Dielectrophoresis (DEP) and electrowetting (EWOD) as …743805/FULLTEXT01.pdf · Anti-fouling process for antibacterial surfaces ... Dielectrophoresis (DEP) and electrowetting (EWOD)

Dielectrophoresis (DEP) andelectrowetting (EWOD) as an

Anti-fouling process for antibacterialsurfaces

STAVROS YIKA

Master’s Degree ProjectStockholm, Sweden June 2014

XR-EE-MST 2014:00X

Dielectrophoresis (DEP) and electrowetting(EWOD) as an Anti-fouling process for

antibacterial surfaces

Stavros Yika

Master’s Degree Project

Supervisor: Fredrik Carlborg

Examiner: Wouter Van der Wijngaart

June 2014

Microsystem Technology

KTH Royal Institute of Technology

Stockholm, Sweden

Abstract

Today the medical field is struggling to decrease bacteria biofilm formation whichleads to infection. Also, biomedical devices sterilization has not changed overa long period of time which has resulted in high costs for hospitals healthcaremanagements. The objective of this project is to investigate electro-dynamic effectsby surface energy manipulation as potential methods for preventing bacteria biofilmgrowing on medical devices.

Based on electrokinetic environments two different methods were tested: re-jection bacteria dielectrophoretic forces feasibility by numerical simulations; andelectrowetting-on -dielectric by the fabrication of golden interdigitated electrodeson silicon glass substrates covered by a Teflon layer.

In the first experiment, numerical simulations of gold electrodes in buffer so-lution and frequencies were carried out to determine the forces required to rejectbacteria. In the second experiment, interdigitated gold electrodes coated with adielectric Teflon layer, were characterized in terms of breakdown voltage, dielectricadhesion and contact angle in terms of applied voltage. Finally the effect of EWODon bacterial adhesion was tested.

The project resulted in promising simulation results for bacteria rejection usingdielectrophoresis due to the wide range of frequency that rejects the modelledbacteria. However, practical experiments such as electrowetting-on-dielectric mustverify this at incubation times larger than 24 hours in spite of the Teflon non-adhesive properties.

Abbreviations

AC Alternate current

CM Clausius-Mossotti

DC Direct current

DEP Dielectrophoresis

EDL Electrical Double-Layer

EP ElectroPhoresis

EW ElectroWetting

EWOD ElectroWetting-on-Dielectric

HCAI Healthcare Associated Infections

M9 M9 minimal medium - minimal microbial growth medium

nDEP negative DEP

OSTE off-stoichiometry thiolene

PDMS PolyDimethylSiloxane

PBS Phosphate buffered saline

pDEP positive DEP

UV Ultraviolet light

VRI Viral respiratory infections

WHO World Health Organization

Contents

1 Introduction 81.1 Project Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.2 Research Idea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2 Background and State of Art 122.1 Biofilm formation and prevention . . . . . . . . . . . . . . . . . . . . 122.2 Theory themes (DEP-EWOD) . . . . . . . . . . . . . . . . . . . . . 15

2.2.1 Dielectrophoresis Force . . . . . . . . . . . . . . . . . . . . . 152.2.2 Dielectrophoresis in DC and AC Fields . . . . . . . . . . . . . 162.2.3 EWOD in Direct current (DC) and Alternate current (AC)

fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.2.4 Theory limitations . . . . . . . . . . . . . . . . . . . . . . . . 19

2.3 Modelling of biological cells . . . . . . . . . . . . . . . . . . . . . . . 192.3.1 Biological cells on DEP . . . . . . . . . . . . . . . . . . . . . 192.3.2 Hydrophobic effect on bacteria adhesion . . . . . . . . . . . . 21

2.4 Modelling of electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3 Design and assembly of the experimental setup 243.1 Chip design and assembly . . . . . . . . . . . . . . . . . . . . . . . . 24

3.1.1 Electrode design . . . . . . . . . . . . . . . . . . . . . . . . . 243.1.2 Chip fabrication . . . . . . . . . . . . . . . . . . . . . . . . . 27

3.2 Gasket fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363.2.1 OSTE Gasket . . . . . . . . . . . . . . . . . . . . . . . . . . . 373.2.2 PDMS Gasket . . . . . . . . . . . . . . . . . . . . . . . . . . 413.2.3 Leakage tests . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

3.3 Bacteria to be used . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453.3.1 Salmonella Typhimurium . . . . . . . . . . . . . . . . . . . . 45

4 Measurements and results 474.1 Numerical simulations of DEP on bacteria model . . . . . . . . . . . 47

4.1.1 MATLAB calculations . . . . . . . . . . . . . . . . . . . . . . 474.1.2 COMSOL analysis and results . . . . . . . . . . . . . . . . . 53

4.2 Experiments of EWOD surfaces on live bacteria . . . . . . . . . . . . 644.2.1 Contact angle tests . . . . . . . . . . . . . . . . . . . . . . . . 644.2.2 Waveform tests . . . . . . . . . . . . . . . . . . . . . . . . . . 68

3

CONTENTS

4.2.3 Teflon adhesion to glass . . . . . . . . . . . . . . . . . . . . . 684.2.4 Teflon breakdown voltage tests . . . . . . . . . . . . . . . . . 71

4.3 Bacteria tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734.3.1 Teflon bacteria adhesion tests . . . . . . . . . . . . . . . . . . 76

5 Discussion and outcome 785.1 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785.2 Outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

5.2.1 Project method summary . . . . . . . . . . . . . . . . . . . . 835.2.2 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

A Biofilm on hospital environments challenges 92

B MATLAB Program 94B.1 Command set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94B.2 Flow Chart Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

C COMSOL analysis and simulations 98C.1 Drift velocity from Einstein– Smoluchowski relation . . . . . . . . . 98C.2 DEP force measured at different heights . . . . . . . . . . . . . . . . 99

D Bacteria formation 100D.1 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

D.1.1 ImageJ macro . . . . . . . . . . . . . . . . . . . . . . . . . . . 100D.2 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

E OSTE tests detailed information 102E.1 Material specification . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

F Chip fabrication detailed information 103F.1 Design / L-EDIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103F.2 Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104F.3 Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

G EWOD attemps 107G.1 Another EWOD tests . . . . . . . . . . . . . . . . . . . . . . . . . . 108

4

List of Figures

2.1 Attachment of bacteria cells mechanism . . . . . . . . . . . . . . . . 132.2 DEP variations by its applications . . . . . . . . . . . . . . . . . . . 152.3 Effect of ~E on particle . . . . . . . . . . . . . . . . . . . . . . . . . . 172.4 Interdigitated electrode distance effect on Electric field[1] . . . . . . 182.5 DEP cell modelling[2] . . . . . . . . . . . . . . . . . . . . . . . . . . 202.6 Hydrophobicity and attach cell number (RP62A) relationship . . . . 212.7 Commercial Electrode-gasket sample for cells and/or proteins . . . . 222.8 Different existing Dielectrophoresis (DEP) devices classification . . . 232.9 ElectroWetting (EW) electric fields and equivalent Electric circuit . 23

3.1 Electrodes configuration . . . . . . . . . . . . . . . . . . . . . . . . . 253.2 Electrodes in square spaces . . . . . . . . . . . . . . . . . . . . . . . 263.3 Mask and expectable wafer design . . . . . . . . . . . . . . . . . . . 273.4 Wafer material layers . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.5 Process of Photo-lithography . . . . . . . . . . . . . . . . . . . . . . 293.6 Metal Mask Measurements . . . . . . . . . . . . . . . . . . . . . . . 313.7 Teflon technique to insulate on only electrodes . . . . . . . . . . . . 333.8 Finished chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343.9 Etching time problem . . . . . . . . . . . . . . . . . . . . . . . . . . 353.10 Electrode bad etching finish . . . . . . . . . . . . . . . . . . . . . . . 353.11 Commercial gasket and design . . . . . . . . . . . . . . . . . . . . . . 363.12 Gaskets designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373.13 OSTE gasket on aluminium mould . . . . . . . . . . . . . . . . . . . 393.14 One hole gasket procedure . . . . . . . . . . . . . . . . . . . . . . . . 403.15 Different made PolyDimethylSiloxane (PDMS) gaskets . . . . . . . . 433.16 OSTE and PDMS gaskets on sealing tests . . . . . . . . . . . . . . . 443.17 OSTE gasket adhesion and leak . . . . . . . . . . . . . . . . . . . . . 443.18 Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . 46

4.1 Excel & Matlab CM plots . . . . . . . . . . . . . . . . . . . . . . . . 494.2 CM on diff. mediums . . . . . . . . . . . . . . . . . . . . . . . . . . . 504.3 Different CM values for biological mediums for yeast and E.coli par-

ticles within various frequency values . . . . . . . . . . . . . . . . . . 524.4 Electric potential intensity . . . . . . . . . . . . . . . . . . . . . . . . 544.5 Electric Field intensity . . . . . . . . . . . . . . . . . . . . . . . . . . 56

5

LIST OF FIGURES

4.6 DEP Force . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584.7 COMSOL DEP simulation - Electric field analysis . . . . . . . . . . 614.8 Maximum bacteria drift rejection velocity based on applied voltage . 624.9 EWOD setup for Contact angle measurements . . . . . . . . . . . . 644.10 Contact angle by evaporation . . . . . . . . . . . . . . . . . . . . . . 664.11 Contact angle within different voltages . . . . . . . . . . . . . . . . . 674.12 Waveform tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684.13 Teflon peel off from glass surfaces . . . . . . . . . . . . . . . . . . . . 704.14 Teflon breakdown tests . . . . . . . . . . . . . . . . . . . . . . . . . . 724.15 Setup used for bacteria tests . . . . . . . . . . . . . . . . . . . . . . . 734.16 Electrodes’ cells numbered and labelled . . . . . . . . . . . . . . . . 744.17 Bacteria tests stages . . . . . . . . . . . . . . . . . . . . . . . . . . . 754.18 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

5.1 DEP force (Newton)at 30 V at the edges . . . . . . . . . . . . . . . . 795.2 Squared Electric field gradient affected by Teflon layer . . . . . . . . 805.3 Droplet contact angle change & electrolysis . . . . . . . . . . . . . . 815.4 Bacteria tests on with interdigitated electrodes . . . . . . . . . . . . 82

B.1 Matlab flow chart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

C.1 DEP forces at different heights . . . . . . . . . . . . . . . . . . . . . 99

D.1 Bacteria biofilm matrix forces . . . . . . . . . . . . . . . . . . . . . . 100

F.1 Different electrodes topology . . . . . . . . . . . . . . . . . . . . . . 103F.2 Electrode design attempt . . . . . . . . . . . . . . . . . . . . . . . . 104

G.1 EWOD variety test . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108G.2 EWOD in in macro-level electrodes . . . . . . . . . . . . . . . . . . . 109

6

List of Tables

1.1 Costs due to antibiotic resistant bacteria[3] . . . . . . . . . . . . . . 91.2 Current Antibacterial surfaces methods . . . . . . . . . . . . . . . . 10

2.1 Bacteria adhesion earlier experiences . . . . . . . . . . . . . . . . . . 14

3.1 Different width of the electrode depending on the case used . . . . . 253.2 Photoresist curing time parameters . . . . . . . . . . . . . . . . . . . 283.3 Different used etching times on lithography . . . . . . . . . . . . . . 303.4 Spindle and Feed speeds for the different materials . . . . . . . . . . 303.5 Glass Blade parameters . . . . . . . . . . . . . . . . . . . . . . . . . 313.6 Silicon Oxide Blade parameters . . . . . . . . . . . . . . . . . . . . . 313.7 OSTE Formulation: Stoichiometry, monomers and curing agent . . . 383.8 Curing time for 2 mm OSTE . . . . . . . . . . . . . . . . . . . . . . 413.9 Curing time for 1 mm OSTE . . . . . . . . . . . . . . . . . . . . . . 423.10 PDMS Formulation: base and curing agent ratio . . . . . . . . . . . 42

4.1 Three-layer particle parameters . . . . . . . . . . . . . . . . . . . . . 484.2 DEP force calculated from COMSOL simulations . . . . . . . . . . . 634.3 Drift velocity for different bacteria . . . . . . . . . . . . . . . . . . . 634.4 Diffusion length of bacteria during one second . . . . . . . . . . . . . 634.5 Bacteria adhesion tests done in Karolinska laboratories . . . . . . . . 76

D.1 M9 medium parameters . . . . . . . . . . . . . . . . . . . . . . . . . 101

E.1 Culture plate parameters . . . . . . . . . . . . . . . . . . . . . . . . 102

F.1 UV exposure and development times - Second lithography attemptwith standard softbake(110◦C) . . . . . . . . . . . . . . . . . . . . . 105

F.2 UV exposure and development times - Second lithography attemptwith lower softbake(90◦C) . . . . . . . . . . . . . . . . . . . . . . . . 105

F.3 Measurements of the electrodes and gaps in the second attempt fromTab.F.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

F.4 Dicing blade parameters about the used Flange . . . . . . . . . . . . 106

7

Chapter 1

Introduction

The aim of this chapter is to introduce the topic of the thesis and explain in furtherdetails the project definition, goals and the motivation behind it.

1.1 Project Motivation

From ancient times until nowadays the absence of antibacterial surfaces has beenpresent, its demand has been increasing and this is a current problem.It is widely known microorganisms defence from other microorganisms’ has been toproduce their own chemical substances, this is used today as antibiotics or antimi-crobial agents. Unfortunately our body is not as resistant as these microorganisms,making us more vulnerable. Indeed, this can be seen in infections caused by trans-mission of resistant microbes that lives in other’s patients systems like in hospitalenvironments.

Antibiotics are becoming less effective while more resistant microorganisms arespreading all over the world. Antimicrobial resistance is mainly driven byhealthcare practices, where antimicrobial are overdosed in patients. Indeed they donot need exactly that, as refereed in [4],[5]. For instance, a multi-drugresistant or-ganism (MDRO), also known as Superbug, is often resistant to fluoroquinolones[6].

Nowadays, places were healthcare is not well supported are still struggling withdeaths caused by infections, and in more supported healthcare places antimicrobialresistance is the major cause of deaths, making antibiotics practically ineffective.

The main area of need is in hospital environments, where bacteria can growalmost on any medical instrument. Operating rooms (OR) must be a very ster-ile environment and antimicrobial resistance make it vulnerable to eventually beunsterilised surgeries; in fact, 80% of most common hospital acquired infectionsare associated to catheters, such as urinary ones[5]. Likewise, in the USA, 5-10%of hospital patients suffers healthcare-associated infections (HAIs), where 35% ofthem are urinary tract infections related to catheters.

In invasive operations, such as Sectio Caesarea, antimicrobial prophylaxis isneeded to be administrated 60 min prior to the operation. This always reduces the

8

1.1 Project Motivation

incidences of infections.

From the public health point of view, due to a misused way of healthcarepractices highly resistant bacteria have been created, which represent high costs inhospital-related infections solutions.

Hospital-acquired infections has become a major cost due to time and logisticinvested in the use of disinfectants in hospital environments as can be seen inTable 1.1.

Hospital-acquired Infections Costs (US$)

USA $10 billion per yearMexico $450 million per yearThailand $40 million per year

Table 1.1: Costs due to antibiotic resistant bacteria[3]

The main problem of antimicrobial resistance lies in the bacteria infection treat-ment dependence. Consequently, new methods to avoid bacterial infections cannotprevent antimicrobial resistance from becoming a bigger problem, but help to stepdown as the most important public health concern[7].

On the other hand, what happens if this antibiotic dependence ends? Therehave been incredible reductions of use in antibiotics in the EU countries due to a banon some of the antimicrobial growth promoters (AGP), which reduce antibacterialresistance on food germs[8].

Bacteria can survive several days on surfaces, and the only way to reduce biofilmformation is cleaning and disinfecting the used surfaces. Time to inactivate viruscan range from five minutes to more than one day depending on the concentrationof the inoculants and the disinfectant[9].Reusable medical devices that are in continuous mucous contact should receivehigh-level disinfection (explained further in section A) between different patients,including endoscopes, Endotracheal Tube or Breathing Tube(ETT), anaesthesiacircuits, and ventilators). In addition, some prosthetics, inner devices and operationtheatres surfaces such as surgical tables need chemical sterilization instead of hightemperature sterilization due to the material degradation. In this cases, betterengineered materials are needed to suppress bacteria growth like covered fibers orwet fabrics with antibacterial substances.

Biofilms, mainly in endotracheal tubes is eliminated mechanically or by silvercoating which is known to kill bacteria. Since the mechanical release of attachedbacteria requires manual cleaning, trained personnel is required adding costs tohospitals bill. The metal coating can also wear off and become dangerous if swal-lowed.

Most of antibacterial surfaces nowadays are passive, such as chemical coatingsthat can change the surface functionalization by the use of polymers or polypeptides[10] or metal alloys [11].

9

1 Introduction

The majority of current solutions release an active ingredient that interacts withthe microorganisms: either inhibiting from spreading or killing them.

CONTACT KILL RELEASE

Polymer graftsAntimicrobialpeptides (AMP)

Silver or copperBactericides(i.e.chlorhexidine)

Antibiotic coatings(i.e. gentamicin)

Mechanism

Covalent attach-ment of polymerswith bacteria re-pellent or toxicgroups.

Disrupts thecell membrane,attaches byelectrostaticattraction.

Silver ions pre-vent formationof enzymesand causecell death.Copper mech-anism is notwell under-stood.

Prevents initialadhesion of bac-teria.

Broad spectrumantibiotics in-corporated intosurface coatings.

Advantage

Covalent bond,no leaching.Prevent surfaceadhesion or killbacteria.

Broad spectrumantibiotic effect.Can be covalentlybonded to sur-face: Leach free.Unknown tocause antibioticresistance

Can kill from a range.

Disadvantage Surface loading.

Difficult tostabilise incoatings.Leaches leadsto detrimentaleffect on theenvironment.

Difficult tocontrol coatingrelease at longterm, leaching.

Cause antibioticresistance.

Table 1.2: Current Antibacterial surfaces methods

From Table 1.2, can be seen that releasing methods become a toxicity problemfor living beings and the environment. The major problem with both techniquesis that killed microorganisms remain in the surface making easy target for newbacteria to attach and blocking surface-active agents from diffusing. Consequentlyantibacterial surfaces become gradually inactive.

Previously all approaches to prevent health care related infections have centredaround removing or killing the bacteria adhering on the surface of the devices.These have several drawbacks such as eventual build up of a biofilm consisting ofdead bacteria and/or long term negative effects such antibiotic resistance caused bycatheters coated with antibiotic substances. Instead the electrokinetic approach in-vestigated in this thesis focuses on creating a sufficiently unattractive environmenton the surface as to completely prevent bacteria from attaching.

The solution would be to create a non-toxic surface that preventsbacteria to attach, reproduce and form biofilm. In fact, once bacteria biofilmis adhered into the surface, detaching it requires an enormous force.Bacteria rejection surface like this is needed and could be applied on medical tubessuch as catheters or ETTs, surgical surfaces, and different operation theatres tools.

The purpose of this project is to investigate how electrokinetic microenviron-ments affect bacteria adhesion, specifically with the interaction of electric fields

10

1.2 Research Idea

gradient, and surface dielectric capacitance in an array of interdigitated electrodes.

The practical motivation behind the project is to investigate possible solutionsfor preventing bacteria biofilm formation in healthcare devices, such us on jointprosthesis (orthopaedic), on urinary catheters and on airways intubation tubes,where bacteria become the cause of Healthcare Associated Infections (HCAI) andViral respiratory infections (VRI).

1.2 Research Idea

The research idea of this thesis is creating antibacterial surfaces by the use ofinterdigitated electrodes methods for preventing the bacteria to attach on medicaldevices or healthcare surfaces thus avoiding biofilm formation without the use ofantibiotics or release of chemicals.

For that reason, different methods will be investigated to evaluate their effi-ciency to prevent bacteria adhesion: Electrophoresis (EP), Dielectrophoresis (DEP)and Electrowetting (EW). For instance, DEP can exert enough forces into particlesdepending on the dielectric values and medium. This rely on a very wide range offrequencies which define an attraction or repulsive force from the surface. Henceprevent bacteria to reach the surface and form biofilm.

On the other hand, ElectroWetting-on-Dielectric (EWOD) can create particledisplacement as it can be done with liquid droplets depending on the applied voltageand the thickness of the dielectric layer on top of the surface. Therefore bacteriabiofilm might be not only disrupted, from adhesion and further biofilm formation,but capable of be manipulated and transported along the surface.

Considering the last shown methods and having a manufactured a device com-posed of interdigitated gold electrodes covered with PTFE (dielectric layer, Teflon);Dielectrophoresis and electrowetting-on-dielectric can be analysed and tested withthe applied voltage as main variable.

11

Chapter 2

Background and State of Art

In this chapter current anti-biofouling technologies history is reported. Further-more, the theory themes behind the experiments are presented. Additionally, thischapter provides a basic theory about EW and DEP.

2.1 Biofilm formation and prevention

Surfaces have an important role in the micro-organism activity; biofilm formation isformed as a defence from adverse conditions (low nutrients, extreme temperatures).

Adherence to the surface happens by the secretion of extracellular polysaccha-ride substance from cells (known as slime).

The biofilm formation sequence starts with the conditioning of the surface,followed by the colonisation of the bacteria and finally its multiplications. Thismicroenvironment can increase bacteria growing if two factors are ruled: either bythe removal of microcolonies parts and future growth in farther places, or increasedby fluids flows that carries nutrients.

Biofilm’s structure is very similar to a pile of Lego R© pieces where bacteria canoverlapped each other in different spots and orientations, making easy to intercon-nect themselves by its flagella and cellulose (like bricks and cement) in a commonsecretion medium. By this, bacteria is allowed to breath and be protected fromouter biocides. Factors that increased the formation come from nutrients presence,to warm temperature, and flows stagnation on fluids channel dead-ends[12].

Microbial adhesion mechanism, DLVO theory In a dielectric medium, ionsare dispersed all over it. But there will be a gradient of ions density where a in-terface is contained. For example, when a charged particle is introduced in thismedium counter ions begin to screen all over its surface by self-organization man-ner however not in an uniform way. In fact, counter ions structured in layerswith different characteristics which behaviour is explained through Debye-Huckeltheory[13].

This is called Debye screening effect[14]. The first layer is always composed ofthe attached counter ions; this together with the ions behind the interface forms the

12

2.1 Biofilm formation and prevention

so called Stern layer[15] which is located in the Inner Helmholtz Plane (IHP). Thesecond layer is composed of a diffuse ion layer, which is attached by Coulomb forces.This two equalled opposite charged layers are defined as an Electrical Double-Layer(EDL). It is important to consider the distance that ions affect beyond the EDL,which is called Debye Length, for phosphate-buffered saline (PBS) is 0.7 nm.

The kinetic stability between energy interactions of particles is explained byDerjaguin-Landau-Verwey-Overbeek (DLVO) theory[13]. This theory combinedthe van der Waals attraction forces and the electrostatic repulsion forces at low sur-face potentials (potential energy <<thermal energy) considering EDL limits[16].First, bacteria cell is transported to the surface by sedimentation forces and hydro-dynamic forces (drag force), until reaching the Diffusive boundary layer illustratedin Fig 2.1. At this point the only main force is the diffusion, where its diffusiontransportation behaviour is purely by Brownian motion; here DLVO forces arepresent between surface and the bacteria cell. However, the cell attachment is re-versible due to still week surface interaction. This interaction range is relative small(< 1 µm), where both surfaces present negative charges until EDL is overcome.Finally stronger irreversible forces arrived at attachment where its range beginsfrom 5 to several hundreds of nanometers[17].

Figure 2.1: Attachment of bacteria cells mechanism by Dickinson[18]

Previous bacteria adhesion experience in different surfaces There areseveral surfaces that have been tested for bacteria adhesion measurements, as wellas new antibacterial surfaces that prevent chemical leaching. As it can be seen intable 2.1.

But overall, chemical free devices are still not attempted for antibacterial sur-faces.

13

2B

ackgrou

nd

and

State

of

Art

Test target Tested surface Bacteria Results Solution

catheter-associatedurinary tract infection:CAUTI.

2 cm-long uri-nary catheterssegments.

Pseudomonasaeroginosa,Stapholoc-cus aureus,CNS,Enterococcusspp., Streptococ-cus spp. andSalmonella ty-phirium strainsMAE52 andMAE50.

•179 bacterial isolates fromcatheters (96) and urine (83)samples.•4 from 5 catheters surfacesdetected coexistence of gram-negative and gram-positivebacteria.•E. coli was the most infectionagent in catheters.

The prevention ofbiofilm, hence preven-tion of infection.[19]

bloodstream infec-tions, fungal infectionrisks in intensive careunit, gastrointesti-nal tract surgery andpower antibacterialagent treatment conse-quences.

SETM: Silatos sil-icone sheet

Candida albicansand non-albicansspp.

•More biofilm formation bynon-albicans Candida speciesbecause its higher capacity•Although C. Albicans Can-dida, a more pathogenicspecie, are the main cause ofbloodstream infections.

Fluconazole is the maincounter agent for bothspecies but resistance toit has been displayed byboth too.[20]

biofilm formation incatheters and bio-prosthetic devices.

SE, Thermanoxcell culturecoverslip andpolystyrene 96-well plate.

Candida species.Antifungal:fluconazole, am-photericin Band caspofungin(Sigma-Aldrich).

•10 from 11 samples wereseen to have highly formedbiofilms.•Amphotericin B is effectiveagainst biofilms.

•The choice of the ma-terial is critical for re-ducing biofilms•Amphotericin B toxic-ity against mammaliancells and kidney damageis a drawback.[21]

characterize the Hfq(RNA) dependency onbiofilm formation.

polystyrene, glassand cholesterolgallstones.

Salmonella en-terica serovartyphimurium.

Hfq dependency for regulatingbiofilm development.[22]

—

antibacterial:”New ma-terial”, leakage free.

polyelectrolytemultilayer(PEM): PVAmand AnionicPAA (polymers);different pulps(fibers).

gram-positiveBacillus subtilis,gram-negativeEscherichia coliATCC11775.

Satisfactory in terms of bacte-rial growth inhibition.

•Varied because of thedependence on polymercontent and cationiccharge of the fibers.•pH used was raisedto physiologic valueswhere low values im-prove antibacterialeffects.[23]

Table 2.1: Bacteria adhesion earlier experiences

14

2.2 Theory themes (DEP-EWOD)


In this part all physical theory is presented and explained. DEP is explained in twoparts, what DEP force is and how it can be an important part in the manipulationof dielectric particles. EWOD is explained by its basic mechanism and applications.Both mechanisms were selected for their ability to repel particles from the surface.

EP Electrophoresis is the motion of particles by the appliance of an uniform Elec-tric field.

DEP Dielectrophoresis is the manipulation force of polarisable (uncharged) dielec-tric particles by the use of non-uniform electric field.

eDEP electrode-less DEP due to a dielectric constriction of the electric fieldon a narrow space.Fig 2.2a

TW DEP Travelling wave DEP produces forces that pushes particles in/offthe direction of wave propagation.(depending on the polarity of imagi-nary factor of CM)Fig 2.2b

(a) eDEP (b) TWDEP

Figure 2.2: DEP variations

This last two are DEP variations that are considered in selected methods asa possible result in the tests purpose.

EW Electrowetting is the surface properties modification due to applied electricfield.

EWOD electrowetting-on-dielectric is EW on top of dielectric-coated elec-trodes.

2.2.1 Dielectrophoresis Force

In this part Dielectric Force is explained, as well the Clausius-Mossotti (CM) factor.Before explain of Dielectrophoresis, electrophoresis must be first defined.Electrophoresis is the coulomb force created due to a electrostatic charged par-

ticle that moves (if it is DC potential) or stay still (if it is AC potential).Electrophoretic mobility is described by Eq.2.1 and considering a “Thick double

layer” is described by Eq.2.2

µe =~υ

~E=εmεoζ

η(2.1)

15

2 Background and State of Art

where εm, εo , are medium and vacuum permittivity, ζ is the zeta potential andη is the dynamic viscosity.

µe =2 εrεoζ

3 η(2.2)

Considering this and restricting it to dielectric particles, the study is focusedon how electric field gradient affect the particle charge.

In order to define Dielectrophoresis (DEP), DEP force must be detailed as it isthe main physical concept that influence this phenomenon.

The main force exerted by a gradient electric field in a general manner is de-scribed by Eq.2.3 where ε∗ stands for complex permittivity.

The time-average force is:

〈 FDEP 〉 = 2πr3εoεm<{ε∗p − ε∗mε∗p + 2ε∗m

}∇| ~Erms|2 (2.3)

where ε∗ is the complex permittivity(vacuum, medium or particle), r is radiusof the particle, and ~Erms is the electric field.

But considering spherical or cylindrical particles the equation can be reducedto Eq.2.4.

FDEP =πr2l

3εoεm<{

ε∗p − ε∗mε∗m

}∇| ~E|2 (2.4)

ε∗ = ε+ σ

ω(2.5)

where l is the cylinder length, ε is the dielectric constant σ stands for conduc-tivity and ω is the frequency domain. Last but not least <{f} is the real part ofClausius-Mossotti(CM) factor {f}.

It can be concluded from above that the force magnitude depends stronglyon the polarizability of the particle in respect of the medium. This motion ofparticles resulting in polarization forces by an gradient electric field was nameddielectrophoresis by Herbert A. Pohl in 1950[24].

2.2.2 Dielectrophoresis in DC and AC Fields

This part shows how DEP works in DC and AC fields. Finally the two cases arecompared for our purpose.

Non-uniform DC electric fields the particle (now charged) will move along theelectric field force.In the light of the particle dielectric property, if it is higherthan the medium it will move towards the highest gradient field area (positiveDEP (pDEP)) otherwise it will move towards the lowest gradient field area (negative DEP (nDEP)) as seen in Fig 2.3.

16


Figure 2.3: Movement of the particle under ~E

Non-uniform AC electric fields Aside from the charged particles (they presentslittle movements due to the exerted opposite forces produced by the ACfield),depending on dielectric particles properties, it will follow the same be-haviour of non-uniform DC. As a consequence, positive DEP(<{CM}> 0)and negative DEP (<{CM}< 0) will work as trapping and rejecting particlemethod respectively.

From above cases, it can be seen that DEP force depends strongly on ~E gradient.Based on it, this information can be deduced:

• FDEP is proportional to particle volume and εm. Its direction is within ∇| ~E|rather than ~E.

• FDEP is inversely proportional to the cube of the electrode distance (gap).Asseen on Fig 2.4.

17


(a) Electric field on 5, 10 and 15 µm gaps

(b) Electric field gradient vs. gap distance

Figure 2.4: Interdigitated electrode distance effect on Electric field[1]

2.2.3 EWOD in DC and AC fields

In this part EWOD is similarly presented and compared not only by DC and ACfields but the difference in frequency values.

To this extent, DC voltage is applied to see how the contact angle change as partof the force created by the electric field and how that force overcome the surfacetension (Gibbs free energy) while the potential increases between the electrodesand the medium (capacitance).

By applying AC voltage the result is the same but depending strongly on thefrequency applied the contact angle changes and return to its original position (zeropotential)in a dynamical speed.

The electrowetting principle reckon on the described Lippmann-Young equationEq.2.6

cos θ = cos θo +1

2

C

γLGV 2 (2.6)

where θo is original contact angle(without electric field), θ is the one with theelectric field, C is capacitance per unit area(between electrodes and liquid), γLGis the surface tension vector(liquid-gas) and V is the applied voltage. It must beconsidered: de-ionized water γLG = 30 mN/m.

18

2.3 Modelling of biological cells

2.2.4 Theory limitations

In this section limitations are displayed and justified, including: the Debye layer,the ion diffusion on Electrical Double-Layer (EDL) as seen on Section2.1, REDOXon electrolysis and joule-heating as part of the inherently happening in the proposedmicroenvironment.

It is important to consider also the limitations presented in every experimentin regard of the theory presented.

• FDEP is proportional to square of voltage. (reversing BIAS does not reverseforce)

• Joule heating effect can be neglected as the resistance and low voltage canreduce the electrical current considerable in this stationary test.

• As long as the Debye layer is able to be present in ionic medium, it must betaken on count but can be neglected if the force is high enough compared toEDL.

• Other limitations not accounted for like taking as constant values for di-electric, permittivity, conductivity, viscosity, resistivity, and more inherentproperties of the particle, medium and surface materials.

2.3 Modelling of biological cells

In this part Biological particles are modelled in a layered approximation, also ispresented the effect of a hydrophobic dielectric for EWOD applications.

The bacteria that was used is Salmonella typhimurium, which is characterizedfor having cylindrical surface and a tail.

2.3.1 Biological cells on DEP

In this part it is illustrated the approximation modelling of bacteria cell as athree concentric layer particle and details of calculation of the complex permit-tivity needed to calculate the CM factor.

As bacteria is not a single particle but a particle made of different conductivitylayer; it has been modelled as followed in Fig 2.5a where bacteria is studied as anspherical shell model.

19


(a) DEP multi-shelled cell modelling

(b) Spheric approximation of multi-shelled particles bysmeared-out sphere approach

Figure 2.5: DEP cell modelling[2]

From Fig 2.5a can be seen that having the conductivity and permittivity datathe right frequency can be calculated.

But considering a wider range frequency where bacteria change its CM factorDEP can be used.

20

2.4 Modelling of electrodes

2.3.2 Hydrophobic effect on bacteria adhesion

In this part is presented a study of how surface energy is directly translated intohydrophobicity, and how adhesion works on that.

Bacteria adhesion is measured in free energy of interaction (∆Giwi). And soit is the hydrophobicity measurement, where if ∆Giwi> 0 it is hydrophilic, and if∆Giwi< 0 it is hydrophobic.

Figure 2.6: Relation between the number of attached cells of Staphylococcus epider-midis ATCC 35984 (RP62A) and the degree of hydrophobicity (∆Giwi) of varioustypes of substrata,ref [25]

From the Figure2.6 it can be seen how linear is the relationship between bothmeasurements, confirming by this way how hydrophobic bacteria interact easierwith hydrophobic surfaces and same with the hydrophilic interactions.


In this part electrodes configuration is selected by its purpose and an electricalmodelling is approximated according to the material electrical values.

About electrode-gasket methods, there are already some samples of DisposableElectrode Arrays built and commercialized like in Fig 2.7, which are able to interactbetween cell proteins, and make cell proliferation measurements[26].

Moreover, it can be found also in the market antimicrobial-impregnated centralvenous catheter which use two antiseptics(chlorhexidine and silver sulfadiazine) toavoid bacteria colonization, which has been used in transplants, septic patients(included burned patients)[27].

After seen the some cell manipulation electrodes in the market, electrodes the-ory and limitations presented for DEP-EWOD phenomenon, the next step is toselect the best electrodes array for the Project goal.

There are several electrode configurations studied and applied in dependenceof the operating strategy.Fig 2.8

21


Figure 2.7: Electrode-gasket sample for Cell-extracellular matrix (ECM) proteininteractions, signal transduction assays,detection of invasion of endothelial celllayers by metastatic cells, barrier function measurements and cell proliferationapplications[26]

The selection of the electrodes array, in the electrode pattern, is interdigitatedbeneficial to levitating the particles in DEP and making the particles flow overthem without reaching the bottom.

By having an open EWOD design, with interdigitated electrodes, rejecting bac-teria can be possible.Fig 2.9

From here having a DC square signal, changing from positive to negative in oneperiod, allow bacteria to stand still in a Electrophoresis manner, while the electricfield is constant at all time.

22


Figure 2.8: Classification of DEP devices according to the configuration of mi-croelectrodes: (A) parallel or interdigitated, (B) castellated, (C) oblique, (D)curved, (E) quadrupole, (F) microwell, (G) matrix, (H) extruded, (I, J) top-bottompatterned, (K) side-wall patterned, (L) insulator-based or electrodeless, and (M)contactless,[28]

(a) EW electric fields affecting bacteria

(b) Electric Modelling of the setup

Figure 2.9: EW electric fields and equivalent Electric circuit

23

Chapter 3

Design and assembly of theexperimental setup

This part describes the wafer fabrication which consist of the assembly of theelectrodes array onto a glass surface using standard MEMS technology processes.

Several aspects of the design had to be considered in order to adapt the ex-perimental device to the measurement setup available; transparent substrate andthin device layer for observation of the bacteria through an inverted fluorescentmicroscope, oil based lenses, culture time, and Teflon layer deposition.

The design and assembly can be summarized to 3 stages: electrodes design,chip fabrication and gasket moulding. The electrodes design was started on L-Edit R© program, where the interdigitated electrodes arrangement resulted to bethe most suitable for the DEP rejecting bacteria application as it is explainedlater on. The chip fabrication was done in the clean room in KTH Kista facilitiesbecause of the micro scale proportions of the electrodes design; metal sputtering,lithography, etching, dicing and finally Teflon spinning were done to build the finaldesired chip.

Finally, it is shown how gasket design and fabrication were made for containingbacteria on top of the produced electrodes.

3.1 Chip design and assembly

This part describes the chip manufacturing which includes electrodes design, metalsputtering, photo-lithography, etching and dicing needed to obtain the wafer withgold electrodes on top of it. Finally a Teflon layer is spun which makes possible toact as an capacitor for EWOD tests purposes.

3.1.1 Electrode design

Fitting an electrode design on a round wafer shape needed to be taken into accountconsidering having squared linear electrodes spaces. Detailed pictures of the waferdesign are shown in Fig 3.3b.

24


The investigated electrode design of interdigitated electrodes was chosen be-cause it was the most suitable and flexible design for DEP and EWOD applicationsaccording to the Fig 2.8 [28] as it was shown in Section 2.4, because this kind ofconfiguration are often found in Travelling wave DEP and DEP applications, whereelectric fields induce movement on floating particles. But also this configuration isused in pulsed DEP, which has a similarity with the pulsed behaviour of EWODactuated with an on-off signal.

Besides the common EWOD configuration, a wire-free configuration[29] is moreuseful because a tip on top of the droplet is not longer needed, and match up withthe selected DEP configuration mentioned in the paragraph before.In this case a wire-free configuration[29] on top of a glass substrate of goldeninterdigitated electrodes is presented, in contrast to the conventional EWOD con-figurations as it can be seen in Figure.3.1a.

The L-Edit software was used to design the electrodes layout, which have beenplanned to have three different widths (a, b in Table 3.1)keeping the length of eachelectrode constant (l=6.450 mm) as seen in Fig 3.1b. The a and b variables werechosen within the bacteria scale size(<10 µm).

a µm b µm

Case 1 5 10

Case 2 10 20

Case 3 20 50

Table 3.1: Different width of the electrode depending on the case used

(a) EWOD conventional configuration vs wire-free configuration[29]

b a

6.450 mm

(b) Interdigitated electrodes design wherethe width ’a’ and ’b’ are variable and thelength is kept constant.(see table3.1)

Figure 3.1: Electrodes configuration

The electrode length is defined by the gasket holes diameter size, which sizeis the same as the commercial laboratory 96– wells bacteria culture (Micro-titre)

25

3 Design and assembly of the experimental setup

plates. For that reason, the gasket should stand on top of each square regions (asseen on Fig 3.2b) where each square is an array of electrodes as seen in Fig 3.2a.From the 9 electrodes squares, 6 are active and the last 3 on the bottom are goingto be used as negative control (no power supply is applied by not connecting to thepower grid).

(a) Gasket on chip to see the electrodes lengthreason

6.450 mm

(b) Interdigitated electrodes spaced on a quarterof a 10 cm diameter wafer.

Figure 3.2: Electrodes in square spaces

One test area is composed of 9 electrodes squares. For the sake of fabricatingas many tests areas (chips) as possible, 4 chips are inserted in one wafer are as itcan be seen in Fig 3.3b. In addition, each chip has been numbered with case 1, 2or 3 as used in Table 3.1 to differentiate each chip from each other, this is shownin Fig 3.3a.Particularly, case 1 was repeated twice on each wafer to be considered more rele-vant because DEP forces act stronger within smaller scale environments as it wasexplained in Subsection.2.2.2.

26


(a) Metal mask design for wafer lithography

(b) mask designing on diced wafer(what isexpected)

Figure 3.3: Mask and expectable wafer design

3.1.2 Chip fabrication

The following subsection describes the process and details involved in the manu-facturing of the wafer. From the layer structures to the Teflon spinning of the finalfinished wafer. All procedures of this fabrication where done on Kista Campus atKTH facilities.

The procedure is outlined below:

1. Glass wafer: 500 µm of thickness

2. Sputtering Metal: TiW (200 A)under Au (1500 A) layer

3. PhotoResist(PR): Type nLOF2070 is used

4. Development: three minutes of duration

5. Etching: First layer (Au)

6. Etching: Second layer (TiW)

7. Striping: Last layer (PR)

Process

The different deposited layers are shown in Fig 3.4. In this part is shown the metaldeposition in a structural order (layers) for a better understanding of the finaldisplay.

27


500 um

20 nm

150 nm

10 um

Figure 3.4: The material layers of the final wafer

Metal Deposition

The metal deposition was made in a metal sputtering machine (KDF R© 844NT,Model 844GT), using DC sputtering system. In order to improve the 150 nm goldlayer adhesion on the glass wafer, 20 nm of titanium tungsten alloy (TiW) wassputtered.

Structuring of gold

Lithography In this part is detailed the photo-lithography method, where usedphotoresist and development times are included. Different development times underUltraviolet light (UV) exposure were tried in order to get the best result.

To define an etch mask for the gold, a photoresist layer (AZnLOF2035 RER600 [2:1]) is spun on the wafer, , in a OPTI R©spin machine model SST20(SSE) at3000 RPM for 30 s with a final soft bake at 90◦ C for 60 s.

Next, the UV exposure dose was optimized through some tests to reach theideal exposure parameters (Table 3.2) on a Karl Suss R©, MA6/BA6KSM modelmachine (Fig 3.3a).

Lamp intensity Lamp power Wavelength

Mode CP

with 10 s of Lo Vac Contact 18,3 mWcm2 350 W 450 nm

and Alignment Gap of 40 µm

Table 3.2: Photoresist curing time parameters

28


Lastly, to finish the lithography process the pattern was developed. After bak-ing on a hot plate to cross-link the photoresist, the wafer is developed for 4:05 min toremove the unexposed areas of photoresist film. Subsequently 1 minof post expo-sure bake is performed. This process is automatically executed by MAXIMUS R©ma-chine.

The photo-lithographic process is shown in Fig 3.5

(a) Wafer with sputtered metals (b) Wafer preparation for lithography

(c) Wafer exposure to UV

(d) Development for removing unexposedPR

(e) Etching of metal (f) Wafer finished after PR remove

Figure 3.5: Process of Photo-lithography

Etching Etching consists in the removal of unwanted metal layers from the glasssurface, such as sputtered metal that does not belong to the electrode pattern.

In this part, each metal etching is detailed with their respective recipe. Havingmany different electrodes widths made this a complex task resulting in some overetching or poor etch finishing.

As the layer of gold is the first one from the top, it is a good idea to start withit.

Au ETCH FORMULA:

• 60 g of I2

• 240 g KI

• 2400 g H2O

In proportion (1:4:40): etch rate 5.8 nm/s.

29


Because our electrodes have different width, different etching times were usedwith the aim of etching more metal without affecting the electrode arranged width.

Au ETCHING METHOD:For every glass wafer, the etch was performed for 30 s at 10 RPM. Additionally,

extra time was tried according to the following Table 3.3 in manual mode(static).

#1 #2 & #3

1st wafer – –

2nd wafer 5 s –

3rd wafer 5 s 3 s

Table 3.3: Different used etching times on lithography

In the same way the TiW layer is etched.

TiW ETCH FORMULA:

• 31% 50 H2O2 @50◦ C: etch rate 1.25 nm/s.

TiW ETCHING METHOD:The etching liquid is poured manually onto the glass wafer during 20 s. This

process is refined by feedback from observation under the laboratory microscope.In this way, time can be measured and see how far the etching can go without

over etching the electrodes. Both methods were used in the actual devices to etch

different material layers, in Au etch method the best time was 5 seconds, and forthe TiW etch method was 20 sand then steps of 5 to 8 s.

Dicing In this part it is explained how the glass wafers are diced and the param-eters used for the saw machine setup DISCOTM DAD 320.

The dicing of the wafer is more delicate in glass wafers. A different speed fromthe silicon wafer dicing should be used to avoid break as on Table 3.4).

Spindle speed Feed Speed

Glass 14’000 RPM 1

Silicon oxide 30’000 RPM 5-10

Table 3.4: Spindle and Feed speeds for the different materials

The parameters used in this process are shown in the tables Table 3.5 andTable 3.6.

Afterwards, metal mask measurements can be compared with final fabricatedchips, as seen in Fig 3.6.

30


P1A851 Work thickness (wafer thickness) Blade height Tape thickness (blue tape) Rnd(round work size) z-axis down speed

Machine Parameters 0,8 mm 0,0650 mm 0,05 mm 110 mm 10 mm/s

Table 3.5: Glass Blade parameters

ZH05-SD2000-N1-70 EE Work thickness (wafer thickness) Blade height Tape thickness (blue tape) Rnd(round work size) z-axis down speed

Machine Parameters 0,55 mm 0,1 mm 0,08 mm 120 mm 10 mm/s

Table 3.6: Silicon Oxide Blade parameters

(a) Electrode bus connection

(b) Contact pads

(c) Electrode # 1 (narrowest)(d) Electrode # 2

(e) Electrode # 3 (widest)

Figure 3.6: Metal Mask Measurements

Teflon layer In this part is described how Teflon layer is formed, and the thick-ness of it. Teflon is needed as an insulating technique for EWOD application, where

31


behaving as an insulator makes possible to create potential difference between thesubstrate and the used medium. Additionally, it makes easy to reuse the surfaceafter bacteria attachment. In an ideal case, Teflon was thought to be covering onlythe top of the electrodes because the gasket could adhere easier to the spaces wherethe square electrodes were not located.

Teflon (C2F4)n is formed with the mix of 0.6 % Teflon R©AF-1600 (DuPontTM)and FC–40 in a dilution ratio of 1/7.

Depending on the thickness of the layer, the spinning speed and accelerationare modified. The spin coater used was SPIN 150– NPP. In order to have 480 nm ofthickness in our samples, a 1200 RPM speed is selected within 60 s at 100 RPM/s ofacceleration.

Subsequently, the chip is heated up in a two-steps procedure on a hot plate at165◦C for 10 min, and then 200◦C for 25 min. The hot plate used was IKA R© C– MAGH57. Teflon fumes may be dangerous and care must be taken to avoid inhalation.Thereafter, the samples were left to cool down for 20 min.

With the intention of hang Teflon only on the electrode’s area, a high tempera-ture patterned adhesive film was carefully stuck to the chip. But this only workedon glass substrate rather than in the silicon glass substrates as is illustrated inFig 3.7

32


Cut pattern in �lm1 Cutted by hand and plotter2

Fit the sticker �lm to the wafer3

Heat it up for Te�on curing5

Press all over to reduce bubbles4

Result in the wafer6

Figure 3.7: Teflon technique to insulate on only electrodes

Finally, after the Teflon-coated chip is ready, macro connectors are soldered foreasier connection with the function generator as seen on Fig 3.8a

33


(a) Diced finished wafer and soldering macro electrodes

(b) Final built test device

Figure 3.8: Finished chip

Comments Some problems arose during the wafer fabrication process from de-sign to the etching part that are worth noticing.

The electrode array design was supposed to mimic the 96– wells plate thatare used in biological laboratories (Fig 3.11a as it is going to be explained inSection3.2). In this case, we used only nine wells design because those were enoughfor our anti-fouling experiments.

During the etching, the major challenge was adapt the etching times definedfor the smallest electrodes and the bigger ones.

For example, it was needed more time for the smallest ones (Case1) so over-etching were seen (red shrinking arrows in Fig 3.9); while for the bigger electrodes(Case 2 and 3), the required time was less and it resulted in under-etching of thesmallest electrodes (green increasing arrows in Fig 3.9).

About the finished wafers, it has been found that due to the different size gapsin the electrodes, the etching times for each electrodes case were different. Thisbecame a major problem since it was very common to see traces of etched goldparticles in the smallest design, which short-circuited the electrodes.

34


Figure 3.9: Etching time problem between Case 1 electrodes and Case 2 electrodes

(a) Broken electrode bus(b) Photoresist residue

(c) Photoresist and non-etched spots(d) Irregularpatterns overelectrodes

Figure 3.10: Electrode bad etching finish

35


This problem was solved opening the short-circuited electrodes with a sharpdiamond needle (same used for cutting the glass slides for the gasket moulding.

Next, the main concern in Teflon spinning was to achieve a thicker layer enoughto act as a capacitor but thin enough to allow electric field to act on particles byEWOD. Several iterations of spinning time and curing temperature were done toachieve the final height (∼500 nm).

Some of the key problems were the way Teflon was spun on the spinning ma-chine. If there were any dust particle or bulk on top of the surface, it got affectedmaking some sections covered by Teflon in a thinner level.

It has been found that using 2.25% of dielectric layer (Teflon) is to thin thatbreakdown becomes easy to happen. In fact, pin holes in the Teflon layer facilitateelectrolysis to occur.

Finally the wafer is covered with Teflon and soldered to copper electrodes as itcan be seen on Fig 3.8b

3.2 Gasket fabrication

In this section a gasket design is developed in order to contain the bacteria mediumon top of our chip (manufactured diced wafer glass). Two materials are analysedto be used as a gasket for the bacterial medium container.

Gaskets were done to mimic the well (Micro-titre) plates that are used by thebacterial tests.

The chip was made based on the size of the wells from the Cell Culture Plate(96 Well) Sterilin R©, which well diameter φ is constant (φ= 6,45 mm), as it can beseen in Fig 3.11a.

(a) Commercial CellCulture Plate (96 Well)Sterilin R©

(b) Design of the gasket to be done

Figure 3.11: Commercial gasket and design

Two versions were fabricated, the first one made of a new material calledOSTE[30] with very flexible properties, and the other version made of PDMS for

36


the sake of practicality which in fact had good adhesion and made-up with one bigsingle well.

The main design limitations presented were the thickness (calculated for themedium quantity), size (covering all the electrodes, giving space for the macroelecrodes connections and enough for improve adhesion to prevent leaking), easydetachment(for fast measure of bacteria after incubation)and evaporation of themedium during the time without affecting the observation procedures.

3.2.1 OSTE Gasket

In this part, off-stoichiometry thiolene (OSTE) was selected mainly for the tuningabilities of the material and its versatility for complex moulding.

The first gasket was a squared shape with nine wells in it, where each wellcorrespond to each square electrode in the chip. Because adhesion of the OSTEwas not uniform all over glass surfaces and some wells leaked due to the usedthickness, a second gasket was made with a single well for better tuning of theproperties. In this case, two thicknesses were tested.

With the intention of calculate the exact amount of UV-curing time, gasketswere made by reducing the layer thickness and also making only one hole for prac-tical measurements: 5 mm, 2 mm and 1 mm respectively(Fig 3.12). The sealingtests were shown in Subsection 3.2.3.

Figure 3.12: OSTE nine hole and one hole gaskets designs

Formulation

In this section the design is sought to fit into the well plate structure but is modifiedfor better clamping and sealing.

The only elements used for the formulation were two types of monomers, a thiolfunctional group and an allyl functional group. Combining the two components,an off-stoichiometric thiol-ene network polymer is fabricated.

The OSTE formulation is based on 100 % thiol excess as can be seen in Table 3.7.

Mixing procedure

Once the right amount of monomers and initiator are selected, the pre-polymeris mixed thoroughly. In this case a vortex was used to have more homogeneousresults. One of the major problems of using this, is the presence of bubbles at the

37


Off stoichiometry Thiol Allyl TPO-L(initiator)

50%PETMA 1.125x TATATO 1x 0.5 wt%PETMP 1.125x TATATO 1x 0.5 wt%

80%PETMA 1.35x TATATO 1x 0.5 wt%PETMP 1.35x TATATO 1x 0.5 wt%

Table 3.7: OSTE Formulation: Stoichiometry, monomers and curing agent

end of the mixing, which can be solved by introducing the sample to vacuum forat least 30 min at a pressure of approximate -72 kPa.

Preparation – moulds

In this part aluminium and glass moulds were used for the gasket shaping as il-lustrated in Fig 3.13 and Fig 3.14 taking in consideration the size of the wells.

The aluminium mold consisted in two squared frames, one base and a glass forlithography. For assembling it only was required to screw the parts together.

In case single hole wells, aluminium mould were not needed as a result of nothaving big samples and trying only a little piece of polymer. Thus suitable glassslides were used for cleanliness and practicality.

In order to made a single hole, a very simple and quick mould should be used.By this, some glass slides were cut with a diamond burr type Flamme, from theHobby Drill 2000 brand,as it can be seen in section 1 of Fig 3.14.

Once many 2 cm x 2 cm are cut, are glued with double side Scotch 3M R© tapeto a single glass slide which was previously covered with a Xerox R© film.(to detacheasily after curing).

Soon after, pre-polymer is poured on the glass mould and covered with the onehole film mask stuck to a glass slide. To prevent any leaking 2 clamps are fixed onthe sides, and then inserted on the UV light chamber for the time is required.

38


Clean mold and equip �lm mask1 Assemble the mold, �lms, mask2

Adjust the screws in it3

UV curing5

Input prepolymer in mold4

Cured Polymer - Gasket6

Figure 3.13: OSTE gasket on aluminium mould

39


Cut and glue the glass slides1 Glass slide with �lm covers2

Prepare the cover slip mask3

Clamping and UV curing5

Input prepolymer and top mask4

Cured Polymer - One Hole6

Figure 3.14: OSTE one hole gasket on glass slide mould

Lithography

In order to cure the pre-polymer, UV curing using an OAI R© UV curing lamp,is used to trigger the allyl reaction while the TPO-L for the respective thiol-ene

40


was used. Obviously, single hole wells used much less quantity of pre-polymer,consequently UV curing time decreased significantly (less than 10 seconds).In bigger gasket case, undoubtedly a nine patterned film mask was used as it canbe seen in the bottom corner of the first sequence of the Fig 3.13.Table 3.8 and Table 3.9 shows the different times used and the polymer finish

characteristics.

One hole gasket test of 2 mm

Thiol monomer Off stoichiometry Time (s) Results

PETMA

50 %

26 too much curing time X20 still over-cured, no hole X15 half well X10 defined well

√

8 more sticky, neat√

6 very clear, uncured, affected by cover film X

80 %

27 too much curing time X26 still overcooked X15 almost defined hole X10 hole, and more clear

√

8 uncured, bottom too raw & soft, clearest X

PETMP

50 %

27 too much curing time X20 over-cured, no hole X15 over-cured X10 still no hole, bottom uncured X8 same as 10 & no adherence X5 half well, blurred X3 almost defined hole, blurred, detachable

√

2 almost defined hole, clearer, softer√

1.5 uncured, very stiff, detachable X

80 %

27 too much curing time X5 almost defined hole, bottom raw, not clear X3 defined hole but stretched, clearer X

(very well defined wells) 2 defined hole & softer, breaks easily√

softer than 50 % 1.5 defined hole, easier to detach√

Table 3.8: Curing time variations for best OSTE 2 mm sample

Development

Finally, the development of the gaskets was possible by the use of solvents such asacetone or butyl acetate. Importantly, de-moulding from the aluminium and glassmoulds were made by detaching the polymer from the Xerox R© films that lied ontop and on bottom of the moulds and helped by the solvents actuation.

3.2.2 PDMS Gasket

In this section a rubbery gasket(PDMS) was selected for rapid manufacturing andfor its stickiness to Teflon.

41


One hole gasket test of 1 mm

Thiol monomer Off stoichiometry Time (s) Results

PETMA50 %

15 too much curing time X10 over-cured X6 defined hole

√

4 defined hole√

80 %8 almost defined hole X6 a bit uncured X

PETMP

50 %

6 too much curing time, no hole X4 over-cured, no hole X2 defined hole

√

1 defined hole, bit uncured√

80 %

4 almost defined hole X2 almost defined hole X1 top face stuck to cover film X

0.5 too uncured, melted√

Table 3.9: Curing time variations for best OSTE 1 mm sample, curing agent forPETMP 80% was 0.1 g for the others 0.05 g

PDMS known as well as Poly(dimethylsiloxane), is an organic polymer widelyused in microfabrication. It was chosen due to its simple formulation; of course thecost of it was to make a simpler moulding than the desired one, but for practicalinstances.Contrary to OSTE, PDMS is not cured using UV, and the patterning must bedefined in the mould. The manufacturing is even more simple because the lack ofspecific curing times.

Formulation

In this part is described the parameters used for the manufacturing of the PDMSgasket. The formulation used is shown in the Table 3.10.

base curing agent

10x 1x %

Table 3.10: PDMS Formulation: base and curing agent ratio

Mixing procedure

Elements mentioned in Table 3.10 are mixed. Both elements are stirred for at least10 min until is fully mixed.Later on, the mix is inserted on a vacuum chamber for 30 min. It must be degassedto get rid of the bubbles.

42


Cast of PDMS films

In this part the glass moulds are specified as a part of the curing step. Afterthe mixing procedure the uncured PDMS is poured very carefully (to avoid anyair bubble) into a glass mould which consist in a glass surface delimited with glassslides depending on the desired height. Carefully a Xerox R© film is extended all overthe liquid PDMS. The prepared filled mould is then introduced in a 75◦C heatedoven for 3 h. Once the PDMS is cured, the well could be cut out using a razorblade to give the adequate shape. Different samples can be seen in Fig 3.15

(a) Chip with gasket

(b) PDMS gasket in use with bacteria

(c) PDMS gasketswithin differentheights

Figure 3.15: Different made PDMS gaskets

3.2.3 Leakage tests

In this part, the properties of the gaskets are discussed how gaskets were resultedand the problems or successful outcomes from the manufacturing process.

This parameter can be reflected on the gasket pictures in Fig 3.16It has been found that OSTE gasket worked fine, but for not enough time for

bacteria incubation as it can be seen in Fig 3.17

43


(a) OSTE gaskets with different sealingtests

(b) OSTE one hole gaskets within dif-ferent curing times

(c) 2 cm PDMS gasket with sealingproperty on Teflon layer

(d) 1 mm PDMS gasket with hydropho-bic property without leaking

Figure 3.16: OSTE and PDMS gaskets on sealing tests

Figure 3.17: OSTE gasket adhesion and leak

44

3.3 Bacteria to be used

Comments In this stage, two gasket materials were tested. In the nine-holeOSTE gasket, the lithography problem was due to very thick layers, the baseremained uncured creating a non-uniform surface due to the unknown exact UVcuring time, while the top face was satisfactory. Even though, sealing tests weremade on nine-holed gaskets which the best sealing was with PETMP 80% but notlast for more than 1 h as it is shown in Fig 3.17

In the single hole OSTE gasket lithography parameters considered mainly: holecircular shape, polymer transparency and sealing, where the best ones were:

2 mm sealing test: PETMA 50% 10 s, and PETMP 80% 1.5 s.

1 mm sealing test: PETMP 50% 1 s (PETMA had not good hole shapes).

There was not enough time to return to the nine-hole test because bacterialtests came out and a gasket was required for it. Nevertheless nine-hole tests shownnot having the right curing time because on sealing tests liquid leaked from underthe gasket.This is displayed in Fig 3.16a

In the PDMS gasket, sealing was quite good, and even with different gasketheights (5 mm, 1 mm) it was capable to retain the bacteria medium on top of themanufactured wafer, taking in consideration that the wafer’s surface was a thinlayer of Teflon. This because of its rubber-texture of the PDMS material that stickeasily to the surface, but with a little help of pressure to keep air bubbles out ofthe bottom surface of the gasket. It is important to mention that for very thingasket films overflow is difficult to occur due to the hydrophobicity of the surfaceas is shown in Fig 3.16d.

3.3 Bacteria to be used

In this part, a brief description of the bacteria used and its morphology and topol-ogy.Regarding the bacteria maturity, they cause infections by sporadic or epidemicmediums[5], these can be divided in several typologies where common ones areGram-negative bacteria and gram-positive bacteria. Generally, the more resistantbacteria against antibacterial agents are the gram-negative because of their severalcellular layers.

3.3.1 Salmonella Typhimurium

Salmonella species are reported as an often bacteria acquired in the community, asin Table 5 of the Infection Control Guide in Hospital Personnel[31].

The main bacterium used was Salmonella Typhimurium because of their well-known adhesion mechanism. Another bacterium used on Teflon-glass slides, Pseu-domonas aeruginosa also used to compare with Salmonella, its adhesion force onthe same surface.

45


(a) Salmonella close up

(b) Salmonella en-tanglements

(c) Biofilm forma-tion

Figure 3.18: Salmonella typhimurium

Comments Some of the problems related to the bacteria was the long periodof time required to see the attachment(at least 60 min), but was not too muchconsidering that in common DEP/EWOD experiments lasted from 3 to 7 days[32].

46

Chapter 4

Measurements and results

This section consists of three parts. The first is a numerical simulation of the DEPforce experienced on particles under the influence of spaced planar electrodes. Dif-ferent models for the bacteria are compared and optimal frequencies are determinedfor repulsion from the surface. Pro and cons are discussed for DEP techniques. Thesecond part is an experimental study of EWOD to influence the bacteria adhesionof Salmonella Typhimurium. This study is carried out in cooperation with thegroup of Prof. Ute Romling from the Karolinska Institute. The third part is anexperimental setup and several experiments for evaluating the bacteria attachment.

4.1 Numerical simulations of DEP on bacteria model

In this part, Clausius-Mossotti(CM) factor plots parameters are calculated for thelater experimental bacteria. Similarly, simulations are done on how the gradientof electric field from two gold electrodes in a liquid solution (water) affects themodelled bacteria.

DEP simulations were studied using COMSOL simulation and complementedby MATLAB for calculations of effective CM values. The system was modelledas: two co-planar gold electrodes under a layer of Teflon with a water medium.MATLAB curves, illustrating the difference of the CM factor by changing theparticles’ medium can be seen in Fig 4.2 (different medium conductivities displayed)as explained in the following subsection.

4.1.1 MATLAB calculations

Bacteria and medium complex permittivity is calculated considering the biologicalapproximation model. Finally, the CM factor plot is derived. From Section 2.3.1,data shown in Table 4.1 is obtained.

47

4 Measurements and results

Three layer particle information

Particle Layers (outer to inner) Radius [µm] σ [ µS/cm] ε [F/m]

Yeast1 (cell wall) 2.5 140 60

2 (cytoplasm) 2.36 0.0025 63 (nucleus) 2.35 2000 50

E. Coli1 (cell wall) 1 500 60

2 (cytoplasm) 0.98 0.0005 103 (nucleus) 0.975 1000 60

Table 4.1: Different parameters to consider on three-layer particle(E. colidensity:ρ = 1100 kg/m3 ± 3% )

By the smeared-out sphere approximation considered in Fig 2.5b formulas inEq 4.1 and Eq 4.2 are solved for the bacteria analysis by MATLAB programming.

ε∗BACT = ε∗3

(r3r2

)3+ 2

(ε∗21−ε∗3ε∗21+2ε∗3

)(r3r2

)3−(

ε∗21−ε∗3ε∗21+2ε∗3

) (4.1)

ε∗ = ε+ σ

ω(4.2)

From above, εx is the permittivity of the layer x, thus x can be one layer(the thirdone) or a combination of layers(the first two ones); then σ stands for conductivityof the medium and ω is the frequency domain of the applied ~E. Last but not least<{f} is the real part of Clausius-Mossotti(CM) factor {f}.

It can be compared the results of the calculated values plotted on excel inFig 4.1b and the plot from Matlab in Fig 4.1c to see how the few values obtainedin Excel was a good approximation but not as detailed as the ones obtained byMatlab complex calculations.

From the MATLAB calculations, using five different medium conductivities (2,10, 17.78, 31.62, 38, 50.62, 100 and 380 µS/cm) where DI water has 2 µS/cm, it isshown how the CM-frequency curves were affected by the medium and how Yeastand E. Coli bacteria change its pDEP and nDEP areas (explained in subsection2.2.2) as a consequence of it.

From Fig 4.2 can be seen how yeast and E.Coli bacteria particles can change theCM values as frequency goes up, so the range where nDEP can be used change forlarger or shorter ranges in the frequency axis depending on the submerged medium.This change of CM values is due to how frequency is directly proportional to theimaginary part of the complex permittivity as can be seen in Eq.4.2.

48


(a) <{CM} value from ap-plied frequency

(b) Excel CM plot from frequency values for general idea

(c) Matlab CM plot vs. frequency for detailed data

Figure 4.1: CM plots from Excel and Matlab calculations with applied frequencies

49


102

103

104

105

106

107

108

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

frequency (Hz)

Re[K

(w)]

Plot of Clausius-Mossoti factor(CM), sm=3.162278e-03

YEAST

E. COLI

102

103

104

105

106

107

108

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

frequency (Hz)

Re[K

(w)]


YEAST

E. COLI

102

103

104

105

106

107

108

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

frequency (Hz)

Re[K

(w)]


YEAST

E. COLI

102

103

104

105

106

107

108

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

frequency (Hz)

Re[K

(w)]


YEAST

E. COLI

102

103

104

105

106

107

108

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

frequency (Hz)

Re[K

(w)]


YEAST

E. COLI

Figure 4.2: CM plots of particles on different conductivity mediums (sm) based ondiverse frequencies

50


These values were selected from different mediums used in different scientificpublications about DEP. From the variety of ranges obtained, yeast and bacteriacan be observed how they are affected by the medium conductivity.

It can be seen from Fig 4.2 that the higher the conductivity of the medium,the smaller the area under the curve that is in the real part of the CM factor.Regarding the negative area under the curve, this means that if you want morerepulsive forces it is convenient to have higher conductive values of the mediumand also shows lower frequency values to work with.

In addition to the selected mediums, additional common mediums related tothe healthcare environment were investigated.

Blood conductivity the conductivity of the blood varies depending on differentblood composites: Hematocrit that reflects the red blood cells/blood volumeratio (the higher of it, the lower the conductivity); plasma is the major com-ponent which contains suspended ions (the higher of it, the higher the con-ductivity); electrolytes change the blood resistivity (the higher of them, thehigher the conductivity); erythrocyte helps the oxygenation of the blood bydiffusion, which orientation define the conductivity (at higher flow rates, thehigher of it)[33]. The average value, by [34] experiments, is 6666.67 µS/cm.

Urine conductivity the conductivity of the Urine is normally around 280 µS/cm[35]

Saliva conductivity its permittivity for low frequency is almost constant (<3GHz):the real and imaginary part are 6.8 and 1 respectively on average[36]. Totranslate to conductivity is: 6280 µS/cm, which is very similar to the blood.

This values are used to see which ranges of frequency and conductivity arethe particles used to be in healthcare environments. As it is shown in Fig 4.3, itseems low frequency is needed and also it is convenient for further uses in EWODapplications.

51


(a) Medium DI water and NaCl solu-tion (b) Ethyl alcohol and human blood

(c) Skin and Urine (d) Medium saliva and gastric juices

Figure 4.3: Different CM values for biological mediums for yeast and E.coli particleswithin various frequency values

52


4.1.2 COMSOL analysis and results

In this section, a complete simulation about how electric field acts on two coplanarwave-guide geometry electrodes is described. Here DEP force is the main activeforce and the direct relation with the frequency is portrayed.

From the COMSOL simulations, there were two measurements of electric po-tential intensity, electric field intensity and DEP force on the medium, as presentedon Fig 4.4, Fig 4.5 and Fig 4.6 respectively.

From the first figure, the electric intensity was tried to be varied with differentfrequency values. This shown electric potential lines increased proportional withthe frequency, and become narrower in the electrodes surroundings. As it can beseen in Fig 4.4a, as long as frequency is low an envelope function is formed abovethe electrodes. With this characteristic, more effective electric potential intensitycan be produce to cover the most of the upper area of the electrodes.

53


(a) Frequency at 1 kHz

(b) Frequency at 1 MHz

Figure 4.4: Electric Potential intensity in 8 µm electrodes width at defined fre-quencies in Cartesian plane

54


From the Fig 4.5, the electric field intensity was shown to see how it changedwith different potential values. This only shown the variation of the amplitude butnot significant change in the intensity was observed. This was useful to know inorder to use lower potential with the same effect of electric field intensity.

55


(a) Vo=1 V (b) Vo=2 V (c) Vo=5 V

(d) Vo=10 V (e) Vo=20 V

Figure 4.5: Electric Field intensity at different potentials in Cartesian plane

56


From the last figure, the purpose was to see if there was any change of theDEP forces while the particle and potential were changed over a range. Therewere no significant changes, but in all pictures the main reject force was theone at 1 kHz and from the attraction forces, three were the main ones which:F(3.33 MHz)<F(6.67 kHz)<F(10 MHz). This was important to set a range offrequencies to work with.

57


(a) Yeast at 1 V (b) E. Coli at 1 V (c) E. Coli at 2 V

(d) E. Coli at 5 V (e) E. Coli at 10 V (f) E. Coli at 20 V

Figure 4.6: DEP Force at different particles and potentials in Cartesian plane

58


Bacteria rejection possibility Based on the presented electrical field simula-tions, it can be calculated if bacteria rejection is viable.

The bacteria simulated in the COMSOL simulation was Yeast at a frequencyof 100 Hz,in order to start with the least visible value from the Fig 4.1c. The realpart of Clausius-Mossotti factor used for this particle was -0.4687 considering thenegative sign as the zone of the attraction for DEP as it can be seen in the samefigure.

The CM factor was calculated using MATLAB, where medium was DI wa-ter (conductivity from COMSOL 38 mS/m) and the yeast was considered as a3-layer permittivity particle, which resulted in complex permittivities 1.1746 ×10−8 − i2.5778× 10−6 and 7.0834× 10−10 − i6.0479× 10−5 (F/m) for yeast andwater respectively.

The possibility of bacteria rejection can be measured by the exerted DEP forcefrom the electrodes, which should be larger enough to stop (and maybe reverse)the speed of the bacteria due to the usual Brownian motion. The start point todo this, is the Einstein– Smoluchowski relation from the Kinetic theory [37] whichdescribes the Brownian motion on a medium.

The general equation is the Eq.4.3.

D = µ κB T (4.3)

where D is the diffusion constant, µ is the mobility, κB is the Boltzmann’sconstant and T is the absolute temperature.

It can be found diffusion bacteria values like: Pseudomonas aeruginosa (2.1 ×10−9 m2/s), Klebsiella pneumoniae (0.9×10−9 m2/s)[38] and Escherichia coli (0.8×10−12 m2/s)[39]. Considering room temperature (T = 300 K)and the Boltzmann’sconstant (κB = 1.3807 × 10−23 m2. kg/s2. K) and replacing the diffusion valueson Eq.4.3 mobilities are obtained: Pseudomonas aeruginosa (5.1 × 1011 m/Ns),Klebsiella pneumoniae (2.2× 1011 m/Ns) and Escherichia coli(1.9× 108 m/Ns).

From the general equation is derived also mobility as particle’s drift velocity(vd)to an applied force (F )[40], where it is demonstrated from Eq.C.4.

µ =vdF

(4.4)

where the acting force is evaluated through the gradient of the squared electricfield on the vertical axis(∇| ~Ey|2), due to see the effective force that is going to acton the bacteria.

Velocity is going to be calculated by the multiplication of mobility and forcededuced from Eq.4.4.

As mobility values are already obtained, it remains only FDEP values to becalculated. The force F y

DEP is obtained from ∇| ~Ey|2 values obtained from thesimulation.

The F yDEP is used because from COMSOL simulations, it can be seen that

in Fig 4.7a ∇| ~Ey|2 keeps the same shape while its values increase as the potential

59


voltage is increased. The highest values of ∇| ~Ey|2 are on top of the electrodes goinginto the diagonal direction while the lowest are very near the electrodes’ corneredges. This electric field intensity analysis can complement also the distance effectseen in Fig 2.4.

In the simulation is illustrated three different distances (as seen in Fig C.1) tosee how FDEP changes with the variation of height. From the three distances, 1and 3 µm were no enough force to cover as much vertical area as it happens withthe last distance. For that reason, from the resulted mesh it can be considered7 µm of distance (height) from the electrodes as an acting DEP force effect forrejecting purposes.

Next, in Fig 4.7b shows the DEP forces at 7 µm of distance along the x axis.This is at 1 V because if potential was increased, the change was only in theamplitude but not the shape of the FDEP force.

60


(a) DEP force in Cartesian plane, with selected distance as space of rejection, 7 µm

(b) DEP forces in distance x-axis (abscissa) at 7 µm above the electrodes

(c) DEP force (blue dotted line)and drift velocity (yellow Pseudomona, ma-genta Klebsiella and green Escherichia) in distance x-axis

Figure 4.7: COMSOL DEP simulation - Electric field analysis

61


Using Eq.2.4 and COMSOL ∇| ~E|2 values, DEP force is calculated in Table 4.2considering radius of the particle (bacteria) r = 0.5 µm, vacuum permittivityεo = 8.854 × 10−12, water medium εm = 80.1, and<{CM} = −0.4687 extractedfrom MATLAB calculations.

Replacing values from Table 4.2 into Eq 4.4, and using the mobilities of eachbacteria, the Drift velocity of the particle (bacteria) is illustrated in Table 4.3

It can be seen in Fig 4.8 that the Drift velocity increases exponentially withthe applied voltage.

0.00E+00

2.00E-02

4.00E-02

6.00E-02

8.00E-02

1.00E-01

1.20E-01

1.40E-01

1.60E-01

0 5 10 15 20 25 30 35

(m/s

)

(Volts)

Bacteria average Drift speed

Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli

Figure 4.8: Maximum bacteria drift rejection velocity based on applied voltage

Comparing the velocity obtained with the Diffusion length, which defines howfar the concentration is dispersed in a medium during a period of time. It is definedby Eq.4.5[41].

L = 2√D t (4.5)

Replacing the Diffusion values of the bacteria can be seen on Table 4.4 thecommon length that moves during one second.

In comparison to the values obtained in Table 4.3, the numerical simulationsprovides more speed due to the DEP force compared with the calculated by controlnegative movement(without any applied force), which means that bacteria rejectionis seemed to be plausible as bacteria can be moved in the rejection areas with theleast applied voltage.

62


Potential DEP force

(V) @max. height(N) max.found value(N)

1 -5.97x10−16 3.2093x10−16

2 -2.39x10−15 1.2837x10−15

5 -1.49x10−14 8.0233x10−15

10 -5.97x10−14 3.2093x10−14

20 -2.39x10−13 1.2837x10−13

30 -5.37x10−13 2.8884x10−13

Table 4.2: DEP force calculated from COMSOL simulations

Potential Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli

(V) min.(m/s) avg.(m/s) max.(m/s) min.(m/s) avg.(m/s) max.(m/s) min.(m/s) avg.(m/s) max.(m/s)

1 -6.75x10−4 -2.49x10−4 1.63x10−4 -2.89x10−4 -1.07x10−4 6.97x10−5 -2.57x10−7 -9.47x10−8 6.20x10−8

2 -2.7x10−3 -9.94x10−4 6.51x10−4 -1.16x10−3 -4.26x10−4 2.79x10−4 -1.03x10−6 -3.79x10−7 2.48x10−7

5 -1.69x10−2 -6.21x10−3 4.07x10−3 -7.23x10−3 -2.66x10−3 1.74x10−3 -6.43x10−6 -2.37x10−6 1.55x10−6

10 -6.75x10−2 -2.49x10−2 1.63x10−2 -2.89x10−2 -1.07x10−2 6.97x10−3 -2.57x10−5 -9.47x10−6 6.20x10−6

20 -2.7x10−1 -9.94x10−2 6.51x10−2 -1.16x10−1 -4.26x10−2 2.79x10−2 -1.03x10−4 -3.79x10−5 2.48x10−5

30 -6.08x10−1 -2.24x10−1 1.46x10−1 -2.60x10−1 -9.59x10−2 6.28x10−2 -2.31x10−4 -8.52x10−5 5.58x10−5

Table 4.3: Drift velocity for different bacteria

Diffusion Length (m) Bacteria

9.17x10−5 Pseudomonas aeruginosa

6.00x10−5 Klebsiella pneumoniae

1.79x10−6 Escherichia coli

Table 4.4: Diffusion length of bacteria during one second

63


4.2 Experiments of EWOD surfaces on live bacteria

In this section EW tests are described. First contact angle measurements in asingle plate with a single tip are used to calculate which voltage is the ideal fora more strong EW effect. Furthermore, different waveforms voltages were used toseeing the difference in that as well. Because sending different waveforms shapesmay change the efficiency of the EWOD effect on the medium, as it can be seenin subsection 4.2.2. Finally, Teflon adhesion and breakdown prevention tests areperformed.

4.2.1 Contact angle tests

In this section several measurements are displayed: Contact angle vs. time toobserve evaporation effect and Contact angle vs. Voltage within different mediumsto see the effect of volts and its difference with theoretical curve.

In order to learn and predict the EWOD behaviour of the particles in generalmediums, potential applications were done to find the best voltage to use. Contactangle measurements were made on a simple setup: from the bottom a conductivemedium (a sheet of double side 12 µm copper in a 8 mm printed circuit board,PCB) then a dielectric layer (two-side Si and SiO2 wafer covered with Teflon)and a very narrow surface with applied potential (metal tip to conduct the appliedvoltage) as shown in Fig 4.9.

(a) EWOD setup design

(b) EWOD setup

Figure 4.9: EWOD setup for Contact angle measurements

Setup used: Camera of 1.3 M Pixel USB2.0 Moticam 1000; CANON TV zoomlens V6x16 16-100 mm 1: 1.9; C-MOUNT COSMICAR TV lens extension tubeset which connect zoom lens with Camera 40 mm; SELTRON PS 1715 DC power

64


supply, 0-25 V 2 A for a 3 V bulb light; a FINNPIPETTE pipette from 0-40 µL;and a SIGNATONE 393-J Tomkins Ct. probe, Model S-725-CRM, tip model 7A.

65


Medium evaporation tests The calculated droplet evaporation over time showedhow contact angle and volume changed. This helped to the evaporation impact oncontact angle measurements during time without considering the effect of EWOD,as shown in evaporation curve of Fig 4.10. Electrowetting droplet during time havealso been studied on parylene HT films rather than Teflon layer, where stability isproven by more dielectric failure resistance compared to parylene C and durability(up to 6 h) in oil medium[42].

Figure 4.10: Contact angle measurement vs. time within evaporation

66


Contact angle tests by applied potential Using different mediums at differ-ent DC potentials, it was discovered how the experimental curve differed from thetheoretical, and reached a limit in which electrolysis was achieved.Fig 4.11

Figure 4.11: Contact angle vs. voltage within different mediums and theoricalcurve

Theoretical curve by Young-Lippmann in wetting capillarity described by equa-tion Eq 2.6 in Subsection 2.2.3 is shown in Fig 4.11, where contact angle saturationis not contemplated by theory, but it is clearly observed in practice until dielectriclayer breakthrough.

67


4.2.2 Waveform tests

In this section contact angle measurements vs. contact angle change steps withindifferent waveforms are displayed and discussed.

Measurements done by a function generator changing between different wave-forms to see the improvements on EWOD applications not only on the AC voltagechange. The purpose of these tests was to see how the EWOD effect can changeas the administrated potential is applied in different ways. It can be applied insteps as positive or negatives square waves (positive and zero level or negative andzero level potentials), in sinusoidal patterns or just in triangular steps. The testswere made monitoring the droplet contact angle changing during time. From onesteady contact angle measurement to a different one, a step was recorded. Froma total of six steps, if it seen which waveform changed more the contact angle asa translation of the maximum EWOD effect on the droplet. The contact angleschanges were connected to each other with a fit curve in order to have a betterdisplay of the contact angle changing over the steps sequence.

80

85

90

95

100

105

110

115

120

0 1 2 3 4 5 6 7

Co

nta

c A

ngl

e (

°)

Steps were CA changes

CA change with different waveforms

Negative

Positive

AC

ACx2

ACx2 (triangular)

Figure 4.12: Highest effect of waveforms on contact angle (CA) measurements

From Fig 4.12, it is shown that the more extreme contact angle change is madeby positive waves, which squared waves were used keeping the negative chargepotential on top of the droplet. This may increase the effect of the electric fieldfrom the electrodes as the voltage is applied.

4.2.3 Teflon adhesion to glass

It is important to mention that it was found out that Teflon has not good adhesionto glass, even worse at thicker layers.Teflon adhesion on glass slides was tested. It was found that pre-treating the glasssurface with Plasma enhance the spreading properties of the Teflon during thespinning process, although there were no significant difference between applying

68


15 or 30 sof plasma. Plasma is applied to degrade glass surface for better Teflonadhesion, but also it might decreases its adhesion durability as well, as it has beenseen easier breakdown of Teflon compared to the ones without plasma exposition.

It has been tested five pieces of glass slides: two with 2.25 wt% of Teflon, whereboth of them were cleaned with Isopropanol: one was rinsed and the other not.And, the other three were spun with 4.5 wt% of Teflon, where only two were rinsed.All the pieces were sunk under DI water for more than 24 h. The rinsed glass slidesshowed faster detachment of Teflon which almost instantly fell out. The non-rinsedones, with spun Teflon while soaked with Isopropanol, had more attachment, butat the end it fell out too as it is shown in Fig 4.13b.In this case, when the glass is pushed underwater because the hydrophobic surfaceprevents the glass slide to sink as seen in Fig 4.13a, the water surface tension forcepulls out the Teflon layer penetrating from the peeled off edges of the glass slides.

69


(a) Glass hydrophilic-hydrophobic ratio and soaking

(b) SU-8 thickness versus spinning speed fromdata-sheet [43]

Figure 4.13: Teflon peel off from glass surfaces

70


4.2.4 Teflon breakdown voltage tests

Teflon breakdown is one problem to struggle with, while doing EWOD tests. Ithappened during the experiments, but varied depending on different parameters:the surface treatment before the Teflon spinning, the spinning speed, the Teflontemperature and time curing steps. For example, some particle of dust can stopTeflon from spreading in the spinning and therefore creating non-uniform layers ofTeflon. Additionally, it can be found some micro-porosity in the Teflon surfaces,which enhance the breakdown during voltage applying (pin holes).In order to know how to produce a smoother, uniform and pin-hole free Teflonsurface of layer, a SU-8 2000.5 photoresist layer was spun at different speeds andthen tested with applied voltages until it breakdown. The tested surfaces wereshort-circuited failed wafers which were used in standard EWOD configuration,such as the one seen in Fig 3.1a but with a gasket. This can be viewed in Fig 4.14c.From the ten used wafers (four made in silicon and six in glass), there were onlytwo resistant enough to not breakdown and at certain voltage EWOD was seen.The results are shown in Fig 4.14a and the thickness of the SU-8 can be calculatedfrom the photoresist datasheet table also shown in Fig 4.14b.Importantly, one of the cases of EWOD showed a ”conductivity of EWOD” throughthe droplets which were connecting the electrodes together and creating a wire-freeconfiguration as seen previously in Fig 3.1a.

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Figure 4.14: Teflon breakdown tests

72

4.3 Bacteria tests

4.3 Bacteria tests

In this part, in order to continue with the proof of anti-fouling process of thechips, bacteria tests were made on Karolinska institute labs, and detailed with theproblems that were found, such as measurement facilities and convenient setup,and the future ideas for better results. All processes are included.

Bacteria tests on adhesion has been studied in other papers, which one of themused electrode plates hanging in a bacteria medium for several hours[32] to see howmuch potential can affect its adhesion. But none of them focused on anti-foulingmethods or antibacterial surfaces. In this case, electrowetting setup is used [29](based on an open wire-free design) (and DEP is disregarded due to the lack ofinformation about the medium and bacteria conductivity to synchronize with theirCM frequency).

Bacteria tests have been carried out on Karolinska labs, using LEICA DMREmicroscope and HAMAMATSU dual mode cooled CCD camera C4880. This mi-croscope uses lens four lenses:∞/0.17/D, 20x/0.5, 63x/1.32-0.6 (oil based) and100x/1.25, where the last one is the used for observing bacteria.

A Filter #7 is used (FITC=SP-101) for bacteria fluorescence viewing. The pic-ture management program is HiPic-32 version6.4.0 (Hi Performance Image controlsystem). This setup can be seen in Fig 4.15.

(a) Function generator, oscil-loscope and multimeter

(b) Fluorescence microscope(c) Bacteria observation onwafer

Figure 4.15: Setup used for bacteria tests

One of the problems from the beginning was to clump the glass wafer into amicroscope glass slide because LEICA R© microscope has a fixed glass slide holder.However, with some PBS as medium has been controlled.

The implemented device has 5, 10 and 20 µm electrodes with gap distances of2.5, 5 and 15 µm respectively.

In the interest of having a better way to refer the chip interdigitated electrodesblocks, it has been assigned a number, and it is shown also which wafers were usedaccording to the size of the “finger”electrodes. Fig 4.16

Bacteria tests were performed using Phosphate buffered saline (PBS) and M9minimal medium - minimal microbial growth medium (M9)(1x) as medium, whereM9(10x) is diluted to obtain (1x). There are presented some effects of voltage onbacteria medium, but not adhesion of bacteria was observed.

Procedure:

1. Turn on the chip by applying the desired voltage.

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(a) Glass wafer active block cells

(b) Assigned number to eachinterdigitated cell block

Figure 4.16: Electrodes’ cells numbered and labelled

2. Stick PDMS gasket

3. Pour the medium with the bacteria

4. Wait for 30 min

5. Wash away bacteria medium and replace with clear one.

6. Pictures are taken in a clockwise order from up to bottom and left to right,taking the reference the up left corner of the electrodes block. (Microscopeslide holder resolution grid of 0.3, 0.6, 1 and 2 mm). Fig 4.17 shows differentstages at the tests procedure.

In this way percentage of bacteria around the block can be calculated andhave a real estimation of the rejection of bacteria by the active electrodes.

7. Count the bacteria for each picture and making a table of the quantitativemeasure per electrodes block. Compare quantities of control negative blocksand active ones.

At the beginning, the bacterial concentration was fixed, while the attachmentof the bacteria on the surface was seen after 30 min of incubation.

To test whether the bacteria can attach to the Teflon surface (negative control),adhesion experiments on glass slides with and without Teflon covered on wereperformed. Two bacteria were tested, Salmonella Typhimurium and PseudomonaAeruginosa.

For 30 min adhesion, there were no bacteria attached to the Teflon surfacecompared to glass slide (without Teflon covered on) which showed many cells ofbacteria attached on the surface.

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4.3 Bacteria tests

(a) Pouring medium on ac-tive chip with gasket

(b) Complete Setup: Oscilloscope, amplifier,countdown timer, multimeters and function gen-erator

(c) Bacteria tests un-der luminescence light (d) Proceeding to take pictures

Figure 4.17: Bacteria tests stages

The adhesion time was increased to 24 h which it shown the bacteria on theTeflon surface. Therefore Salmonella Typhimurium and Pseudomona Aeruginosabacteria can attach to the Teflon surface at 24 h but not at 30 min. This maysuggest that incubation time for only 30 min may not be enough for creatingnegative control biofilm adhesion on Teflon covered surfaces as it can be observedin Tab.4.5.

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Surface Time for adhesion After washing Results

Chip 30 minInconclusive interms of adhesion

Active with potential Negative control

Glass slidecoveredwith teflon

30 min No bacteria attached

Salmonella Typhimurium Pseudomona Aeruginosa

24 h Attached bacteria

Salmonella Typhimurium Pseudomona Aeruginosa

Table 4.5: Bacteria adhesion tests done in Karolinska laboratories

4.3.1 Teflon bacteria adhesion tests

Bacteria adhesion was not enough in earlier experiments as seen in Table 4.5. Forthat reason, more experiments with bacteria incubation times on Teflon surfaceswere done, to obtain the most approximate time bacteria needs to attach on Teflonsurfaces and have more reliable control negative tests. The main problem was thatTeflon detached very easily from several cover glasses, also due to the very thickTeflon layer, but fortunately detached in different periods.Incubated bacteria, Salmonella Typhimurium was seen attached to the Teflon sur-face after 20 h as it can be seen in Fig 4.18a, using a photo shoot pattern shownin Fig 4.18b. In this pattern the quantity of photos were not the same for all incu-bation times, neither in all the done experiments.Finally, the quantification of bacteria was done by imageJ program, using a Macroinserted in a created Batch for multiple images in a folder. The results of thecounted bacteria can be seen in Fig 4.18c.

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4.3 Bacteria tests

(a) Bacteria incubation on Teflon coveredglass slides

(b) Bacteria pattern for tak-ing photos

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Figure 4.18: Bacteria

77

Chapter 5

Discussion and outcome

5.1 Discussion

In this part, a final discussion of all the measurements and tests is presented toindicate a final state outcome.

DEP Numerical simulations From the MATLAB calculations the best curveselected for the simulation was at a 380 µS/cm medium conductivity with yeastand E. Coli bacteria multi-shell value parameters using the smeared-out sphereapproach Subsection 2.3.1. It was the best because it presented better differencebetween negative (rejection) and positive (repulsion) values of the real part of theCM factor, where made easier to choose a frequency to start with.

From COMSOL simulations, electric field intensities rejected particles usingvery high frequencies (more than 100 kHz) with different values.It has been seenthat the higher the frequency, the smoother the electric potential over the elec-trodes because increased in the corners. In the Fig 5.1, the simulation of DEPforces is displayed, where the axes are the coordinates of electrodes (representedas rectangles) spacing. It is appreciated that the force is focus more at the cornersof the electrodes rather than the on top of them, making the near corner reallystrong in forces but just micrometres later a lower display of forces. The force isdisplayed in Newton.

78

5.1 Discussion

Figure 5.1: DEP force (Newton)at 30 V at the edges

The 100 nm Teflon layer on top of the electrodes reduced completely forcesdirectly on top, but did not qualitatively change the field, which is shown in Fig 5.2.

There, an electrode is displayed (red rectangle) in a Cartesian plane. Thepotential is set to 10 V and the squared Electric field gradient (grey lines),whichdirectly proportional to the DEP force, can be seen attenuated by the layer of theTeflon.

Even when was not used as an antifouling method, it has been demonstratedthat DEP is viable way to repel bacteria by keeping medium synchronized to widerange of frequency, in order to embrace most of the bacteria types.

From picture Fig 4.7a, the gradient of the squared electric field is repulsivebetween both electrodes, but not over them. Rejection over a larger area of thesurface can be achieved by better configuration as explained later on.

Furthermore, Fig 4.7b shows the DEP force along the x-axis at a constantdistance from the electrodes (7 µm); this clarifies the behaviour of the forces thatcan reject the bacteria, as the distance become much shorter the forces increaseexponentially, as well as for the voltage applied as it is seen in Table 4.2.

From DEP numerical simulation it is possible to reject bacteria because shownforces along horizontal axis can hold an almost uniform rejecting force value, andcan be increased by increasing the applied voltage; but it is needed to improvethe configuration by closer spaced and narrower electrodes, allowing forces to exertrepulsion more uniformly along the surface.

EWOD surfaces experiment Before any EWOD test was made, droplets wereused to understand the setup and determine a range of voltage to work on. Contactangle measurements were used to see the effect of EWOD on the droplets. Dropletsbehaviour and experiments discussion can be seen on Appendix G.

The discovering of the experimental curve above the theoretical one in Fig 4.11,give as the highest voltage value to obtain the larger change of contact angle during

79

5 Discussion and outcome

(a) Squared Electric field gradient without Teflon

(b) Squared Electric field gradient with Teflon

Figure 5.2: Squared Electric field gradient affected by Teflon layer

EWOD and with the suitable medium which was M9 medium. Even though DIwater has better contact angle change, M9 was similar to it and works better forbacteria grow-enhancing medium. Nevertheless, droplet on EWOD has the samebehaviour within different mediums, but strong change in contact angle.

The voltage used in the tests were obtained from differential potential from agenerator, where the ground is obtained from the same equipment.

Applied voltage in electro-wetting can be said to be time-dependent, becauseas time goes by, higher voltage is needed to see some effect on the droplet, as inFig 5.3b. This may be due to the capacitive charged potential inside the dropletwhich needs to be increased to force the droplet to change the contact angle as itcan be deduced from Eq.2.6. Beyond the evaporation limits, the droplet reduces

80

5.1 Discussion

its size making a higher surface tension more difficult to overcome.

(a) Happened electrolysis on chip

(b) Changing of Contact angle by thechange of potential

(c) Droplet difference on Teflon break-down

Figure 5.3: Effects of EWOD on tests

Teflon breakdown has been difficult to deal with because of inevitable small-scale pin holes and poor Teflon adhesion as the Teflon layer thickness become larger.This turn into a cyclical problem where to prevent breakdown needs thicker Teflon,and the thickest the Teflon less is the adhesion to the glass.

The problem of the Teflon adhesion can be solved with the use of Fluorosilanesto improve adhesion to glass and silicon as it is recommended by DuPont R© ’sproduct datasheet. Thus no pre-treatment of the glass slides surfaces should berequired, neither a thicker spun Teflon layer.Furthermore, this solution ought to make a better layer in terms of uniformity andpinhole free. Consequently, decreasing the Teflon breakdown chances.

Bacteria tests Bacteria tests were done in the medium, and during the timethere were floating ElectroPhoresis (EP) and DEP effects might be displayed. Someof the inconvenient was not starting with a detailed planning observation method,as the electrodes were too little, taking pictures with the fluorescent microscopewas too sensitive at movements.

Regarding the used microscope, the fact it was oil-based lens exert some pres-sure over the chip and gasket. This become a problem because seeing through achamber of filled medium changed the microscope’s lens focus and made the livebacteria observation from the top more difficult because of the diffraction.

From the pictures in Fig 5.4 can be seen that bacteria get trapped on someelectrodes maybe due to the EP effect from the electrodes. This can help also tosediment bacteria biofilm in areas where electrodes are not covering.

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It should be considered how chip’s surface started to deteriorate as electrolysisoccurs at higher levels of voltage.

(a) nDEP in electrodes withbacteria

(b) nDEP in electrodes withstronger luminescence bacte-ria

(c) Electrophoresis line ofbacteria in PBS medium

Figure 5.4: Bacteria tests on with interdigitated electrodes

More experiments of bacteria adhesion are waiting to be done, once the Teflonattachment is solved with the use of Fluorosilanes. An additional strain of bacte-ria can be proved after obtaining results for Salmonella Typhimurium and Pseu-domonas aeruginosa bacteria.

5.2 Outcome

In this part the most important points and acquired knowledge are shown.

The purpose of this project was to study the electrokinetic micro-environmentseffect on bacteria adhesion to prevent its growth from medical surfaces. Althoughthis thesis was not able to answer the question if Dielectrophoresis and Electrowetting-on-dielectric can be used as antibacterial methods, we made considerable progresstowards a complete measurement setup for analysing the effect of electrodynamicalfields on bacterial attachment. First of all, an electrodes array deposited by metalsputtered in a translucent medium was manufactured successfully with lithographyand following dicing process in a cleanroom laboratory.

Gaskets were manufactured in two materials: OSTE and PDMS, where thesecond turn out to be the most suitable material due to its elastomeric sealingproperties. In order to finish the assembly of the final testing device, a Teflon layerwas uniformly spun on the top surface until approximately 240 nm of height (4.5%wt.), and covered with the PDMS gasket. This gasket material sealed perfectlywhen the selected bacteria and its medium were poured. DEP had strong repul-sion forces for particles using relatively low voltage, but more electrical propertiesof different bacteria are needed. Regarding the numerical simulations, computa-tion of complex values was executed with a complementary program besides themicroenvironment simulations. Only DEP physics where studied in this virtual

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5.2 Outcome

environment. Contact angle measurements were made to predict the EWOD be-haviour and approximate field strengths.

By experiments on surfaces, contact angle measurements where crucial forknowing the EWOD effect for M9 medium, DI water and PBS where contact anglechange dramatically and remain almost constant until breakthrough happens onthe droplet (electrolysis).

As time goes by during electro-wetting tests, higher voltage was needed tosee some effect on the droplet. Hence, it can be inferred that applied voltage inelectro-wetting is also time-dependent.

From the bacteria tests can be seen bacteria placed on some sections of theelectrodes because of the entropy of the bacteria, but it may be also implied EPand DEP behaviour were observed from the electric field effects due to the similaritywith the pattern of DEP effect on particles. One of the biggest challenges was toapply dielectrophoresis and electro-wetting theories and mixed them in a simpletest based on the surface energy of the manufactured chip.

5.2.1 Project method summary

• Device assembly

1. Electrode array over a translucent wafer manufacturing (lithography anddicing).

2. Cover it with an enough thin Teflon layer for EWOD applications (480 nm).

3. Select the suitable gasket for bacteria tests, define UV-curing times andsealing tests.

• Measurements

1. Numerical simulations, COMSOL complemented to MATLAB calcula-tion for the smeared-out sphere approximation. (DEP behaviour)

2. Experimental tests, contact angle measurements and waveform tests inan basic EWOD setup to predict the behaviour in a the chip setup (openwire-free design).

3. Bacteria tests, in cooperation with Department of Microbiology, Tu-mor and Cell Biology from KI(Karolisnka Institute), Salmonella Ty-phimurium tests were made on PBS and M9 medium with EP and DEPresults. No adhesion was noticed.

5.2.2 Outlook

In this part some future ideas and propositions are exposed; jointly with the out-come, further studies and projections are being discussed.

The electrodes structure of the finished wafers kept the mask outline, but dur-ing the etching process some electrodes width changed radically. New stable andgood structured electrode array can be done with more time in the cleanroom.

83


Additionally, the spacing and width of the electrodes simulated in DEP needs tobe optimized for larger repelling forces areas.

Although DEP experiments to avoid biofilms were not taken place, more testsshould be made at different frequencies, as it has been seen in the simulationsdifferent forces arrangement appeared as a result of the electrical field exerted onthe medium. This fixed frequencies can be (100 Hz, 1 kHz, 100 kHz) due to theirstrong effect on EWOD contact angle changes and also on bacteria.

Prevention of Teflon breakdown became a challenge because of pin holes andadhesion, probably can be solved with new lithography and adhesion promoter.

Although some tests on resistance of materials has been modelled [44], but sometests on resistance of the materials used can give the project more tools for nextgenerations of anti-fouling techniques, mainly a curve of impedance versus voltageand impedance versus frequency to narrow the frequency range simulated on thenumerical simulations section.

84

Acknowledgements

I am very glad to have had the opportunity to do my thesis in the biomedicalresearch area at Micro and Nanosystems (MST) department. It has been a longjourney through new topics and experiences, and lot of people helping me in theway with its professionalism and support. Firstly, I would like to thank for theopportunity to my supervisor, Fredrik Carlborg and the support he give me dur-ing the thesis process, and my sincere gratitude to my examiner Wouter van derWijngaart for being a professor with a wide mind for criticism and ambition. Ienjoyed having a challenged like this, where new topic was tested in the group.I worked very hard to have for the first time DEP and EWOD developed in theMST group. I would like to thank my family, that without their support from thedistance this Master program and thesis wouldnt be possible. My deepest thoughtsare on my amazing sisters and especially on my mother from who I felt it as shewas here all the time. To my right hand, Carmen, for support me and walk withme through this rocky path. You have become the most important part in my life,thank you for your endless patience and kindly perseverance and determination.Furthermore I would like to thank the MST group personnel who supported mein different moments, in no particular order: Stemme Goran for welcomed me inthe group and sharing its thoughts every Tuesdays meetings; Niclas Roxhed forhanding me the wafers I need for my tests, Hans Sohlstrom for helping me with theelectrical equipment and some ideas for my setup; Kristinn Gylfason for sending meupdate news about the research topic; Erika Appel and Ulrika Pettersson for theirhelp in handing out administrative tools and papers; Cecilia Aronsson and NikolaiChekurov for helping me in the clean room with my wafer designs as well as MikaelBergqvist for helping me in my hardware setup; and PhD students which some ofthem are not students anymore: Andreas Fischer for helping me with my etchingin the clean room; Fredrik Forsberg for help in the milling machine, the dedicatedhours to my wafers fabrication aid and concern about its results later on; TommyHaraldsson for recommendations about Teflon covering and funny comments dur-ing the lunch; Umer Shah and Zargham Baghchehsaraei for sharing the room withme and helping me with my electrical and electromagnetical hypothesis doubts;Gaspard Pardon for its helped in some contact angle software; Simon Bleiker forits interesting chats during Fika times and its advices for standard proceedings;Valentin Dubois for his spontaneously funny jokes; Carlos Errando Herranz forbeing the only Spanish speaking in the group who was nice to talk with and itsunselfishly help when he was a master student; Xuge Fan for sharing his Chinese

85


culture and doubts about electronic devices, and its love for basketball; HitheshGatty for being the fisrt person who could helped me in the clean room to fabricatemy first wafer lithography attempts; Maoxiang Guo for her funny questions dur-ing meetings; Jonas Hansson for cheering up when sometimes was needed; StaffanJohansson for sharing his research problems; Mikael Karlsson for helping me inthe laboratory and advising me in the spinning machine; Farizah Saharil for herhelped with her high temperature tape; Laila Ladhani for her kind invitations;Gabriel Lenk for being a nice labmate; Floria Ottonello for sharing her Italianstown culture; Mina Rajabi for her nice chats; Niklas Sandstrom for helping me inthe small milling machine even when he was running out of time for other tasks;Stepahn Schoder for his sudden hi when I found it at the office; Fritzi Topfer for hercountless invitation to many activities which most of them I couldnt go; AlexanderVastesson for his advices and amusing jokes; Xiamo Zhou (Chianty) for her help inthe laboratory and her funny situations. Also my sincere thanks to Mathias Kvickand Fredrik Lundell from the Microluidics laboratory, Wallenberg Wood ScienceCenter, for their help with the fluid flow flakes they provided me. Lastly I wouldlike to thank the Department of Microbiology, Tumor and Cell Biology (MTC)where Ute Romling helped me and advised me in every meeting we had, also Iwould like to thank her group personnel who helped me with the bacteria tests:Marco Schottkowski for introducing me to their lab; Srisuda Pannanusorn for herhelp in the bacteria culture and finally Irfan Ahmad and Florian Salomons for thelatest bacteria tests and its patience to be done with it.

Thanks to all people I knew and give me a little bit of theirs that help me inthis journey to pursue a life as a researcher. From Italy, Sweden and even Peru,my total gratitude is for you as well.

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[40] K. Dill and S. Bromberg, Molecular Driving Forces: Statistical Thermody-namics in Chemistry and Biology. ISBN: 0815320515, 9780815320517, Gar-land Science, 2003. The Einstein-Smoluchowski Equation relates Diffusion andFriction.

[41] R. B. Bird, W. E. Stewart, and E. N. Lightfoot, Transport phenomena. JohnWiley & Sons, 1976.

[42] M. Dhindsa, S. Kuiper, and J. Heikenfeld, “Reliable and low-voltage elec-trowetting on thin parylene films,” Thin Solid Films, vol. 519, no. 10, pp. 3346– 3351, 2011.

[43] MICROCHEM, “Su-8 2000 datasheet - permanent epoxy negative photore-sist,” in PROCESSING GUIDELINES FOR: SU-8 2000.5, SU-8 2002, SU-82005, SU-8 2007, SU-8 2010 and SU-8 2015 (www.microchem.com, ed.), (1254Chestnut St. Newton, MA 02464 Boston USA), p. 5, microchem, 2014. [email protected].

[44] J. Illergrd, U. Rmling, L. Wgberg, and M. Ek, “Tailoring the effect of an-tibacterial polyelectrolyte multilayers by choice of cellulosic fiber substrate,”vol. 67, pp. 573–578, March 2013.

[45] N. C. f. E. Division of Healthcare Quality Promotion (DHQP) and Z. I. D.(NCEZID), Sterilization or Disinfection of Medical Devices. Centers for Dis-ease Control and Prevention, 1998.

[46] S. Department of Communicable Disease and Response, “Infection preventionand control of epidemic- and pandemic-prone acute respiratory diseases inhealthcare.” webpage, May 2007.

[47] Cathay General Hospital, Home care for trachea treatment patients. CathayFinancial Holding Co., Ltd., 2011.

[48] M. L. Hemani and H. Lepor, “Skin preparation for the prevention of surgicalsite infection: which agent is best?,” Reviews in Urology, vol. 11, no. 4, p. 190,2009.

[49] D. of Blood Safety and C. Technology, Surgical care at the district hospital.World Health Organization 2003, 20 Avenue Appia, 1211 Geneva 27, Switzer-land, isbn 92 4 154575 5 ed., September 2003.

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[50] G. H. O. (GHO) and N. diseases (NCD), eds., The local small-scale prepara-tion of eye drops, WHO/PBL/01.83, World Health Organization, Vision 2020actionplan, 2002.

[51] S. Z. Yoon, Y.-S. Jeon, Y. C. Kim, Y. J. Lim, J. W. Ha, J. H. Bahk, S. H. Do,K. H. Lee, and C. S. Kim, “The safety of reused endotracheal tubes sterilizedaccording to centers for disease control and prevention guidelines,” Journal ofClinical Anesthesia, vol. 19, no. 5, pp. 360 – 364, 2007.

[52] Tapia-Jurado&Reyes-Arellano&Garca-Garca&Jimnez-Corona&Pea-Jimnezand Len, “Comparison of cost/effectivity of surgical wash with variousantiseptics,” CIRUGA Y CIRUJANOS, vol. 79, no. 5, pp. 415–420, 2011.

[53] D. of Diarrhoeal and A. R. D. Control, Guidelines for the control of epidemicsdue to Shigella dysenteriae type 1 (Sd1). World Health Organization, 1994.

[54] Y. S. Nanayakkara, H. Moon, and D. W. Armstrong, “A tunable ionic liquidbased rc filter using electrowetting: A new concept,” ACS Applied Materials& Interfaces, vol. 2, no. 7, pp. 1785–1787, 2010.

[55] J. Berthier, P. Clementz, O. Raccurt, D. Jary, P. Claustre, C. Peponnet, andY. Fouillet, “Computer aided design of an {EWOD}microdevice,” Sensors andActuators A: Physical, vol. 127, no. 2, pp. 283 – 294, 2006. ¡ce:title¿MEMS2005 Special Issue¡/ce:title¿ ¡ce:subtitle¿Special Issue of the MicromechanicsSection of Sensors and Actuators (SAMM), based on contributions revisedfrom the technical digest of the {IEEE} 18th International Conference onMicro Electro Mechanical Systems (MEMS-2005).¡/ce:subtitle¿.

[56] J. J. Pak, “Simplified ground-type single-plate electrowetting device for droplettransport,” Journal of Electrical Engineering & Technology, vol. 6, no. 3,pp. 402–407, 2011. Publisher : The Korean Institute of Electrical Engineers.

91

Appendix A

Biofilm on hospitalenvironments challenges

In order to consider a surface disinfected, a chemical procedure (germicide) must beused to eliminates all pathogenic microorganisms. From the three existing levels,the highest kills all bacteria present by sterilants, the intermediate kills most ofviruses , and bacteria by “turberculocide”(the killing of tuberculosis bacilli); andthe lowest level kills only some viruses and bacteria by hospital disinfectants.

One short step before disinfection is always cleaning the surfaces to rise thegermicide effect.[45]. This carry on costs on disinfectants (hypochlorite, quater-nary ammonium) , laundry, technical personal training, waste management asit is described in the infection prevention manual from World Health Organiza-tion (WHO)[46]. In addition it must be taking on count different disinfectionprotocols and disinfectants worldwide (upon the period of time needed to disinfect,known as “contact times”) which also can lead to material degradation problemsif is not correctly used (i.e. the rubber cracking caused by several alcohol applica-tions) Annex H in [46].

On top of that high-level disinfection for semi critical items also add costs likesterilization equipment(autoclave, ethylene oxide EO gas sterilizer) which are notcompatible with all used medical materials neither, Annex J in [46]. But if homecare is required like Trachea tube sterilization,it needs to be sterilized until threetimes a day if necessary, where also needs to be dissembled, sterilize for a periodof time (high temperature) and reassembled again for use it[47].Preoperative skinpreparation is also critical, where common disinfectant agents are less probable ef-fective due to evaporation, neglected hair removal and non-uniform results over thecovered area[48]. Wound healing can be impaired by infection if it is not prevented;bacteria cannot be eliminated but is reduced by aseptic measures. Disinfections’solutions only inactivate infections agents and sterilization kills microbes[49].

Even in very common prosthetics like contact lenses, autoclave, high pressurechambers and steamers are needed by specialists to sterilize them and eye drops;they are also working with antimicrobial agents like gentamicin, neomycin andpovidone iodine when autoclaving cannot be done which can lead to skin irritation

92

or a basic knowledge of administration doses[50].A latent problem in developing countries is the way to reuse daily sterilized

endotracheal tubes (ETTs) to reduce the hospital costs. As a consequence, Centersfor Disease Control (CDC) guidelines sterilizations methods as ethylene oxide gas(EO) or alkaline glutaraldehyde (GA) solution may affect the mechanical propertiesof tube’s material(mainly the ETT’s cuff), specifically the tensile strengths as aresult of continue reprocess of them[51].

It can be seen that costs vary from more than 5 Euros up to 20 only in washinghands, Table 5 in [52]. Antimicrobials need to be administrated more than once,and for a period of time to have an effect, and depending on the variation of it canbe more sensitive to store and transport to others, Table 1 in [53]. Consequentlydepending on the used antiseptic some advantages or disadvantages can be pop out:alcohol can have a immediate effect but would produce skin irritation or dryness,on the contrary hexachlorophene can have good effect but it takes more time toact), Table 6 in [52].

Another fact to be taking on consideration is the period of time the material todisinfect are in used, like Legionnaires’ disease patients needed more than others.[12]

93

Appendix B

MATLAB Program

In this part all program code is written.

B.1 Command set

Program code is completely shared.clear all;j = sqrt(-1);%E permittivity, cE complex permittivity%cE = compl permt(e, s, w)% e = permittivity (F/m)% s = conductivity (S/cm)% w = angular frequency (Hz)%e0 = 8.8542e-12;%vacuum permittivity (F/m)% %w=1.3; % Khz Specific frequency%f=10*106;%f = logspace(2,8,1000); % Hz frequency rangew = 2*pi*f; % angular frequency range%—————————————————-%YEAST - 3 layer%Radius from inner to outer (1...)r1 = 2.35*10-6;%particle inner radius (m)r2 = 2.36*10-6;%particle middle radius (m)r3 = 2.50*10-6;%particle outer radius (m)

%Conductivity from inner to outer (1...)sr1 = 2*10-1;%particle inner radius conductivity(S/m)sr2 = 25*10-8;%particle middle radius conductivity(S/m)sr3 = 140*10-4;%particle outer radius conductivity(S/m)

%Permittivity from inner to outer (1...)

94

B.1 Command set

er1 = 60*e0;%particle inner radius permittivity(F/m)er2 = 6*e0;%particle middle radius permittivity(F/m)er3 = 50*e0;%particle outer radius permittivity(F/m)

%Particle Permittivity (bacteria) - calculation%complex permittivity by layersce1 = compl permt(er1, sr1, w);ce2 = compl permt(er2, sr2, w);ce3 = compl permt(er3, sr3, w);

%complex permittivity between layers 1&2CM21 = (ce1-ce2)./(ce1+2*ce2);r21 = r2/r1;ce21 = ce2.*(((r213)+2*CM21)./((r213)-CM21));

%complex permittivity between layers 3&21 (particle)CM321 = (ce21-ce3)./(ce21+2*ce3);r32 = r3/r2;

cep = ce3.*(((r323)+2*CM321)./((r323)-CM321));

%—————————————————-%—————————————————-%MEDIUM (water)%sm = 2*10-4;%medium conductivity (S/m)% 2(uS/cm) DI watersm = 38000*10-6;%medium conductivity (S/m)%from COMSOL, wikipediaem = 80*e0;%medium permittivity (F/m)cem = compl permt(em, sm, w);% Clausius-Mossotti factorCM = (cep-cem)./(cep+2*cem);output = real(CM);%semilogx(f,output);%xlabel(’frequency (Hz)’);%ylabel(’Re[K(w)]’);%grid on;%///////////////////////////////////////////////////////////////%///////////////////////////////////////////////////////////////%—————————————————-%E. COLI - 3 layer%Radius from inner to outer (1...)r1b = 0.975*10-6;%particle inner radius (m)r2b = 0.98*10-6;%particle middle radius (m)r3b = 1*10-6;%particle outer radius (m)

%Conductivity from inner to outer (1...)

95

B MATLAB Program

sr1b = 10-1;%particle inner radius conductivity(S/m)sr2b = 5*10-8;%particle middle radius conductivity(S/m)sr3b = 500*10-4;%particle outer radius conductivity(S/m)

%Permittivity from inner to outer (1...)er1b = 60*e0;%particle inner radius permittivity(F/m)er2b = 10*e0;%particle middle radius permittivity(F/m)er3b = 60*e0;%particle outer radius permittivity(F/m)

%Particle Permittivity (bacteria) - calculation%complex permittivity by layersce1b = compl permt(er1b, sr1b, w);ce2b = compl permt(er2b, sr2b, w);ce3b = compl permt(er3b, sr3b, w);

%complex permittivity between layers 1&2CM21b = (ce1b-ce2b)./(ce1b+2*ce2b);r21b = r2b/r1b;ce21b = ce2b.*(((r21b3)+2*CM21b)./((r21b3)-CM21b));

%complex permittivity between layers 3&21 (bacteria)CM321b = (ce21b-ce3b)./(ce21b+2*ce3b);r32b = r3b/r2b;

ceb = ce3b.*(((r32b3)+2*CM321b)./((r32b3)-CM321b));

% Clausius-Mossotti factorCMb = (ceb-cem)./(ceb+2*cem);outputb = real(CMb);semilogx(f,output,f,outputb,’–’);%blue yeast, green E. COLIxlabel(’frequency (Hz)’);ylabel(’Re[K(w)]’);title(’Plot of Clausius-Mossotti factor(CM)’);legend(’YEAST(CM)’,’E. COLI(CMb)’);grid on;

%88888888888888888888888888888888888888888888888888888888888888888888888888

B.2 Flow Chart Diagram

In this part sequential a flow chart diagram for matlab code is drawn.

96

B.2 Flow Chart Diagram

Yes

No

START

j = sqrt(-1); vacuum permittivity;

frequency;

r1, r2, r3; s1,s2,s3; e1,e2,e3;

General values

Yeast layers values

r1, r2, r3; s1,s2,s3; e1,e2,e3;

E. Coli layers values

e, s, w; cE = (e-j*s./w);

START

CMxy=(cEy-cEx)/(cEy+2cEx); rxy=rx/ry;

cexy=cEx*(((rxy^3)+2*CMxy)/((rxy^3)-CMxy));

x=2; y=1;

x=3; y=21;

CMxy=CM321

compl_permt

END

sm, em

medium values

x=m; y=321;

cE1, cE2, cE3, cEm

real (CM321m)

CMxy=(cEy-cEx)/(cEy+2cEx);

real (CM321m)

Yeast E. Coli

END

Figure B.1: Matlab flow chart

97

Appendix C

COMSOL analysis andsimulations

In this part extra information from the calculation and simulations are shown.

C.1 Drift velocity from Einstein– Smoluchowski rela-tion

It is needed to obtain velocity and force from the same relation fo the generalformula for Brownian motion. Considering the formula:

D = µ κB T (C.1)

It is decomposed in an dimensionless quantities to obtain mobility (µ) units.

[L2

T] = µ [

L2 · MT 2 · Θ

][Θ] (C.2)

µ = [T

M] (C.3)

then

µ = [T · ( L

T 2 )

M · ( LT 2 )

] = [LT

M · LT 2

] =vdF

(C.4)

98

C.2 DEP force measured at different heights

C.2 DEP force measured at different heights

In this part can be contrasted the force intensity while the measured line, usingCut Line 2D of the COMSOL, move closer to the bottom electrodes.

Figure C.1: DEP forces at different heights, 7 µm(blue), 3 µm(green), 1 µm(red)above electrodes (21-29 and 51-59 µm in x-axis)

99

Appendix D

Bacteria formation

In this part further information of the bacteria and results are exposed, which canbe divided in the particle and its floating medium.

D.1 Bacteria

In this part the biological information and results are set out.

Figure D.1: Bacteria biofilm internal bonding forces

D.1.1 ImageJ macro

Macro used for counting bacteria in a general mode and inserted in a created batchfor applying it onto multiple images contained in a folder.

100

D.2 Medium

%—————————————————-CountBact.ijmrun(”Subtract Background...”, ”rolling=15”);setAutoThreshold(”Default dark”);//run(”Threshold...”);setAutoThreshold(”Otsu dark”);setOption(”BlackBackground”, false);run(”Convert to Mask”);run(”Watershed”);run(”Analyze Particles...”, ”size=5-Infinity pixel circularity=0.00-1.00 show=Nothingdisplay include summarize”);%—————————————————-

D.2 Medium

In this part medium characteristics are mentioned.

(5x) M9 medium 1l

168µmol 37.6 g Na2HPO4 −H2O110µmol 15 g KH2PO4

94µmol 5 g NHO4Cl43µmol 2.5 g NaCl

(1x) M9 medium 500ml

100mL 5xM910µmol 5mL Glucose(1M)1µmol 500µL MgSO4

25µL Thiamin(1%)0.027µmol 13.5µL CaCl2(1M)

400mL H2O

Table D.1: M9 medium parameters and dilution

101

Appendix E

OSTE tests detailedinformation

In this part OSTE properties and information is delineated.

E.1 Material specification

In this part all materials used on the OSTE are considered and displayed.

From Sterilin.co.uk is obtained the following information:

Item Code Description Well Capacity(µL) Material Sterility611F96 96 well plate, Flat bottom 400 PS NS

Table E.1: Culture plate information from catalogue(Microtitre Plates, Clear fromSterilin http://www.sterilin.co.uk/

102

Appendix F

Chip fabrication detailedinformation

In this part further information about glass wafer electrodes manufacturing is laidout in different scenarios.

F.1 Design / L-EDIT

In this part, first attempts of designs are described and explained why they couldnot work.

Even with the choice on hand to use the interdigitated array for the electrodesmany topologies came to pop up and the simplest one was chosen.Fig F.1

Figure F.1: Different designs for the electrode array topology: (A)basic array, (B)tree array and (C)top-bottom tree array

Since in every test the most important aim to is try to use as much as the it

103

F Chip fabrication detailed information

could be the resources that we count on; the first attempt of the design was thoughtto cover almost all the wafer in order to have more test areas Fig F.2; but later on,the cost by each wafer make me to re-design the mask and maximized the area tobe used in a (10 mm) diameter wafer, 9 interdigitated electrodes are chose to befit in a quarter of wafer.Fig 3.2b

Figure F.2: First attempt to design the wafer mask allowing to have more testspaces.

F.2 Procedures

In this part, a more detailed information about the procedures used in the lithog-raphy process which are too redundant to be in the thesis.

Feedback from the lithography was useful to not“overcured” the lithography byapplying too much intensity of light or too much time under the UV exposure.Such process can be shown in Fig 3.9.

Steps:

• nLOF: spin speed 3000 RPM for 30 s

• soft bake 110◦C for 60 s

• exposure for 22 s: 440 mJ/cm2

• Dev: MST AF 4in Dev nLOF 2070 (loaded recipe)

First attempt on lithography:

Coating sample was a proportion of AZ nLOF 2035:RER600 by (2:1) preparedon the year 2010. This coating was spun at 3000 RPM for 30 s.

104

F.2 Procedures

It has been made 4 attempts with different UV exposures and developmentperiods as it can be seen on Table F.1 and Table F.2. The type of contact was ’LoVac’ with a Al.Gap(µm)of 40.

Times on wafer1st 2nd 3rd 4th

UV exposure 40 s 80 s 120 s 80 s

Development 3+1+2+3 min 3+2+4+5 min 5+4 min 5 min

Table F.1: UV exposure and development times - Second lithography attempt withstandard softbake(110◦C)

Second attempt Times on wafer1st 2nd 3rd 4th (more intense UV,ch2[20W]) 5th

UV exposure 10 s 5 s 8 s 10 s 10 s

Development3+1+2+3 min 3+2+4+5 min 5+4 min 5 min –

handmade Maximus 804 ATM sse (Coating and develop of PR film)

Table F.2: UV exposure and development times - Second lithography attempt withlower softbake(90◦C)

Times of UV exposure by Tab.F.2

5 s 8 s 10 s

# 1 (in µm)

Whole gap (2 gaps 1 elect) 10,67 > 10,18 > 9,69Electrode width 4 < 4,76 < 4,98Elec-bus gap 5,42 > 4,76 > 4,60Elec-elec gap 3,40 > 2,79 > 2,13

# 2 (in µm)

Whole gap (2 gaps 1 elect) 20,52 > 20,03 > 19,37Electrode width 8,50 < 9,03 < 10,02Elec-bus gap 6,57 > 5,91 > 4,76Elec-elec gap 6,30 > 5,66 > 5,01

# 3 (in µm)

Whole gap (2 gaps 1 elect) 55,55 > 50,73 > 49,25Electrode width 17,90 < 19,05 < 20,19Elec-bus gap 6,40 > 5,58 > 4,43Elec-elec gap 16,64 > 15,76 > 14,70

Table F.3: Measurements of the electrodes and gaps in the second attempt fromTab.F.2

105

F Chip fabrication detailed information

F.3 Parameters

In this part some parameters that are worth to be kept but are not fundamentalto be shown. Some parameters of the dicing blades are described.

Some other parameters that are worth to mention, like Dicing blade measure-ments illustrated in Table F.4.

OD(Out diameter) Flange diameter (mm) Blade exposure Blade thickness (mm)

P1A851 Flange 56 50,3 2,85 0,3

27HEEE Hub (Flange) 56 50,3 0,89 0,3

Table F.4: Dicing blade parameters about the used Flange

106

Appendix G

EWOD attemps

sectionEWOD droplets tests Droplets evaporation was important to notice a trendline and discard future possible effect of evaporation on EWOD contact anglesdue only to the applied voltage and lasted time. During the first 60 s of the test,evaporation can be neglected. Also, this control negative test helped to know thatthe electrode tip has an effect on contact angle measurements, but still can beneglected because of the very little contact angle change compared to the ones onthe experimental curve.

From the contact angle measurements, I have discovered that electrolysis canbe avoided for voltages higher than 16VDC by inserting the tip before it is charged,otherwise the surface tension acts as a conductive medium breaking the Teflon layerdown.

In the other hand, every angle measurement requires a new Teflon spinningbecause of the breakthrough of it. Dielectric failure (dielectric breakdown) as inFig 5.3a can occur even at low voltages due to the use of ionic solutes (as well ofthe Double layer effect); this can be avoided with the use of larger ions size (addingspecific surfactant or larger polar molecules solutions than water) or increasingthe thickness of the Teflon layer. It is important to highlight that the dielectricstrength of the dielectric layer become higher if the thickness increases; in the otherhand, more voltage would be needed to have the same EWOD effect.

Hysteresis have been seen, so after applying voltage to reach higher voltagepotential takes less time than arriving from zero potential, this due to dropletkeeps charged and uncharged very slowly (capacitor behaviour).

At lower range of voltages (< 100 V) using +DC, -DC, 60 Hz, 1 kHz or10 kHz has not much difference in the contact angle changing. But for sure thesaturation point can be shortly achieve firstly in the first cases and the latter atlower frequencies[54].

As for the waveform test, the selected waveform was obviously the step squarewith the negative potential on top of the surface as can be seen on Fig 4.12, butneeded to be confirmed by these tests. In addition to that, it has been demonstratedthat changing Duty cycle in a square signal reduces EWOD effect on the contactangle, and considering that test, it has been observed that droplet behaviour is moreeffective with different duty cycles independent of their applied voltage. (Studied

107

G EWOD attemps

Duty cycles: 1%, 40%, 60% and 100%).New designs at macro level were built, but were also used as a reference because

at those distances DEP forces and E field intensity are very low, as seen in thefollowing section.

G.1 Another EWOD tests

Another test run followed but the idea of DEP on catena[55, 56]. For the powdertests is that used Iriodin R© 120, Lustre Satin colored (fluid flow flakes) which itsparticles size is in the range of 5 - 25 µm. It was provided by Mathias Kvickand Fredrik Lundell from the Microluidics laboratory, Wallenberg Wood ScienceCenter, Department of Mechanics, KTH.

(a) Catena test on gasket(copper wire) (b) Merge of droplets tests

(c) Electrolysis and powder tests

Figure G.1: EWOD variety test

Milled electrodes

108

G.1 Another EWOD tests

(a) Milled PCB in a macro level

(b) Smallest size obtained by milling ma-chine

Figure G.2: EWOD in in macro-level electrodes

109

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