bioparticle manipulation using acoustophoresis and...

Bioparticle Manipulation using

Acoustophoresis and Inertial Microfluidics

Muhammad Asim Faridi

Royal Institute of Technology

School of Biotechnology

Stockholm 2017

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© Muhammad Asim Faridi Stockholm 2017 Royal Institute of Technology School of Biotechnology Science for Life Laboratory SE-171 65 Solna Printed by Universitetsservice US-AB Drottning Kristinas väg 53B 114 28 Stockholm Sweden TRITA-BIO Report 2017:4 ISSN 1654-2312 ISBN 978-91-7729-264-7 Cover Image: Schematic illustration of elasto-inertial separation of blood cells and bacteria in a microchannel.

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Abstract

Despite the many promising advances made in microfluidics, sample preparation remains the single largest challenge and bottleneck in the field of miniaturised diagnostics. This thesis is focused on the development of sample preparation methods using active and passive particle manipulation techniques for point of care diagnostic applications. The active technique is based on acoustophoresis (acoustic manipulation) while the passive method is based on inertial microfluidics (hydrodynamic manipulation). In paper I, acoustic capillary-based cavity resonator was used to study aggregation of silica and polystyrene particles. We found that silica particles show faster aggregation time (5.5 times) and larger average area of aggregates (3.4 times) in comparison to polystyrene particles under the same actuation procedure. The silica particles were then used for acoustic based bacteria up-concentration. In paper II, a microfluidic-based microbubbles activated acoustic cell sorting technique was developed for affinity based cell separation. As a proof of principle, separation of cancer cell line in a suspension with better than 75% efficiency is demonstrated. For the passive sample preparation, inertial and elasto-inertial microfluidic approach that uses geometry-induced hydrodynamic forces for continuous size-based sorting of particles in a flow-through fashion were studied and applied for blood processing (paper III-V). In paper III, a simple u-shaped curved channel was used for inertial microfluidics based enrichment of white blood cells from diluted whole blood. A filtration efficiency of 78% was achieved at a flow rate of 2.2 ml/min. In paper IV, elasto-inertial microfluidics where viscoelastic flow enables size-based migration of cells into a non-Newtonian solution, was used to continuously separate bacteria from unprocessed whole blood for sepsis diagnostics. Bacteria were continuously separated at an efficiency of 76% from undiluted whole blood sample. Finally, in paper V, the inertial and elasto-inertial techniques were combined with a detection platform to demonstrate an integrated miniaturized flow cytometer. The all-optical-fiber technology based system allows for simultaneous measurements of fluorescent and scattering data at 2500 particles/s. The use of inertial and acoustic techniques for sample preparation and development of an integrated detection platform may allow for further development and realization of point of care testing (POCT) systems.

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Did they not reflect in their own selves. (Surat Ar-Rum 8, Al-Quran)

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ا"ب

#م! ! ا! اور ا!

vi

!"ہ ! ! ذرے %$ذرے ! اس "ر "

!"! ! #ا! ! ! #اں ! ! !

" ! ا!

For the sake of it I searched through the core of each particle

But

It fanned the sparks of my everlasting inquisitiveness into flames.

Faiz Ahmad Faiz

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List of publications and manuscripts

This thesis is based on the following articles or manuscripts, referred to in the text by their Roman numerals (I-V). The articles can be found in the appendix.

Paper I: Glass Capillary based cavity resonator for particle trapping study and bacteria up-concentration Muhammad Asim Faridi, Ida Sadat Iranmanesh, Harisha Ramachandraiah, Martin Wiklund and Aman Russom. Biomedical Microdevices (submitted). Paper II: Microbubble activated acoustic cell sorting: BAACS Muhammad Asim Faridi, Harisha Ramachandraiah, Ida Sadat Iranmanesh, Dimitry Grishenkov, Martin Wiklund and Aman Russom. Biomedical Microdevices (submitted). Paper III: Dean Flow Inertial Coupled Forces in Curved Channels Harisha Ramachandraiah, Sahar Ardabili, Muhammad Asim Faridi, Sergey Zelenin and Aman Russom. Biomicrofluidics. 2014;8(3):034117. Paper IV: Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics Muhammad Asim Faridi, Harisha Ramachandraiah, Indradumna Banerjee, Sahar Ardabili, Sergey Zelenin and Aman Russom. J.Nanobiotechnology. 2017 15:3. Paper V: Optical Fiber inertial focusing based micro Flowcytometer Sebastian Etcheverry, Muhammad Asim Faridi, Harisha Ramachandraiah, Walter Margulis, Fredrik Laurell and Aman Russom. Nature Communications (submitted). All papers are reproduced with permission from the copyright holders.

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Contributions to the Publications/Manuscripts

Paper I Major parts of all experiments and writing. Paper II Major parts of all experiments and writing. Paper III Minor parts of the experiments and writing. Paper IV Major parts of all experiments and writing. Paper V Major contribution to the experiments and parts of writing.

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Table of Contents Abstract ............................................................................................... iii List of publications and manuscripts .............................................. vii Contributions to the Publications/Manuscripts ............................ viii Thesis outline ....................................................................................... 1

1. Introduction .............................................................................. 2

1.1 Background ................................................................................... 3 1.2 Focus of thesis work ...................................................................... 5

2. Microfluidic particle manipulation ......................................... 6

2.1 Microfluidics .................................................................................. 6 2.1.1 Microfluidic Physics ................................................................ 6

2.2 Inertial microfluidics theory ........................................................ 7 2.2.1 Dean flow based particles positioning ..................................... 8 2.2.2 Elasto-Inertial based Particle Positioning ................................ 9

2.3 Acoustic particle manipulation .................................................. 10 2.3.1 Ultrasonic Standing Waves .................................................... 11 2.3.2 Acoustic Radiation Forces ..................................................... 12

3. Particle manipulation methods .............................................. 15

3.1 Active Separation ........................................................................ 16 3.1.1 Dielectrophoresis .................................................................... 16 3.1.2 Magnetic ................................................................................. 16 3.1.3 Optics ..................................................................................... 17 3.1.4 Acoustophoresis ..................................................................... 17

3.2 Passive Techniques ...................................................................... 19 3.2.1 Deterministic Lateral Displacement ....................................... 19

x

3.2.2 Pinched Flow Fractionation .................................................... 19 3.2.3 Filtration ................................................................................. 20 3.2.4 Inertial microfluidics .............................................................. 21

4. Present Investigations ............................................................. 24

Aim of thesis ...................................................................................... 24 Paper I: Glass Capillary based cavity resonator for particle

aggregation study and bacteria up-concentration ............................ 25 Paper II: MicroBubble activated acoustic cell sorting: BAACS ..... 32 Paper III: Dean flow-coupled inertial cell focusing in curved

channels ........................................................................................... 36 Paper IV: Elasto-inertial microfluidics for bacteria separation from

whole blood for sepsis diagnostics .................................................. 42 Paper V: The integration of inertial and elasto-inertial based

particles focusing techniques with fluorescent and scattering

detection system: Optical Fiber inertial focusing based micro Flow

cytometer ......................................................................................... 46

5. Concluding remarks ................................................................ 50

Acknowledgements ...................................................................... 55

References .................................................................................... 59

Publications & Manuscripts ....................................................... 68

Thesis outline

This thesis consists of five chapters that reports on the methodologies and the results

of a comprehensive study presented in five papers/manuscripts. The first chapter

briefly introduces and discusses the background. In the second chapter the relevant

physics and governing equations in the microfluidics especially theory of inertial and

acoustic particle manipulation is discussed. Third chapter discusses different particle

manipulation techniques. The fourth chapter summarizes the research and results

presented in five papers/manuscripts. Finally, in the fifth chapter conclusive remarks

and future possibilities are highlighted.

Bioparticle Manipulation using Acoustophoresis and Inertial Microfluidics

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1. Introduction

Microfluidics has provided enhanced control over the dynamics of the particles and

suspending fluids and facilitated their manipulation in minute volumes1,2. It allows

for the development of efficient, cost effective, rapid, miniaturized and integrated

diagnostic tools3,4. The implementation of microfluidic particle manipulation

technologies to develop rapid processing and integrated diagnostic devices

comparable to conventional methods is yet to be achieved2. However, recent

advances in lab on chip devices facilitate faster, more efficient and integrated sample

pre-treatment setups suitable for point of care devices5. The particle manipulation

techniques such as focusing, separation6 and isolation7 have been investigated for the

clinical sample preparation in lab on chip devices. A challenge in sample preparation

is the large variety of biomolecules and their suspending fluids e.g., bacteria, cells,

virus, blood, sputum etc. Bioparticles vary in size and scale from approx. 1µm (e.g.,

bacteria) to 30µm (e.g., blood and cancer cells), in Fig. 1.1 the relevant length scale

of bioparticles and lab on chip devices are presented. Particle manipulation can be

roughly categorized into two different approaches i.e., passive (e.g., inertial

microfluidics) and active techniques (e.g., acoustophoresis)6. The inertial

microfluidics can be used for particle manipulation in a wide range of flow rates by

tuning geometry and fluid6,8,9. The acoustophoresis is also a useful technique for

rapid and controlled manipulation of bioparticles without affecting their viability6,10–

14. In this thesis inertial and acoustic techniques are utilized to improve sample

preparation processes such as cell separation, bacteria up-concentration, cell

focusing, etc.


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Figure 1.1. A scale of microfluidic devices and bioparticles used. (Inspired from Ida Iranmanesh15 and Barnknob16 thesis)

1.1 Background

Sample preparation. Regardless of the technological advancement in diagnostics

and treatments, mortal diseases such as blood stream infections and cancer

continuously affect millions of people around the world17–20. Early diagnostics is

directly related to in-time provision of treatment and can significantly reduce the

number of deaths. Samples for diagnosis are mostly received in complex forms such

as blood, sputum, etc. and are needed to be prepared in terms of the end goal

analysis. The sample preparation involves separation of bacteria, up-concentration of

cells/bacteria and isolation of cancer cells from contaminating parameters such as

white blood cells (WBCs), red blood cells (RBCs) and platelets.

Sepsis in Sweden causes ~1000 deaths per year21. In sepsis diagnostics delay in

appropriate antibiotic therapy increases the mortality by 7.6% with each passing

hour22. To date, blood culturing is considered as the gold standard in identifying the


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type and the class of microorganisms for infectious diseases such as sepsis. In spite

of the automation in blood culturing methods the time required to obtain the results

ranges from 24-72 hours23. This loss of precious time used for identification process

has been a motivation for the utilization of the molecular diagnostics techniques such

as polymerase chain reaction (PCR) for detection of pathogens in blood24–29.

Although PCR techniques decrease the time parameter considerably, they have low

sensitivity in comparison with blood culturing due to the improper sample

preparation. The sample preparation for molecular diagnostics such as PCR to

diagnose sepsis consists of multiple steps such as isolating bacteria while avoiding

PCR inhibitor factors i.e., DNA30,31 from nucleated blood cell and haemoglobin32.

For cancer, which cause millions of deaths worldwide17 , early detection increases

the efficiency of treatment33. Cancer cell separation and enrichment is a major

preliminary step before performing diagnostic and analysis of cancer cells34. More

specifically circulating tumour cells (CTCs) are of particular interest in scientific

community35 since they are not only a biomarker for early cancer detection but also

represent efficacy of the treatment36.

Flow cytometry is a well-established technique for study of physical and chemical

properties of cell37. It has been used for applications such as counting,

immunophenotyping38, cancer diagnostics39, etc. This technique is costly, requires a

bulky setup and is limited to highly equipped centralized laboratories. Traditional

cytometers consists of fluidics i.e., for cell transport, optics i.e., detection of signal

and a signal processing unit. Efforts have been made over the last decade40 to

miniaturize the cytometer so that it can be used in point of care testing (POCT).

Advent of modern optics (e.g., fibres) and detection systems (e.g., micro

photomultiplier tubes41) in combination with microfluidic particle manipulation

techniques have now made it possible to design and develop a micro-cytometer

device.


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1.2 Focus of thesis work

This thesis focuses on improving and developing microfluidic sample preparation

methods using acoustic and inertial particle manipulation techniques. The methods

developed are used for sample preparation applications such as up-concentration,

separation, isolation etc. The particle manipulation technique is integrated with

detection platform to develop a micro flow cytometer.


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2. Microfluidic particle manipulation

2.1 Microfluidics

Advancement of micro fabrication technologies have acted as a foundation for the field of microfluidics42. Microfluidic systems have provided possibilities to perform chemical, biological and analytical analyses in micro/Nano scales. These platforms have made it possible to design and develop point of care (POC) diagnostic devices. Some of the advantages of microfluidic devices for clinical applications are cost efficiency, use of lower reagent and sample volumes, shorter processing time, multiplexing and parallel operations1,43,44.

2.1.1 Microfluidic Physics

Microfluidics deals with handling and manipulating of fluids and suspended particles in microliters to picoliters volumes. Generally, in a microfluidic channel the most significant effects are3:

(i) The inertial effect and gravity are usually negligible and flow is mainly laminar.

(ii) The surface related forces are dominant due to the large surface to volume ratio.

(iii) Diffusion is the main transportation mechanism. (iv) The dimensions are larger than the intermolecular distance and the

continuum hypothesis is applicable. (v) In non-Newtonian fluidics i.e., microfluidics normal stress differences

are dominant.

The governing differential equations are expressed as continuity, momentum and energy45. Continuity equation is46:

∂t ρ + ł � (ρν) = 0 (2.1)


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where ρ is fluid’s mass density and ν is the velocity vector of fluid. Conservation of momentum is indicating that the change of momentum within certain volume equals the summation of all forces acting over it. The momentum equation is written as47:

ρ [∂/∂t (ν) + ν � ł (ν)] = ρg - łp + ηł2 (2.2)

For Poiseuille flow with no slip boundary condition3 in a microchannel the simplified

Navier-Stokes equation is:

- łp + ηł2 = 0 (2.3)

2.2 Inertial microfluidics theory

In the transient flow regime for finite inertial and viscous forces9, the particles suspended in a Newtonian fluid flowing through a straight channel experience the shear induced lift forces (FL,S)9 that push the particles towards the wall48. In addition, the wall induces lift forces opposite to the shear induced forces pushing the particles away from the wall i.e., towards the centre of flow stream (FL,w)49. As a result of the balance of FLS

48 and FLW49 particles attain equilibrium positions within a dynamical

system09,50. Segré & Silberg have shown that the particles are migrated to 0.6 radius of a circular tube51,52(Fig 2.1a). In the case of square and rectangular micro channels particles attain four and two equilibrium positions 0.3 times the width of the channel away from the wall, respectively8,9,50,53–55(see Fig2.1b,c).

Figure 2.1. Inertial migration of particles in circular (a), square (b) and rectangular (c) microchannels.


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The net lift force (FL) on a particle is expressed by combination of FLS and FLW:

FL = ρU2 a4/D2h .fc (Re, Xc ) = Rp2 fcµ2 / ρ 48 (2.4)

where, ρ, U, µ, a and Dh are density, mean velocity, dynamic viscosity of the fluid, particle diameter and hydraulic diameter of microchannel, respectively. The hydraulic diameter of straight rectangular microchannel is:

Dh =2wh/(w + h) (2.5)

where, w and h are the width and height of micro channel, respectively.

The coefficient of inertial lift force fc (Xc, Re) is dependent on the position of particle in the microchannel (Xc) and Reynolds number (Re) i.e., defining the scale of net forces within microchannel3. Re is the dimensionless number defining the ratio of inertial to viscous forces used to predict the flow regime3:

Re = ρ ν Dh / µ (2.6)

Particle’s Reynolds number Rp is another important dimensionless number that is a ratio of inertial to viscous forces with dependence on the particle’s diameter8,9.

Rp= Re (a2/D2h) (2.7)

2.2.1 Dean flow based particles positioning

In curved micro channels, a centrifugal force proportional to the curvature of the channel takes into account and induces secondary flows across the cross section. In such channels the fluid pressure is higher near the inner wall in comparison to the outer wall. As a result, fluid is pushed from the centre towards the outer wall, and to satisfy the conversation of mass fluid from top and bottom moves towards the inner wall. Therefore, two counter rotating vortices are developed across the cross sectional plane of the microchannel56,57. The particles are trapped in these vortices and due to the net effect of inertial and Dean forces find new equilibrium positions58 i.e., closer to the inner wall. Particles distribute according to their sizes such that the relatively larger particles are positioned closer to the inner wall59. It has been


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reported that by using low aspect ratio microchannels (height<<width), the particles are focused into two positions along the height of channel56. The effect of Dean force on the flow is characterized by Dean number (De)8, which is60:

De =Re (H/2R)1/2 (2.8)

where H is height and R is the radius of curvature.

2.2.2 Elasto-Inertial based Particle Positioning

The elastic forces (Fe) are developed and exerted on the particles suspended in the

non-Newtonian fluids flowing in microchannels. The effect of elastic forces is

defined by a dimensionless parameter known as Weissenberg number (Wi). After

simplifications it can be defined as a product of shear rate (ϒ) to the relaxation time

(λ) of the non-Newtonian fluid (λ)61–64:

Wi = λ ϒ (2.9)

This dimensionless number represents the strength of shear rate65 and is related to the

presence of normal stress differences in a flow66. The elastic forces propel the

particles towards the centre and four corners of the micro channel in a creeping flow

(Stokes flow)50,67. This is due to the weakness of the first normal differences (N1) at

the four corners and centre of the channel50,62. The first normal differences arise from

the strain-induced anisotropy in polymeric non-Newtonian fluids i.e., change of

polymeric fluid molecules from their isotropic equilibrium shape68. N1 is expressed

as42,54:

N1 = τxx – τyy (2.10)

where τxx is the stress in the shearing direction (flow) and τyy is in the direction

perpendicular to the flow69. Wi may also be defined as the ratio of N1 (τxx – τyy) to

the shear stress (τxy)70.


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Fe on the particle is scaled with effect of N1, which varies depending on the size of

particle62,71:

Fe ~ a3 ∇N1 (2.11)

In a non-Newtonian fluid flowing through a straight rectangular channel at certain

flow rates both the inertial and elastic forces act on the particles i.e., known as elasto-

inertial effect. At optimized flow rates according to the particle size and geometry

when Wi and Re both are greater than zero, these forces cooperate to focus relatively

larger particles (≥ ~2µm). The net elsto-inertial effect focuses the particles into a

three dimensionally single position at the channel centre. These forces cooperate to

focus particles based on size67 (see Fig 2.2).

Figure 2.2. Forces acting on particles in elasto-inertial microfluidics

2.3 Acoustic particle manipulation

One application of sound waves typically ranging from 1MHz-10MHz is to

manipulate particles and fluids i.e., acoustofluidics. The acoustic force interacting


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and exerting pressure on particles was first reported by August Kundt72. A sound

wave induces time-averaged radiation pressure (p) and displacement (s) can be

written for one dimension as:

p(x,t) = pa sin(ωt ± kx) (2.12)

s(x,t) = so sin (kx ± ωt) ; k = 2π/λ, λ = wavelength (2.13)

where pa , so, x, t, ω and k are pressure amplitude, displacement amplitude, position,

time instant, angular frequency and wave number, respectively. Sound waves can be

categorized into travelling waves and standing waves (stationary waves)73. Travelling

waves have also been utilized for particle manipulation74–76 however, most of the

acoustic actuation is performed by ultrasonic standing waves (USW)10,12,77–81.

2.3.1 Ultrasonic Standing Waves

The longitudinal ultrasonic waves travel through fluid in the form of rarefaction and

compressions. Ultrasonic standing waves (USW) are most often generated when two

travelling waves of same magnitude and frequency but opposite directions are

superimposed. USWs are generated by continuously superimposing the propagating

sound wave with its own reflection wave. The total displacement (stotal) field of USW

is:

stotal(x,t) = 2so cos(ωt) sin(kx) (2.14)

In the eq. (2.14) the positions (x) where the value of x = nλ/2, (n = 0,1,2…),

amplitude is zero and are referred as nodes. The positions where x = nλ/4, (n =

1,3,5…) are the maximum amplitude positions and known as antinodes15,73,82,83.


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2.3.2 Acoustic Radiation Forces

2.3.2.1 Primary Radiation Forces

The acoustic forces generated by travelling and standing waves using an ultrasonic

transducer can be utilized to manipulate the particles. These forces can be

categorized into primary and secondary radiation forces. An important assumption

for defining the role of acoustic radiation force for particle manipulation is that the

diameter of the particle is much smaller than the wavelength (a<<λ). The primary

acoustic radiation force (PRF) acts on particles due to scattering of the acoustic

waves depending on the differences in properties of particles material i.e.,

compressibility and density84. Primary radiation forces can be described by change of

pressure (p) and velocity (v) as expressed by Gor’kov85:

Frad = - 4/3 × πa3 [(f1 κ ł p2)/2) – (f2ł3ρv2)/4] (2.15)

where Frad, a, p, v and κ are primary radiation forces, particle diameter, pressure,

velocity and the compressibility of the particle, respectively. f1 and f2 are

dimensionless contrast factors, given by:

f1 (κ) = 1- κ, where κ = κp/ κmed (2.16)

f2(ρ) = [2(ρ-1)]/[2ρ+1], where ρ=ρp/ρmed (2.17)

where κp, κmed, ρp, ρmed are particle compressibility, medium compressibility, particle

density and medium density, respectively. The contrast factors f1 and f2 are deduced

from pressure gradient and the velocity gradient83,86–88, respectively. The particles are

trapped under USW at local minima (pressure anti nodes/nodes) that are formed by

three-dimensional interaction of pressure and velocity gradients. The quantification

of difference between density, compressibility of particles and fluid is defined as the

acoustic contrast factor (Φ). In one-dimensional USW the relation between the

pressure and the velocity field is used to calculate acoustophoretic contrast factor

(ACF).


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The ACF is calculated as16,87:

Φ (κ, ρ) = 1/3 f1 (κ) + 1/2 f2 (ρ) = 1/3 [(5ρ-2)/(2ρ+1)- κ] (2.18)

The particles encounter Frad in the direction of pressure node or antinodes depending

upon the sign of Φ i.e. for +Φ, particles are trapped at nodes and for -Φ particles are

trapped at anti-nodes (see figure 2.3)56,81–83.

Figure 2.3. Primary radiation Force pushes negative acoustophoretic particles towards pressure antinodes whereas positive acoustophoretic particles are pushed towards pressure nodes.

Table 2.1 Calculated acoustophoretic contrast factors (ACF) using density and compressibility values of different particles reported in the literature89–92:

Particle Type

Density Kg m-3

Compressibility

10-10 Pa-1

Acoustophoretic

Contrast Factor Φ

1.

Polystyrene Particle

1050

2.49

+0.165

2 Silica Particle 2200 0.27 +0.536

3 RBCs 1099 3.31 +0.12

4 WBCs 1019 3.995 +0.044

5 Colon Cancer Cell 1077 4.04 +0.06

6 Polyvinly air filled

Microbubbles

429.6 819.67 -60.7


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2.3.2.2 Secondary Radiation Forces

The scattered waves will interact with neighbouring particles when there is more than

one particle exposed to an acoustic field. It gives rise to the secondary radiation

forces (SRF) or Bjerknes forces93. Provided the size of particle is much smaller than

wavelength of acoustic field94. SRF is:

SRF = 4πa6 [(ρp – ρo)2 3(cos2 θ – 1)]v2(x)/6 ρod4 – [ω2 ρo(κp- κo)] p2(x)/9d2 (2.19)

In the case of negative SRF, particles move towards each other while in the case of

positive SRF the force between two particles is repulsive15,73,95.

2.3.2.3 Acoustic Streaming

Acoustic waves while propagating through fluid get attenuated and generate streams

that drag the suspended particles. There are three different acoustic streaming

patterns, which are Eckart96,97, Rayleigh97,98 and Schlichting97,99,100. The Eckart

streaming (quartz wind) is caused by viscous attenuation of acoustic wave in bulk

fluids. A fluid stream due to this attenuation propagates in the direction of acoustic

wave propagation. According to the law of conversation of mass and due to the

boundary conditions of the channel a back flow begins that creates vortex alike

recirculating fluid steams96. The Rayleigh streaming is caused due to the presence of

USW between two parallel plates giving rise to vortices scaling around λ/415,83,101.

The Schlicting streaming are contained only in the viscous boundary layer of the

channel and is in the range of submicrons i.e. <<λ15,73,99.


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3. Particle manipulation methods

Microfluidic based particle manipulation methods can be roughly categorized into

two types i.e., active and passive6 (Figure 3.1). Active techniques use external force

fields while passive techniques use fluid’s flow field and the geometrical aspects of

microchannels. Active particle manipulation techniques are based on particles

properties such as dielectric, optical, magnetic, acoustics etc. Passive particle

manipulation techniques are based on particle properties such as size, deformability,

shape, density etc.

Figure 3.1 Different microfluidic particle manipulation techniques, categorized according to active and passive approaches.


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3.1 Active Separation

3.1.1 Dielectrophoresis

Dielectrophoresis technique uses the movement of cells due to their property of being

polarized when they are subjected to non-uniform electrical field. The non-uniform

field is generated in a microchip by fabricating arrays of insulating posts. The

applications such as trapping, focusing, fractionation of bioparticles and cell up-

concentration have been reported6,7,102–104. Using dielectrophoresis Hu et al.105 sorted

the cells by labelling them with polymeric beads. They showed bead-bound cells

have DEP response amplitude significantly different than unbounded cells that

increases the efficiency105. This technique was further modified (i.e., iso-dielectric

separation) where cells are displaced to their equilibrium position i.e., the point

where cell polarization reaches zero value while passing through conductivity

gradient106. This technique has some limitation such as joule heating (disturbing

bioparticles viability) and physics of collide electrophoretic mobility7,15.

3.1.2 Magnetic

The magnetic separation and sorting of cells was first reported by Miltenyi’s107. The

cells of interest with specific antigens are attached to magnetically active antibody-

coated beads. The microfluidic channel is usually actuated with permanent magnets

or electromagnetic coils7. The cells selectively attached to larger beads were

separated from cells attached to smaller beads and then the unattached cell are

separated from the cells attached to smaller beads using the magnetic field. The

separation is due to the difference in the magnitude of magnetic field which is

dependent on the bead size and magnetic susceptibility108,109. This technique has been

reported for various applications6,7such as CTC capture110 and isolation111,

lymphocyte isolation112, cell capturing for single cell analysis113and removal of

malaria infected RBCs114. The use of magnetic labels provide increased control and

high throughput cell sorting in a large array113.


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3.1.3 Optics

Fluorescence activated cell cytometry and sorting is a gold standard for cell analysis

in clinical laboratories37. The fluorescent antibody labelled cells are differentiated

and sorted based on their fluorescence and scattering signal. Recent advancement in

micro fabrication, microfluidic particle manipulation and miniaturization of detection

systems have greatly increased the opportunities to develop micro cytometry115

system that can sort cells116,117. Optical tweezer is a direct optical particle

manipulation technique which uses focused beam of photons to trap particles at

certain positions83,118. When the incident photons interact with the particles their

momentum is disturbed and due to the difference of refractive index between

particles and submerging medium, two forces are produced i.e., scattering and

gradient forces7,119,120. Optical tweezers are utilized to manipulate particles ranging

from 10nm to 100µm by altering parameters such as wavelength power and geometry

of micro channel6. They can be used for single cell, molecule manipulation121 and

sorting122,123.

3.1.4 Acoustophoresis

Acoustophoresis is the utilization of ultrasonic sound waves to manipulate the

position of particles79,124–126. The aggregation of red blood cells into stationary

clumps in the radiation field under exposure to ultrasound was first reported by

Dyson et al127. The acoustic particle manipulation does not affect the viability of

bioparticles (e.g., cells)14. There are two approaches to manipulate particles with

ultrasonic standing sound waves i.e., standing surface acoustics waves

(SSAW)6,7,117,128 and bulk acoustic waves (BAW)6,7,117,129. Different devices such as

silicon etched glass chips117,129 and glass capillaries11 have been used for particle

manipulation by ultrasonic sound standing acoustophoresis applications. The

standing waves are generated by mounting an ultrasound transducer i.e., lead

zirconate titanium (PZT) over and under the chips/capillaries15,73,129. The devices

using BAW mostly operate at frequencies ranging between 0.5 to 5MHz117.


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Depending on the frequency, there is a size limit i.e., critical diameter for dominating

radiation forces over acoustic streaming, given that the streaming direction is

opposite to the radiation force130,131. Barnknob et al.131 calculated this critical

diameter as 1.4µm for polystyrene particles in water at 2MHz. The devices that use

SSAW operate at rather higher frequencies (20 to 40MHz) enabling sub-micron

particle manipulation132. The micro channels in these devices are fabricated from

polydimethylsiloxane (PDMS) and bonded with Lithium Niobate (LiNbO3)

substrates. The LiNbO3 substrate is patterned with interdigital transducers (IDTs) that

are generating SSAW in the channel117.

To date acoustophoresis has been used in wide range of applications such as bead-

based assay133, concentrating bacteria 11, size-based cell separation10,13,78–81, in cell

sorting 116 and flow cytometery117. Hammarström et al.11 have shown that by trapping

10µm polystyrene particles (seed area) the bacteria were trapped and enriched with

an efficiency of 95% while flowing through the seeding area. Recently Ohlsson et

al.134 have presented an acoustic integrated system for sepsis diagnosis. They have

demonstrated separation of bacteria from red blood cells integrated to seed particle

based bacteria enrichment capillary. The enriched bacteria is washed and transferred

to PCR chip containing stored dry reagents. In comparison with blood culture

method their device has a rapid processing time but failed to detect 50% of the sepsis

positive patients134. The density and compressibility parameters have also been

investigated for cell separation and sorting by acoustophoresis135–140. Johnson et al.139

have shown size independent sorting of bio-functionalized polystyrene particles (+Φ)

by attaching them to bio-functionalized silicone particles such as PDMS (-Φ) at

stationary fluid. Recently Augustsson et al.138 have developed an iso–acoustic

technique for particle manipulation that allows for size independent cells separation.

They have altered the acoustophoretic contrast of cells i.e., neutral, negative and

positive by creating acoustic impedance gradient in the suspending fluid flowing

through a microchannel.


19

3.2 Passive Techniques

3.2.1 Deterministic Lateral Displacement

Deterministic Lateral Displacement (DLD) is a technique introduced by Huang et

al.141 in 2004. DLD employs microchannel geometry i.e., asymmetric pillar

(obstacles) matrices to separate particles. Particles are separated by moving through a

matrix of obstacles according to their dimensions and deformability. The particles of

relatively less deformability and dimensions larger than a certain critical diameter

based on inter obstacles gap, continuously change their path when they collide with

obstacles. Therefore, particles that possess similar dimensions and deformability

move towards the same outlet. The particles smaller than inter obstacle gap, travel

through the array of obstacles in a straight line. There are various patterns and

geometries of the obstacles used for particles separation. This technique has been

employed for blood cell fractionation142,143, isolating cancer cells144 and separation of

parasites from blood145. Loutherback et al.146 have shown the separation of cancer

cells from blood by developing a high throughput system using flow rate of

10ml/min. The possibility of clogging in the micro channels used for cell separation

in DLD system is relatively high147. The recent advances have shown that

programmable particle separation is possible by using thermally tunable poly-N-

isopropylacrylamide (PNIPAM) made obstacles that can reduce the clogging in the

micro channels140.

3.2.2 Pinched Flow Fractionation

In a laminar flow regime particles suspending in a fluid flowing through a

microchannel have tendency to travel along the streamline passing through its centre

of mass. In such flows Pinched Flow Fractionation (PFF) technique can be used to

separate particles according to their dimension. In this method, there are usually two

inlets that simultaneously supply particle-laden and particle-free fluids into a

common channel. The particle-free fluid is pumped with a relatively higher flow rate


20

that pinches particle-laden fluid along the wall while passing through a pinched

section that is connected to wider section. Therefore, the smaller particles travel

through streamlines closer to the micro channel walls and the larger particles travel

through a streamline closer to centre of the channel148. The range of particle sizes that

can be separated is dependent on the ratio of the flow rates of particle-free and

particle-laden fluids. The efficiency and the resolution of separation depend on the

geometrical shape of pinched segment and the expansion. The resolution of

fractionation also depends on the difference between particle sizes, diameter of

particles and the aspect ratio of the micro channel6,148. The technique has been used

for cell sorting and its efficiency can be improved by adding multiple asymmetric

outlets149 altering spatial orientation of microchannels150. It is also useful for sorting

droplets which is difficult to achieve with mechanical methods e.g. filtering,

centrifugation etc.,151,7.

3.2.3 Filtration

Micro filtration devices are a class of size based passive separation methods that use

different techniques such as micro obstacles (i.e., pillars and weirs), cross flow,

hydrodynamic, etc.,6. Pillar and weir filtration microstructures are mainly used for

blood cells separation. These devices consist of an inlet for whole blood, a main

channel consisting of three sub-channels containing obstacles and outlets for

separating white blood cells (WBCs) from red blood cells (RBCs)6,152. The increase

in the length of microchannel containing micro obstacle improves the separation

efficiency6. In the cross flow filtration, the direction of flow is perpendicular to the

obstacles or a porous membrane153. Cross flow filtration has been used to separate

WBCs and plasma from whole blood and myocytes from monocytes7,152,154–157. In

hydrodynamic filtration particles are separated according to their size into multiple

side-outlets. This principal was demonstrated first by Yamada et al.158,159 and later

reported by other researchers for applications such as leukocyte enrichment158–160.

The main channel is connected to the number of side channels where the flow rate is


21

substantially smaller than that of the main channel. The range of particles diameter

separated from the side channels is determined with the width of the side channels

and the flow rate in the main channel158.

3.2.4 Inertial microfluidics

3.2.4.1 Straight channels

The particles suspended in a Newtonian fluid flowing through a straight channel

experience the shear induced lift forces (FL,S)9 that push the particles towards the

wall48. In addition, the wall induces lift forces opposite to the shear induced forces

pushing the particles towards the centre of flow stream (FL,w)49. Inertial particle

manipulation is performed in transient flow regime for finite inertial and viscous

forces9. Segre and Sillberg51,52 experiments showed that particles in a cylindrical tube

experienced lateral migration to a position of 0.6 of the tube radius. The technique

was further investigated in straight square and rectangular microchannels53,55,161–163.

The particles find four and two equilibrium positions in square and rectangular

channels at 0.3 times the width of channel, respectively53,55,161–163. The inertial

microfluidics for straight channels have been reported164 for applications such as

focussing8, sorting165, filtration166 and separation167. Hansson et al.168 have reported

the use of parallel micro channels to achieve high throughput particle separation. A

straight channel is used by Wu et al.167 to separate bacteria from diluted red blood

cells with the separation recovery 62% and purity of 99.7% using inertial

microfluidics in combination with asymmetrical sheath flow i.e., soft inertial

microfluidics. Mach et al.166 performed scaled up separation of bacteria from diluted

red blood cells using 40 single parallel straight channels placed in a radial array.

They achieved 80% removal of bacteria after recirculating the samples twice through

the device.

3.2.4.2 Dean separation

Dean inertial microfluidics is an extended form of inertial microfluidics where

curvature is introduced to the geometry of the microchannel8,56,169. The Dean forces


22

and inertial lift forces in curve channels interact to focus particles, such that the

larger particles are focused closer to the inner wall170. Russom et al.56 previously

showed that particles migrate to two equilibrium positions along the height of a low

aspect ratio channel. The curved geometries (e.g., spiral, U-shaped) have been used

for separation of WBCs8,56,169171,172 and circulating tumor cells (CTCs) from blood

samples173,174. They have also been used for focusing of cells175,176, ordering of

cells177 and blood filtration178. Sun et al.174 increased the efficiency of separation of

5µm from 15µm particles by using longer length of spiral channels. They have

shown separation of spiked HeLa cells from 20x-diluted blood with efficiency of

90%. The most significant benefit of spiral microfluidics is the possibility of

conducting high throughput particle separation by employing higher flow rates58. The

throughput can also be increased by the use of parallel channels and design and

development of PDMS multilayer spiral microchannel179.

3.2.4.3 Elasto-inertial separation

The viscoelastic lateral particle migration was first experimentally reported by

Mason et al.180–182. Leshanky et al.71 reported that particles suspended in a non-

Newtonian fluid flowing through a straight rectangular microchannel experience

elastic forces (Fe). Their results show that due to these elastic forces particles are

pushed to four corners and centre of the channel. The net effect of inertial and elastic

forces, achieved by optimizing channel geometry and flow rates, has been used to

focus particles in 3D in a single stream183. A number of investigations have recently

focused on optimizing and varying the different parameters such as flow rate and

concentration of non-Newtonian fluid184. The ability to focus in 3D has been used for

separation of blood components48,49. Furthermore, elasto-inertial microfluidics has

been used by Liu et al.187 to separate bacteria (E. coli) from red blood cells with an

efficiency of 99.9%. Recently, Lim et al.188 demonstrated 3D particles focusing using

hyaluronic acid in a straight microchannel at extremely high flow rate of 50ml/min.

The 3D particle focusing in circular microchannels has also been demonstrated using

non-Newtonian viscoelastic fluid189,190 such as polyethylene oxide (PEO). The elasto-


23

inertial technique has been recently used in a spiral geometry demonstrating high

throughput 3D single stream focusing of particles191.

Summary

In this chapter, different active and passive sample preparation techniques were

reviewed, as summarized in table 3.1. Among these, inertial microfluidics offers

versatility and high throughput particle manipulation as well as separation of very

small particles (e.g., bacteria) by geometric alterations. Similarly the acoustic

technique offers both size based and size independent possibilities for controlled

particle manipulation. These techniques can be integrated or improved for integration

with miniaturized detection systems. This integration can facilitate realization of

decentralized laboratories and introduction of Point of Care Testing (POCT).

Table3.1. Summarized discussed particle manipulation techniques15,192.

Method Marker Mechanism Throughput (µl min-1)

Passive

Inertial

Size and Shape

Lift forces and Dean Forces

1000-4500158,193

DLD

Size, Deformability

Laminar Flow

20158-10,000146

Hydrodynamic Filtration

Size

Hydrodynamic

forces

20 155

Pinched Flow Fractionation

Size

Laminar Flow,

Parabolic velocity profile

0.33 149

Active

Acoustic

Size, Density, Compressibility

Primary Radiation Forces, Secondary Radiation Forces

1000-400012

Magnetic

Size, Magnetic susceptibility

Magnetic field

0.41-0.66 194

Dielectrophoresis

Size, polarizability

Electric field

0.83-3.3 105


24

4. Present Investigations

Aim of thesis

The aim of this thesis is to employ different sample preparation steps, with an

emphasis on bacteria up-concentration and cell sorting. The work behind this thesis

consists of the development of two particle separation techniques, namely

acoustophoresis (papers I-II) and inertial microfluidics (papers III-V).

Paper I: In this study, the effects of particles material and suspending media are

investigated for acoustophoresis based particle manipulation in a glass capillary

cavity resonator.

Paper II: In this study, a microfluidic-based microbubble-activated acoustic cell

sorting (BAACS) method that rely on the specific binding of target cells to

microbubbles conjugated with specific antibodies on their surface is introduced for

continuous cell separation using ultrasonic standing wave.

Paper III: In this study, the introduction of curvature to straight channel for inertial

microfluidics based high throughput cell separation is studied using U-shaped

channel geometries.

Paper IV: In this study, label-free passive bacteria preparation in flow through

straight channels using elasto-inertial microfluidics is presented for sepsis

diagnostics.

Paper V: In this study, miniaturized flow cytometer is presented where inertial 3D

focused and sorted cells are simultaneously measured with fluorescent and scattering

in an all fiber optics device.


25

Paper I: Glass Capillary based cavity resonator for particle aggregation study and bacteria up-concentration

Particles aggregation using acoustophoresis have been used in several applications,

such as sample preparation (i.e., up-concentration, sorting, separation) and

immunoassays6,7. Bacteria up-concentration can facilitate the detection of multiple

bacterial strains and removal of matrix-associated assay inhibitors. It also enables the

detection of low level pathogens195 e.g., in sepsis diagnosis11,134. Hammarström et

al.11 have performed bacteria up-concentration in the glass capillaries using seed

particle-enabled acoustic trapping. This trapping technique was recently used by

Ohlsson et al.134 for developing a sepsis diagnosis integrated platform called

ACUSEP. The capillary-based trapping can be used for integrating immunoassays

with on chip detection platform.

Acoustophoresis is mostly performed in silicon-glass based microfluidic chips,

requiring sophisticated and expensive fabrication procedures. Commercially

available glass capillaries can be used to resonate acoustic waves for generating

ultrasonic standing waves. In this work, the patterns and trapping behaviours of

different materials i.e., polystyrene and silica particles for various PBS

concentrations and water was studied in commercial glass capillaries. Furthermore,

bio-functionalized silica particles were used for bacteria up-concentration in a glass

capillary-based cavity resonator. The principle of silica-particle based bacteria up-

concentration is shown in Fig. 4.1. The bacteria are trapped only with the assistance

of seed when the capillary was actuated with ultrasound. Fig 4.1b shows that the seed

particles were trapped at the pressure node positions, resulting in trapping and up-

concentration of the bacteria.


26

Figure 4.1. Schematic representing the capillary resonator and the seed particle trapping method for bacteria up-concentration. a) The bacteria is not trapped under acoustic actuation (b) the bacteria are trapped attached to seed particles.

Summary:

The acoustic setup was characterized by investigating different parameters i.e., time

to trap, trapping pattern and area of trapped particles in a single aggregate of silica

and polystyrene particles. Water and PBS of concentrations 0.01M and 0.005M were

used as suspending media. 0.2mm×2mm glass capillary was mounted over a lead

zirconate titnate (PZT) ultrasound transducer and the ultrasonic transducer was

driven at 1.97 MHz with various potentials i.e., 4, 8, and 12 Vpp. Fig. 4.2 shows the

aggregation for water and PBS. It shows that the silica particles formed aggregates

approximately 5.5 times faster than polystyrene particles. The shorter aggregation


27

time of silica particles can be explained by the higher magnitude of primary radiation

forces and particle-particle interaction i.e., secondary radiation forces for silica

particles.

Figure 4.2. The accumulation time of polystyrene and silica particles to pressure nodes in water, 0.01M PBS and 0.005M PBS at 4, 8 and 12 Vpp. The system is driven 1.973MHz.

Fig. 4.3 shows the comparison between aggregation pattern of silica and polystyrene

particles exposed to the same acoustic field. Fig4.3a, b shows the random distribution

of polystyrene and silica particles before the activation of acoustic field. Fig. 4.3d, f

shows that the silica particle aggregate with a striated pattern unlike the polystyrene

particles (see Fig4.3c, e). The pattern of silica particles aggregates confirms the

strong influence of secondary radiation forces.


28

Figure 4.3. 5µm polystyrene and silica particles, randomly distributed without acoustic field, respectively (a, b). The silica (d, f) and polystyrene (c, e) particles aggregate under the acoustic actuation.


29

Figure 4.4. The 5µm silica and polystyrene particle aggregation in water of (a) average area of single aggregate, (b) number of particles trapped and (c) the up-concentration factor.


30

Fig. 4.4a,b shows that the average aggregation area and the number of trapped silica

particles in a single aggregate were 3.4 times higher than that of polystyrene

particles. Fig. 4.4c shows the comparative up-concentration factor of silica was 3.2

times higher than that of polystyrene. The up-concentration factor was calculated by

comparing the number of particles in the same area before and after the acoustic

actuation. The silica particles were bio-functionalized with anti-lipid

Lipopolysaccharide antibody and were attached to bacteria. The bacteria were up-

concentrated by trapping the silica particles attached to bacteria. Fig. 4.5a shows that

the bacteria aggregation is insufficient without the silica particles during the acoustic

actuation. In contrast, a strong fluorescence signal was obtained from the silica

particle-bacteria aggregate (see Fig. 4.5b, c).

This study demonstrated the use of commercially available disposable glass capillary

as three-dimensional cavity acoustic resonators. Silica particles with shorter

aggregation times and better up-concentration factor were used to demonstrate

bacteria up-concentration. The developed method is generic and can be used for

different purposes such as in immunoassays, cell sorting and bacteria up-

concentration with integrated detection.


31

Figure 4.5. Demonstration of silica particle assisted bacteria trapping. a) No bacteria trapping without seed silica particles. b) Acoustophoretic silica particles assisted bacteria trapping and up-concentration after washing. c) Zoom out of the image of trapped bacteria in (b).


32

Paper II: MicroBubble activated acoustic cell sorting: BAACS

Cell sorting is a critical part of sample preparation especially for diagnostic analyses for pathological conditions such as cancer diagnostics196. Selective cell sorting is conventionally performed in fluorescent activated cell sorters (FACS) and magnetic-activated cell sorters (MACS) that use fluorescent antibody and antibody coated magnetic beads conjugated to the cells, respectively. These systems are costly and are often limited to equipped laboratories. Efforts have been made to develop miniaturized, cost effective cell sorters since the advent of microfluidics40. Different microfluidic particle manipulation techniques such as dielectrophoresis197, optics123 and more recently acoustics116,198 have been used to develop miniaturized cell sorting techniques. Acoustophoresis is a robust and gentle14 microfluidic particle manipulation technique129 that has allowed separation10,12,81, enrichment13 and up-concentration11 of the samples in the microfluidic chips. Acoustophoresis is highly dependent on the magnitude of radiation forces (primary acoustic radiation forces) and size of the particles. The selective cell sorting often requires isolation of cells of narrow size distribution. Recently, Augustsson et al.138 developed a method called iso-acoustic particle manipulation to separate similar sized cells by differentiating them according to their ratio of relative density and compressibility to their suspending medium. This was achieved by developing the acoustic contrast gradient of the medium using diffusion transport mechanism. However, a complex fluidic setup is required for generation of this diffusion-based gradient in the channel.

The particles under the acoustic radiation field migrate to either pressure nodes (positive acoustophoretic contrast particles) or pressure anti-nodes (negative acoustophoretic contrast particles) depending on their relative density and compressibility131. Cells and most particles e.g., polystyrene, silica (see table 2.1) have positive acoustophoretic contrast factors and migrate to pressure anti-node under the acoustic field. In this work, we have developed a simple approach to use air filled polyvinyl alcohol microbubbles (negative acoustophoretic contrast particles) for size independent selective sorting of cells.


33

Figure 4.6. Schematic illustration of flow through acoustophoresis based cell sorting using immunoaffinity cell capture with antibody-coated microbubbles.

The principle of microbubble activated acoustic cell sorting is shown in Fig. 4.6. Cells attached to microbubbles flowing through a channel actuated with ultrasonic transducer migrate to pressure anti-nodes and are isolated from unbound cells that remain at the pressure nodes.

Summary:

The air filled polyvinyl alcohol microbubbles were synthesized and their positions

under acoustic radiation forces were characterized in a flow through condition using

glass-silicone microfluidic chip actuated by a PZT transducer. At pressure < 120kPa

and flow rates up to 80µl/min microbubbles (MBs) flow through the antinodes. The

streptavidin-coated MBs labelled with red fluorescent dye were attached with

HCT116 colon cancer cells labelled with green fluorescent dye (see Fig. 4.7). The

MBs-cell complexes were positioned on antinodes on the stationary flow condition.


34

Figure 4.7. Cell–MBs binding: a) Bright field and b) fluorescent images where MBs are red and HCT116 Cells are green.

Fig. 4.8a shows bright field (left) and fluorescent (right) images of acoustic

positioning of MBs-cell complex on antinodes in no flow condition. Fig. 4.8b shows

that MBs-cell complex were separated and positioned on antinodes (centre and along

the walls) from their mixture with unbounded cells (nodes).

Figure 4.8. Acoustic contrast based sorting in stationary condition. a) The MBs-cell complex at antinodes under acoustic field, b) the mixture of cells (green) and MBs-cell complexes (red-green) where cells are at nodes and MBs-cell complex at antinodes.


35

Figure 4.9. Flow through streptavidin coated MBs assisted cell sorting. a) Merged image of frames shows that MBs (red streaks) and Mb-cell complex (red-green overlapping streaks) were flowing through the centre of the capillary via antinodes under acoustics whereas green streaks passing through nodes are cells. b) The fluorescent intensity peaks representing lateral distribution of microbubbles (red), cells (green) and MBs-cell complex (red-green overlap) at nodes and antinodes, c) Sorting efficiency of 75% of MBs-cell complex at flow rate of 180µl/hr.

In continuous flow condition, the MBs-cell complexes were sorted from a mixture of

MBs-cell complex and unbounded cells at 180µl/hr. Fig. 4.9a shows that MBs (red

streaks) and MB-cell complex (red-green overlapping streaks) were flowing through

the centre of the capillary (antinode) whereas unbounded cell (green streaks) were

flowing through nodes. Fig. 4.9b shows the lateral positioning of MBs (red), cells

(green) and MBs-cell complex at nodes and antinodes. Fig. 4.9c shows that all the

MBs were positioned on antinodes (100%) whereas the MBs-cell complex was

sorted with efficiency of 75%.

This study proposes microfluidic-based microbubble-activated acoustic cell sorting

(BAACS) method as an alternative to FACS and MACS methods. The size

independent selective cell sorting ability of this method in flow through systems can

be used to develop miniaturized, cost effective cell sorters.


36

Paper III: Dean flow-coupled inertial cell focusing in curved channels

In the current sample preparation protocols cells are separated from the surrounding bioparticles e.g., blood cells for further analysis. Existing cell separation techniques such as centrifugation are often laborious, time consuming, require clinical equipment and may alter the immunophenotype of sensitive leukocyte196,199,200. Label free particles and cells manipulations have interested researchers around the world during the last decade6,7,196. Separation and isolation of selective cells e.g., white blood cells (WBCs) without using biochemical markers (labels) reduces the risk of sample alteration that increases the diagnosis accuracy196. Inertial forces have been employed to conduct label free particle separation in straight and curved micro channels6,9,196. The inertial forces are consisted of shear induced and wall lift forces that push particles towards centre and walls of the channel, respectively. The curvature of the channel induces the secondary dean forces that can enhance the separation of cells. The balance of these forces determines the new equilibrium position for particles. In this study, the effect of curvature was systematically analysed and reported that randomly distributed particles entering a simple u-shaped curved channel are focused to a fixed lateral position exiting the curvature (Fig. 4.10). We apply this for throughput filtration of white blood cells from diluted whole blood.


37

Figure 4.10. Schematic illustration of U-shaped channel, highlighting the forces involved on particle focussing while flowing through curvature.

Summary:

A comprehensive study of the effect of curvature on the particle positioning is conducted by investigating the effect of dean forces on particles in U-shaped channels. The height and channel straight length (i.e., connecting the inlet) were fixed at 50µm and 20mm, respectively. The width of channels were varied in order to fabricate micro channels of aspect ratios 1:1, 1:2, 1:5, 1:10 and 1:20. The effect of different aspect ratios (ARs), the radius of the curvature and the range of Dean numbers i.e., 2<De<80 have been experimentally studied. The results show that the particles are focused on a single streak in ARs 1:1 to 1:10. Due to channel dimensions the resultant forces were not enough to effect on the particles in AR1:20. The particles were pre-focused before entering the curvature for AR1:1 and 1:2 due to the additional lateral forces in aspect ratios below AR 1:3201. Therefore the effect of curvature on particles migration was studied using AR 1:5 and 1:10 since particles were randomly distributed before entering the curvature. The particles are focused adjacent to the inner wall of the channel right after the curvature i.e., where the dean forces are dominant. The flow rate was varied to determine the optimum Dean numbers that can be used to enhance the focusing of the particle. It was shown in


38

Fig.4.11a, b that particles are focused on single lateral position at 1.5ml/min (De 36) and 3ml/min (De 41), for AR 1:5 and 1:10, respectively. The effect of the radius of the curvature on the particles migration was investigated for AR 1:5 and 1:10 by varying radius of curvature from 2mm to 9mm. As can be seen in Fig.4.12a, b that the focusing positions of particles are independent of the radius of curvature.

Figure 4.11. Fluorescent images of 10µm particles flowing through U-shaped channel with aspect ratios a) 1:5 and b) 1:10.


39

Figure 4.12. Fluorescent intensity of 10µm particles at the cross section of U-Shaped channel exit point over a range of radius of curvature for aspect ratios a) 1:5 and b) 1:10, respectively.

A different U-shaped microchannel having an inlet and four outlets was used to demonstrate the separation of 10µm particles at flow rate of 4.25ml/min with efficiency of 92%. Figure 4.13 shows the separation of 10µm from a suspension of 10µm and 2µm particles. The Fig 4.13a shows that 10µm particles are separated away to the outlet number 2 using the balance between Dean and inertial forces. After the characterization of U-shaped channel it was used for separation of WBCs from diluted whole blood at 2.2ml/min with efficiency of 78% (see Fig 4.13c). Their


40

efficiency is comparable to spiral channels for particle separation, however for cells separation a spiral channel is considerably higher. The difference between efficiency of cells separation can be due to the larger effect of Dean forces for the longer curvature length. Cells separation can be improved in the U-shaped channel by increasing the curvature radius.

Figure 4.13. Separation of particles and cells in single-inlet four-outlet U-shaped channels.a) Separation of 10µm fluorescent particles at high flow rate (4.5ml/min) with 92% efficiency. b) The 10µm particles are separated with 96% efficiency and c) WBCs are successfully separated with 78% efficiency at flow rate of 2.2ml/min.


41

Using a simple u-shaped geometry, we have shown in this study that particle can be focused into a fixed lateral position largely independent of the radius of curvature. Furthermore, particle and cell separation is demonstrated at high throughput. The simple geometry provides an ideal system for systematic analysis of the influence of curvature in future studies and to perform simple and cost effective cell focussing and separation applications.


42

Paper IV: Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics

Bacteria separation is a critical step in the diagnostic of sepsis infection. The diagnostic methods available for bacteria identification require blood culturing for the identification of bacteria to determine the effective antibiotic therapy20. The blood culture is highly time consuming and usually needs skilled operators and even the automated blood culturing systems takes 24 to 72h202. The molecular diagnostics such as PCR (e.g., SEPTIFAST kit) can decrease the time to identify the bacteria to ~6h. However the molecular diagnostic techniques encounter issues such as relatively low sensitivity, amplification inhibition, etc.134. Sepsis infection molecular diagnostics can benefit from a microfluidics particle manipulation platform that separates bacteria from the contaminating blood components in a shorter time. The microfluidic platform can be integrated with PCR platforms and detection systems to present a sample to answer sepsis diagnostic system134. Bacteria do not respond to the inertial and Dean forces since size of the bacteria is relatively small approx.1µm. Therefore, in order to separate bacteria from blood cells, elasto-inertial method is employed in this study. Here, the optimized resultant of elastic and inertial forces is used to three-dimensionally focus particles according to their size in a non-Newtonian fluid i.e., polyethylene oxide (PEO). A schematic illustration of the elasto-inertial based particle separation is shown in Fig. 4.14. A mixture of small and larger particles pumped along the sidewalls in a straight channel and a non-Newtonian fluid is pumping from the centre. The larger particles migrate towards the centre of the channel while smaller particles remain un-affected and keep travelling along the sidewalls.


43

Figure 4.14. Schematic illustration of size based elasto-inertial particles focussing in a straight channel. The synergetic combination of forces involved is highlighted.

Summary:

The elasto-inertial microfluidic platform constituted of a straight channel was experimentally characterized. The effect of particle size (2µm, 5µm and 10µm), length of the channel (5mm to 35mm), different concentrations of a non-Newtonian polymeric fluid (250, 500, 750 and 1000ppm of PEO polymer) and different aspect ratios were investigated. The platform used consisted of straight rectangular microchannels made from PDMS by soft lithography. The height of microchannels was kept at 65µm and the width was varied in order to fabricate microchannels of aspect ratio 1:1, 1:2 and 1:3. Fig. 4.15a shows that when 2µm and 5µm particles were pumped from the inlet (see the inset shown by yellow square) 5µm particles migrate to single stream at centre of the channel but 2µm particles were collected at the side outlets (see the inset shown by red square). Fig. 4.15b shows that at low flow rates (Rp < 0.006) no focusing is observed due to insufficient elastic forces. It is also shown in Fig. 4.15c that when the flow rate is increased to 30µl/hr for 5µm particles and to 360µl/hr for non-Newtonian fluid (Rp > 0.007), the minimum of 25mm channel length is required for focussing using 500ppm PEO non-Newtonian fluid. Fig 4.15d shows that using the optimized aspect ratio and flow rate the 2µm and 5µm particles were separated with efficiencies of 93% and 95%, respectively.


44

Figure 4.15. Elasto-inertial particle separation. a) 2µm and 5µm mixture introduced at inlet [yellow box] and separation is shown at outlets [red boxes]. b) 5µm particles focussing profile with respect to particle’s Reynold number [Rp] and channel length; red is no focussing, yellow is partial and green is complete focussing. c) Lateral displacement of 5µm particles with respect to width at Rp=0.08. d) 2µm particles are separated yielding 93% while 5µm particles were separated with yield of 95%.

The blood cell separation using Newtonian fluid was compared with that of non-Newtonian by pumping whole blood in PBS and PEO, respectively. The blood cells were focussed into a single stream in the non-Newtonian solution and were not focussed for the Newtonian fluid. We have demonstrated that 92% of WBCs can be migrated into middle outlet from the whole blood. In addition, bacteria were separated from PBS and the blood. Fig 4.16a shows confocal microscope image of collection from side and middle outlet after elasto-inertial processing showing no traces of bacteria in the middle outlets. Fig. 4.16b shows that on the optimized conditions bacteria is separated from PBS and whole blood with efficiencies of 82% and 76%, respectively.


45

Figure 4.16. Bacteria separation from PBS and whole blood. a) Confocal microscope image of collection from side and middle outlet after elasto-inertial processing. b) Bacteria separation from PBS and whole blood with efficiencies of 82% and 76%, respectively.

This study demonstrated bacteria separation from whole blood based on elasto-

inertial microfluidics. The label free passive separation method is promising.

However, for clinical implementation, the throughput needs to improve. Towards

this, we are currently working on the design of parallel-channel microfluidic systems.

Hence, the method has future promise as a stand-alone sample preparation or as

integrated lab-on-chip system for molecular and phenotypical based sepsis

diagnostics.


46

Paper V: The integration of inertial and elasto-inertial based particles focusing techniques with fluorescent and scattering detection system: Optical Fiber inertial focusing based micro Flow cytometer

Flow cytometery is preferred usually for qualitative and quantitative multi-parameter analyses of cells, particles, nuclei, bacteria and chromosomes, due to its excellent accuracy and throughput. It is a complex device consist of fluidic unit for cell focussing/aligning, optical detection unit and the analysis system203. Flow cytometer is bulky, expensive and limited to equipped centralized laboratory settings. To use flow cytometer in point of care testing (POCT) there is a need of miniaturized robust, sensitive and less expensive cytometer. Recent advances in integration of optical, detection systems41 and microfluidic manipulation techniques6,7 have facilitated the development of the miniaturized flow cytometer.

The Inertial and elasto-inertial techniques have been used to separate, isolate and focus particles according to their size9,204. The integration of inertial focussing with optical detection systems to develop miniaturized cytometer is hampered due to the complexity of alignment205 and auto-fluorescence206 of PDMS. Therefore instead of PDMS, silica-microcapillary was used for the inertial and elasto-inertial particles focussing to design and develop a miniaturized flow cytometer. The microcapillaries were integrated with a double cladding optical fibre (DCF) to handle two colours excitation Laser and signal detection. The optical fibre system was connected to a compact detection and analysis system enabling differential counting of cells using fluorescence labels and scattering. Fig. 4.17 shows this integrated opto-fluidic system where the excitation light travels through the DCF and exits from the fiber-tip towards the fluid striking on the cells focussed in micro-capillaries. The combination of DCF and the detection system with micro-capillaries enable the differential counting of cells by processing fluorescence and scattering signals.


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Figure 4.17.Schematic (left) and microscopic image (right) of the opto-fluidic components.

Summary:

The micro capillaries are experimentally characterized using inertial (using PBS solution) and elasto-inertial (using PEO solution) for focusing of particles. The particles of different sizes (2µm, 10µm and 15µm) were focused using micro-capillaries (25µm, 56µm and 90µm). Fig. 4.18a shows inertial and elasto-inertial focusing of 10µm particles in a 56µm capillary to an annulus at 0.6 time the radius of capillary51,52 and a single stream at the centre of micro capillary, respectively. The lateral positions of particles are shown by fluorescent streak (top view image) and normalized fluorescent intensity graph of lateral particles positions. Figure 4.18b shows a single stream elasto-inertial focussing of 2µm and 15µm particles in 25µm and 90µm capillaries, respectively.

The 56µm micro-capillary opto-fluidic system was used for fluorescent and scattering signal detection of green fluorescent 10µm polystyrene particles. At low flow rates ≤ 25µl/min the inertial forces are not strong enough and particles are randomly distributed which decreases the detection sensitivity. The inertial (PBS) and elasto-inertial (500ppm PEO) focusing using flow rate of 120µl/min demonstrated that the amplitude of the detected signal was rather uniform for both techniques but for elasto-inertial it is ~2.5 times higher. The 10µm green and red fluorescent particles were pumped in opto-fluidic component at 120µl/min to investigate the systems capability of differential particles counting.


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Figure 4.18. Capillary based Inertial and elasto-inertial focussing characterization. a) 10µm particles annular inertial focussing in 56µm capillary and 3 dimensional 10µm particles focussing by elasto-inertial technique in 56µm capillary. b) Elasto-inertial focussing of 15µm particles in 90µm capillary and 2µm particles in 25µm capillary, respectively.

Fig. 4.19 shows that green fluorescent, red fluorescent and blue scattering peaks were detected and differentially enumerated. The 8003 green and 2210 red fluorescent particles were counted, whereas total scattering peaks of 10113 were detected that showed 99% agreement with total red and green particles. The 90µm microcapillary opto-fluidic system was used for fluorescently tagged mammalian cells (approx. size range 15µm) counting. Fig 4.20a shows that at relatively low flow rate (100µl/min) cells were not focused resulting in poor detection efficiency. Fig 4.20b demonstrates that the elasto-inertial focusing at 400µl/min notably enhances the detection. Fig.4.20c shows the expanded 0.04 seconds time-slot of measurements to verify that


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Figure 4.19. Detection and counting of green fluorescent and red fluorescent particles and scattering (blue) signal.

the fluorescent and scattering signals were coinciding. The total numbers of 30719 cells were counted along with their scattering peaks.

Figure 4.20. Detection of fluorescent and scattering signals from cells. a) Unfocused and b) focused cells in a 90µm capillary.. c) Zoom in of scattering (left) and fluorescent signals (right).

This study presented an integrated all optical fiber based opto-fluidic platform that is

capable of focusing particles and cells and allows for simultaneous measurements of

fluorescent and scattering data at 2500 particles/s. This integrated platform has the

potential to replace bulky and expensive flow cytometers in applications at clinical

and point of care testing (POCT).


50

5. Concluding remarks

Microfluidic biochips have been successfully utilized for the detection of entities

such as proteins in serum, yet remain to be fully developed as integrated POC

detection and identification of microorganisms from complex fluidics, such as blood.

This is mainly because it is virtually impossible to detect and quantify the

aforementioned analytes without first removing blood’s high concentration of

erythrocytes, leukocytes, and platelets. In addition, despite the many promising

advances made in microscale cell separation technology, most microfluidic sorting

devices for mammalian cells are limited by their incompatibility with complex,

heterogeneous biological fluids and their extensive sample preparation requirements.

A major challenge that continues to confront microfluidic cell manipulation is its

inability to match the sample throughput of traditional large scale FACS and MACS

systems, while maintaining the specificity of sorting, high yield and reduced cost of

microscale system.

This thesis has focused on the development of different sample preparation

technologies. Towards this, two fundamental different technologies were employed

and applied to up-concentrate bacteria for infectious diseases applications (paper I

and IV) and separation and sorting of mammalian cells (paper II, III, and V).

Antibiotic resistance among bacteria leading to the increasing failure to prevent and

treat infections has become increasing threat to human health. Towards this,

diagnostics are more and more seen as an important tool to safeguard the future of

our antibiotics. In this thesis, as part of a EU project called RAPPID, we have

developed two different bacteria up-concentration methods, one for blood stream

infection (paper IV) and one for lower tract infections (paper I). In paper 1, we

introduced an acoustics based sample manipulation technique using silica beads to

up-concentrate and deliver the capture intact bacteria-beads to an optical detection

site for rapid diagnostics. This work, as part of the EU RAPPID project, aiming at


51

develop an ambitious and novel lower tract infection point-of-care test (POCT),

capable of providing rapid species identification of a panel of respiratory pathogens

within 30 minutes. Current state of the art, nucleic acid based methods, requires

complex sample preparation and cannot provide answer within the given time frame.

The novel approach takes intact bacteria for direct immunofluorescence detection in

a multiplexed manner. The final outcome of the acoustic sample preparation method

is to maximize and speed up the uptake of analyte and fluorescent antibodies onto the

capture beads, and to arrange the capture beads into a closely packed monolayer

positioned in the field of view and focus of the microscope used for the optical

detection. Acoustophoresis techniques are mostly performed in silicon-glass based

microfluidic chips that requires sophisticated and expensive micro-fabrication

procedures like dry/wet etching. To reduce the price and complexity, in this thesis we

have explored off-the-shelf rectangular-shaped glass capillaries to act as a resonator

for generating ultrasonic standing wave. In the current paper, we evaluated the

performance of the acoustic based preparation method, we used GFP-expressing

E.coli as a model organism and anti-LPS coated silica particles were pre-incubated

with E.coli to enable binding. We were able to confirm the bacteria are enriched and

trapped into silica bead clusters. Future work will include developing an acoustic

trapping capillary with side-mounted transducers in each end of the capillary that is

capable of handling up to 1ml sample. Some initial crude filtration to remove host

cells/debris is required prior the bead-sample introduction into the capillary to avoid

clogging. Finally, there will be a need to include integration of the capillary trap with

upfront sample solubilisation and downstream fluorescence based detection for

multiplexing detection of bacteria for respiratory infection disease detection.

In paper IV, we developed elasto-inertial based bacteria isolation method. Initially,

we evaluated the use of inertial microfluidics especially the introduction of curvature

to straight channels in an effort to understand the underlying particle focusing

phenomena (paper III). Inertial microfluidics is a powerful passive separation method

and show great promise for precise positioning and ordering of particles. However,


52

because of the technique is recently developed several basic studies would be useful

in determining the optimal operating conditions. Currently, it is well established that

dominant inertial forces (wall-and shear-induce lift forces) cause particles to move

across streamlines and occupy equilibrium positions along the faces of walls in flows

through straight microchannels. However, exactly how these forces interact with the

particles is currently not fully understood. Even less understood is the introduction of

curvature. In flow through curved channel geometries, curvature amplifies a lateral

instability that drives a secondary cross-sectional flow field (Dean flow)

characterized by the presence of two counter-rotating vortices located above and

below the plane of symmetry of the channel. Towards this, paper III has focused on

the systematic analysis of what happens when curvature is introduced into a straight

channel. Surprisingly, we found that particle entering a curvature do focus into as

pre-determined lateral focusing position independent of the radius of curvature. As a

proof of principle we performed high throughput filtration of white blood cells.

Similarly, there is currently limited theoretical understanding in “elasto-inertial

microfluidics” where another force i.e. elastic force acts on suspended particles as a

result of non-uniform normal stress differences in the flow. It is known that non-

Newtonian fluids including polymers in the carrier fluid results in new equilibrium

positions and more distinct focusing regions for different particles compared to

inertial microfluidics. As regards the equilibrium position of a single particle in

channel flow of viscoelastic flow, it has been shown that the elastic effects drive the

particle towards the channel centreline. We use this fact to selectively migrate larger

blood cells to separate bacteria for sepsis diagnostics in paper IV. We have shown

that we can process whole blood and separate bacteria by selectively migrating blood

cells away from the streamline while bacteria are remained in the stream-line. While

this approach can process undiluted whole blood, the total flow rate is extremely low

(less than 10 µl/min). Future work will include parallelization to increase the

throughput to enable minimum of mL/h of whole blood to be realistic in sepsis

applications.


53

The second application has been on mammalian cell sorting, using both the active

and passive technologies developed within the thesis. The multi-parametric,

qualitative and quantitative analyses of cells for research and diagnostics of

pathological conditions such as cancer, require isolation and separation of cells from

complex biological fluid as a critical sample preparation step. The cell analyses and

sorting based on immunomagnetic (MACS) and immunofluorecent (FACS) methods

are routinely used in clinical laboratories. These methods exploit the difference in

surface protein markers for differences between cells for specificity. Typically, an

antibody is selected to identify a single (or a few) cell surface markers and the

antibody is conjugated with fluorescent molecules for FACS sorting or attached to

magnetic microparticles to separate target cells. MACS uses magnetic beads coated

with specific antibodies as is faster, but less specific and is better suited to process

larger cell numbers using both positive and negative selections. Due to the high

specificity of antibody—antigen binding, using FACS it is possible to able to sort

cells with extremely high purity. It is also possible to perform multiparameter sorting

and cell analysis. Consequently, FACS is one of the most powerful cell sorting

techniques that is based on flow cytometry. In Paper II we have developed micro-

Bubble activated acoustic cell sorting (BAACS) method that uses the strong negative

acoustophoretic contrast factor of microbubbles that can drag and sort cells captured

with antibody-coated microbubbles under acoustic field in a flow through setup. This

method has the potential to address a number of the current limitations in MACS cell

sorters including purity since the BAACS sort the cells under continuous flow

condition similar to FACS, but without the need to interrogate each cell optically.

Hence, with further development the BAACS is ideal as a miniaturized and cost

effective cell sorter for point of care applications.

In flow cytometry, cells are hydrodynamically focused to a narrow stream and pass

optical interrogation points one by one where laser beams illuminate individual cells.

The fluorescent and scattered light signals are detected by multiple detectors. Modern

flow cytometers can offer throughput in the range of 10,000 cells/s, while bulk


54

sorting techniques like MACS offers higher throughput but less specificity. Despite

flow cytometers provide good sensitivity and impressive throughput of thousands of

cells per second, commercial systems remain bulky, costly and require trained

personnel for operation and maintenance. In Paper V inertial and elasto-inertial based

cell focussing techniques were used continuously focus cells for miniaturised flow

cytometry. The opto-fluidic system combines micro capillaries and optical fibres to

deliver a truly miniaturised and low cost micro flow cytometer. This micro flow

cytometer is capable of enumerating two fluorescent colour cells and detect their

scattering signals for further analyses. Different aspects of this cytometer can be

enhanced such as scattering analyses and microcapillaries geometry to increase its

cells differentiation ability and the system throughput, respectively. The compact

system has the potential to replace bulky and expensive flow cytometers in many

applications at clinical and POC settings.

In summary, novel microfluidic separation cell sorting technologies have been

developed in this thesis. The bacteria and cell sample preparation has shown their

potential to realize integrated miniaturized platforms with detection and analyses

setups such as micro cytometer. The techniques explored shall be further investigated

and developed to offer solutions not only to increase reach of clinical diagnostic but

also expand the research horizon. Integration of separation with downstream analyses

is a natural future direction in order to develop a truly integrated point of care testing,

especially within the field of infection disease.


55

Acknowledgements

I would like to express my gratitude foremost to Almighty Allah SWT and can never thank and praise enough for countless blessing and making it possible.

Aman Russom, you are not just a supervisor for me, you literally played the role of mentor for PhD project and beyond. I can never forget when I was stuck in a project and we met in Shanghai for a Summer School and there we had a meeting during dinner on a boat. I am really thankful to taking me on board in your group and pull me out of that project. There have been lot of occasions that may have tested your patience and I can say you always dealt them with great positivity. I learned a lot from you including passion for research and can never thank enough.

I want to thank Helen Andersson Svahn for accepting me in the Nanobiotechnology group as a PhD student and providing me with great opportunity to work in great environment. Martin Wiklund, it is a great honour for me that you have been my co supervisor, I really am thankful for exchange of really useful ideas, a few minute discussion with you always provided great help. I perfectly understand why you have won the best teacher award lately. Sergey Zelenin you have been an inspiration and great source of knowledge and discipline in work. I am really thankful for your contributions and things I have learnt from you. Dmitry Grishnkov I am really thankful to you for the collaborative time we spent for microbubble paper and the extremely enthusiastic feedbacks I received from you. I want to thank Fredrik Laurell and Walter Margulis for very interesting discussions and guidance for optical detection for microflow cytometer project. Afshin Ahmedian I am grateful to you for reviewing this thesis work. Håkan Jönsson it was great experience conducting the N5T summer school under you thanks for that.

Harisha, I would not hesitate to say you stand out in whole group for your great intellectual support. I am really thankful to you for teaching me the inertial work. You are a problem solver with great ideas and numerous short meetings that usually occur without any prior notice from my side for different projects. Ida Iranmanesh what perfect timing you have joined our group as a postdoc and even before that in spite of the fact that you were expecting your role in all the acoustic work can never be forgotten. Sebastian for being such a great collaborator I really enjoyed your company while developing micro cytometer with your expertise. Amin Kazemzadeh


56

it is not quite long ago that you have joined our group but you brought a great impact in reshaping my thesis writing. Your dedicated feedbacks, intense discussions and friendly advices can never be forgotten. Sahar Ardabli, I remember when I joined the group your support really made me comfortable and off course the discussion we had in pendalbus. Also the breakfast and movie club that we have started out of your ever-imaginative ideas was rocking. Indra Banerjee it is always great to have a discussion in my mother tongue with you. Your simulation work for the elasto paper was a great contribution and I really had a great time with you friend. Zenib Aljadi for friendly discussions and off course for source of providing blood for a lot of experiments. Wang thanks for very nice discussions and I will remember the lunch we had in a Chinese restaurant. Thragan Kumar what can I say about you, you are a brother and have been really a great help. I always used the non-Newtonian fluid you prepared for your own work and you never complained. Tagrid Saleh during your short stay we really had good time with you specially the lunch we had with Ida and Tharagan. Satya brother I will never forget the time we spent in China thanks for being such a good friend, I remember you were the one who pushed me to talk to Aman when I was stuck in a complex situation. Hamed Muhammed thanks for being very friendly and polite. Mathias Ohlin thanks for the discussions and help in Martin’s Lab. I really admire your inventive approach. The past nanobio group member Ali, Emilie, Staffan, Thiru, Narendar, Nilay and Prem thanks for all the good times at Scilife lab. The present members Frida, Karl, Petter, Maria, Vamakshi, Lovisa, Sara, Lara, Jorge, Gustav, Lourdes, Marta, Jesper, Martin, Philippa, you all make this group a great place, it is a pity that last two years we had very less interactions since I have been mostly running between Albanova and Scilifelab.

I have not forgotten the friends who have played very important role and made my life easier and pleasant in Sweden especially Moeed Hussain and Bhunesh Kumar for being so helpful and great friends. Sajjad bhai, Abrar bhai, Umer and Fasih I am really grateful for your hospitality and helping me settling in Sweden. Ghufran Khalid and Adnan Faqui you are brothers and time we spent at Flemingsberg is filled with so many great memories that I will remember for life. Rashid bhai and Naveed bhai you guys brought Pakistani flavour to my life in Sweden. All of you have been here at different phases but the time I have spent with you guys has so many rich memories that I kept them as a treasure.


57

I would like to mention my really dear friends back home Shahid Latif and Asad Nafis from whom I have been away for long time but we kept in touch and they were always there for me. I can never thank much for their selfless friendship. My cousin brother Tayyeb Siddiqui I can never forget your role when I got the scholarship for Sweden and I need very important guarantee paperwork that was not possible without your and your friend Abdur Rab bhai’s generous help. My cousin sister Javeria I am thankful for so many very enthusiastic discussions and friendly fights. I would like to thank all my teachers from SSUET, Linköping and KTH universities. Muneer bhai I remember the crash course in mathematics of SSC that you taught just in one week so brilliantly that I got distinction.

I would like to thank my uncle Late. Dr Abdul Samad for being an inspiration and teaching me to critically and logically think. I would love to thank my Late Paternal grand Father Abdul Razzaq Faridi who always wanted me to take education seriously. I want and thank and mention my Maternal Grand father Late Ibrahim Khan and my maternal grand mother Late Hashmi for being so proud of me when I started my PhD I can never forget their love. My paternal grand mother Habib-u-Nisa, I am her favourite grandchild and she really took great care of me. I know now you are in a condition unaware of everybody but I keep you really dear. I would like to thank all my extended family. I thank my parent in laws for believing in me and giving your beloved daughter’s hand to me whom I have taken far away from you. You have given a great companion for the journey of my life.

Finally last but not least, my beloved family. It would have never been possible without unconditional and ever loving support of my mother (Shamim Ara) and father (Abdul Wahab Faridi), I love you and I love you for being such selfless and most loving parents, thanks Ammi thanks Abbu for everything and I can never thank enough. My sisters Mano (Sarah) and Saadi (Sadia) your love and support especially bearing with my ever-irritating discussions deserve a salute. Both of you have been playing the role of my best friends and my advisor so brilliantly that you are hired for life. Mano and Saadi you are important part of this work and I really mean it, thanks Mano my bacha you deserve a chunk of PhD degree, thanks Saadi my elder yet younger sister you are greatest sister ever. My brother Anas I knew and I know if I ever need to depend on anybody it is you, you have taken all the responsibilities on your shoulders while I have been away, whenever I visited back home it is you who made sure I get to do and eat anything and everything that I missed, love you brother. My brother in law Adil Khan for being great support and always extraordinarily


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understanding. My niece Aimal and nephew Ammar for bringing so much happiness to me. My wife Maddo (Madeeha), my friend my strength my love you have been there for me when I was down you never complained and always been the light in darkness. Together we have seen lot of ups and downs in short time but you always stood besides me and being a great source of strength. I have to thank the almighty Allah again for the blessed gift we got during last phase of PhD work my son Hussain Ahmad who brought so much positivity, blessings and hope in my life, thanks Maddo for being a great wifey and closest friend. Thanks Hussain for your most pleasant entry at this phase of my life. Maddo you always say this PhD work should be transferred to your name, here you go I make you one of the major shareholders of this work along with my Ammi, Abbu, Mano, Saadi, Anas and Hussain J.


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