, 99 particles were captured out of 100 targeted particles. The particles

were counted manually and images are presented in Figure 4.9 with constant time gap after

increasing the size of the particles in ImageJ software for the sake of better visualization.

(a)10 m

20 m

30 m

40 m

50 m

70 m

80 m

90 m

100 m

110 m

60 m 120 m 0

5

10

15

20

25

30

35

40

0 20 40 60 80 100 120Time (m)

Inte

nsi

ty(a

.u)

(b)

1 particle/µL

32

Figure 4.9. Efficient particles trapping by the vertical component of ARF from extremely low concentrated

particles solution. The concentration of sample solution is 10 particles/ml. 99 out of 100 targeted particles were

captured in 2 hour.

4.2.3. A pumpless device for the concentration and separation of particles

In addition to characterization of particles trapping and concentration, we proposed a

pump-free acoustofluidic device for the concentration and separation of different sized particles

solution, which can be operated without a microscope. A mixture of microspheres was

concentrated and separated using two parallel placed IDTs beneath the straight microfluidic

channel: contained an inlet well and an outlet created by slicing the PDMS microchannel at the

opposite side of an inlet (see Figure 4.10). Experimental images showed the extent of particles

accumulation and isolation under the applied vertical force component of TSAW. After an

injection of 20 µL ethanol, a 100 µL solution contained three different sized particles (red, 12

µm; green, 4.8 µm & blue 2.1 µm) were filled in the inlet well with the help of pipette. The

particles flowed through the microchannel at a certain velocity which depends on the height of

the inlet well. When the device was off, the particles mixture flowed through the microchannel,

which results in no concentration or separation of particles. When the device was actuated 12

µm (red) particles were concentrated beside first IDT (F = 73 MHz) due to the vertical

component of ARF as 𝜅>1 for red particles and 𝜅<1 for green and blue particles. Similarly,

4.8 µm (green) particles were concentrated alongside second IDT (F = 140 MHz) due to the

vertical component of ARF as 𝜅>1 for green particles and 𝜅<1 for blue particles. The results

demonstrate that red and green particles were concentrated beside first and second IDT

respectively and the blue (smallest) particles leave through the microchannel. Figure 4.10(a)

shows the separation of blue, 2.1 µm particles through the chromatography of red, 12 µm and

1 min

10 min 20 min 30 min 40 min



Top view

OutletInlet

Frequency = 73 MHz

Power = 0.3 W

Flow rate = 5 mL/hrNumber of trapped particles = 1

= 13 = 18 = 29 = 37

= 45 = 52 = 61 = 68

= 72 = 82 = 89 = 99

Particle size = 9.9 µm

Sample concentration

= 10 particles / mL

33

green, 4.8 µm particles. After the process of 100 µL sample, a same quantity of ethanol was

filled with the pipette to wash the inlet well followed by the microchannel. Figure 4.10(b)

shows the concentration of red, 12 µm and green, 4.8 µm and particles at specific locations

inside the microchannel.

Figure 4.10. Photographic images of the particle concentration and separation experiment based on the stronger

force component of an applied ARF imposed by TSAWs. (a) Power on: blue particles passed through the

chromatography of red and green particles. (b) Power on: after washing with ethanol red and green particles

concentration is displayed. (c) Naked eye view of the concentrated (different diameter) particles inside the

microchannel. (d) Hemocytometer images obtained displaying the particle size compositions of concentrated and

separated particles.

4.2.4. Naked eye view of device operation, sample collection and separation

analysis

The proposed device can be functioned properly without a microscope and thus to reduce

the number of external equipment. The acousto-chromatography of different sized particles can

be seen with the naked eye similar to the pregnancy test kit in which two colored lines can be

seen on the piece of paper. As shown in Figure 4.10(c) red (12 µm) and green (4.8 µm) particles

were concentrated inside the single-layered microchannel producing a purified sample of 2.1

µm (blue) particles which was collected manually by placing a micro-pipe at the outlet as

shown in the photographic image of experimental setup (see Figure 4.11). The collection of

concentrated red and green particles was done after washing the microfluidic channel with the

ethanol as described earlier. Firstly, second IDT of 140 MHz frequency was turned off and the

green particles colony was collected at the outlet in the similar fashion as blue particles were

collected during the process. Secondly, first IDT of 73 MHz frequency was turned off and the

red particles colony was collected at the outlet. As a result, red, green and blue particles were

(A) Power on (During process)

(B) Power on (After process)

(D)

12 µm 4.8 µm 2.1 µm

(C)

750 µm

140 MHz73 MHz 70% 95% 100%

34

collected at the outlet for separation analysis. A hemocytometer was used to measure the purity

of each collected sample (see Figure 4.10(d)). Blue particles were collected with 100% purity,

followed by the collection of green and red particles with 95% and 70% purity respectively.

The reduction in the purity of concentrated samples, green and red particles can be attributed

to the secondary acoustic radiation force field formed due to the vibration of microspheres

which trapped the undesired particles resulting in the decreased of purity.

Figure 4.11. Photographic image of the experimental setup for naked eye view of the particles chromatography

and sample collection from the outlet for separation analysis.

4.3. Conclusions

We demonstrated a novel acoustofluidic technique for the concentration and separation

of different diameter particles inside a single layered microchannel without the assistance of

microfabricated membrane. Particles from an extremely low concentrated solution can be

detected, trapped and concentrated in highly efficient manner inside the microchannel. This

characteristic is particularly useful for the concentration and isolation of specific analyte such

as circulating tumor cells (CTCs) which are present in very small number (1-10/ml) inside the

blood of an infected person. The major component of ARF generated by TSAWs acted as a

wall for the concentration of selective particles inside the microchannel while allowing other

Collection outlet

outlet

Experimental setup Inletwell

AC signal

AC signal sliced

35

particles to filter through it. The proposed method is non-contact, noninvasive and label free.

These features are particularly significant for biochemical assays. The present acoustofluidic

chip does not require the induction of external pumping to inject the sample solution inside the

microchannel. Moreover, the operation of this device (trapping, concentration, separation) can

be visualized without a microscope. These characteristics of the device result in a reduction of

external tools and could be useful for point-of-care testing applications based on biological

sample preparation.

36

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