Automated Vitrification of Mammalian Embryos on a Digital … › bitstream › 1807 › 65597 ›...

Automated Vitrification of Mammalian Embryos on aDigital Microfluidic Platform

by

Derek Geoffrey Pyne

A thesis submitted in conformity with the requirementsfor the degree of Master of Applied Science

Graduate Department of Mechanical and Industrial EngineeringUniversity of Toronto

c© Copyright 2014 by Derek Geoffrey Pyne

Abstract

Automated Vitrification of Mammalian Embryos on a Digital Microfluidic Platform

Derek Geoffrey Pyne

Master of Applied Science

Graduate Department of Mechanical and Industrial Engineering

University of Toronto

2014

This thesis presents the development of a digital microfluidic system to achieve auto-

mated sample preparation for the vitrification of mammalian embryos for clinical in

vitro fertilization (IVF) applications. This platform included micro devices fabrication,

an imaging system, a high voltage control system, and a LabVIEW interface. Individual

micro droplets manipulated on the digital microfluidic device were used as micro-vessels

to transport a single embryo through a complete vitrification procedure. The device

showed cell survival and development rates of 77% and 90%, respectively, which are

comparable to the control groups that were manually processed. Technical advantages

of this approach, compared to manual operation and channel-based microfluidic vitrifi-

cation, include automated operation, cryoprotectant concentration gradient generation,

and feasibility of loading and retrieval of embryos.

ii

Acknowledgements

I would like to thank my advisers, Professor Yu Sun and Professor Mohamed Abdelgawad,

for without their guidance and encouragement this thesis would not have been possible.

I wish to express my sincere appreciation and thanks to all the members of the Advanced

Micro and Nanosystems Laboratory at the University of Toronto. I would also like to

thank my collaborators Jun Liu (University of Toronto) and Waleed Salman (Assiut

University).

Finally, I would like to thank my parents for their continuing support, advice and

being my biggest fan.

iii

Contents

1 Introduction 1

1.1 Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Digital Microfluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.3 Merging Digital Microfluidics and Vitrification . . . . . . . . . . . . . . . 5

1.4 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.5 Dissertation Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2 System Setup 10

2.1 Device Fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.1.1 Photoresist Coating . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.1.2 UV Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.1.3 Developing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.1.4 Chromium Etching . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.1.5 Photoresist Removal . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.1.6 Dielectric Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.1.7 Teflon Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.1.8 Assembled Devices . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.2 Electrical System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.3 Control and Software System . . . . . . . . . . . . . . . . . . . . . . . . 20

2.4 Imaging System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

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3 On-Chip Embryo Vitrification 26

3.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.2 Device Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3.3 Embryo Loading and Retrieval . . . . . . . . . . . . . . . . . . . . . . . . 28

3.4 Cryoprotectant Mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

3.5 Thawing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

3.6 Measurement Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.7 Vitrification Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.8 Volume Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.9 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3.10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4 Partially Filled Electrodes 47

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

4.2 Modelling and Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . 48

4.3 Experimental Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

4.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

5 Summary 57

5.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

5.2 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

A Detailed Fabrication Recipe 60

B Selected LabVIEW Code 66

Bibliography 66

v

List of Tables

3.1 Summary of vitrification results. . . . . . . . . . . . . . . . . . . . . . . . 35

4.1 Parameters used in numerical simulations. . . . . . . . . . . . . . . . . . 49

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List of Figures

1.1 Vitrification process involves sequentially bathing the embryo/oocyte in

cryoprotectant baths according to a strict timing protocol. Cryoprotec-

tant concentration increases in later baths and the embryo is plunged

immediately after exiting the final bath. The first bath is typically called

Equilibrium Solution (ES), and later baths called Vitrification Solution

(VS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2 Overview of fundamental digital microfluidic operations. Droplets can be

transported around the device for mixing or movement to another seg-

ment of the device. Smaller droplets can be dispensed from larger holding

reservoirs. Droplets of larger volume can be split into two daughter droplets. 4

1.3 Droplet mixing is achieved by first merging droplets by actuating on to

a common electrode. The merged droplet is then transported around the

device until mixing is homogeneous. Red dye was used to help visualize

this process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.4 Schematic showing differences between manual vitrification approach, which

requires manual pipetting between mediums in cryoprotectant stage, and

the digital microfluidic (DMF) approach, which moves the embryo be-

tween mediums on chip. The chip automates the high skill portion of the

procedure providing labor cost savings and opportunities for parallelization. 7

2.1 Overview of system elements and central LabVIEW interface. . . . . . . 10

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2.2 Overview of device fabrication. Photolithography was used to pattern

chromium electrodes, chemical vapour deposition was used to deposit pary-

lene C, and Teflon wass finally spin coated on the device. . . . . . . . . . 11

2.3 Photoresist is spin coated on the device at 3000 rpm for 30s. . . . . . . . 12

2.4 Photoresist is patterned by exposing to UV light through a photomask for

10s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.5 During development areas of photoresist exposed to UV light are washed

away in the developer. The desired pattern is then left in the photoresist

layer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.6 During etching, areas of the chromium layer not protected by photoresist,

are dissolved in the etchant. This transfers the design pattern from the

photoresist layer to the chromium layer. . . . . . . . . . . . . . . . . . . 15

2.7 Photoresist is removed using a stripper solution in an ultrasonic bath. The

ultrasonic bath helps removes photoresist from all sections of the device. 16

2.8 Parylene C is used as a dielectric layer and deposited using a chemical

vapour deposition process with a dedicated coater. . . . . . . . . . . . . . 17

2.9 Teflon is applied by spin coating a uniform layer, and then baking to

remove solvents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.10 Assembled device is held in place using a set of 3D printed parts. Top

ITO slide is connected to ground using an alligator clip. . . . . . . . . . . 18

2.11 Schematic of electrical system showing relay array control and high voltage

generation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.12 Relay array containing 24 mechanical relays, connection to LabVIEW sys-

tem, and connection to ribbon cables to device. . . . . . . . . . . . . . . 20

2.13 Electrodes are interfaced to relay system using an edgeboard connector.

Allows for fast changing between devices. Parylene is removed from elec-

trodes to ensure good connection. . . . . . . . . . . . . . . . . . . . . . . 21

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2.14 Overview of LabVIEW programming environment. The Front Panel is the

user interface and holds controls and indicators usable by the operator.

The Wiring Diagram controls data flow and is done using a graphical

programming paradigm where data flows through wires instead of local

variables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.15 Overview of droplet control LabVIEW elements. A pattern of buttons

is used for manual control in the shape of the present device. Droplet

sequence control is accomplished by reading electrode sequences from an

excel file. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.16 Overview of imaging apparatus. Zoom and focus are motorized allowing

them to be controlled from within LabVIEW. . . . . . . . . . . . . . . . 24

2.17 LabVIEW front panel highlighting imaging components. Zoom and focus

are controlled either by sliders on the right side of the screen, or by a toggle

allowing quick switching between predefined low and high zoom settings.

Videos are recorded with a toggle and files saved using timestamps as their

name. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.1 Chip design showing regions for vitrification medium dispensing and em-

bryo inlet/outlet. The top ITO slide is placed on the device in a manner

that exposes portions of the top electrodes in the dispensing reservoir and

the leg of the T-shape to allow for medium and embryo loading respectively. 27

3.2 Embryo is input and extracted by actuating electrodes at edge of top glass

slide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.3 Overview of general approach for mixing on a digital microfluidic platform. 30

3.4 Schematic showing implementation of mixing protocol using this device

design. Daughter droplet containing embryo in step 4 is identified manually. 31

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3.5 Mixing profile showing generation of ES medium and VS medium. Exper-

imental droplet concentrations were found by using image processing to

measure the droplet volumes before and after each mixing step. . . . . . 32

3.6 (a) Embryo (red circle) contained in culture medium (CM) droplet. (b)

Embryo droplet mixed with VS droplet. (c) Droplet split into two droplets

(left contains embryo). (d) Droplet containing embryo is kept and other

droplet is sent to waste. Process is repeated to increase VS concentration. 33

3.7 Sample healthy and failure cases for survival rate based on morphology

before and after freezing. . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.8 Sample healthy and failure cases for development rate based on culturing

for an additional 24-48 hours after freezing. . . . . . . . . . . . . . . . . . 35

3.9 Embryo cell volume measurements for a typical (a) human and (b) mouse

protocol on chip. Volumes were normalized to initial volume. The initial

volume dip in the human protocol matches the volume dip over the mouse

protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3.10 Comparison of mouse and human vitrification protocols. [38, 47–52] . . . 38

3.11 Implementation of common vitrification protocols on a digital microfluidic

chip with a single dispensing reservoir. Timings and concentrations are

shown in (a), and the generalized mixing curve is shown in (b). . . . . . . 39

3.12 A storage ring could be used to store multiple embryos each in their own

droplet. This would allow embryos to be loaded all at once, and then

individually processed when needed. . . . . . . . . . . . . . . . . . . . . . 42

3.13 Schematic showing droplet transfer to removable freezing device. (a) Em-

bryo initially inside device, (b) droplet moved outside of closed structure,

(c) droplet moved using open digital microfluidic electrodes, (d) droplet

transported onto freezing device, and (e) freezing device removed and di-

rectly plunged into liquid nitrogen. . . . . . . . . . . . . . . . . . . . . . 44

x

3.14 (a) Droplet is in contact with substrate in air and (b) floating over a small

oil layer when in an oil bath. . . . . . . . . . . . . . . . . . . . . . . . . . 45

4.1 (a) Schematic of partially filled electrodes providing space for additional

on-chip tools or as a window for imaging. (b) Example designs of partially

filled electrode configurations considered. Electrodes are 1mm x 1mm (c)

Image of droplet on series of partially filled electrodes. . . . . . . . . . . 48

4.2 Simulation results. (a) Droplet half space mesh and swept uniform mesh

on droplet surfaces. (b) Actuation force on droplet for conventional elec-

trode on leading and trailing droplet surfaces. Reverse actuation force is

generated on trailing droplet surface as backward interface begins to move

onto electrode. (c) Induced forces increase linearly with electrode fill ra-

tio, which was changed by varying the width of the horizontal bars in the

electrode. (d) Force is independent of vertical location of removed area

from electrode. The leading edge of the droplet is fixed at the midpoint of

the electrode for panels (c) and (d). . . . . . . . . . . . . . . . . . . . . . 51

4.3 Simulation results: electrode design with crescent-like filled areas at the

entrance and exit of the electrode produces increased force at beginning

and end of droplet motion to create initial force to ensure droplet motion

is generated. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

xi

4.4 Experimental comparison of electrode designs. In experiments, maximum

droplet actuation frequency was measured at different fill percentages for

normal and improved designs. Number of horizontal bars in the electrode

was kept constant, thus reducing the bar width, reduces electrode fill per-

centage. Please note that reduction in maximum actuation frequency is

almost proportional to the reduction in the bar width similar to the force

reduction simulations. Experiments were conducted with deionized water

at 75 V rms and 15 kHz by actuating droplet back and forth across a series

of 5 electrodes at increasing speed until droplet motion could not keep up

with actuation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.5 (a) Single mouse embryo morphology on partially filled Cr electrodes. Up-

per image uses bright field transmission DIC imaging showing superior

detail compared to reflection microscopy imaging used in lower image.

(b) RBC viability measured with different osmolarity by counting cells

through unfilled regions on chip. N = 150 600 for each osmolarity point. 56

B.1 Camera communication is opened using IMAQ commands. Some initial

options are written such as packet size, gamma control, and exposure time.

This camera feed is fed into the image capturing loop. . . . . . . . . . . . 67

B.2 To simplify camera adjustments, color balancing is done automatially us-

ing a single button on the front panel. When the ’Auto Balance’ is pressed

the gain and white balance are automatically adjusted using functions on

the camera. These settings can be rebalanced during experimentation if

lighting or sample conditions change. . . . . . . . . . . . . . . . . . . . . 68

xii

B.3 Inside the main imaging loop, frames are taken from the camera and dis-

played on an imaging window in the front panel. When the ’Record’ button

is initially pressed, a AVI container file using an MPEG compressor is cre-

ated using the current timestamp as the filename to ensure that there are

no filename conflicts. After this initially loop, if the ’Record’ button is

still pressed, individually frames are added to the AVI container file. A

timer is also used to display the frame rate on the front panel. . . . . . . 69

B.4 Communication with the signal generator is achieved over USB communi-

cation. Standard VISA commands are used to set the function, voltage,

and frequency values. Communication is sent to the signal generator when-

ever either the voltage, or frequency control values on the front panel are

changed. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

B.5 Macro control is initiated by reading in excel files containing the list of

droplet actuations for each command. These files are named to match their

function and each button on the front panel corresponds to an individual

excel file. Once the file is opened it is fed into the main droplet control loop. 71

B.6 In the main droplet control loop, references to each electrode button are

first build into an array so that they are indexed. Each line of excel file

is then read and the corresponding electrodes turned on. Options are

available to leave the reservoir electrodes on at all times to ensure that the

large reservoir droplet is not moved. The time between actuations can also

be set using a front panel control. Communication with an LCR meter

was also initiated to sense droplet locations after each step. However, the

droplet motion was found to be very robust, making the extra complexity

of droplet sensing not necessary. . . . . . . . . . . . . . . . . . . . . . . . 72

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Chapter 1

Introduction

1.1 Cryopreservation

Cryopreservation is a key technology in biology and clinical practice. It is a process

where substances, cells, or even whole tissue are cooled to low enough temperatures

that all enzymatic and chemical activity is essential stopped, alowing the preservation

of the sample for an indefinite amount of time. The first successful pregnancy following

cryopreservation was reported in 1983 [1]. Stem cells [2], sperms [3, 4], and embryos [5]

are now routinely frozen and preserved for use at a later time. Patients who undergo

therapeutic procedures that can place their fertility at risk, such as chemotherapy, have

the option of preserving their reproductive cells, such as sperms or oocytes, for future

use through in vitro fertilization techniques (IVF) [6–9]. Furthermore, extra fertilized

embryos after an IVF procedure can also be frozen for use at a later time. The length

of time frozen has been shown not to have a significant impact on clinical pregnancy,

miscarriage, implantation, or live birth [10, 11]. However, in these cases the number of

viable cells for preservation can be low making the survivability rate and reproducibility

of cryopreservation techniques critical. In particular, preservation of embryos or oocytes

is challenging since these cells are highly sensitive, and the cell number is very small.

1

Chapter 1. Introduction 2

Fig 1: Vitrification schematic

ES VS1 VS2 Freeze

Figure 1.1: Vitrification process involves sequentially bathing the embryo/oocyte in cry-oprotectant baths according to a strict timing protocol. Cryoprotectant concentrationincreases in later baths and the embryo is plunged immediately after exiting the finalbath. The first bath is typically called Equilibrium Solution (ES), and later baths calledVitrification Solution (VS).

The two commonly used cryopreservation techniques for freezing embryos are the

slow freezing method and the vitrification method. Both techniques aim to minimize the

damage caused by freezing that is largely due to the formation of intracellular ice crystals

that can produce a mechanical shear and rupture the cells [12]. Conventionally, cells are

frozen through the slow freezing method where cells are placed in a large freezer that

can accurately control the freezing rate down to liquid nitrogen temperatures, with low

concentrations of cryoprotectants [13]. During slow freezing extracellular water freezes

away from the embryo, using a seeding technique, which creates an osmotic gradient that

draws water out of the cell until it finally freezes without the formation of intracellular ice

crystals [14]. This procedure requires sophisticated equipment to control the freezing rate,

which ranges between 0.3 and 1.0◦C/min, and produces a relatively poor survivability

rate [15, 16].

On the other hand, vitrification offers an alternative approach in which cells are frozen

at extremely high rates, usually by directly plunging the sample into liquid nitrogen, af-

ter bathing them in a sequence of high concentration cryoprotectants [17]. Vitrification

reduces intracellular ice formation, which is the primary cause of cell death, by freez-

ing the sample in a glass-like state before the molecules have a chance to form crystal

structures. This results in a higher cell survival rate after thawing compared to conven-


tional slow freezing without the need for a seeding procedure or a programmable freezer

[15, 18]. However, vitrification requires precise washing sequences and timings in each

cryoprotectant medium since higher concentrations are used and there is a significant

risk of toxicity if overexposed (Fig. 1.1). The process is expensive in terms of technical

skills required. In IVF clinics, processing an embryo/oocyte in cryoprotectant medium

typically costs a highly skilled embryologist 10 to 15 minutes.

Three factors are essential when implanting and designing a vitrification protocol;

cooling rate, viscosity of mediums used, and volume of medium frozen [14, 19]. The

cooling rate needs to be as high as possible to ensure that vitrification occurs before

crystal formation. The viscosity of the medium also works to enable vitrification as

with higher concentrations of cryoprotectants the glass transition temperature is raised,

allowing vitrification to happen earlier in the cooling process. The volume of the medium

surrounding the embryo while freezing is also critical as smaller volumes allow faster heat

transfer and thus a higher chance of vitrification. Surrounding containers and the thermal

conductivity of the holding devices can also work to slow the cooling/warming rate.

1.2 Digital Microfluidics

Traditionally, microfluidic research has largely centered around channel-based systems in

which fluids are controlled through microchannels using pumps, valves, and mechanical

mixers [20]. These continuous systems are well suited for applications such as diagnos-

tics for blood, urine and saliva [21]. Digital microfluidics, or electrowetting on dielectric

(EWOD), uses an alternative approach in which individual liquid droplets are manipu-

lated in discrete steps. This creates a new set of design opportunities better suited for

handling low sample number applications with a high degree of control. Larger cells, or

solids can also be handled without the risk of clogging channels [22]. An array of elec-

trodes is used to control droplet movement. Electrode configurations are not application


ON ON

ON ON ON ON ON

ON ON ON ON

Transport

Dispensing

Splitting

1 2 3

1 2 3

1 2 3

Figure 1.2: Overview of fundamental digital microfluidic operations. Droplets can betransported around the device for mixing or movement to another segment of the device.Smaller droplets can be dispensed from larger holding reservoirs. Droplets of largervolume can be split into two daughter droplets.

specific and thus can be generalized and reconfigured for different applications. Elec-

trode widths typically vary from 0.5-2.0 mm (or larger for reservoir droplets) creating

droplets in the nL to mL range [23]. These small liquid volumes require small reagent

consumptions, and allow for faster reaction rates. Loading and unloading of samples can

also be easily done by simply pipetting droplets on the edge of the device or by building

the device with a capillary tube inside the structure [24].

Droplet motion in a digital microfluidic system is typically created by applying a high

voltage across a dielectric coated gap in which the liquid droplet is contained. This creates

a large electric field within the insulating dielectric layer, which in turn produces a surface

charge on the leading edge of the droplet. This creates a net horizontal force that moves

the droplet towards the actuated electrode [23, 25, 26]. This force is only generated when

a section of the droplet overlaps the actuated electrode. This leads to the requirement

that the gap between adjacent electrodes be as small as possible to ensure droplet motion.

However, this has the added benefit that droplet force is generated locally on the device

allowing droplets to be isolating from another with no dependence on each others motion.

More details modelling the force generation in a digital microfluidic system are shown in


Chapter 3.

Similar to the evolution of the logic gate in the digital world, using the binary oper-

ation of a single electrode, basic operations can be built and performed to accomplish a

large amount of liquid handling tasks. Transport is the basic operation allowing droplet

transfer between sections or modules on the device. Merging of droplets is achieved by

actuating multiple droplets on the same electrode, and mixing is achieved by transport-

ing the droplet in a pattern until its composition is homogenous. Larger droplets can

be split into two daughter droplets by simultaneously stretching a droplet in opposite

directions, and dispensing is similarly performed by stretching a single side of a larger

reservoir droplet until a smaller daughter droplet is dispensed. These basic actions are

shown in Figure 1.2 and are the fundamental operations used to build more complicated

laboratory sample processing tasks. Figure 1.3 also shows a mixing operation using dyes

to visualize droplet mixing.

1.3 Merging Digital Microfluidics and Vitrification

Digital microfluidics is a power tool for sequential sample processing and has been used

in tasks such as PCR, cell culture, and immunoassays [27–29]. In this work, digital mi-

crofluidics is used, for the first time, to automate embryo preparation for the vitrification

procedure, aiming to lower the high labour cost and ultimately helping further spread

the use of vitrification in IVF clinics.

The key to automating the vitrification process is to replicate the washing and timing

steps of a given protocol while also keeping complete control of the embryo (as the

sample population may be quite small making each individual cell very critical). Digital

microfluidics is proposed to be uniquely positioned to address this task since droplets on

the digital microfluidic platform can act as micro-vessels to move an embryo and subject

it to a series of cyroprotectants of different concentrations, as required by IVF vitrification


Figure 1.3: Droplet mixing is achieved by first merging droplets by actuating on toa common electrode. The merged droplet is then transported around the device untilmixing is homogeneous. Red dye was used to help visualize this process.


Manual C

ryop

rote

ctan

t S

tage

DMF Chip

Ext

ract

ion

Sta

ge

Free

zing

S

tage

ES

VS VS

VS

embryo

micropipette

liquid nitrogen

Figure 1.4: Schematic showing differences between manual vitrification approach, whichrequires manual pipetting between mediums in cryoprotectant stage, and the digitalmicrofluidic (DMF) approach, which moves the embryo between mediums on chip. Thechip automates the high skill portion of the procedure providing labor cost savings andopportunities for parallelization.


protocols (Figure 1.4). Compared to channel-based microfluidic vitrification [30–32], the

device reported in this work does not undesirably park the embryo to a confined area

and has less intricacy of embryo introduction and retrieval onto and from the device.

By keeping the embryo in a single droplet, as opposed to microchannels or wells, the

system is also able to constantly track and control the embryos locations throughout the

procedure to avoid cell loss.

1.4 Motivation

Overall, by creating a standardized platform for embryo vitrification several majors ben-

efits are realized. Firstly, this platform would drastically lower the cost of fertility preser-

vation hopefully opening up this procedure to a wider range of patients. This is especially

relevant as with the development of new cancer treatments creating an increase in the

number of surviving patients, greater attention is being focused on providing a high

quality of life to the survivors [7]. Secondly, by eliminating the need for a highly skilled

embryologist, a higher number of clinics and labs could perform this procedure open-

ing the geographic availability. Finally, by automating the procedure, variability from

manual operation would be eliminated. This would standardize the implementation of

vitrification protocols allowing for stronger comparisons between timings and mediums.

If adopted, this would significantly aid researchers in progressing cryopreservation work.

1.5 Dissertation Outline

This thesis is organized into the following chapters. Chapter 2 describes the digital

microfluidic platform, including electronics and software, and the fabrication process used

to produce the devices. Chapter 3 presents a system for automation of the vitrification

process for mammalian embryos along with initial mouse embryo testing. Chapter 4

presents a study on partially filled electrode designs including finite element simulations,


preliminary experimental results, and suggestions for improved designs. Finally, Chapter

7 summarizes the impact of this system.

Chapter 2

System Setup

Building a digital microfluidic platform involves coordinating several different systems

and pieces of equipment together in order to generate precise, fast droplet controls. On

top of this basic function, systems also need to be incorporated to help automate other

elements of the users tasks to quicken and streamline operation, as certain experiments

are time sensitive. The microchips themselves also require cleanroom fabrication tech-

niques, which posses an additional challenge. Here we outline our platform, software

and fabrication techniques, which aim to address this issues and create a robust, efficient

experimentation environment. Figure 2.1 shows an overview of the platform including

Fig 1: Overview system

LabVIEW controller

digital microfluidic chip

high voltage signal generation

imaging system

Figure 2.1: Overview of system elements and central LabVIEW interface.

10

Chapter 2. System Setup 11

Figure 1.5: Chip fabrication overview

top glass plate

bottom glass plate

embryo

Teflon

parylene C

Cr electrode

ITO

liquid droplet

embryo

top glass plate

bottom glass plate

Teflon parylene C Cr electrode ITO

liquid droplet Figure 2.2: Overview of device fabrication. Photolithography was used to patternchromium electrodes, chemical vapour deposition was used to deposit parylene C, andTeflon wass finally spin coated on the device.

sample holder, electrical system, imaging system, and LabVIEW control interface.

2.1 Device Fabrication

Since digital microfluidic chips require a micro scale gap between adjacent electrodes to

create droplet motion, cleanroom fabrication techniques were used. A cleanroom is a

low particulate laboratory that reduces the amount of particulate that can settle on a

sample during fabrication and create defects in the device. Lithography is the major

cleanroom procedure in our chips and involves the transfer of a geometric pattern from

a photomask to a chromium layer through the use of photosensitive materials [33]. Our

chips contain an electrode gap of 20 µm, which is smaller then can be produced in printed

circuit board processes, yet is relatively large on lithography standards. The Emerging

Communications Technology Institute (ECTI) facilities at the University of Toronto were

used which is a class 1000 cleanroom (maximum 1000 particles ≤ 0.5 µm per cubic foot of

air). Glass slides pre coated with 100 nm of chromium were used as the starting substrate

(Deposition Research Labs Inc., St. Charles, MO). This eliminated the need for metal

evaporation of chromium saving both time and money. The lithography process, which

patterns the chromium layer, and the additional layer deposition processes, is outlined


below.

2.1.1 Photoresist Coating

Photoresist is the key component in the lithography process. It has the unique property

that after exposure to UV light, its solubility to a developer compound changes. This

means that after developing, either the exposed, or unexposed, areas will dissolve in

the developer depending on the type of photoresist used. Positive photoresist references

photoresist in which the exposed areas are washed away by the developer, and negative

photoresist references photoresist in which the unexposed areas are washed away by

the developer. This enables patterns to be transferred onto a photoresist coating by

selectively exposing different areas of the photoresist by using a photomask. Initially, the

substrate was primed with Hexamethyldisilazane (HMDS) before spin coating Shipley

S1811 photoresist (3000 rpm, 30s). The sample was then soft backed at 115◦C for 2

minutes to harden the layer by removing solvents. At this point a uniform thickness

layer of photoresist has been deposited on the chromium coated glass slides (Fig. 2.1.1).

Fig 2: Spin coater

bottom glass plate

chromium positive photoresist

(a) (b)

(a) Photoresist Coating

Fig 2: Spin coater

bottom glass plate


(a) (b)

(b) Spin Coater

Figure 2.3: Photoresist is spin coated on the device at 3000 rpm for 30s.


2.1.2 UV Exposure

The sample is next exposed to UV light for 10s through a photo mask printed on a

transparency at high resolution (Pacific Arts and Design, Markham ON). The areas of

photoresist that are exposed become more soluble to the developer and are thus removed

during developing. For our devices, the exact dimension of the gap is not critical, only

that is small enough to create reliable motion. If the gap is larger, motion can be slightly

slower, and may not be reliable in some cases. However, if the gap is too small, and there

is a connection between adjacent electrodes, a short circuit is formed. In this case these

two electrodes act as a single electrode and the device is not functional. Thus to avoid

this risk the sample is overexposed, making the gap larger then on the photomask, to

ensure no short circuits are formed (Fig. 2.1.2).Fig 3: Mask aligner

bottom glass plate

UV EXPOSURE


photomask

(a) (b)

(a) UV Exposure

Fig 3: Mask aligner

bottom glass plate

UV EXPOSURE


photomask

(a) (b)

(b) Mask Aligner

Figure 2.4: Photoresist is patterned by exposing to UV light through a photomask for10s.

2.1.3 Developing

To remove the exposed areas of photoresist, the sample is submerged in MF-321 developer

for approximately 2 minutes. The timing in this step is forgiving as sufficient time only

needs to be given to remove all the photoresist from the exposed areas, and the unexposed

areas will only slowly start to dissolve themselves. After some experience development


completion can be judged by eye. The sample is rinsed with deionized water to remove

any residual developer. The sample is then hard baked at 115 ◦C for 60 s to further harden

the photoresist by removing solvents. At this point the pattern has been transferred from

the photomask to the photoresist and is visible to the user (Fig. 2.1.3).

Fig 4: Developer

bottom glass plate


(a) (b)

(a) Developing

Fig 4: Developer

bottom glass plate


(a) (b)

(b) Photoresist Developer

Figure 2.5: During development areas of photoresist exposed to UV light are washedaway in the developer. The desired pattern is then left in the photoresist layer.

2.1.4 Chromium Etching

An acidic solution, CR-4 etchant, is used that is specifically designed to etch chromium

patterns. The sample is etched for approximately 3 minutes in CR-4 chromium etching

solution, with some gentle agitation to speed up the process. Similar to developing,

the timing in this process is not critical. Over-etching can result in undercut features

and wider gaps, which dont affect the devices. This etching removes chromium from

all areas not protected by photoresist, exposing the glass substrate underneath. The

sample is rinsed with deionized water to remove any residual etchant. The samples can

now be investigated under a microscope to ensure that the devices are defect free. If

short circuits or deformed electrodes are found, the devices should be disposed off and

the fabrication process restarted as there is no point in moving forward with defective

devices (Fig. 2.1.4).


Fig 5: Etching

bottom glass plate


(a) (b)

(a) Chromium Etching

Fig 5: Etching

bottom glass plate


(a) (b)

(b) Chromium Etchant

Figure 2.6: During etching, areas of the chromium layer not protected by photoresist,are dissolved in the etchant. This transfers the design pattern from the photoresist layerto the chromium layer.

2.1.5 Photoresist Removal

Finally, photoresist still remaining on top of the chromium pattern is removed with AZ-

300T stripper. Samples are placed in a beaker of stripper and placed in an ultrasonic

bath to ensure complete removal of photoresist. Samples are then well rinsed with deion-

ized water, as the stripper is viscous and can leave residue if not rinsed properly. The

lithography process is now complete and the desired chromium pattern produced. The

lithography process should be completed in a single session as the properties of the pho-

toresist can change if left for even a couple of days, due to solvent evaporation. However,

the samples can now be left in this state for as long as needed before continuing on to

the next coatings (Fig. 2.1.5).

2.1.6 Dielectric Coating

Parylene C is used as a dielectric coating to stop electrolysis. It is a conformal coating and

applied in a vapour deposition process at room temperature using a dedicated coating

machine. Parylene C is purchased as a solid in small pellets. A weighed amount of

Parylene is placed into the coater and the thickness of the resulting layer is dictated


Fig 6: Ultrasonic bath

bottom glass plate

chromium

(a) (b)

(a) Photoresist Removal

Fig 6: Ultrasonic bath

bottom glass plate

chromium

(a) (b)

(b) Ultrasonic Bath

Figure 2.7: Photoresist is removed using a stripper solution in an ultrasonic bath. Theultrasonic bath helps removes photoresist from all sections of the device.

by this weight, as the coater will automatically run until all the available Parylene is

deposited and cannot deposit a partial coating. A two stage heating process is used to

heat the Parylene into a monomer before it enters the chamber with the samples and is

deposited as a clear uniform layer. Since this deposition is at room temperature, there are

no concerns about melting of any components of the sample. This means that tape can

also be placed over the outer electrode connections to keep these areas free of Parylene.

Parylene in these regions would need to be removed anyway to insure a strong connection

with the edge board connector, so using tape during this coating speeds up the process.

Overall, a 2 µm thick layer of dielectric is produced (Fig. 2.1.6).

2.1.7 Teflon Coating

Teflon AF1600 is used as a final hydrophobic coating and applied by spin coating at 1600

rpm for 60s and then baking at 160 ◦C for 10 minutes. The Teflon mixture is a liquid

composed of 1 % w/w Teflon AF1600 in FC-40. Teflon AF1600 is purchased as a solid

and dissolved in the liquid FC-40 by baking on a hotplate at 80 ◦C for several hours

beforehand. The top ground slide of the devices is a glass slide pre coated with indium

tin oxide (ITO). ITO is used, as it is a clear conductor and a robust coating. These slides


Fig 7: Parylene C coater

bottom glass plate

chromium parylene C

(a) (b)

(a) Parylene C Coating

Fig 7: Parylene C coater

bottom glass plate

chromium parylene C

(a) (b)

(b) Vapour Deposition Coater

Figure 2.8: Parylene C is used as a dielectric layer and deposited using a chemical vapourdeposition process with a dedicated coater.

are also coated in Teflon using the same technique (Fig. 2.1.7).Fig 8: Teflon spin coating

bottom glass plate

chromium parylene C Teflon

(a) (b) top glass plate

(a) Telon Coating

Fig 8: Teflon spin coating

bottom glass plate

chromium parylene C Teflon

(a) (b) top glass plate

(b) Teflon Spin Coating

Figure 2.9: Teflon is applied by spin coating a uniform layer, and then baking to removesolvents.

2.1.8 Assembled Devices

Finally, the devices are assembled by placing the top ITO slide, which acts as a ground

plane, over the patterned device using two pieces of double sided tape as a spacer

(Fig. 2.10). There is a balance to achieve strong droplet motion while also being able

to dispense and split droplets. A large gap can produce stronger motion but make dis-


Fig 9: Assembled device

ground connection

digital microfluidic device

double-sided tape spacers

sample holder

Figure 2.10: Assembled device is held in place using a set of 3D printed parts. Top ITOslide is connected to ground using an alligator clip.

pensing very difficult, as the droplet surface is harder to split. The ITO slide is offset

so that one end is hanging and can be connected to ground using an alligator clip. The

other edge of the slide is positioned so that the outermost device electrode is half covered

and half exposed. This allows droplets to be pipetted on the edge of the ITO slide and

actuated into the device either to fill reservoirs, or input samples.

2.2 Electrical System

The electrical system is the core component in a digital microfluidic system as it drives

the functionality of the system; compared to other components built around control and

monitoring of the device. Droplet actuation is created by creating a large electric field

across the device. This causes charge to build up in the dielectric layer underneath the

contact line of the droplet. A corresponding surface charge is then formed in the droplet

creating a net electrostatic force in the horizontal direction. Very little current flows

through the device, by design, yet a large voltage is needed to generate the necessary

electric field. The signal is created using a function generator (Agilent 33522A) and a high

voltage amplifier able to provide up to 100 Vrms at kHz frequencies (Trek PZD350A).

The driving signal is typically 15 kHz with an amplitude of 50-80 Vrms. Care needs to be


Fig 10: Electrical system

function generator

voltage amplifier

relay array edgeboard connector

device electrodes

LabVIEW controller

Figure 2.11: Schematic of electrical system showing relay array control and high voltagegeneration.

made when dealing with high voltage as there is the potential for significant harm. The

voltage amplifier current limit should be set low to minimize this risk, especially since

high current is never needed in the functioning device. Lowering the current limit also

reduces the damage caused by electrolysis in a malfunctioning device or electrode.

It is essential that the voltage is computer controlled to allow for reliable, fast droplet

motion. An array of mechanical relays was used for this task. Two state relays were used

connecting each electrode to either ground or high voltage. In their off state the relays

were connected to ground. Since these relays required more then 10 V and significant

current (approximately 50 mA per relay when on) a transistor was used to connect

the relay control switch to a DC power supply. The transistors were controlled with a

digital signal provided by the LabVIEW DAQ. An overview of this system is shown in

Figure 2.11.

A total of 24 relays were used which provided ample electrodes for this papers ap-

plications (Fig. 2.12). Also, in certain chip designs multiple electrodes can be connected

together to conserve relays if either parallel motion is desired, or each area of the device

is sufficiently distanced to not interfere with each other. Electrodes were connected to

the relays by an edge board connector and a ribbon cable (Fig. 2.13). This allowed for

chips to be quickly connected by simply sliding them into the connector. The connector

Chapter 2. System Setup 20Fig 11: Relay circuit

connection to ribbon cables

relay circuit boards (24 relays total)

NI DAQ connector

Figure 2.12: Relay array containing 24 mechanical relays, connection to LabVIEW sys-tem, and connection to ribbon cables to device.

had a pitch of 2.54 mm allowing for 27 electrodes to be placed on each edge of the chip.

This allowed for 2-3 devices to fit on a single glass slide depending on the number of

electrodes used.

2.3 Control and Software System

LabVIEW, the graphical programming environment, was chosen as the programming

language for our platform. LabVIEW excels in interfacing with laboratory equipment,

quickly creating a graphical user interface, and integrating with National Instruments

array of physical tools (such as analog/digital input and output devices). For digital

microfluidics, this fit exactly the needs of the platform and allowed more time to be

spent on device design and experimentation as apposed to a control system built in a

more traditional environment, such as C++, where the specifics of device control and

the user interface would have to be dealt with.

Programming in LabVIEW is done through two windows; the front panel and the


Fig 12: Edgeboard connector

(a) (b)

open connected (a) Open

Fig 12: Edgeboard connector

(a) (b)

open connected (b) Closed

Figure 2.13: Electrodes are interfaced to relay system using an edgeboard connector.Allows for fast changing between devices. Parylene is removed from electrodes to ensuregood connection.

wiring diagram as shown in Figure 2.14. The front panel is the user interface and is

where all buttons, controls, and imaging windows are placed. Elements are added to

this window from a tools palette and are resizable and movable on the front panel.

The wiring diagram is where all logic and programming is done. All components on

the front panel have a corresponding block in the wiring diagram. Data flows through

the wiring diagram by connecting these blocks together with wires, instead of the more

conventional approach of using named variables. Data is manipulated through function

blocks that have both inputs and outputs for wires. Loops or case structures are created

by surrounding sections of code in a rectangular fence. Wires can also run into and out

of these structures. This style of programming allows for additional tools to be quickly

added to the front panel and to be ran in parallel in the wiring diagram. This allows the

system to quickly shrink and expand without spending time building a scaling interface

and infrastructure.

Droplet motion is controlled using a National Instruments Data Acquisition (DAQ)

board, which is connected to the relay array. The LabVIEW control interface is high-

lighted in Figure 2.15. Control of each electrode is done through 24 buttons on the front

Chapter 2. System Setup 22Fig 13: Front panel, wiring

(a) (b)

front panel wiring diagram (a) Front Panel

Fig 13: Front panel, wiring

(a) (b)

front panel wiring diagram (b) Wiring Diagram

Figure 2.14: Overview of LabVIEW programming environment. The Front Panel is theuser interface and holds controls and indicators usable by the operator. The WiringDiagram controls data flow and is done using a graphical programming paradigm wheredata flows through wires instead of local variables.

panel. This allows the user to manually actuate selected electrodes and also move, scale,

and colour the buttons to better represent the layout of the current device. Although

these buttons are controls, their state can also be programmatically controlled within the

wiring diagram. This allows the user to also design automated sequences of droplet actu-

ations. This system reads in excel files that contain an ordered list of droplet actuations

(more than one electrode can be actuated at once) and executes them with a fixed time

interval, which can also be adjusted on the front panel. The idea is to break down droplet

protocols into a series of smaller operations such as mixing, dispensing, transport, and

rearranging. This allows the user to execute the complete protocol by simply executing

these smaller segments in order. This has allowed for quicker testing and debugging as

the user is able to assess the situation after each segment and make any small manual

adjustments needed, or even repeat a segment if necessary. Electrodes are left on after

each segment to prevent the droplets from floating away. Segments can also be stopped

if needed using the Stop button.

Control over the electrical signal is also performed by communication over USB with


Fig 14: Front droplet control

signal control

manual droplet control

droplet sequence

control timing

interval

Figure 2.15: Overview of droplet control LabVIEW elements. A pattern of buttons isused for manual control in the shape of the present device. Droplet sequence control isaccomplished by reading electrode sequences from an excel file.

the function generator. LabVIEW contains pre made functions for standard protocols

over USB, Ethernet, and GPIB. The user is able to turn the function generator on and

off, change the voltage level, and change the frequency. This allows for quick changes

to be made on the fly during experimentation as different liquids and geometries require

different voltage strengths to achieve motion while avoiding electrolysis.

2.4 Imaging System

Different imaging systems can be used in a digital microfluidic platform. Transmission

microscopy often allows for higher resolution imaging and even the use of fluorescent

imaging. However, if conventional metal electrodes are used, these block the imaging

path and prevent the imaging of anything in the droplet over an electrode. This can be

circumvented by fabricating transparent electrodes, usually out of ITO, although this is

not common practice. An alternative is to use reflection microscopy in which the light

source is normal to the electrodes; passing through the objective lens, reflecting off the

device, and passing back into the objective lens. This produces a strong metallic reflection

off of the electrodes allowing for imaging of its contents. In our platform reflection


Fig 15: Imaging overview

camera

motorized zoom/focus

2x objective lens

Figure 2.16: Overview of imaging apparatus. Zoom and focus are motorized allowingthem to be controlled from within LabVIEW.

microscopy was used with a motorized optical system allowing for computer controlled

adjustment of both the zoom and focus as shown in Figure 2.16. Since this papers

primary application dealt with mouse embryos, this allowed quick switching between

magnifications during experiments to either monitor droplet motion at low magnification

or embryo morphology at high magnification.

All of these controls were built into the LabVIEW platform as shown in Figure 2.17.

Video recording was also provided with each video saved using a timestamp as its file

name. This allowed the user to quickly record multiple videos during an experiment

and then organize and change the name of important videos after experimentation was

complete.


Fig 16: Labview imaging

imaging window

video recording

motorized zoom control

focus control

Figure 2.17: LabVIEW front panel highlighting imaging components. Zoom and focusare controlled either by sliders on the right side of the screen, or by a toggle allowingquick switching between predefined low and high zoom settings. Videos are recordedwith a toggle and files saved using timestamps as their name.

The sample was also held in place with a set of 3D printed parts. These parts

were made to work with a salvaged microscope stage. This allowed for devices to be

quickly secured to the stage using only two screws and to be manually moved during

experimentation in the horizontal plane for imaging different areas of the device.

Chapter 3

On-Chip Embryo Vitrification

3.1 Materials

Mouse embryos were gathered from the Canadian Mouse Mutant Repository (CMMR;

Toronto, ON). Embryos were produced by superovulating a female and were gathered

2.5 days past conception, which corresponds to most embryos being in the 8-cell stage.

Vitrification solution usually contains antifreezing agents or cryoprotectant, such as

dimethyl sulfoxide (DMSO), some small molecular size glycols (e.g., ethylene glycol), or

sucrose [34]. A mixture of several cryoprotectants is often used to reduce the individ-

ual specific toxicity as well as using both permeable and impermeable mediums [15]. A

combination of DMSO and sucrose was used to follow the protocol used by the embryo

suppliers. The vitrification solution (VS) was made by diluting DMSO in serum-free

KSOM medium (EMD Millipore, Billerica, US) at 33% concentration, with 1.0 M su-

crose. The equilibrium solution (ES) was at half concentration of VS (i.e., 16.5% DMSO

+ 0.5 M sucrose). VS was preloaded on the DMF chip before each experiment. The first

mixing step, which mixes the VS with embryo culture medium (i.e., serum-free KSOM),

generates the ES.

26

Chapter 3. On-Chip Embryo Vitrification 27

Fig 4: Overview chip design

waste#reservoir#

embryo#inlet/outlet#

dispensing#reservoir#

reservoir#inlet#

mixing/spliOng#

waste&reservoir&

embryo&inlet/outlet&

dispensing&reservoir&

reservoir&inlet&

mixing/spli6ng&

edge#of#top#ITO#slide#

edge#of#top#ITO#slide#

Figure 3.1: Chip design showing regions for vitrification medium dispensing and embryoinlet/outlet. The top ITO slide is placed on the device in a manner that exposes portionsof the top electrodes in the dispensing reservoir and the leg of the T-shape to allow formedium and embryo loading respectively.

3.2 Device Design

Voltages applied to actuate droplets were 55-75 Vrms at 15 kHz. Cyroprotectant droplets

were actuated inside silicone oil (2.0 cSt, Gelest Inc., Morrisville, PA) to reduce friction

and evaporation. Different regions of the device were designed to achieve the general

digital microfluidic fucntions of transporting, mixing, dispensing, and splitting droplets.

A large reservoir was used to hold and dispense the high concentration cryoprotectant

medium, as shown in Figure 3.1. This reservoir was split up into many sections to

handle variations in liquid volume in the reservoirs as droplets are dispensed during the

vitrification protocol. The second reservoir was used as a waste reservoir and, thus, was

split into two large electrodes only as less control was needed.

A central inverted T-shaped array of electrodes was used for droplet transport, mixing,

and splitting. Electrodes in this array were interdigitated to allow droplet overlap with

adjacent electrode and increase electrodynamic forces applied on droplets. Top electrode

in the leg of the T-shape was an input/output region where half of the edge electrode was


Fig 5: Droplet input/output

embr

yo

inpu

t em

bryo

re

triev

al

2 1 3

2 1 3

top

slid

e ed

ge

mic

ropi

pette

Figure 3.2: Embryo is input and extracted by actuating electrodes at edge of top glassslide.

exposed out of the ITO slide to enable embryo loading. For embryo loading, the embryo-

carrying droplet was pipetted on the exposed half of the electrode and then actuated

into the device through the covered half [35]. For extraction, the embryo-carrying droplet

was moved to this edge electrode where it bulges out of the device and is retrieved by

a standard micropipette. This same mechanism was used to fill the dispensing reservoir

before the embryo and reagents were loaded.

3.3 Embryo Loading and Retrieval

As shown in Figure 3.2, to input an embryo, a small embryo-containing droplet was

pipetted onto the loading electrode and then actuated into the device. This technique

minimized exposure of the embryo to outside air. Extraction of the embryo was completed

in the opposite manner by transporting the embryo-containing droplet to the edge of the

device and retrieved by a micropipette. Once the embryo was extracted from the device,

it was directly frozen in the micropipette inside liquid nitrogen.


3.4 Cryoprotectant Mixing

An embryo is input into the device in a small droplet of embryo culture medium, and

100% cryoprotectant is input into the device in larger volumes (reservoir inlet in Fig. 3.1).

The cryoprotectant bathing procedure is then performed through a serial mixing/splitting

process (Fig. 3.4 and Fig. 3.5). This is accomplished by mixing the embryo-containing

droplet with a vitrification solution droplet (VS), thus increasing the concentration of

cryoprotectant around the embryo. The resulting droplet is then split into two smaller

droplets with the daughter droplet containing the embryo identified and kept, while

the other droplet is moved to the waste reservoir. After the first mixing step the droplet

reaches 50% cryoprotectant concentration (i.e., equilibrium solution or ES), the embryo is

kept in the ES droplet for 10 minutes. Then the cryoprotectant concentration is increased

again by droplet mixing and splitting. Contrary to ES medium, embryo volume sharply

decreases in VS medium and does not recover (Fig. 3.9). The overall mixing profile

generated with a single dispensing reservoir is shown below in Eqn. 3.1.

C(n) = 100

(1− 1

2n

)%, (3.1)

where C(n) is the concentration of the droplet, and n is the number of mixing steps. This

protocol mimics a typical two-step human embryo/oocyte protocol. However, mouse

embryos are typically frozen with only a single step protocol where the embryo is directly

transferred to VS medium and then frozen. On our chip this corresponds to simply

removing the 10-minute waiting period in the ES medium step. Both protocols were

performed but the results presented were done with the mouse embryo timings to follow

precisely the protocol provided to us by CMMR.

After complete transfer of the embryo into the VS medium, the droplet containing

the embryo is moved toward the edge to be collected by a micropipette (Fig. 3.2), and

then plunged into liquid nitrogen. To verify success of the vitrification process using


Fig 2: Droplet mixing

1. Initial droplets

2. Droplets merged

3. Droplet mixed

4. Droplet Split

Figure 3.3: Overview of general approach for mixing on a digital microfluidic platform.

digital microfluidics, embryos vitrified on device were thawed back and confirmed to

have recovered in volume and have healthy morphology.

Contrary to conventional vitrification protocols and manual operation, which subject

embryos to sudden changes in medium concentration, the digital microfluidic approach

gradually increases the VS medium concentration, (Fig. 3.5), which is generally accepted

by IVF practitioners to be more benign to embryos due to lower osmotic stress [36]. This

gradual medium concentration increase is not feasible to achieve in manual operation.

3.5 Thawing

The thawing procedure for embryo vitrification is much simpler then the freezing process.

Embryos were thawed by plunging in a bath of culture medium with 1.0 M of sucrose.

The sucrose helps to draw the cryoprotectants out of the embryo by osmotic pressure


1.  Embryo input using edge of top ITO slide

2.  Droplet dispensed from reservoir

3.  Droplets merged and mixed until homogeneous

4.  Droplet split into two daughter droplets

5.  Embryo containing droplet kept, remaining droplet moved to waste

Figure 3.4: Schematic showing implementation of mixing protocol using this device de-sign. Daughter droplet containing embryo in step 4 is identified manually.

Chapter 3. On-Chip Embryo Vitrification 32Fig 3: Mixing Curve

(b)

(d)

VS

(a)

(c)

CM

0102030405060708090

100

0 2 4

drop

let c

onc.

(%)

number of mixing steps

theoretical

measured

ES VS

(b)

(d)

VS

(a)

(c)

CM Figure 3.5: Mixing profile showing generation of ES medium and VS medium. Exper-imental droplet concentrations were found by using image processing to measure thedroplet volumes before and after each mixing step.


Fig 3: Mixing Curve

(b)

(d)

VS

(a)

(c)

CM

0102030405060708090

100

0 2 4

drop

let c

onc.

(%)

number of mixing steps

theoretical

measured

ES VS

(b)

(d)

VS

(a)

(c)

CM

Figure 3.6: (a) Embryo (red circle) contained in culture medium (CM) droplet. (b)Embryo droplet mixed with VS droplet. (c) Droplet split into two droplets (left containsembryo). (d) Droplet containing embryo is kept and other droplet is sent to waste.Process is repeated to increase VS concentration.

to minimize toxicity. The embryo is left in this bath for approximately 10 minutes over

which its volume slowly increases back to its original size. After this point the embryo

is transferred to culture medium without sucrose and is returned to the incubator. It

is generally advised that the embryo not be used for any additional applications until it

has had several hours to equilibrate in the incubator.

The thawing temperature gradient is equally important to the freezing gradient and

should be as quick as possible. The micropipette containing the embryo in liquid nitrogen

is thus transferred to the initial sucrose bath in one smooth, quick motion. The sucrose

bath is also of sufficient size to have a large enough thermal mass to quickly warm

the micropipette tip. The same thawing procedure was used for manual and digital

microfluidic operation. Since this procedure is much simpler and not the main focus of a

vitrification protocol, it was held constant to put more emphasis on the cryoprotectant

preparation protocol prior to freezing.


Fig 7: Embryo morphology

Healthy

16 cells

Sept 27 continued

Sept 30 - S: 0/2 (0%)

Oct 7 - S: 4/5 (80%), D: 3/5 (60%)

Oct 9 - S: 1/1 (100%), D: 1/1 (100%)

Sept 27 - S: 4/5 (80%), D: 2/4 (50%)Oct 11 - S: 2/2 (100%), D: 2/2 (100%)

Unhealthy

16 cells compacted

8 cells

shrunken

ruptured cells

darkened cells

Figure 3.7: Sample healthy and failure cases for survival rate based on morphology beforeand after freezing.

3.6 Measurement Scheme

Two measures were used to evaluate the performance of digital microfluidic vitrification,

including survival rate and development rate. Survivability was measured by examining

the morphology of the embryo before and after freezing [37] (Figure 3.7). Embryos

were considered unhealthy if they had an abnormal shape, membrane damage, leakage

of cellular content or degeneration of their cytoplasm [38]. The development rate was

determined by culturing survived embryos for an additional 24-48 hours after freezing

and thawing (Figure 3.8). If the cell number within the embryo increased or it developed

to the blastocyst stage, it was counted as developed. Control samples of non-vitrified

embryos were also cultured to identify the base development rate of the mouse embryo

population. Only embryos morphologically judged to be healthy were used for either

manual or digital microfluidic testing. Only embryos that had healthy morphology after

freezing were cultured following similar procedures to other vitrification studies [39].


Fig 8: Development embryo

Oct 9 - S: 1/1 (100%), D: 1/1 (100%)Oct 9 - S: 1/1 (100%), D: 1/1 (100%)

Sept 27 continuedSept 27 continued

Sept 27 - S: 4/5 (80%), D: 2/4 (50%)Sept 27 - S: 4/5 (80%), D: 2/4 (50%) Oct 7 continuedOct 7 continued

Healthy Unhealthy Before

Freezing After 1-2

Days Before

Freezing After 1-2

Days

developed to 16 cell compact

stage

developed to blastocyst

all cells dead

cells no longer growing and

coloring is dimmer

Figure 3.8: Sample healthy and failure cases for development rate based on culturing foran additional 24-48 hours after freezing.

3.7 Vitrification Results

Table 3.1 summarizes the results, showing comparable survival and development rates

between manual and automated trials. However, with a larger population size I hypoth-

esize the digital microfluidic chip to produce a higher survival rate due to its gradient

generation.

Table 3.1: Summary of vitrification results.Survival Rate Development Rate

Control (Non vitrified) 100% (14/14) 93% (13/14)Manual 73% (11/15) 91% (10/11)DMF Chip 77% (10/13) 90% (9/10)

3.8 Volume Monitoring

Additionally, since the embryo is constantly imaged on video, its volume can be measured

throughout the procedure and used to measure the quality of both the embryo and the

protocol. Figure 3.9 shows one such assessment.


0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1

0 2 4 6 8 10

volu

me

(nor

mal

ized

)

time (minutes)

liqui

d ni

troge

n fre

ezin

g

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1

0 0.25 0.5 0.75 1 1.25

volu

me

(nor

mal

ized

)

time (minutes)

ES# VS# VS#50µm

liqui

d ni

troge

n fre

ezin

g

(a) (b)

Figure 3.9: Embryo cell volume measurements for a typical (a) human and (b) mouseprotocol on chip. Volumes were normalized to initial volume. The initial volume dip inthe human protocol matches the volume dip over the mouse protocol.

3.9 Discussion

Previous vitrification studies for embryos (4-16 cell stages) reported survival rates in the

range of 80-100% [5]. Vitrification using digital microfluidics in this work showed a sur-

vival rate of 77% that is similar to the manual vitrification trials (survival rate of 73%).

This lower survival rate, compared to the results in the literature, can be mainly at-

tributed to our use of a micropipette (vs. vitrification straw) inside liquid nitrogen. The

micropipette tip is a standard plastic pipette for embryo manipulation and had an inner

diameter of 125 µm (The STRIPPER micropipetter, Origio). Much research has gone

into developing different mechanical structures (e.g., straw-type carriers [40], cryotube

[41], Cryotop [42], and mesh-type carriers [43]) to increase the heat transfer rate [14].

Structures have also been developed and modeled with the specific measurement of suc-

cess being a high heat transfer coefficient [44]. In these designs materials are chosen with

a higher heat transfer coefficient and the liquid volume containing the embryo is mini-

mized. Some researchers have also directly dropped the embryo containing droplet into a

liquid nitrogen bath to maximize the freezing rate [45]. Using these devices would involve

transferring the embryo from the DMF chip manually onto the device (e.g., vitrification

straw) with minimal liquid volume. Since this would introduce an extra manipulation


step by hand and this work focuses on proving the feasibility of using digital microfluidics

for embryo processing, the embryos were directly frozen inside the micropipette tip in

this work.

Using micropipette tips in liquid nitrogen was not ideal and negatively affected the

survival rate; however, since it was held constant between manual and DMF trials, the

relative survival rates can still be compared. Additionally, the development rate, which

was measured using embryos that survived freezing, was high and comparable with other

vitrification studies. This measurement in some ways removes the effect of the limited

manual embryo handling skills and shows the potential of the microfluidic device for

automated processing of embryos for vitrification.

Due to the programmable characteristic, one key benefit to the digital microfluidic

approach is the ability to implement/test a number of vitrification protocols for efficacy

comparisons. Table 3.10 lists example human and mouse vitrification protocols, all using

different cryoprotectants, number of mediums, and timings. However, at their core, all

of these protocols involve the controlled increase in cryoprotectant concentration over

a given period of time with the initial equilibrium solution typically containing half the

cryoprotectant concentration as the full strength vitrification solution. This is convenient

for DMF device design as it always produces a 50% concentration after the first mixing.

This means that a typically two-step protocol involving an initial 50% concentration ES

step, following by a short 100% VS step, can be easily realized by following the mixing

curve shown in Figure 3.5 with a pause after the first mixing step to allow the embryo

to reach equilibrium.

Figure 3.9 shows how most two-step protocols follow the same mixing curve, only

differing by their timings. This shows that although the protocols in Table 3.10 involve

significantly different amounts of manual manipulation, when performed on the digital

microfluidic chip, they follow the same mixing procedure with the only difference lying

in their timings. For our current trials, a mouse protocol provided by CMMR was used


CMMR DMSO, sucrose 8-cell VS

Dhali et al. DMSO, EG, sucrose zygote to morulae VS

Khosravi-Farsani et al. EG, sucrose oocyte VSi

Irvine DMSO, EG, sucrose 2PN to blastocyst

Origio EG, PG, sucrose ii 4-cell to 16-cell VS

Kitazato DMSO, EG, sucroseii 2PN to 8-cell VS1iii VS2iii

iOnly step containing sucroseiiExact concentrations not giveniiiSame medium but different bath

ES3ES2ES1

15time (minutes)Protocol Cryoprotectants 1 3 5 7 9 11 13Stage

ES

ES

ES VSi

ES

mou

se&

human&

Figure 3.10: Comparison of mouse and human vitrification protocols. [38, 47–52]

which involved a single mixing step. This allowed us to better conduct our manual trials

as it required less manipulation; however, more complicated protocols can be readily

performed by adding more dispensing reservoirs on chip and filling them with lower

concentrations of cryoprotectant. This would allow for a high number of producible

concentrations, especially in the low concentration range. Multiple reservoirs could also

be used to implement protocols using different cryoprotectant compositions throughout

their procedure [46].

One limitation of the digital microfluidic platform was handling culture mediums

containing high serum concentrations. Serum contains a mixture of proteins that can

absorb on the Teflon coated surfaces of the device, eventually accumulating to the point

that the surface becomes hydrophilic, making droplets immovable [53]. Some strategies

have been developed to help overcome this problem, such as the use of Pluronic additives

[54], silicone oil baths [55], and superhydrophobic surfaces [56]. Pluronic additives were

avoided in our work as embryos are highly sensitive to additives. Superhydrophobic

surfaces were also avoided as they require significant additional fabrication efforts. A

silicone oil bath was used which did increase droplet movability; however, this approach

did not work with sufficient effectiveness for conventional embryo culturing mediums that

contain high serum concentrations. Therefore, in this work, a serum free culture medium

was chosen for this proof-of-principle study. Further work will focus on droplet movability

improvement for serum-containing culture medium and the automation of transferring

embryos onto vitrification devices (e.g., straw or Cryotop).


c(t)

t t1 t2 t3

100%

c1

Protocol c1 t1 t2 t3 t4

CMMR 100% 0.5 1 - -

Dhali et al. 50% 1 3 3.5 4

Irvine 50% 1 8 9 10

Origio 50% 1 10 10.5 11

Kitazato 50% 1 12 13 14

(a) (b)

t4

(a) Mixing curve c(t)

t t1 t2 t3

100%

c1

Protocol c1 t1 t2 t3 t4

CMMR 100% 0.5 1 - -

Dhali et al. 50% 1 3 3.5 4

Irvine 50% 1 8 9 10

Origio 50% 1 10 10.5 11

Kitazato 50% 1 12 13 14

(a) (b)

t4 (b) Protocol timings

Figure 3.11: Implementation of common vitrification protocols on a digital microfluidicchip with a single dispensing reservoir. Timings and concentrations are shown in (a),and the generalized mixing curve is shown in (b).


Additionally, some platforms have also incorporated temperature control during the

vitrification process to improve embryo culture conditions [46]. Due to the planar nature

of the digital microfluidic platform, and the relatively thin glass substrate used, a warming

plate could easily be integrated in the future. This is especially critical if the system is

expanded to handle multiple embryos at once as this may involve storing multiple embryos

on the chip initially, before vitrifying each embryo individually. This would require these

embryos to remain in storage on the device for longer periods of time in which case

maintaining optimal culture conditions would be critical.

Overall, a platform was designed and tested to automate the liquid handing and

timing portion of a vitrification procedure. The platform is general in its design allowing

clinicians and researchers to easily change protocols or develop new ones. This is of

particular importance as vitrification protocols are not presently standardized making

a platform that could do this highly valuable. Once a common platform is adopted,

it is easier for clinicians to develop protocols by comparing their findings with another

clinicians using the same platform. Embryo quality is also more easily assessed when

the same tools are used. Manual vitrification involves a large amount of manipulation

by pipette which leads to high variance between clinics. The manner that washing is

performed, the amount of liquid aspired with the embryo and the speed transferring

between baths all affect the outcome. In order for vitrification to be used widespread, a

system like this would be highly beneficial.

The programability of the platform also enables researchers to attempt more compli-

cated protocols that were not realistic when the embryo needed to be manually transferred

between baths. Typical procedures now consist of at most 4-5 steps, with this platform

procedures can be easily broken down into smaller segments and the complexity greatly

increased. In addition, continuous video monitoring could allow for closed-loop feedback

protocols. In other words, if image processing tools were added such that the embryo

volume is monitored in real-time in the LabVIEW interface, this information could be


used to adjust protocol timings on the fly. For instance, as the embryo volume shrinks,

this information could be used to forecast the time at which it will reach minimum vol-

ume and be removed from the device at this point. This modeling could be done without

modeling the physiology of the embryo but instead by fitting a curve to previous em-

bryo experiments and using the same mathematical function on the real-time embryo

approach. LabVIEW does contain extensive image processing tools as it is often used for

monitoring of process lines in industrial settings, so this proposal is likely buildable, but

beyond the scope of this project.

The obvious benefit of this system is the cost savings. Presently, vitrification requires

a large labour cost as an embryologist or highly skilled technician is needed for embryo

handling. This system eliminates the need for a high dexterity user and replaces it with

computer control. The infrastructure for the system is relatively inexpensive requiring

only signal generation equipment, an imaging system, and a computer for control. The

microfluidic chips are presently made in a cleanroom at significant cost. However, since

the devices only consist of a patterned chromium layer and two coatings on top, they

are easily manufactured at a large scale at a drastically lower cost. These chips would

be disposable in a clinical setting and switched between patients to ensure zero cross-

contamination.

Presently, the system reduces the skill required to complete the vitrification protocol,

yet the required time remains the same and cannot be reduced as it is dictated by

the embryo physiology leading to the protocol timings. However, the system can be

parallelized to reduce the time needed to freeze a large embryo sample. One easy way to

accomplish this would be to simply use multiple devices. The clinician could pipette a

single embryo onto each device, perform the protocol using a fully computer controlled

procedure, and then remove each embryo from the device. This would allow the device

design to remain simple, although if volume monitoring were needed, the imaging system

would be much larger to simultaneously monitor each device. Alternatively, the device

Chapter 3. On-Chip Embryo Vitrification 42Fig 1: Storage ring

embryo storage ring

Figure 3.12: A storage ring could be used to store multiple embryos each in their owndroplet. This would allow embryos to be loaded all at once, and then individually pro-cessed when needed.

design could be grown to handle multiple embryos at once. Embryos should still be

handled individually as with a human patient the embryo count can be very low (¡10),

so a storage system could be used to allow the user to load all the embryos at once,

then individually take each embryo from the storage system and through the vitrification

protocol.

A key addition necessary in scaling this technology up is an automated tool for trans-

ferring the embryo from the digital microfluidic device to the liquid nitrogen. This is

essential, as it would first eliminate variability and error from the user manually pipetting

and transferring the embryo off the device and plunging it into the liquid nitrogen. The

timing during the high concentration portion of the protocol is very critical so any added

time can have a significant effect. Also, in terms of cost reduction, the user presently

needs to load the embryos on the device and then return to transfer them into the liquid

nitrogen. If the user did not need to be present for this task the time savings would be

significantly larger. One method proposed would be to build a device that could be en-

tirely plunged into liquid nitrogen. This would completely eliminate the issue of embryo


extraction but posses new problems. First, by freezing a larger device the heat transfer

will be significantly slower in both freezing and thawing. This is known to decrease the

survivability of the embryos. Secondly, after the embryo has been frozen it needs to go

through a thawing protocol and be extractable. Reconnecting a thawing device is not

easily possible and if attempted would require a large amount of robotic tools. Overall,

freezing the device seems initially attractive, due to its simplicity in freezing, but makes

the task of thawing extremely difficult.

An alternate method would be to automate the entire embryo extraction procedure

and then robotically transfer the embryo into liquid nitrogen. The simplest method would

be to use a computer controlled robotic pipette and syringe pump to mimic what the

user is presently doing. However, this requires significant robotics and image processing

to achieve so ideally a solution easier to implement would be desired.

Another approach would be to add to the microfluidic system. Since droplets can be

moved inside and outside of the top ITO slide depending on the coverage of the ITO slide

on the exit electrode, the droplet could first be removed from the closed device. Secondly,

electrodes could be continued to be patterned on the open section of the device. Open

digital microfluidics is a commonly used approach although it suffers from evaporation

problems and the inability to split droplets. The embryo droplet could be moved on the

open device onto a detachable section of electrodes. This section of the device could

then be removed and plunged in liquid nitrogen. This is illustrated in Figure 3.13. This

method of transferring from a closed to open digital microfluidic platform has recently

been used to integrate a digital microfluidic chip with a mass spectrometer [57].

This proposed mechanism raises significant feasibility questions yet if the microfluidics

are possible, would produce a simple device that fully automates the process. Addition-

ally, in the design of the detachable device carrier, a flexible substrates can also be used

to guide droplet movement and open new design possibilities [58].

A silicon oil bath was used on the device to aid in droplet motion by lowering the


Fig 2: Open DMF

(a) (b) (c)

(d) (e)

top ITO slide

detachable section

Figure 3.13: Schematic showing droplet transfer to removable freezing device. (a) Embryoinitially inside device, (b) droplet moved outside of closed structure, (c) droplet movedusing open digital microfluidic electrodes, (d) droplet transported onto freezing device,and (e) freezing device removed and directly plunged into liquid nitrogen.


Fig 3: Oil floating

(a) (b)

droplet in air droplet in oil

Figure 3.14: (a) Droplet is in contact with substrate in air and (b) floating over a smalloil layer when in an oil bath.

effective friction of the system [59]. For basic liquids, like deionized water, this is not

normally needed but can still aid in high speed applications. For vitrification mediums,

which were of higher viscosity, the oil bath was found to be necessary for strong, reliable

motion. However, this also has a second set of benefits particularly useful for critical

biological applications. First, evaporation is eliminated from the device. This is critical

as if the concentration of vitrification mediums is altered the entire process becomes

unusable. Typically when the procedure is performed manually a mineral oil bath is

used to cover each well of liquid. Secondly, electrode contamination is reduced. Without

oil small amounts of contaminates have been found to remain on electrodes after use

making multiple uses of a single device not advisable [60]. With an oil bath, the droplet

is actually floating in the device with a small oil layer above and below the device,

eliminating residue [55].

Since an electric field is present in the device to drive the droplets, this also raises

the concern that this may pose negative effects on the health of the embryo. However, it

has been shown that for small electrodes and low driving frequencies that no significant

damage is done to mammalian cell lines [61]. Additionally, architectures requiring much

lower voltages are have been developed which may result in even lower cellular stress due

to electric effects [62].


3.10 Conclusion

This chapter described a digital microfluidic device for automated embryo processing for

vitrification applications. The results demonstrated that a high embryo development rate

can be achieved using the automated approach. Advantages of this approach, compared

to manual operation and channel-based microfluidic vitrification, include automated op-

eration, cryoprotectant concentration gradient generation, and feasibility of loading and

retrieval of embryos. The device permits one to readily modify/test vitrification proto-

cols with significant reduction in labor costs. Further development can possibly facilitate

new vitrification protocol development and clinical IVF practice.

Chapter 4

Partially Filled Electrodes

4.1 Introduction

Digital microfluidic devices, as a platform technology, enable programmed manipulation

of small droplets on arrays of microelectrodes [63, 64] for performing tasks such as PCR,

cell culture, and immunoassays [27–29]. In digital microfluidic devices, conductive or

polar droplets are moved under the effect of electrodynamic forces. These forces are

generated by the electric field induced from the energized electrode beneath the droplet

[65].

To increase capabilities of digital microfluidic devices, researchers have begun inte-

grating additional elements within electrodes such as impedance spectroscopy [66], elec-

trophoresis electrodes for particle separation [67], absorbance detection windows [53],

heaters [68], field effect transistor-based biosensors [69] and cell culture patches [27].

As these elements are added, electrode area for droplet actuation is reduced, and force

generation becomes weaker. Electrodes cannot simply be scaled larger to compensate

for this lower actuation force since this would also increase the droplet volume thereby

compromising miniaturizing advantages and increasing device foot print. Therefore, un-

derstanding the effect of reducing electrode area on generated forces and droplet speed

47

Chapter 4. Partially Filled Electrodes 48

(a) (b)

(c)

partially filled electrode

filled/opaque chromium region

removed/transparent region

Figure 4.1: (a) Schematic of partially filled electrodes providing space for additional on-chip tools or as a window for imaging. (b) Example designs of partially filled electrodeconfigurations considered. Electrodes are 1mm x 1mm (c) Image of droplet on series ofpartially filled electrodes.

is necessary.

Vitrification in previous chapters was done under reflective imaging, while transmis-

sion microscopy is the most often used imaging platform. Therefore, this chapter presents

a detailed study evaluating different partially filled electrode designs and suggesting de-

signs that combine a high actuation force with a large reduction in electrode area that

permits integration of large elements within the electrode. As a sample application, a

non-ITO, partially filled Cr electrode design that permits the imaging of droplet contents

using standard transmission microscopy is presented.

4.2 Modelling and Simulation

To quantify the effect of removing electrode area, actuation forces on different designs of

partially filled electrodes were simulated using finite element analysis (COMSOL Multi-

physics). Relevant dimensions used in simulation are summarized in Table 4.1. Droplet

was modelled as a non-deformable lossy dielectric and hence, the conservation of charge


Table 4.1: Parameters used in numerical simulations.

summarized in Table I. Droplet was modelled as a non-deformable lossy dielectric and hence, the conservation ofcharge [Eq. (1)] and Laplace [Eq. (2)] applies12

rðrrVÞ ¼ 0; (1)

rðerVÞ ¼ 0; (2)

where e ¼ ere0 (er is the relative permittivity of the medium,and e0 is the permittivity of free space), r is the electricalconductivity of the droplet, and V is the electric potential.Actuation forces are then calculated by integrating theMaxwell stress tensor over the surface of the droplet, assum-ing negligible magnetic fields,13 according to

F ¼ð

ST $ nds; (3)

Tij ¼ eðEiEj % 0:5 dijE2Þ; (4)

where Tij is the Maxwell stress tensor. Actuation forces werecalculated over a series of droplet locations along its actua-tion path. Since the integration of the Maxwell stress tensoris dependent on mesh geometry and density, a uniform sweptmesh is used to allow for a fine mesh on the droplet leadingand trailing faces without considerably increasing the totalnumber of elements, Fig. 2(a). Swept layers are distributedin the vertical direction following a geometric sequence sothat layers are denser near the underlying dielectric layer toavoid a large jump in element size at the droplet and dielec-tric interface.

Fig. 2(b) shows force curves as a function of dropletposition. This approach enables quantifying the quality ofany arbitrary electrode design. Forces on the leading surfaceof the droplet are dominant except at the end of dropletmotion when the trailing surface produces a reverse force onthe droplet, which leads to droplet motion stopping on thecenter of the electrode. Position is defined as the distancefrom the left edge of the electrode to the leading edge of thedroplet.

The goal of simulations was to address two questionsin the design of partially filled electrodes: how theremoved electrode area affects actuation forces andwhere electrode removal is most critical to force genera-tion. To answer the first question, a horizontally stripedelectrode design with variable strip width was simulated,and the relationship between electrodynamic force andelectrode fill percentage was determined [Fig. 2(c)].Force was found to have a linear relationship with fillpercentage, which is expected as the majority of force isgenerated at the advancing three phase contact line of thedroplet. In these simulations, the length of the contact

FIG. 2. Simulation results. (a) Droplethalf space mesh and swept uniformmesh on droplet surfaces. (b) Actuationforce on droplet for conventional elec-trode on leading and trailing dropletsurfaces. Reverse actuation force isgenerated on trailing droplet surface asbackward interface begins to moveonto electrode. (c) Induced forcesincrease linearly with electrode fill ra-tio, which was changed by varying thewidth of the horizontal bars in the elec-trode. (d) Force is independent of verti-cal location of removed area fromelectrode. The leading edge of thedroplet is fixed at the midpoint of theelectrode for panels (c) and (d).

TABLE I. Parameters used in numerical simulation.

Parameter Value

Electrode dimensions 1& 1 mm2

Dielectric thickness 10 lm

Droplet radius 740 lm

Droplet contact radius 600 lm

Droplet height 140 lm

Droplet conductivity (r) 5.5& 10%6 S/m

Relative permittivity of droplet 80

Relative permittivity of air 1

Relative permittivity of dielectric 2.5

Actuation voltage 100 V

024103-2 Pyne et al. Appl. Phys. Lett. 103, 024103 (2013)

This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP:142.150.190.39 On: Wed, 16 Oct 2013 02:31:57

(Eq. 4.1) and Laplace (Eq. 4.2) applies [70].

5(σ5 V ) = 0 (4.1)

5(ε5 V ) = 0 (4.2)

where ε = εrε0 (εr is the relative permittivity of the medium, and ε0 is the permittivity of

free space), σ is the electrical conductivity of the droplet, and V is the electric potential.

Actuation forces are then calculated by integrating the Maxwell stress tensor over the

surface of the droplet, assuming negligible magnetic fields [71], according to

F =

∫S

T · nds (4.3)

Tij = ε(EiEj − 0.5δijE2) (4.4)

where Tij is the Maxwell stress tensor. Actuation forces were calculated over a series of

droplet locations along its actuation path. Since the integration of the Maxwell stress

tensor is dependent on mesh geometry and density, a uniform, swept mesh is used to allow

for a fine mesh on the droplet leading and trailing faces without considerably increasing

the total number of elements, Fig. 4.2(a). Swept layers are distributed in the vertical


direction following a geometric sequence so that layers are denser near the underlying

dielectric layer to avoid a large jump in element size at the droplet and dielectric interface.

Fig. 4.2(b) shows force curves as a function of droplet position. This approach enables

quantifying the quality of any arbitrary electrode design. Forces on the leading surface

of the droplet are dominant except at the end of droplet motion when the trailing surface

produces a reverse force on the droplet, which leads to droplet motion stopping on the

center of the electrode. Position is defined as the distance from the left edge of the

electrode to the leading edge of the droplet.

The goal of simulations was to address two questions in the design of partially filled

electrodes: how the removed electrode area affects actuation forces; and where electrode

removal is most critical to force generation. To answer the first question, a horizontally

striped electrode design with variable strip width was simulated, and the relationship

between electrodynamic force and electrode fill percentage was determined (Fig. 4.2(c)).

Force was found to have a linear relationship with fill percentage, which is expected as

the majority of force is generated at the advancing three phase contact line of the droplet.

In these simulations the length of the contact line in contact with the electrode increases

approximately linearly as the fill percentage is increased (i.e. as the horizontal strips are

widened), which leads to the linear increase in force.

To answer the second question of assessing the location effect of electrode area removal

on the induced force, an electrode with a single horizontal window was studied. The

distance between the window and the centerline of the electrode was then varied to test

the position dependence in the perpendicular axis (Fig. 4.2(d)). Vertical position of

the removed portion of the electrode was found to have no effect on force generation.

Since most of the actuation force is generated on the contact line, this result can be

explained by considering the amount of force lost in the horizontal window. As the

window moves farther from the centerline and the contact line curves, a larger length of

it is placed inside the window. However, the generated force per length is normal to the


(b)

Forc

e (µ

N)

Fill Ratio (%) 30 40 50 60 70 80 90 1

1.5

2.0

2.5

3.0

3.5

Varied bars’ heights

Forc

e (µ

N)

Horizontal Bar Gap Position (µm) 50 100 150 200 250 300 350 400

2.5

2.7

2.9

3.1

3.3

3.5

3.7

3.9 4.0

Gap Position

(c) (d)

(a)

Forc

e (µ

N)

Position (µm)

Total Leading Surface Trailing Surface

0 200 400 600 800 1000 1200 -1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

Figure 4.2: Simulation results. (a) Droplet half space mesh and swept uniform mesh ondroplet surfaces. (b) Actuation force on droplet for conventional electrode on leadingand trailing droplet surfaces. Reverse actuation force is generated on trailing dropletsurface as backward interface begins to move onto electrode. (c) Induced forces increaselinearly with electrode fill ratio, which was changed by varying the width of the horizontalbars in the electrode. (d) Force is independent of vertical location of removed area fromelectrode. The leading edge of the droplet is fixed at the midpoint of the electrode forpanels (c) and (d).


droplet surface; therefore, the component of the actuation force parallel to the centerline is

proportional to the vertical projection of the contact length. Since this projection remains

constant as the window moves away from the centerline, there is no force dependence

on the position of the removed portion. These results lead to the conclusion that force

generation is dominated by total electrode area and at any particular position, by the

vertical projection of the three phase contact line in contact with the filled area of the

electrode.

In addition to its effect on the generated force on the droplet, filled areas of the

electrode should be distributed in a manner that does not compromise the initial pulling

force at the beginning of the motion where generated forces are at its lowest. This is

critical to produce a large enough force to induce motion and overcome line pinning.

Large initial pulling forces can be achieved by making sure that the entrance area of the

electrode is always completely filled as a crescent that matches the droplets leading edge,

as shown in Fig. 4.3. Removal of a portion of the electrode, in the form of horizontal

stripes or any other form, can then follow to allow for the integration of various elements

into the electrode. This shape guarantees that maximum force is applied to the advancing

contact line at the crucial stage of initiating droplet motion. This filled crescent is

mirrored at the other side of the electrode to provide strong droplet motion in both

directions, but it can be removed if droplet motion is desired only in one direction allowing

for an even larger area for the integration of other device elements.

4.3 Experimental Evaluation

To experimentally evaluate the effect of reducing the energized electrode area, the max-

imum droplet actuation frequency (i.e., how many electrodes can the droplet cross per

second) on partially filled and solid electrodes is compared. Droplet motion was con-

trolled using a custom LabVIEW program controlling an array of 24 relays connected to


0

10

20

30

40

50

60

Max

imum

Act

uatio

n Fr

eque

ncy

(Hz)

Crescent design

Horizontal bars

Entrance/exit fill increase initial force

Forc

e (µ

N)

Position (µm)

Improved Entrance/exit Design Horizontal Bars

0 200 400 600 800 1000 1200 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8 (a) (b)

100 µm 75 µm 40 µm 100 % Filled

Horizontal Bar Width (d)

unfilled

d

Figure 4.3: Simulation results: electrode design with crescent-like filled areas at theentrance and exit of the electrode produces increased force at beginning and end ofdroplet motion to create initial force to ensure droplet motion is generated.

a high voltage amplifier (Trek PZD350A, Medina NY). Motion was then recorded using

a CCD camera (Basler acA1300-30gc, Exton PA) connected to a motorized zoom lens

(Navitar 12X Body Tubes, Rochester NY). As expected, partially filled electrodes showed

a decrease in maximum actuation frequency and droplet motion speed, since only seg-

ments of the contact line are exposed to high electric fields [65]. Nevertheless, actuation

frequencies of over 10 electrodes per second were achieved on devices with partially filled

electrodes at a low electrode fill area of 40%, Fig. 4.4. This speed is sufficient for many

biology and clinical applications of digital microfluidic devices [72].

Removed areas on partially filled electrodes can be used for integrating other elements

(e.g. heaters, detectors and hydrophilic patches) into the electrodes; furthermore, they

can be useful for on-chip imaging of droplet contents using transmission microscopy.

Transmission microscopy imaging, particularly on inverted microscopes is a standard

platform used in biology labs and clinics. Since completely filled, non-transparent elec-

trodes made of metallic materials (e.g., Cr or gold) are not compatible with transmission

microscopy imaging, ITO is typically used to construct electrodes [24, 28, 68]. However,


0

10

20

30

40

50

60

Max

imum

Act

uatio

n Fr

eque

ncy

(Hz)

Crescent design

Horizontal bars

Entrance/exit fill increase initial force

Forc

e (µ

N)

Position (µm)

Improved Entrance/exit Design Horizontal Bars

0 200 400 600 800 1000 1200 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8 (a) (b)

100 µm 75 µm 40 µm 100 % Filled

Horizontal Bar Width (d)

unfilled

d

Figure 4.4: Experimental comparison of electrode designs. In experiments, maximumdroplet actuation frequency was measured at different fill percentages for normal andimproved designs. Number of horizontal bars in the electrode was kept constant, thusreducing the bar width, reduces electrode fill percentage. Please note that reduction inmaximum actuation frequency is almost proportional to the reduction in the bar widthsimilar to the force reduction simulations. Experiments were conducted with deionizedwater at 75 V rms and 15 kHz by actuating droplet back and forth across a series of 5electrodes at increasing speed until droplet motion could not keep up with actuation.


ITO is more expensive in materials and fabrication than metallic electrodes, and the

invisible ITO electrodes can pose challenges in device debugging. In comparison, par-

tially filled metallic electrodes can be easier to construct while being compatible with

transmission microscopy imaging.

Therefore, intentionally constructed electrodes using metallic materials were used in

this study to demonstrate their compatibility with standard transmission microscopy

imaging. Partially filled Cr electrodes were used to actuate single mouse embryos. Mor-

phology of the mouse embryos on partially filled electrodes was imaged under transmis-

sion differential interference contrast (DIC) microscopy, which revealed detailed embryo

morphology and cell structures (Fig. 4.5(a)). In applications, if the droplet medium is

relatively viscous, the cells position would remain relatively constant within the droplet.

In this case, a set of partially filled electrodes negative of each other can be used to ensure

that the cell can be imaged across these electrodes. If the cells position does not remain

constant with a less viscous droplet, the droplet can be easily actuated back and forth

on partially filled electrodes until the cell rests on a non-filled region. Since it is viable to

remove half of the electrode area without affecting generated forces significantly, a few

actuations is typically sufficient to achieve cell imaging with this technique.

Also droplets containing human red blood cells (RBCs) were actuated using partially

filled Cr electrodes. PBS droplets containing RBCs were mixed with differing amounts of

DI water (i.e. varied osmolarity). RBC viability as a function of medium osmolarity was

measured on chip. Lysed RBCs were easily identifiable on our partially-filled electrodes

under transmission microscopy, Fig. 4.5(b). The non-filled section on a single electrode

was large enough to sample a significant portion of the population to gather meaningful

statistics.


0 10 20 30 40 50 60 70 80 90

100

75 125 175 225 275

% R

BC

s ly

sed

Osmolarity (mOsm/L)

(b)

intact cell

lysed cell 100µm

!!

!!

100 µm

(a)

Figure 4.5: (a) Single mouse embryo morphology on partially filled Cr electrodes. Upperimage uses bright field transmission DIC imaging showing superior detail compared toreflection microscopy imaging used in lower image. (b) RBC viability measured withdifferent osmolarity by counting cells through unfilled regions on chip. N = 150 600 foreach osmolarity point.

4.4 Conclusion

In summary, this chapter investigated, numerically with preliminary experimental verifi-

cation, the effect of removing sections of electrodes in digital microfluidic devices on the

generated electrodynamic forces and the maximum achievable droplet speed. Generated

electrodynamic forces were found to be linearly dependent on electrode fill percentage

and independent of position of the horizontal non-filled areas. To maintain high initial

pull for the droplet, entrance and exit areas of the electrodes were left completely filled.

As application examples, partially filled metallic electrodes were constructed and their

compatibility with standard transmission microscopy imaging demonstrated. These re-

sults are meaningful for guiding the design of digital microfluidic devices that require

the integration of other elements, such as detectors, heaters, and cell culture patches, on

electrodes.

Chapter 5

Summary

5.1 Conclusions

In this work, a digital microfluidic platform was first built, including device fabrica-

tion, an imaging system, a high voltage control system, and a LabVIEW interface. The

main purpose of this system was to automate the cryopreservation process of vitrifi-

cation, which can achieve a higher survival rate then conventional methods yet has a

high labour cost requirement. This system automated the liquid handling and timing

tasks in embryo vitrification. Technical advantages of this approach, compared to man-

ual operation and channel-based microfluidic vitrification, include automated operation,

cryoprotectant concentration gradient generation, and feasibility of loading and retrieval

of embryos. The device permits researchers to readily change/test protocols. Significant

labor costs were also reduced by eliminating the need for highly skilled operators. The

device showed cell survival and development rates of 77% and 90%, respectively, which

are comparable to the control groups that were manually processed.

As a secondary task, the effect of removing sections of electrodes in digital microflu-

idic devices on the generated electrodynamic forces and the maximum achievable droplet

speed was studied through simulation and experimentation. Generated electrodynamic

57

Chapter 5. Summary 58

forces were found to be linearly dependent on electrode fill percentage and independent

of position of the horizontal non-filled areas. To maintain high initial pull for the droplet,

entrance and exit areas of the electrodes were left completely filled. As application ex-

amples, partially filled metallic electrodes were constructed and their compatibility with

standard transmission microscopy imaging demonstrated. These results are meaningful

for guiding the design of digital microfluidic devices that require the integration of other

elements, such as detectors, heaters, and cell culture patches, on electrodes.

5.2 Future Directions

The following are examples of future works that can be undertaken:

1. Testing the physiological effects of Pluronic additives on mammalian embryos and

whether it increases droplet movement enough to allow the use of conventional

embryo culture mediums which contain serum.

2. Development of a system to transfer an embryo from the digital microfluidic device

into liquid nitrogen by either using a detachable open digital microfluidic segment

or by robotically pipetting the embryo containing droplet off of the device.

3. Development of an image processing algorithm to monitor embryo volume in real-

time to provide physiological feedback and allow the development of closed loop

vitrification protocols.

4. Establishment of an on-chip storage system to decrease required technician work-

load in vitrifying a large sample of embryos. Integration of a warming platform

may also be required to provide the culture conditions to allow embryos to safely

remain on the device for longer periods of time.

5. Integration of capacitance measurements to detect droplet locations and enable

closed loop droplet motion protocols.

Chapter 5. Summary 59

6. Design of a system to automate the thawing portion of the cryopreservation proce-

dure.

7. Testing of the current system’s ability to vitrify other biological samples of interest

such as cells, tissues and sperms.

Appendix A

Detailed Fabrication Recipe

Fabrication protocol for digital microfluidics chips:

1. Process is started with precoated 100 nm chromium glass slides (50 x 75 mm). If

these are not available chromium can be deposited by metal evaporation. However,

using precoated slides was found to save significant amounts of time and was even

cost effective, as metal evaporation requires 3-4 hours.

2. Slides are cleaned by rinsing in acetone, isopropanol, and de-ionized water (DI

water). Wafer handling tweezers are used to hold slides and rinsing is done into

a beaker so that solvents can be properly disposed of after cleaning. Acetone and

isopropanol are kept in squeeze bottles while DI water is available through the wet

bench taps by a DI water generation system in the cleanroom. The isopropanol

is used to remove residue left by the acetone, and the DI water provides the final

clean. Ensure that while rinsing the slide is repositioned in each step to reach the

area of the substrate underneath the tweezers.

3. Substrates are blown dry with a nitrogen gun. To ensure complete dehydration,

slides are placed on a hotplate at 115◦C for 5 minutes. Slides are then removed

and given another 5 minutes to cool to room temperature.

60

Appendix A. Detailed Fabrication Recipe 61

4. Slides were then liquid primed with P-20 (20% HMDS) primer. Slides are placed

on a chuck in the spin coater one at a time and the vacuum is engaged, which

holds the slide to the chuck. It is a good idea to first test the vacuum strength

by performing a dry run with a disposable glass slide. This avoids the possibility

of damaging one of the more valuable cleaned chromium slides. Primer is applied

to the entire wafer with a soft pipette. A reminder that a new pipette should be

used for each medium to avoid cross contamination. Allow primer to remain for

10 seconds, and then spin at 3000 rpm for 30s with an acceleration setting of 8.

When opening the spin coater lid after coating, it is a good idea to hold a clean

room tissue over the sample to protect it from any drips off of the lid.

5. Keeping the slide in the spin coater, photoresist S1811 is dispensed over the slide

using a new pipette. Photoresist should be poured into a small beaker and dispensed

from this beaker to avoid contaminating the supply bottle. Ensure that ample

photoresist is used to coat the entire area of the slide. Since the photoresist has

a high viscosity, uncoated areas may leave streaks during spin coating. Spin using

the same settings of 3000 rpm for 30s with an acceleration setting of 8. Be extra

careful when lifting the lid after this step as due to the large amount of photoresist

used there is a higher chance of drips.

6. Samples are then soft baked at 115◦C for 2 minutes on a hot plate to remove some of

the solvents inside the photoresist. This partially hardens the photoresist coating.

7. Samples are now ready for UV exposure using the mask aligner. Slides should be

brought to the mask aligner station one at a time in case there is any stray UV

exposure while the aligner is running. Firstly, ensure that the nitrogen, vacuum,

and compressed air lines to the aligner are open. This is critical as they provide

cooling to the UV bulb, which can overheat and crack without it. The mask aligner

and UV bulb can then be turned on. Wait for approximately 10 minutes until the


UV bulb has warmed up, it will display its power usage once it is warm. The flood

exposure setting is used which simply exposes the entire sample without vacuuming

the sample against a glass plate or glass photomask. This is the quickest setting to

use, compared to hard or soft contact, yet produces the lowest resolution. However,

it is sufficient for this application as the smallest feature size is 20 µm. Once the

flood exposure setting is engaged, the exposure time is set to 10 s. This slightly

over exposes the sample to ensure that no short circuits are present as the failure

case for over exposing is only slightly slower motion, which is much better then

the failure case for under exposure, which is short circuits producing an unusable

device. Place a single slide into the mask aligner and lay the photomask directly on

top of it. Align the photomask with the edges of the glass slide. Alignment is not

very critical as the only concern is that the entire design is on the slide and that

it is roughly parallel with the slide edge. Exposure can now be performed. While

exposing, make sure to not look directly at the machine, as the UV light can be

harmful. This process is then repeated for each slide, after which they should be

returned to the wet bench.

8. To prepare for development and etching, it is best to prepare the mediums in a line

across the wet bench to produce an organized process flow. This is more important

as multiple slides can be processed in parallel and establishing a process line can

help reduce the chances of error. The exposed samples are grouped on the far

right side of the wet bench. The DI water tap is on the left side of the referenced

wet bench so the process works from right to left in this description. Moving left,

MF-321 developer is poured into a wide, low beaker. Enough liquid should be

dispensed to cover the slides by approximately 1 cm of liquid. Next a hot plate is

placed at 115◦C. A cleanroom tissue is placed to the left of the hot plate to act

as a cooling station. Chromium etchant, CR-4 is dispensed in a similar wide, low

beaker, also with enough liquid to cover slides by approximately 1 cm of liquid. A


clean room tissue is then the last item and acts as a finishing area for the etched

devices. One sample should only be placed in each bath at a time to prevent slides

from scratching each other.

9. Slide is next placed in developing solution for 2-4 minutes. Some small agitation

can be done to speed up the process by either swirling the solution with the beaker,

or lifting one end of the slide up and down with a pair of tweezers. Development

is complete when photoresist is no longer clinging, or dissolving off of the surface

of the slide. The risk of over development is much smaller then the risk of under

development for these large feature sizes. This makes it a good idea to keep the

slide in the development solution for an additional 30 seconds after development is

judged to be complete.

10. Development solution is rinsed off with DI water. Again, make sure to adjust the

tweezers position to rinse the area under the initial tweezers placement. Dry the

sample with the nitrogen gun. Hard bake the slide at 115◦C for 1 minute to remove

the last remaining solvents in the photoresist. Place the sample on the cooling

station to the left of the hotplate and allow cooling to room temperature.

11. Etching is performed by placing the slide in the chromium etchant solution. Slight

agitation can be performed in the same manner as during development. Etching

requires approximately 2 minutes and it is also suggested to keep the sample in

the solution for an additional 30 seconds after etching is deemed to be complete.

During etching the chromium not protected by photoresist will dissolve so that

the final device will be visible. Rinse in DI water and dry with the nitrogen gun.

Samples are then placed in the finishing area.

12. Devices should now be inspected under microscope to ensure that lithography pat-

terns look good and no short circuits are present. If the devices look accurate, the

remaining devices can be etched without inspection.


13. Parylene C coating is performed using a dedicated chemical vapor deposition ma-

chine. Samples are placed on a rotating rack inside the vacuum chamber. Ap-

proximately 10 samples can fit in the machine during a run. To prepare samples,

fold a piece of tape over the long edge of each slide to cover the edge electrodes.

This prevents parylene from depositing on these areas, which makes it quicker to

assemble devices, as parylene needs to be removed in these areas to ensure a strong

connection to the edge board connector. Make sure that the tape is pressed well

on the glass slide as parylene is a conformal coating and will coat any exposed

surfaces. Parylene C is initially in solid pellet form and the deposition thickness

determined by the weight of parylene loaded into the machine. Parylene is loading

on an aluminum foil boat that is placed in an initial heating chamber at the bottom

of the machine. 3 grams of parylene was found to produce an appropriate coating

of approximately 1.5 µm. The chamber can now be closed and deposition initiated.

If the vacuum seal is strong enough, the machine will automatically heat and de-

posit the parylene inside the vacuum chamber. If proper vacuum is not reached,

the chamber needs to be vented and seals checked for debris. Deposition occurs at

room temperature so there is no risk of melting any materials on the sample. After

deposition is complete, solvents should not be used on the devices.

14. Teflon solution is prepared by placing 0.25 grams of Teflon AF 1600 into a glass

vial with 25 grams of FC-40. This produces a 1% Teflon solution. The vial is then

placed in an oven or hotplate at 80◦C for several hours until all Teflon has dissolved

in the solution. This solution can be prepared beforehand.

15. The final Teflon coating is then applied by spin coating. Samples are placed back

in the spin coater and the Teflon solution dispensed with a soft pipette. Samples

are spun at 2000 rpm for 1 minute. Lastly, solvents are removed by hard baking

devices at 160◦C for 10 minutes. Slides can then be allowed to cool and are now


complete.

16. Precoated indium tin oxide (ITO) slides are used for the top ground plate. These

slides are also coated in Teflon using the same spin coating technique.

Appendix B

Selected LabVIEW Code

66

Appendix B. Selected LabVIEW Code 67

Fig

ure

B.1

:C

amer

aco

mm

unic

atio

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IMA

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loop

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ure

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pre

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are

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funct

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ions

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Fig

ure

B.3

:In

side

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fram

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Figure B.4: Communication with the signal generator is achieved over USB communi-cation. Standard VISA commands are used to set the function, voltage, and frequencyvalues. Communication is sent to the signal generator whenever either the voltage, orfrequency control values on the front panel are changed.


Figure B.5: Macro control is initiated by reading in excel files containing the list ofdroplet actuations for each command. These files are named to match their function andeach button on the front panel corresponds to an individual excel file. Once the file isopened it is fed into the main droplet control loop.


Fig

ure

B.6

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the

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