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Experimental study of droplet actuation,splitting and particles manipulation using a cross scale digital… Hassan, Md. Fuhad 2013

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EXPERIMENTAL STUDY OF DROPLET ACTUATION, SPLITTING AND PARTICLES MANIPULATION USING A CROSS-SCALE DIGITAL MICROFLUIDICS PROTOTYPE  by MD. FUHAD HASSAN B.Sc. (M), Islamic University of Technology (IUT), 2008 M.Eng., Kongju National University (KNU), 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Mechanical Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) March 2013 © Md. Fuhad Hassan, 2013  ABSTRACT Digital microfluidics (DMF) system or device can be used as a significant and useful tool for chemical, bio-chemical and bio-medical applications because of its capability to perform basic fluidic functions and sequential chemical reactions on miniscule (nanolitre) droplets with better precision and accuracy. Application of sufficient electric potential in a sequence of electrodes manipulates the droplet in an electro wetting on dielectric (EWOD)-based DMF system. The successful implementation and the reliability of an EWOD-based DMF system depends on perfect design and fabrication of the lab-on-a-chip or DMF chip and also on the successful demonstration of basic fluidic functions including droplet actuation, splitting, mixing and separation within the DMF system. This thesis focuses on the study of the efficacy and feasibility of cost-effective fabrication of a crossscale electrode design for EWOD-based digital microfluidics lab-on-a-chip (LOC) systems. The intended microfluidic operations on this LOC include droplet actuation, splitting and particles manipulation (separation). The main features of the proposed cross-scale digital microfluidics prototype include one or more reservoirs, a linear array of square and rectangular electrodes forming the channel for liquid droplets, and a separation site consisting of multiple strip electrodes known as the high density electrodes or the strip electrodes. The fabrication process introduces newer dielectric materials (Cyanoethyl Pullulan (CEP) and S1813 positive photoresist) which result in a simpler fabrication of the proposed DMF prototype. The newer dielectric materials also enhance the functional quality by reducing the required voltage for droplet actuation and increasing the breakdown voltage. Successful droplet splitting has been demonstrated on the proposed cross-scale DMF prototype, and droplet actuation is investigated as the precursor for any operations including splitting. Dielectrophoresis (DEP) is applied to the strip electrodes of the proposed prototype to enhance the movement of particles. The non-uniform electric field generated by the DEP controls the motion of the particles. The strip electrodes are used to enhance the particle trapping and one-side (within the droplet) movement of the particles. A flawless cross-scale digital microfluidics prototype is built by optimizing the fabrication recipe.  ii  Table of Contents ABSTRACT…………………………………………………………………………………...........ii TABLE OF CONTENTS………………………………………………………………………..... iii LIST OF TABLES…………………………………………………………………………............vi LIST OF FIGURES……………………………………………………………………………….vii ACKNOWLEDGEMENTS……………………………………………………………………….i x DEDICATION……………………………………………………………………………………...x CHAPTER 1 INTRODUCTION.......................................................................................................1 1.1 Overview………………………………………………………………………1 1.2 Objectives……………………………………………………………………..3 1.2.1 Design the mask………………………………………………………..3 1.2.2 Fabrication of the Proposed Cross-scale DMF Prototype……………..3 1.2.3 Droplet Actuation……………………………………………………...4 1.2.4 Droplet Splitting……………………………………………………….4 1.2.5 Particles Manipulation………………………………………………....4 1.3 Digital Microfluidics Devices………………………………………………..5 1.4 Literature Review………………………………………………………….....6 1.4.1 Droplet Actuation…………………………………………………….7 1.4.2 Droplet Splitting…………………………………………………….17 1.4.3 Separation of Particles in Digital Microfluidics Systems…………...22 1.5 Contributions………………………………………………………………25  iii  1.6 Thesis Organization………………………………………………………..26 CHAPTER 2 MICRO-CHIP PROTOTYPING ....................................................... ...............27 2.1 Mask Development………….........................................................................27 2.1.1 Electrode Design..................................................................................27 2.1.2 Mask Design........................................................................................28 2.1.3 Features of the Proposed Mask............................................................30 2.2 Micro-fabrication through Wet Etching…………………………………...34 2.2.1Wet Etching Process.............................................................................35 2.2.2 Coating of Material on top of the Patterned Substrate.........................35 2.2.3 Limitations of Micro-fabrication through Wet Etching………………37 2.3 Optimal Fabrication Recipe for the Proposed Cross-Scale DMF Prototype.38 CHAPTER 3 EXPERIMENTAL SETUP AND RESULTS………………………………..42 3.1 Experimental Setup………………………………………………………..42 3.2 Experimental Results……………………………………………………...46 3.2.1 Droplet Actuation…………………………………………………..46 3.2.2 Droplet Splitting……………………………………………………48 3.2.3 Major Factors: Affect Droplet Splitting…………………………....54 3.2.4 Manipulation of Particles or Separation in a Cross-scale DMF……59 3.3 Limitations and Sources the of Error……………………………………..64 CHAPTER 4 CONCLUSIONS AND FUTURE WORK…………………………………...67 4.1 Conclusions……………………………………………………………….67 4.2 Future Work………………………………………………………………69 iv  BIBLIOGRAPHY...................................................................................................................71  v  List of Tables TABLE 2.1:  OPTIMAL FABRICATION RECIPE FOR THE PROPOSED CROSSSCALE DMF PROTOTYPE………………………………………………….  TABLE 3.1:  DROPLET ACTUATION VOLTAGE FOR DIFFERENT GAP SIZES BETWEEN TWO PLATES…………………………………………………..  TABLE 3.2:  38  48  CONDITIONS OF THE PARTIAL SPLITTING INCLUDING THE AMOUNT OF APPLIED VOLTAGE AND THE EFFECT OF THE APPLIED VOLATGE………………………………………………………..  TABLE 3.3:  DROPLET SPLITING EXPERIMENT: APPLIED VOLATGE, SPLITTING TIME, VOLTAGE RATIO AND GAP SIZE…………………………………  TABLE 3.4:  49  54  ELECTRIC FIELD DISTRIBUTION STAGES FOR ELECTRODE SIZE VARIATIONS………………………………………………………………..  55  TABLE 3.5:  CONDITIONS APPLIED FOR PARTICLES MOVEMENT………………..  61  TABLE 3.6:  LIST OF SOURCES FOR MEASUREMENT UNCERTAINTIES………….  66  vi  List of Figures FIGURE 1.1:  OPEN AND CLOSED EWOD-BASED DIGITAL MICROFLUIDICS PLATFORM (BERTHIER , 2008)…………………………………………..  FIGURE 1.2:  DROPLET  SPLITTING  GEOMETRY  (CHO  ET  AL.  2001,  BHATTACHARJEE AND NAJJARAN, 2012)…………………………… FIGURE 2.1:  DIFFERENT  ELECTRODE  SHAPES  (A)  5  RECTANGULAR  21  OR  SQUARE, (B) INTERDIGITATED, (C) ONE WAY CRESCENT AND (D) TWO WAY CRESCENT………………………………………………  28  FIGURE 2.2:  EFFECT OF POSITIVE PHOTORESIST………………………………….  29  FIGURE 2.3:  SCHEMATICS OF THE DESIGNED MASK FOR THE PROPOSED CROSS-SCALE DIGITAL MICROFLUIDICS PROTOTYPE……………..  FIGURE 2.4:  31  SCHEMATIC OF THE PROPOSED MASK A) FULL MASK, B) WORKING OR CONTROL ELECTRODES AND C) ON CHIP SEPARATION  SITE  OR  THE  HIGH  DENSITY  ELECTRODE  REGION…………………………………………………………………….  32  FIGURE 2.5:  PHOTOLITHOGRAPHY PROCESS FOR MICRO-FABRICATION…….  34  FIGURE 2.6:  DIFFICULTIES  FACED  DURING  FABRICATION  (A)  OVER  EXPOSURE AND (B) UNDER EXPOSURE………………………………  37  FIGURE 2.7:  FABRICATION PROCESS STEP BY STEP……………………………….  40  FIGURE 2.8:  DIGITAL MICROFLUIDICS CHIP SHOWS (A) FULL SCHEMATIC OF THE  CHIP  WITH  ALL  CONNECTORS  AND  WORKING  ELECTRODES, (B) ONLY WORKING ELECTRODES AND (C) ON CHIP SEPARATION OR PARTICLES MANIPULATION SITE OR THE  FIGURE 3.1:  HIGH DENSITY ELECTRODE REGION…………………………………  41  FULL EXPERIMENTAL SET UP………………………………………….  43  vii  FIGURE 3.2:  ENLARGE VIEW OF THE DMF PROTOTYPE………………………….  FIGURE 3.3:  DROPLET MOVEMENT FROM THE MIDDLE STRIP ELECTRODE TO THE RIGHT SIDE ELECTRODE………………………………………….  FIGURE 3.4:  48  PARTIAL SPLITTING: (A) DROPLET ORIGINAL POSITION, (B) DROPLET  PARTIAL  SPLITING  AND  (C)  SPLITTING  BOUNDARY………………………………………………………………. FIGURE 3.6:  47  DROPLET MOVEMENT FROM THE MIDDLE ELECTRODE TO ITS LEFT SIDE ELECTRODE………………………………………………….  FIGURE 3.5:  44  50  UNSUCCESSFUL ATTEMPTS OF DROPLET SPLITTING DURING INITIAL INVESTIGATION………………………………………………..  52  FIGURE 3.7:  FIRST SUCCESSFUL DROPLET SPLITTING…………………………….  53  FIGURE 3.8:  DROPLET SPLITTING-LESS ELECTROLYSIS ON THE SURFACE…...  54  FIGURE 3.9:  EFFECT OF THE ELECTRIC FIELD DUE TO THE ELECTRODE SIZE VARIATIONS……………………………………………………………….  57  FIGURE 3.10:  ELECTROLYSIS - SPREADING IN THE WHOLE ELECTRODE………  58  FIGURE 3.11:  ELECTROLYSIS BURNS THE WHOLE CHIP…………………………..  59  FIGURE 3.12:  µ-M PARTICLES IN THE DROPLET……………………………………..  61  FIGURE 3.13:  MOVEMENTS OF MICRO-PARTICLES IN A DROPLET……………….  62  FIGURE 3.14:  PARTICLES TRAPPING - IN BETWEEN ELECTRODES……………….  63  FIGURE 3.15:  SEPARATION OF PARTICLES IN ONE SIDE OF THE DROPLET……..  64  viii  Acknowledgements I would like to begin by expressing my gratitude to my supervisor and mentor; Dr. Homayoun Najjaran for providing me the opportunity to work on the field of Digital Microfluidics. His continuous support, encouragements, valuable advices, patience and expertise helped me to formulate this thesis. His constant guidance helped me to stay focused on achieving the primary goal of this research work. Moreover, I would like to thank him for providing me the opportunity to work with a talented team of researchers in the Advanced Control and Intelligent Systems (ACIS) laboratory at the School of Engineering, UBC (Okanagan). I am grateful to Dr. Hoorfar and Dr. Holzman for their guidance during the course work. I would like to thank Dr. Holzman for allowing me to use the mask aligner, gold etchant, chromium etchant, Microposit remover-1165 and the deposition station (sputtering). I would like to thank Dr. Roberts for providing the micro-particles for my experimental analysis. I would like to thank Dr. Sikandar Gill, Aurora Biomed Inc. and Natural Sciences and Engineering Research Council (NSERC) for their financial support to accelerate this research work. Finally, I would like to take the opportunity to thank my wife, Shaila Sharmin Rupali, for her constant encouragement, support during the most difficult times.  ix  Dedication  …….All praise goes to the Almighty Allah  x  CHAPTER 1 INTRODUCTION 1.1  Overview  Microfluidics is a significant and emerging technology contingent upon closed and micro-channels which is adopted as a way of miniaturization of basic fluidic functions. In the early 90’s, the sustainability of microfluidics, especially in analytical chemistry, first confirmed its popularity (Manz et al. 1992). Microfluidics integrated the operations of various fields (e.g., chemical, biological, biotechnology, engineering, micro-engineering and bio-medical) in a single platform. Incorporation of multi-disciplinary operations in a single platform provides the enlightenment of this technology (Trietsch et al. 2011). Currently, the main focus is to build a miniature-device for the analysis of laboratory-based analytical processes within the microfluidics platform with low sample consumption. The field of microfluidics will involve the majority of laboratory-based analytical processes to be conducted in the miniaturized microfluidics platform using low sample volumes in near future. Lowsample consumption decreases the analysis period as well as the associated costs and sample wastages. Integration of the simplified platform for laboratory-based analytical processes provides high accuracy and precision as well as more controllability on particles maipulation and dilution (Daw and Finkelstein, 2006). The miniaturized microfluidics platform can change the state of clinical diagonstics. The majority of advanced clinical diagonstics procedures in medical science need to integrate costly instruments, which are preffered to be operated by highly skilled personnel. In most cases, it is not feasible to have an easy access to such a facility. The improved miniaturized microfluidics platform will provide this facility for diverse medical applications (Yager et al. 2006). From the architectural and operational view point, microfluidics platform can be divided into three categories: i) continuous microfluidics, ii) multiphase flow -the droplet based analysis in microchannels, and iii) digital microfluidics. Continuous microfuidics depends on confined, interrelated and µ-size channels. It also depends on external pressure source, micro-valve, micro-pumps and other auxilliary setups. Up to the date, continuous microfluidics is the most commonly accepted fludic platform in terms of generating microfluidics operations. Multiphase flow in microfluidics is a mixture of discrete and continuous phases of fluidic operations where the movement of a liquid droplet shows the discrete phase and surrounding immisible filler fluid shows the continuous phase. Multiphase flow platforms depend on µ-size channels. Digital microfluidics uses centralized actuation phenomena to manipulate a discrete droplet on a lab-on-a-chip which enables independent manipulation of samples and reagents. Although all the three microfluidics platforms enable the 1  analyses in a very smaller range than the macro-scale methods (Wheeler, 2008). In some instances, continuous and discrete phases are incorporated within the same platform to increase the capability of the platform as well as integrate multi-disciplinary operations (Abdelgawad et al. 2009, and Miller and Wheeler, 2009). Digital microfluidics is a phenomenon to convert the basic macro-scale fluidic operations to micro-scale fluidic operations. So, the scaling law is much more effective than other macro-scale affects. As an example, in digital microfluidics interfacial or surface tension forces are prominent than body forces such as the force of gravity. Digital microfluidics lab-on-chip is designed in a simple array of electrodes which is used to manipulate picolitre to nanolitre size droplets (Berthier, 2008). The main purpose of digital microfluidics (DMF) platform is to design a miniaturized lab-on-a-chip which provides low sample consumption, high throughput i.e., fast reaction rates and quick analysis, less waste production and most notably, needs no auxilliary instruments (Pollack et al. 2002, Fair et al. 2007, and Teh et al. 2008). DMF comprises few distinct characteristics in comparision with other microfluidics platforms: i) independent and controlled sample manipulation, ii) reagent isolation, iii) electrodes are designed in arrays and iv) reconfigurability (Crabtree et al. 2001). A DMF device manipulates µ-droplets on the surface of the patterned substrate. Basic microfluidics operations are performed on a electro-wetting-on-dielectric (EWOD) based digital microfluidics lab-on-a-chip (DMF-LOC). Basic fluidic operations include droplet dispensing, droplet generation, droplet transport, droplet merging and mixing, droplet splitting and separation of different species (i.e., chemical or biological) within the droplet. Outlined in the following, the distinct features of DMF provides an enhanced and flexible platforrm for fluidic operations.  2  1.2  Objectives  The major motivation of this research is to perform droplet actuation, splitting and particles manipulation inside a droplet using an electro-wetting-on-dielectric (EWOD)-based cross-scale digital microfluidics lab-on-a-chip (DMF-LOC) device or prototype. Initially, the materials and processes are investigated to find a novel and cost effective design, and fabrication phenomenon. Commonly used dielectric materials are also replaced by newer materials such as: Cyanoethyl Pullulan (CEP) (Bhattacharjee and Najjaran, 2010), and S1813 positive photoresist (Murran and Najjaran, 2012). The experimental analysis is performed by designing and fabricating the digital microfluidics chip. Errors are occurred due to material impurities and subsequent surface imperfections, and manual assembly of the equipment. Experiments are accomplished to validate the basic fluidic operations within the proposed cross-scale digital microfluidics prototype. So, the particular objectives for the current thesis are as follows: i)  Design the mask  ii)  Fabricate the proposed cross-scale digital microfluidics (DMF) prototype  iii)  Droplet actuation  iv)  Droplet splitting  v)  Particles manipulation  1.2.1 Design the Mask The mask is the major component of micro-fabrication using photolithography. In photolithography process, the mask transferred the desired pattern of the proposed digital microfluidics lab-on-a-chip prototype on top of the pre-coated substrate (Cu or Au coated). So, the geometry of the design including electrodes, connecting pad, connecting wire and inter electrode gap should be designed with preferred preciseness and accuracy. K-layout is commercial mask design software which is used for mask designing. The mask should be printed with high resolutions so that the micron level features can be patterned successfully from the mask to the substrate. This research proposed a novel design for implementing the fluidic operations within the digital microfluidics prototype. So, the first objective is to design and print the mask. 1.2.2 Fabrication of the Proposed Cross-Scale DMF Prototype Photolithography and wet etching are used as the fabrication methodology. The equipment and chemicals used during fabrication need to be optimized through trial and error. So, the second  3  objective is to fabricate or build a defect-free low cost cross-scale digital microfluidics chip successfully and find out the optimal fabrication recipe for repeatability and reproducibility. 1.2.3 Droplet Actuation A novel and cost-effective cross-scale digital microfluidics prototype is built successfully through micro-fabrication process. Droplet actuation is a method to verify the performance of digital microfluidics device or prototype. To verify the performance of the proposed prototype, electric potential is applied to the sequence of electrodes to transport or move the droplet from one electrode to the other electrode. Droplet actuation also determines the minimum and maximum voltages need to actuate a droplet within the prototype. So, third objective is to implement the droplet actuation successfully with the application of sufficient electric potential. 1.2.4 Droplet Splitting This novel and cost effective cross-scale digital microfluidics lab-on-a-chip prototype is built to enhance the capability of liquid handling. Another significant motive is to implement separation of particles within the prototype. So, droplet splitting is an intermediate process during separation. Droplet splitting is also a basic fluidic operation. The fourth objective is to demonstrate droplet splitting in the proposed prototype using deionized (DI) water as the experimental fluid. 1.2.5 Particles Manipulation The proposed cross-scale digital microfluidics prototype contains a linear array of square and rectangular electrodes. It consists of a high density electrode or a strip electrode region and this high density or strip electrode region also consists of multiple strip electrodes. The high density electrode or strip electrode region is designed for particles manipulation including particles movement, particles trapping and particles separation. Particles movement or motion will also observe on the regular square electrode in application of both sine and square wave signal. So, the fifth or the final objective is to implement particles manipulation in the cross-scale digital microfluidics prototype including particles movement, particles trapping and particles separation (particles movement in one side of the droplet).  4  1.3  Digital Microfluidics Devices  In general, two types of digital microfluidics (DMF) device configurations are found in the literature. The two DMF configurations include i) open platform and ii) closed platform. In an open platform both ground and control electrodes are fabricated on a single plate. Primarily, a catena wire was used as the ground electrode to apply the electric potential required for actuation. In a closed platform, a bottom plate with the control electrodes and a top ground plate are hold in a parallel configuration by forming µ-sized gap (few hundred microns) in between where droplets are dispensed and manipulated by applying voltages to the control electrodes adjacent to the droplets. Both configurations of digital microfluidics use patterned arrays of electrodes for manipulation of the droplets. The bottom plate acting as the substrate is coated with a dielectric and hydrphobic layer. The dielectric layer protects the surface from electric breakdown and electrolysis, and the hydrophic layer provides a higher static contact angle to move the droplet at lower electric potential (Berthier, 2008). Figure 1.1 shows the two known configuration of electro-wetting-on-dielectric (EWOD) based DMF prototypes.  Figure 1.1: Open and closed EWOD-based digital microfluidics platform (Berthier, 2008) The closed platform is typically more capable for implementation of droplet generation, splitting and separation by providing a considerably greater actuation force (Berthier, 2008; Cho et al. 2003). On the other hand, the open platform can perform mixing more favourably than the closed platform. Although it is also possible to implement droplet generation and splitting in the open systems (Berthier, 2008 and Cooney et al. 2006).  5  1.4  Literature Review  Digital microfluidics lab-on-a-chip (DMF-LOC) technology is a substantial and emerging research field in which the main target is to miniaturize a full analytical system into a single (monolithic) chip by obtaining a vast range of advantages over existing technologies. DMF-LOC has gained tremendous advancement in the last two decades after its introduction in 1979 (Terry et. al. 1979).  About  10,000 papers on the topic of microfluidics have been published since 2000, and the number of publications is increasing tremendously per annum. As reported by ISI Web of Science, about 4000 citations per year are currently received. Moreover, almost 1000 patents on microfluidics have been issued by the United States Patent and Trademark Office (USPTO) (Trietsch et al. 2011). Mark et al. (2010) reported that digital microfluidics (DMF) demonstrated as a powerful toolbox both in academia and industry to develop new methods and products in multidisciplinary fields. However, commercial DMF devices, neither quantitatively nor qualitatively, meet the expectations yet. Now, the remaining question is whether microfluidics will continue to be limited to academic research, or will it become a robust and reliable industrial platform for biological and biochemical laboratories (Jebrail et al. 2012 and Mark et al. 2010)? The focus of DMF-LOC is to integrate multidisciplinary skills into a minuscule device. To date, several science and technology fields including chemistry, biochemistry, biotechnology, electronics, biomedicine, and micro- and nano-electromechanical systems (MEMS and NEMS) have benefited from advances in digital microfluidics (Trietsch et al. 2011 and Jebrail et al. 2012). The DMF-LOC platform provides faster analyses, effective separation, reduced sample, reagent and solvent ingestion, a programmable and reconfigurable testing ground. However, DMF-LOC has a challenging objective to fabricate the whole testing ground in the minuscule devices. Complex micro-scale features and phenomena are involved in digital microfluidics to realize this objective. The micro-scale components such as: electrodes and micro-channels and the micro-scale phenomena such as: micro-actuation, splitting, separation and on chip detection protocol. Pollack et al. (2002) reported that many fields including chemistry, biology, biotechnology, and engineering will be influenced by the miniaturization which can lead to highly integrated and highly automated DMF-LOC devices or systems. These DMF-LOC platforms are able to provide advantages such as i) reduced reagent ingestion, ii) reduced sample volume, iii) faster analyses in high throughput processes, and iv) automation and portability. Although microfluidics is the main thrust for DMF, discrete micro droplet handling and control techniques are still at the archaic stage in comparison with other similar microfluidic systems ( Pollack et al. 2002 and Fair, 2007).  6  The current work provides information about the research advancement in this field and the future aspects of the DMF technology. This literature review reports about the basic microfluidic operations pertinent to the scope of this thesis including droplet actuation, splitting and particles manipulation or separation in the field of DMF. 1.4.1 Droplet Actuation Several methods of droplet actuation have been investigated by the researchers in microfluidics devices. Chaudhury and Whitesides (1992) reported in a study that 15 degree tilted silicone surface was used to actuate a water droplet vertically. The droplet actuation was done using a continuous steepness of hydrophilicity. The alkyltrichlorosilanes (RSiCl3) is a form of polysilyne. Polysilyne (RSiCl3) contains undiluted gradient of concentrated silane. Due to the reaction of silicone surface and the vapour phase of polysilyne, the concentrated gradient of vapour modified the characteristics of the surface. In addition, due to this modification for the reaction, the concentrated gradient of vapour acted as a hydrophobic surface. This hydrophobicity generates unbalanced surface tension within the droplet when the droplet was dispensed in the hydrophobic surface. Thus, the surface tension at the front end of the water droplet was different than the lower end of the water droplet. The water droplet was transported successfully to the other end of the surface. Limitations of this water droplet actuation phenomenon lie in droplet alignment, control of droplet movement and formation of the gradient was irreversible (Chaudhury and Whitesides, 1992). Gallardo et al. (1999) reported an extensive study on controlling and aligning of liquids of submillimetre size using electrochemical phenomenon. As like the other electro-wetting phenomenon, “electrochemical” also uses electrodes to transport the liquid. Gallardo used an active oxidation reduction surfactant as the experimental substance on top of two electrodes. The oxidation reduction surfactant can be divided in two parts: i) active component and ii) inactive component, according to the applied voltage. The electrodes were used to reform the active oxidation reduction surfactants into two parts in each side of the electrode. Active and inactive components produced different pressure in the surface of the droplet. Active components were determined the movement of the droplet. Droplet velocity and applied voltage to the electrodes were linearly interrelated or co-related. A droplet with a higher concentration of active component was moved to the electrode of high voltage. A micro-fabricated device or system was built to transport nano to pico litre size liquid droplets. This microfluidics system or device consisted of an upper and a lower plate. Actuation phenomenon was based on thermo capillarity. In this system, channel, heater and temperature sensor were fabricated both in the upper and the lower plates. Though the heater was used to heat one side of the droplet, the capillary pressure was produced throughout the droplet. So, the capillary pressure was varied in the 7  two ends of the droplet as well as an automatic temperature variation was also developed. Contact angle hysteresis depends on the temperature variation in the droplet ends. So, surface tension automatically becomes a depending parameter of the temperature variation. Droplet actuation in this microfluidics platform proved that the temperature variation is proportional to the droplet flow rate. Moreover, contact angle and the minimal temperature to actuate or transport a droplet shows a linear relationship. Droplet with high contact angle requires higher temperature variations at the ends of the droplet. Higher temperature variation at the ends of the droplet can be reached at the boiling point for that specific liquid. Gravitational force, pressure source and changes in channel design are outlined as proposed solutions for above mentioned difficulty by the authors (Sammarco and Burns, 1999). Thermo capillary droplet actuation method was used in Darhuber et al. (2003). In this study, the authors implemented individually operated or controlled series of micro heaters to demonstrate the movement of the droplet and programming of the heaters can be done electronically. The microheaters were patterned on top of the pre-coated metallic surface. All the heaters were covered by insulation or dielectric layer. As a result, the generated heat cannot heat or warm up the droplet on their boiling temperature. In this study, the variations of surface tension at the end of the droplet were the driving forces to actuate the droplet. The major drawbacks of this study were i) capability to transport completely and partially wetted water droplet, ii) low droplet velocity, and iii) enhanced operation capability under continuous application of high voltage. Jiao et al. (2007) also implemented the thermo capillary droplet actuation phenomenon. In their study, silicone oil was used as a droplet. Four heaters were introduced at the four ends of a micro-channel. The driving force for actuation was the surface temperature at a certain position in the channel where the droplet was supposed to transport. The surface temperature at the desired location can be controlled by controlling the electric potential strengths of the heaters. Droplet translocation and the flow rate were in the acceptable range according to the theoretical concordance. Limitations of this study were i) evaporation of the droplet and ii) lower surface tension. Recently, Liu et al. (2012) reported about the two dimensional thermo-electric device or system for the thermo capillary droplet actuation. This study proposed that variations in the temperature are capable of modulating the interfacial force or the surface tension which helps the droplet to move or transport towards the cooler areas. Mostly, resistor heaters provided the essential temperature gradient in a typical thermo capillary droplet actuation. The temperature increment in a typical thermo capillary droplet actuation method depends on the passing currents whereas the cooling process depends on the natural convection or conduction. But, the thermo-electric chip developed by Liu et al. can control the temperature (rise or down) by modifying the input current (direction and magnitude). Closed loop feedback control system manipulates the direction and magnitude of the input current. This results a successful implementation 8  of the thermo-electric chip in a 2-dimensional array for droplet routing, splitting and merging in a preprogrammed thermal map. Ichimura et al. (2000) reported the actuation of droplet by exposing a substrate surface under a photoisomerizableazobenzene monolayer, to change the characteristics of the substrate surface. Due to the exposure of a photoisomerizableazobenzene monolayer, energy difference developed in the surface of the substrate. The variations of energy level in the substrate surface worked as the driving force for the droplet actuation. An aqueous droplet like oil can be used as the liquid droplet or working fluid. But, a water droplet was not possible to use in this study. Magnetic manipulations of droplets have been demonstrated by Nguyen et al. (2007). The droplet of ferro-fluid was actuated by the magnetic field and the desired design was patterned on a printed circuit board (PCB). Permanent magnet and coils were used to create the magnetic field and the polarization effect. Four planner coils were used to motivate or accelerate the actuation phenomenon. Two of them were used to create the same polarity and vice versa. The magnetic force is influenced by the size of the substances. As a result, droplet velocity in the bigger droplets was higher than the small droplets. Application of high electric potential in the coils also accelerated the droplets movement. Droplet translocation and ability to execute the fluidic functions were validated. But, the heating problem was not resolved properly, due to the application of high electric potential to the coils. Lehmann et al. (2006) demonstrated an enhanced two-dimensional microfluidics platform to actuate the droplets with magnetic particles. Both the magnetic field and a printed circuit board (PCB) were used to implement the required parameters to the droplet. Basic fluidic functions were compatible with in this device. The limitations were i) incompatibility of working with low volume liquid droplets and ii) dependence of droplet velocity on the amount of magnetic particles present in the droplet. Renaudin et al. (2006) suggested that, the surface acoustic wave (SAW) can be used as a droplet actuation method, where a transducer is used to determine the droplet movement as well the droplet position. The droplet velocity and the droplet position depend on the SAW discharged from the transducer. The duration of SAW application determines the droplet velocity and the duration of transmission and echo of the signal determines droplet position. To achieve higher efficiency in droplet positioning, target position can be used as a reference to modify the time of the SAW emission or transportation. Sub-millimetre level of accuracy can be achieved for the droplet positioning on this platform. In addition, it does not consist of any electrodes or electrical connections. The major drawback of this platform is the multi droplets handing and the independent operations of the droplets. Latterly, Renaudin et al. (2009) have developed an enhanced platform using the SAW, 9  incorporating a piezoelectric transducer, where the multi-dimensional droplet positioning is attained. The level of accuracy of the droplet position has also been increased in micron (µm) level. Bennes et al. (2007) have been reported about the SAW based droplet actuation phenomenon. A set of transducers have been used in different matrix formation to determine the location of the droplet and the flow rate of the droplet. All matrix formation used in this microfluidics system was 2×2. Accuracy level of the droplet manipulation was shown in the micron (µm) range. Wixforth et al. (2004) suggested a newer SAW driven approach to transport µ-droplets. Wixforth suggested a piezoelectric based SAW, where the transducer was patterned on the substrate. Due to the patterned transducer design, interaction of the liquid droplet was stronger with the SAW. In addition, an acoustic pressure was built in the system; this pressure helped the droplet to transport. This system can manipulate only specific size of droplets. Basic fluidic operations were successfully implemented in this system. Recently, Li et al. (2012) demonstrated a microfluidics system by integrating the surface acoustic wave (SAW) and the electro wetting on a dielectric (EWOD) technology. The combination of these two methodologies provides enhanced fluidic functionality and the combined device was fabricated using a single mask photolithography. The working principle of this combined device was as follows: i) EWOD used for accurate and precise position control of the droplet and ii) SAW was used as the actuation phenomenon. The SAW systems or devices were applied for particle concentration, mixing and merging, acoustic steaming and sensing. The SAW induced forces also enhanced the EWODbased droplet splitting. Basu and Gianchandani (2007) have been demonstrated a 2-dimentional µ-droplet actuation phenomenon where a set of heaters were used to control the fluid motion. The set of heaters were programmed and controlled independently. µ-droplets were suspended in the oil medium. The droplet velocity was controlled by the Marangoni flow. The suspension of droplets in oil medium results the surface and the sub-surface flows within the droplets. Due to this reason droplets were transported. The droplet was placed in between the layers of the oil medium. Evaporation was also controlled by this method. Heat generation was the limitation of this system (Basu and Gianchandani, 2007). Gascoyne et al. (2004) designed a dielectrophoresis based droplet actuation which incorporates a CMOS structure within the microfluidics device. The first implementation of a successful integration of CMOS and microfluidics operation was reported by Hunt et al. (2008). This studied actuated the droplet at considerably reduced voltage. The liquid droplet was surrounded by low-density oil and high-density oil layers. This platform was able to transport, mix and split nanolitre to picolitre size liquid droplets.  10  Washizu (1998) showed a droplet actuation method using the electrostatic field in a microfluidics device or chip. Washizu used a number of electrodes to manipulate the droplet. In this device, the electrodes were designed in arrays below the hydrophobic and dielectric layers. When the droplet was placed into an electrode, the next electrode of the droplet (adjacent to the droplet position) was excited by the sufficient electric potential required to move the droplet. So, the application of the proper electric potential in the arrays of electrodes accelerated the continuous movement of a droplet in this device. Several methods have been proposed to resolve the evaporation of the liquid droplets and to attain 2dimensional transportation capacity or capability. Taniguchi et al. (2002) have proposed two separate designs of the micro-reaction systems to resolve the above mentioned problem in microfluidics devices. Both of these designs showed unique characteristics for the certain fluidic operations. Matrix arrangement of the six-stage electrodes is used to control evaporation. The electrodes are embedded beneath the polypropylene tape. The second design used arrays of electrodes to manipulate and control the droplets individually. To minimize the friction of the droplet and the substrate surface edible oil is performed as filler fluid. Droplet actuation, as well as the chemical analysis was implemented successfully in this proposed micro-reaction device. Lebrasseur et al. (2006) also suggested electrostatic µ-droplet actuation. In the study of Lebrasseur, the electrodes are arranged in such way that the 2-dimensional µ-droplet transportation is attained. Transparent plastic thin sheet is directly used on top of the electrodes. The dielectric or insulation and hydrophobic layers are coated on top of the plastic thin film. The 2-dimensional droplet movement is observed in this platform. This microfluidics platform can control the individual unidirectional droplet movement. Kawamoto and Hayashi (2006) showed droplet transportation using a non-uniform or travelling electric field applied on top of the dielectric coated electrodes. Substantial or considerable modification has been attained in droplet actuation using electro-wetting on dielectric (EWOD) method proposed on Pollack et al. (2000 and 2002). On EWOD-based DMF device, a liquid droplet is placed between the pre-coated glass slides, where the upper slide or plate is used as the ground electrode and the lower slide or plate which consists of the desired patterned electrodes are used as the energized electrodes for a specified microfluidics operation. Working electrodes in the bottom plate are embedded underneath a dielectric and a hydrophobic layer of Parylene C and Teflon AF 1600. The hydrophobic coating is used to maximize the static contact angle of the liquid droplet in the substrate surface. The top plate is coated with the transparent indium-tinoxide (ITO). On top of the ITO coated plate, a thin layer of Teflon AF 1600 is also coated. When a droplet is placed on the surface and an electric voltage is applied on the electrode, the surface tension 11  decreases to accommodate the applied electric voltage in the solid-liquid region. This phenomenon follows   sL   sL0    0 RV 2  (1.1)  2T  where γSL , ε and t are the surface tension of the solid-liquid region, the permittivity and the dielectric layer thickness, respectively and γoSL is the surface tension at 0-voltage. Reduction of the surface tension also affects the contact angle of the substrate surface between the droplet and the energized electrode. This surface tension reduction is determined by  cos  (V )  cos  (0)    0 RV 2 2t LG  (1.2)  The effect described above helps the droplet to move towards the energized electrode. Moon et al. (2002) analyzed the effect of the dielectric material to reduce the actuation voltage of EWOD. This investigation proves that the reduction in the contact angle accelerated by reducing the thickness of the insulation or dielectric layer. An immiscible liquid can be used as the filler fluid in the droplet actuation method to reduce evaporation. Required electric potential can be decreased by modifying the contact angle hysteresis. Friction between the solid-liquid surfaces can also be improved by applying a thin lubrication layer. Pollack et al. (2000) demonstrated the droplet actuation on a digital microfluidics device using a single direct current (DC) pulse. In the study of the single DC pulse, a micro-reactor has been used for the rapid actuation of the discrete µ-droplets. The actuation phenomenon was completed successfully by holding a direct electrical control over the surface tension using the fabricated electrode on top of the substrate. This device consists of a linear array of seven electrodes. Controlled droplet transportation in the single DC pulse study establishes itself as an alternative method over the continuous and the electro-kinetic flows. Another significant advantage was the higher rate of droplet transfer or transport at the lower voltages. The disadvantages of the single DC pulse include i) abrupt movement of the droplet to the applied voltage, ii) no control on the droplet position and the droplet velocity, iii) higher droplet deformation, and iv) fragmentation. Later on, Murran and Najjaran (2012) reported the continuous droplet actuation or transportation with the application of the direct current (DC) pulse train. The DC pulse train actuation is the combination of a series of DC pulses which help the droplet to move or transport continuously from one electrode to the other electrode. The DC pulse train provides more control over the droplet position and the droplet velocity. The DC pulse train modulated the actual applied signal. Each individual pulse in the DC pulse train comprises a high 12  voltage and a low voltage stages or states. The correlation between the response time of the high voltage and the response time of pulse width determine the duty cycle of the applied pulse or the DC pulse duty cycle. In the proposed design of Murran and Najjaran, actuation voltage is applied in between the pause periods or intervals of the high voltage state which helps the droplet to move to the adjacent electrode. The resistance forces create a static or stable droplet in the electrode between the pause periods or intervals of the low voltage state and the actuation force extinguishes or disappears at this state. Literature does not provide further information about the DC pulse train and the DC pulse duty for droplet actuation except this study. Noh et al. (2012) reported about the use of the pulse towards an active matrix lab-on-a-chip device. In their study, they have demonstrated the programmable electro-fluidic control enabled with the arrays of the thin film transistor. Lin et al. (2012) demonstrated the low-voltage picolitre (pl) droplet actuation using an EWOD-based digital microfluidics device. This study demonstrated several outcomes include i) scaling the EWODbased actuation on pl range, ii) multiple layers of dielectric materials (Parylene C and Titanium Pentaoxide), iii) lower gap sizes between the top and the bottom plate (3 µm to 20 µm), and iv) dispensing of droplet from macro-scale pipette. Tsai et al. (2013) reported about the low voltage manipulation  of  an  aqueous  droplet  in  a  micro-channel  via  tunable  wetting  on  dodecylbenzenesulfonate doped polypyrrole (PPy(DBS)). This study has shown droplet actuation with an ultra-low voltage (<1 V). This study shows that reduction oxidation of PPy(DBS) in a filler or an immiscible fluid can manipulate and actuate an aqueous droplet by applying an ultra-low voltage (>1 V). The movement of the droplet within this device is a controlled movement with the application of square pulse redox potential. Recently, Gao et al. (2013) reported that multiple droplet can be manipulated using an intelligent digital microfluidics system with fuzzy-enhanced feedback. Droplet manipulation in a typical or common digital microfluidics device deals with the complex electrohydro dynamics. These complexities create uncertainties to adopt this technology as a strong source in the field of chemical or biological micro-reactors. The intelligent digital microfluidics system with fuzzy enhanced feedback resolves these complexities. The advantages of this system include i) profiling of the droplet electro-hydro dynamics under a real time tracking of the droplet trajectory, ii) the droplet trajectory elaborated from the capacitance, iii) precise and accurate multi-droplet positing without using any visual setup and heavy image processing signal, iv) saves charging time, enhances the high-throughput and the life time of the digital microfluidics chip, and v) expert droplet manipulation capability. This modular digital microfluidics system has its own built-in electronic control software which establishes this system as an intelligent system. This built-in intelligence provides enhancement in liquid handling and a reliable droplet-based fluidic operations. “The  13  programmable electrochemical reduction oxidation process using smart polymers will open a new way for the ultra-low voltage digital microfluidics platform.” Until now, digital microfluidics is unable to represent itself as a complete replacement of the continuous microfluidics. Though the continuous microfluidics uses few auxiliary accessories to implement the fluidic operations in the system, it is still an effective phenomenon to play with microdroplets. Digital microfluidics has some obvious advantages over the continuous microfluidics as mentioned previously (Teh et al. 2008 and Choi et al. 2012). Though a few methods of droplet actuation were described in this section, EWOD is the one and only phenomenon which is able to perform all the basic fluidic operations. EWOD is an effective and efficient method to implement in DMF-LOC device. Design and Fabrication of a Digital Microfluidics Device: Design and fabrication of a digital microfluidics (DMF) device are the most significant factors for attaining the desired objective from the research on the field of digital microfluidics. In this section, EWOD supported design and fabrication of the digital microfluidics (DMF) devices or systems are taken into considerations. A novel design is proposed for this research. A cost effective and easily fabricated digital microfluidic (DMF) device performs an efficient droplet actuation by bringing down the actuation voltages and producing uninterrupted droplet transportation considered as a convenient prototype forever. An actual digital microfluidics device involves arrays of working electrodes in a chip where the electrodes are programmed independently. But, the complexity increases with the increasing number of electrodes in the device. This leads complicated design and expensive fabrication of the DMF devices. Fan et al. (2003) suggested a substitute design where the electrodes are patterned on the both plate. With the help of this design, the connection complexities are improved in the arrays of electrodes on the plates. The limitations of this design are i) overlapping reduced the number of individual electrodes, ii) complication occurs in times of the multiple droplets positioning and iii) reduced the controllability over the electrodes. To fulfill the need of the cost effective and an easy fabrication method in a DMF device, printed circuit board (PCB) technology is embraced. The use of PCB also helps to achieve re-configurability within the DMF device (Gong and Kim, 2005). A rough fabricated surface of the PCB introduced high tolerance in the droplet. So, the droplet actuation needs fairly high electric potential (more than 500 V). This complexity is improved by modifying the surface fabrication phenomenon of the PCB (Gong and Kim, 2008). Increasing the number of electrodes in the design of DMF device creates a complicated and costly device. Chiou et al. (2003 and 2008) investigated on reducing the number of electrodes in the 214  dimensional design by incorporating the opto-electro-wetting (OEW) concept for the liquid droplets manipulation. In this investigation, the droplet velocity was observed higher in times of the droplet movement. In this design, photoconductive material is used beneath the actuation electrodes. The limitation was the use of multiple illumination sources to transport multiple droplets. Later on, Chuang et al. (2008) evolved the open opto-electro-wetting (OEW) where the ground and the energized electrodes are placed in a series. This actuation method used the amorphous Si as the photoconductive substrate. Silicon dioxide and Teflon were used as the dielectric and hydrophobic materials. Krogmann et al. (2008) also observed the light based droplet actuation. The selection of the frequency and appropriate voltages are the driving forces for droplet actuation in this platform. The contact angle in the brighter region of droplet is higher than the darker region of droplet. This platform is advantageous for the droplet transportation because the surface resistance is low. In addition, Li et al. (2008) developed a digital microfluidics device in combination of the complementary metal-oxide-semiconductor (CMOS) architecture within the platform. Special attention should be taken in the multiple layer coating in the substrate. Morgan et al. (2012) integrated detection within the EWOD based DMF platform. An array of thin film transistors were used to manipulate the droplet in this platform. The manual alignment of the plates in EWOD creates some errors and manipulation difficulties in this digital microfluidics device. To resolve this installation problem of plates in EWOD, Nelson and Kim (2011) demonstrated a monolithic chip which is built by the surface micro-machining. The thin films are used to achieve the desired accuracy. Brennan et al. (2011) reported that injection molding can be used as a substitute of the micro-fabrication to build DMF-LOC, capable of performing biological or bio-related assays within the device. Dielectric material is an efficient parameter in a DMF device. It is a major contributor to determine the success of the droplet transportation. Dielectric material has various effects on the electric attributes of a droplet actuation method such as i) determination of minimum actuation voltages, ii) dielectric constant determines the breakdown voltages, and iii) distribution of electric field. Cost, fabrication time and required voltage to transport a droplet are also varies due to the chosen material. Several studies have been conducted to choose the proper dielectric materials. Each of the material has some advantages and disadvantages of using in a DMF platform. Droplet actuation voltage also varies between 50 V to 100 V from system to system in digital microfluidics. Moon et al. (2002) used the Barium Strontium Titanate (BST) as the dielectric material in their digital microfluidics device. The BST has a dielectric constant of 180. A DI-water droplet was actuated successfully by applying 15 V. Kim et al. (2006) exploited the RF-sputtered Bismuth Zinc Niobate (BZN) as the dielectric material in their proposed DMF platform. A liquid droplet was successfully manipulated using 14 V. Berry et al. (2006) used the sodium dodecyl sulfate (SDS) surfactant in the 15  liquid droplet. The droplet was manipulated using 3 V. Dodecane oil was used as the filler fluid. The contact angle can be improved more than 100O due to the application of the SDS surfactant. The contraction between the liquid or the aqueous droplet and the oil is not an appropriate method to meet the objective of DMF system. Generation of the high temperature and high heat create evaporation in the microfluidics device. Chemical vapour deposition can be used as the dielectric layer deposition method. Chemical vapour deposition uses high temperature (>600o C) as well as expensive auxiliary equipment. The high temperature in chemical vapour deposition is forbidden the integration of the CMOS technology and the application polymer coating in the device. Li et al. (2008) exploited the anodic titanium pentaoxide (Ta2O5) as the dielectric material. Dielectric constant of Ta2O5 is 18 and actuation was done using 14 V. This also requires some special and expensive setup. Later on, Bhattacharjee and Najjaran (2010) proposed Cyanoethyl Pullulan (CEP) as the dielectric material which has used 20 V as droplet actuation potential and the dielectric constant of CEP is 15. Advantages are i) deposited by spin coating, ii) no need of any expensive setup, iii) low actuation voltage, and iv) no hazardous chemical handling. Electro-Mechanical Processes in EWOD: Jones (2005 and 2009) and Chatterjee et al. (2009) described the electromechanical involvement in the process of droplet manipulation using EWOD method which provides a general system to improve the coagulum-elements of the electro-fluidic device. EWOD or DEP was the basis of electro-fluid-dynamics of a droplet. The conductivity of the liquid droplets should be taken into consideration. In experimental analysis, a low-permittivity droplet should be used and a low permittivity scale is considered relative to the dielectric material (Zeng and Korsmeyer, 2004 and Chakrabarty et al. 2010). Typically, an electro-wetting method assumed that the change in the contact angle hysteresis moves the droplet. Dielectric droplet transportation, suffusion of contact angle and low-surface tension fluid (the contact angle apparently does not change) is not agreed with the traditional wettability dependency (Chatterjee et al. 2006 and Abdelgawad and Wheeler, 2008). Jones (2003) reported on the electro-mechanics of a general water droplet and the electro-mechanics are providing based on the conductivity of the droplet, dielectric constant of the droplet as well as the dielectric material, resistance of the droplet and circumference of the medium. Both the EWOD and the DEP changed their behaviour in a certain range of frequency. Later on, Kumari et al. (2008) did an extensive investigation on the effect of the frequency in droplet transportation using EWOD. Chatterjee et al. (2009) reported a droplet can be manipulated by applying AC voltages in the chip. Moreover, the effect of gap sizes in between two plates is also proven in this platform. Later on, Bhattacharjee and Najjaran (2009) proposed closed loop EWOD based droplet manipulation where the droplet position determination method was also integrated. Droplet size is an important factor to define the response of 16  the droplet to the applied electric potential. Ahmadi et al. (2009) reported a numerical model for investigating two dimensional flow dynamics in DMF systems. In this numerical study, the contact line forces, shear forces and filler effects were considered for fluid flow in a micro-droplet. The results of this study were characterized the DMF systems for higher Reynolds number. Later on, Ahmadi et al. (2011) showed a numerical investigation of the electro-hydrodynamics on an EWODbased digital microfluidics device. Synthesis of Digital Microfluidics device or system: The architecture of a digital microfluidics device predicts the success rate of the fluidic operations and fluid manipulations including droplet movement, mixing and merging, droplet splitting, and multiple droplet transportation to the desired position. Su and Chakrabarty (2008) improved automatic synthesis software which can be able to design a digital microfluidics system as per the requisition of the user. This synthesis tool also navigates the route of biochemical assays in the system. Fluidic functions and the architecture of the device are also modified to achieve a higher level accuracy (Rickets et al. 2006 and Yuh et al. 2006). Ground level of the synthesis tool determines the optimal algorithm to design and manipulate multidroplets (Griffith and Akella 2005). Investigation of damaged unit and alternative method to implement the desired fluidic operation in the non-faulty region bypassing those faulty units can be achieved (Xu and Chakrabarty, 2007, and Su and Chakrabarty, 2006). Recently, Murran and Najjaran (2012) reported about a capacitance-based droplet position estimator for the digital microfluidics (DMF) device. This study proposed an estimator which determines the continuous translation of the droplet between the electrodes in a digital microfluidics system. The determination process of the droplet position used a dimensionless ratio of capacitance. The result of this study provides a precise control over the droplet position which enhances the accuracy of liquid handling capability of the DMF device. Lin et al. (2012) implemented picolitre droplet actuation with a few micron gap sizes (3 µm to20 µm) between the top and the bottom plates by employing the multiple layers of the dielectric materials. This study of picolitre droplet actuation also uses a lower voltage. This study has lower down the scalability of the EWOD-based digital microfluidics system and opens a new path for the future investigation. This study also introduces an improved sealing in the EWOD-structure by coating a soft material (e.g., Norland Optical Adhesive (NOA)). 1.4.2 Droplet Splitting Droplet actuation, mixing and merging, and splitting are the basic fluidic operations implemented on the digital microfluidics device. All the basic fluidic operations were successfully implemented on the EWOD-based device. Capacity of performing the fluidic operations on the EWOD-device is to establish this technology as a realistic digital microfluidics lab-on-a-chip (DMF-LOC). Pollack et al. 17  (2002) demonstrated the droplet merging and splitting by applying electric potential on the patterned electrodes, successfully. A droplet can be actuated from the reservoir electrode by energizing a couple of electrodes. When the electric potential is applied to the electrode beside the reservoir electrode, electric field pulls the droplet. A neck is created due to the applied voltage. When the neck pinches off a droplet is manipulated from the reservoir. Cho et al. (2002) reported about a droplet manipulation phenomenon where 2-duplicate perpendicular electrodes were used. The 2-duplicate perpendicular electrodes accelerated the process of neck fomentation or initiation. The advantages of this platform are i) no reservoir required for initial droplet actuation and ii) 2-duplicate perpendicular electrodes accelerated the neck creation and the pinch off process. Droplet generation by applying AC voltages of a travelling wave was proposed in a similar research work by the authors. The travelling wave defined a changing pattern of frequencies. The travelling wave application increased the droplet velocity during droplet manipulation (Cho et al. 2003). Paik et al. (2003) demonstrated a mixing method using an active way to perform the operation. In this study, merging of two individual droplets and mixing of particles in the droplet were implemented. To verify the difference two types of droplets (i.e., the fluorescence droplet and the liquid droplet) were used. Frequency and mixing time were related. Ren et al. (2003) proposed an interpellation concentration of the droplet on the EWOD-based digital microfluidics device. Two non-uniform dilutions of the droplets were merged in the system and split in the system. Droplet splitting generated the two droplets of the same concentration. An arbitrary level of dilution or concentration was produced due to the continuous merging and the splitting of the droplets. But, it failed to provide expected level of dilution. Later on, the mixing process is improved with a high accuracy level and low mixing time by Paik et al. (2003). In this study, droplet mixing and droplet splitting are successfully implemented. Symmetric (1:1) droplet splitting has shown on this device. The EWODbased DMF device is designed on basis of the arrays of linear electrodes. Recently, Murran and Najjaran (2012) showed a feedback controlled droplet actuation, droplet mixing and sensing in a synchronized DMF-device with a pre-programmed micro-controller. Droplet splitting is one of the most important fluidic functions implemented on digital microfluidics lab-on-a-chip technology. Success rate of a droplet splitting procedure can have a great impact on the functionality of a digital microfluidics lab-on-a-chip device. Droplet splitting has been initiated by droplet generation from reservoir electrode (Cho et al. 2002 and Pollack et al. 2002). Sequential droplet splitting of different concentration of the liquid can be also be implemented. Droplet splitting process involved a complex electro-hydro-dynamics. So, the accuracy of the droplet splitting depends on having a better apprehension towards the physics and the electro-hydro-dynamics. Cho et al. (2001) demonstrated droplet splitting both theoretically and experimentally. This theoretical study 18  involved the physics of the droplet splitting process. This study has proposed that, according to all the factors associated with the Young-Lippmann and the Laplace equation determine the success of droplet splitting. Cho et al. (2001) derived a relationship between the electric potential and the contact angle combining both the Laplace and the Lippmann-Young equations. Success rate of the droplet splitting depends on the relationship between the applied electric potential and the radius of curvature of the distorted droplet. The equation as follows  R2 R   V2  1 2 0 R d R1 d 2t LG where  ,  , ,  and  (1.3)  are radius of the neck formation, radius of the deformed droplet, the  gap size, the required electric potential, the dielectric layer thickness and the dielectric constant, respectively. The above empirical equation, describes the factors of the droplet splitting. The neck formation must   0 RVd2 be started in the center electrode. in the equation should greater than 1 which generates 2t LG higher and  is defined by the electrode dimension. Droplet splitting can be achieved with a smaller  gap size when the electrode and the droplet dimensions matched with each other. In another way, the higher electro-wetting value can enhance the droplet splitting phenomenon. The electro-wetting number is defined as EWN    0 RVd2 . The higher electro-wetting number is possible to generate 2t LG  using an insulation or dielectric material of high insulation or dielectric constant, lower thickness, low surface tension and higher breakdown voltage or potential during the continuous application of electric potential. The cut-off electric potential of the dielectric or insulation layer was taken into account during the simulation modeling. The surface tension increases the amount of the required voltages. Special attention was taken during designing the gap size between the two plates. The electrode size and gap size ratio was designed as low as possible because the lower ratio provides higher possibility of splitting a droplet successfully. The desired gap between two plates estimated from the values of the radius of curvature of both the original and the distorted shape of the droplet. The disadvantages of the above study are the following: i) unknown deformed droplet radius and ii) no clue of saturation point. Later on, Berthier et al. (2005) proposed an analytical model for prediction of micro-droplet extraction and splitting in digital microfluidics systems. In this study, the modification of Lippmann equation was done based on Langevin’s function. This model established a proper relationship between applied electric potential and major factors of the EWOD-based systems 19  including the electrode size, vertical gap, the non-actuated contact angles and the volume of the liquid in the reservoir. Link et al. (2004) suggested that droplet splitting can be implemented successfully using T-junction design. Clime et al. (2009) have been successfully implemented the fluidic functions including droplet manipulation and splitting in their proposed device. The liquid droplet used in this study includes particles within the droplet. Murran and Najjaran (2012) have been demonstrated an automatic controlled digital microfluidics device where droplet manipulations are successfully implemented. The feedback control is provided by integrating a micro-controller in their proposed digital microfluidics system. Two kinds of simulation can be adopted to evaluate the free-interfacial fluid dynamics. The Arbitrary Lagrangian method has been used by several researchers to explain the droplet interfacial changes using the surface-oriented meshing during simulation (Charabarty et al. 2010 and Furlani and Hanchak, 2011). The meshing blocks are deformed in times of verifying meshing and re-meshing quality of the blocks. Another approach can be the Eulerian method. This provides advantages during the meshing (fixed meshing), and electro-hydro dynamics determination. Recently, Bhattacharjee and Najjaran (2012) proposed an extensive study of droplet splitting both theoretically (simulation) and experimentally. In this study, both the symmetric and the asymmetric splitting of droplet have been shown through simulation and experimental analysis. The simulation model is implemented in commercial computational fluid dynamics package FLOW-3D®. FLOW-3D® uses a methodology including the free-surface interfacial fluid dynamics which provides the required information about the dominant physical parameters associated with the droplet splitting. Mostly, digital microfluidics devices deal with droplet actuation where water-air and water-water surfaces are considered. These intermediate surfaces are related to fluid motion, electro-hydro-dynamics, electrostatic forces, electro kinetics and fluid deformation. A design with extensive information on these physical parameters reduced the period of preliminary investigation and provides an accurate and a reliable model. According to the study of Bhattacharjee and Najjaran, FLOW-3D® uses the Eulerian method using a controlled volume-of-fluid (VOF) to solve the droplet splitting problem. The scalar function is defined by F in this software. F value lies between 0 and 1 where 0 and 1 are used to define the air and the liquid droplet interfaces, respectively. The inactive transportation equation is calculated the surface development. The equation is expressed as  F  .V F  0 t where , , and  (1.4)  represents the time, the velocity-vector and the operator gradient, respectively. The  value of F in every cell is taken into account while the modeling of this simulation. FLOW-3D® 20  involves various physical factors during simulation. But, electro-mechanics, surface tension, and viscosity and turbulence are only employed in this simulation model by Bhattacharjee and Najjaran. Gravitational effect is not accounted for this simulation environment. The main motivation behind this simulation model was to understand the physical phenomenon of the droplet splitting. A vital challenge was to design the model as like as the realistic experimental platform. The simulation was also implemented to observe at the ideal conditions and the response of the electric potential for the droplet splitting process. In the simulation, the dielectric layer thickness is designed as 1 µm. The thickness of hydrophobic layer is not integrated in the simulation model. In both theoretical and experimental analysis suggested that voltage drop throughout the hydrophobic layer is either zero or a negligible value of closest to zero (Kumari et al. 2008). The meshing blocks are divided into six regions. The droplet height from the bottom to top plate determines the gap size and it was fixed as 71 µm. The numerical results are verified through the experimental analysis on both the symmetric and the asymmetric splitting of the droplet. Banerjee et al. (2012) showed a novel procedure or process of droplet splitting by progressively decreasing the applied electric potential where sudden withdrawal of the voltage from the electrode pad was used in a typical digital microfluidics platform. The progressive decrement in the applied electric potential extinguishes the hydrodynamic uncertainties. The hydrodynamic uncertainties are accountable for the fluctuations of the droplet volume. Lin et al. (2012) demonstrated a picolitre (pl) droplet manipulation in a multi-layer digital microfluidics lab-on-a-chip. In this study, a 12 pl droplet is generated two sister droplets of 6 pl. The electrode size and the applied voltage for this study are 33 µm and 18.7 Vrms, respectively. The gap size between two plates is too small (approximately 3 µm-20 µm). Fan et al. (2012) also reported about the droplet splitting process using dielectrophoresis in an oil medium. In this study, droplet splitting has been successfully demonstrated using 2 mm X 2 mm electrode with a gap size of 75 µm in between the top and the bottom plates. Li et al. (2012) also demonstrated an integrated device of the surface acoustic wave (SAW) and the electro wetting on a dielectric (EWOD) technology for particle concentration, droplet splitting and droplet sensing. This study uses the SAW induced forces for enhancement of the EWOD-based droplet splitting.  21  Droplet Neck  Energized Electrodes Figure 1.2: Droplet splitting geometry (Cho et al. 2001; Bhattacharjee and Najjaran, 2012) 1.4.3 Particles Manipulation or Separation in Digital Microfluidics Systems Digital microfluidics lab-on-a-chip has been established as much more enhanced, automatic and flexible liquid handling platform since the origination of this technology in early nineties (Manz et al. 1992 and Ahn et al. 2004). Pohl et al. (1978) first reported on dielectrophoresis-based liquid handling system where the living cell particles were used in the aqueous solution. The major focus was on the isolation of the living cell particles. In addition, separation and characterization of the particles were also performed. Chou et al. (2002) demonstrated that a single and a double DNA- molecule can be trapped and detected by applying the appropriate dielectrophoretic reflex of the DNA molecule in a (fairly) lower frequency. No separate electrode is required to implement this procedure. This methodology is defined as the “electrodeless dielectrophoresis”. Prinz et al. (2002) modified a fluidic device to lyse E. coli which incorporates the dielectric layer in lower frequency. In addition, Huang et al. (2002) demonstrated manipulation of various solid and biological cells using the dielectrophoretic affinity. The DEP-field flow fractionation (DEP-FFF) is an alternative phenomenon of particles manipulation or separation. Particles are actuated by the polarizability of the orthogonal dielectrophoretic influence and the external pumping of the liquid. The liquid density is used for the manipulation of the particles associated with the liquid (Wang et al. 2000). Masuda et al. (1987) first introduced the travelling wave dielectrophoresis (tw-DEP), which is used to manipulate various particles in the non-uniform electric field of various phases in a series of electrodes. Later on, the tw-DEP was used for separation or manipulation of various cells and molecules (Hughes et al. 1996, Morgan et al. 1997, and Cui et al. 2000). Separation and droplet splitting both can provide a numerous advantages in bio-assay applications. Separation is a process of separating various particles in the droplet to their desired position and  22  droplet splitting helps to create the sister droplets of high concentrated particles of the similar shape and size. Cho and Kim (2003) demonstrated that electrophoresis can be used as a successful particle manipulation method in an EWOD based digital microfluidics device. A series of electrodes were used as the separation electrode where electrophoresis was implemented. In this process, DC voltages were used as the actuation potential. Two types of particles were used such as: i) carboxyl modified latex and ii) polystyrene particles. Carboxyl modified latex particles were negatively charged. So, the electrophoresis can attract the charged particles close to the anode or the cathode. When a concentrated droplet of two particles was under the application electrical potential, the anode attracts the negatively charged particles and the other particles were attracted by the cathode. In this study, a symmetric droplet splitting was experimented. But, the concentration of the particles in each sister droplet was different due to the separation of two particles into two sister droplets. In another study, Cho et al. (2007) reported that the separation can be done by using a tw-DEP of changing phase pattern for binary separation. This study was a frequency based observation where high voltage and high frequency were used for particles manipulation or separation of droplet as well as droplet splitting. A complex design of chip was implemented in this study. Zhao et al. (2006) developed a travelling wave dielectrophoresis (tw-DEP) based method where only one types of particles were used for manipulation. Later on, Zhao et al. (2007) suggested a simple design of travelling wave dielectrophoresis (tw-DEP) based DMF-LOC for an effective particle separation and concentration control of the particles in the sister droplets. The aldehyde sulfate beads and the glass beads were used as the experimental particles in the droplet. Moon et al. (2006) associated a Matrix Assisted Laser Desorption or Ionization Mass Spectrometry (MALDI-MS) with the DMF- LOC device. This study shows that the particles can be separated and then the droplet splitting can be done to control the concentration ratio. Srinivasan et al. (2004) demonstrated a DMF-LOC for glucose detection which is one of the most used DMF devices in the clinical diagnostics. Pollack et al. (2003) showed polymerised chain reaction (PCR) can be successfully manipulated the particles surrounded by an oil medium.  Chang et al. (2006)  implemented an enhanced PCR application in the EWOD-based DMF platform. This study manipulated the cDNA molecules and the PCR reagents. Fan et al. (2008) reported on the manipulation of neuroblastomacells and polystyrene beads on a EWOD-based cross-scale digital microfluidics (DMF) device using dielectrophoresis (DEP). Like all other AC voltage based manipulation, this study also developed a frequency dependent system. A series of electrodes was designed in this LOC to modulate the applied electric signal. The droplet was sandwiched between the two plates. The applied electric signal was evaluated as a function of the resistors and the capacitors. The electric signal was applied to a droplet to observe the response of 23  electric field inside the droplet. The response showed an amplified frequency inside the droplet which was more than the critical frequency of the applied signal. Due to their size variations, a non-uniform electric force or field was created. The particles were separated on chip by the DEP relying on the Clausius-Mossotti factor. Srigunapalan et al. (2011) reported on the primary cell culture on a DMF platform. The integration of the DMF and the primary cell culture rendered a way of the future experimentations involved co-culture and high resolution microscopy. Chen et al. (2012) proposed a manipulation or separation process to separate the T cells and the DCs successfully using an EWODbased cross-scale DMF-LOC device by applying dielectrophoresis. Lin et al. (2012) also demonstrated manipulations of the paramagnetic beads and the buffer solutions with protein. Schertzer et al. (2012) reported about the automatic device for particle concentration and chemical reaction in EWOD-based digital microfluidics lab-on-a-chip. Pamme et al. (2012) demonstrated on chip bio-analysis of the magnetic particles. Park et al. (2012) described about the high throughput on chip leukemia diagnosis. Ng et al. (2012) reported that the magnetic particles can be separated by the particle-based immunoassays using a digital microfluidics device. This study implemented without any immiscible environment. Recently, Nejad et al. (2013) reported about the characterization of the negative dielectrophoresis (n-DEP) traps for the particles immobilization in digital microfluidics device. The geometric dimension of n-DEP is analyzed both numerically and experimentally. The particles trapping in the specific region of the DMF-LOC has been implemented. Their study has also shown single particle manipulation. In this study, positive dielectrophoresis (p-DEP) and n-DEP have also been discussed based on the Clausius-Mossetti factor. This factors follows  f CM    *p   m*  *p  2 m*  (1.5)  where  *p and  m* are the complex permittivity’s of the particles and the filler medium, respectively. Later on, Alshareef et al. (2013) demonstrated separation of tumor cell using dielectrophoresis. Integration of detection method into LOC platforms provides a unique feature in the analysis of different particles. In general, a detection method on LOC should have these characteristics: high sensitivity, ability to detect micro volumes, lower sound to noise ration and faster analyses. Sometimes integration of a detection method makes LOC platform very complex. In addition, the detection method is very important in particles trapping within the droplet. The detection methods used in DMF include electro-chemical detection (Karuwan et al. 2011 and Poulos, 2009), colorimetric dectection (Srinivasan et al. 2003, Srinivasan et al. 2004, and Aizenberg et al. 2006), UV-absorbance 24  detection (Sista et al. 2008), parallel scan like test (Chakrabarty, 2007). There are several other methods of detection implementation in a digital microfluidics EWOD-based lab-on-a-chip (LOC) technology. This literature review of digital microfluidics lab-on-a-chip operational system does not claim completeness. It comprises examples of different digital microfluidics operational based platform and their successful implementations which are suitable in terms of system definition. This literature review moreover, convey a considerable details about the fluidic operations (droplet actuation,splitting and particle manipulation) of this research field to the reader and indicate the reader to use the appropriate platform based on the application requirement.  1.5  Contributions  Digital microfluidics lab-on-a-chip (DMF-LOC) technology has evolved to integrate multidisciplinary operations into a single lab-on-a-chip. Specifically, chemical, biological, bio-chemical, bio-technology and bio-medical are the research fields which are taking advantages of µ-total analysis of digital microfluidics lab-on-a-chip device. Evenly, researchers also focused on modeling and simulation of complex fluidic operations to realize and predict the effect of the prominent physical parameters. Modeling and simulation can remarkably facilitate the study of the capabilities of the DMF platform. Subsequently, experiments with DMF prototypes will be used to validate those results. The first contribution of this research is to develop and fabricate a novel and cost-effective DMF design and fabrication method. The proposed design integrates various fluidic operations, such as droplet actuation, droplet splitting and cross-scale manipulation of particles inside the droplet. Another important contribution of this thesis is the use of the dielectrophoresis (DEP) as the particles manipulation method for the proposed cross-scale digital microfluidics lab-on-a-chip prototype. The non-uniform electric field provides the particles motion or movement in the droplet which significantly contributes to the manipulation of particles for the proposed cross-scale digital microfluidics prototype. Droplet splitting is investigated in this thesis through the experimental analysis in the proposed DMF prototype. Droplet actuation is also implemented in the proposed cross DMF prototype where different gap sizes and actuation voltage were used to transport the droplet. Another substantial contribution of this thesis is to implement high density electrode or strip electrode region for particles manipulation within the chip. The main purpose of particles manipulation is to observe the movement of the particles and to manipulate them. This manipulation can be established 25  this cross-scale digital microfluidics prototype as a source of analysis for medical and biochemical applications. During the process of particles manipulation, particle trapping was also observed. Particle trapping can be applied to readout application which opens a new path to motivate the research in future. Finally, a DMF prototype is built without the need for complex fabrication facilities such as a cleanroom. Common dielectric material is also replaced by more effective material which can be spin coated (e.g., photoresist and CEP).  1.6  Thesis Organization  Overview of the research field of digital microfluidics (DMF) and the objectives of this research are presented in Chapter 1. Chapter 1 also represents the literature review on different fluidic operations in microfluidics systems. Chapter 2 describes the fabrication procedure including the mask design and printing, pattern development and material coating. Chapter 3 describes the experimental results and discussions, limitations of this research and sources of error. Finally, concluding remarks and future scope of work in the similar applications are reported in Chapter 4.  26  CHAPTER 2 MICRO-CHIP PROTOTYPING In this chapter, the materials, chemicals, methodology and technique and equipment used for microchip prototyping are described. First, the design and the development of the mask for the cross-scale digital microfluidics lab-on-a-chip (DMF-LOC) prototype are described. Second, the microfabrication technique including the wet etching and the materials coating is also described. Third, opinions or the observations are also done by commenting on the limitations and the sources of error. Finally, the optimal fabrication recipe is provided to build the proposed cross-scale digital microfluidics prototype successfully.  2.1 2.1.1  Mask Development Electrode Design  Electrodes are the basic components of a digital microfluidics (DMF) system. Electrode design can determine whether a DMF device or system performs its operation successfully or not. A designer should be very precise and careful about all the factors associated with the electrode design. Special attention should be given to the factors: electrode shape, size, inter-electrode gap and connecting wire design. The ability of a droplet to move in a DMF system depends strongly on the actuation force in between the top and the bottom plates. More precisely, the droplet movement depends on the distribution of electric field in between the grounded top plate and the energized bottom plate. In literature, various shapes of electrodes have been shown. Examples include square and rectangular (Barbulovic-Nad et al. 2008), interdigitated (Cho et al. 2003), one way crescent (Rajabi and Dolatabadi, 2010) and both way crescent (Abdelgawad et al. 2009). Figure 2.1 shows different shapes of electrodes mentioned in literature.  27  Figure 2.1: Different electrode shape (a) rectangular or square, (b) interdigitated, (c) one way crescent and (d) two way crescent (Murran and Najjaran, 2012) The square or the rectangular shape electrode is easier to design and fabricate in comparison with other shapes of electrode because of its sharp corners and straight edges. In square or rectangular electrode droplet is overlapped with adjacent electrode which is very important to drive the droplet from one electrode to another electrode. For designing the rectangular or square shape electrode, inter electrode spacing or gap size is one of the most important factors to be considered. A smaller interelectrode gap can provide a better droplet positioning accuracy and use a lower voltage for actuation. However, the inter-electrode gap size is typically limited by the fabrication precision. In this thesis, square and rectangular shape electrodes have been used to conduct the experimental analysis. These kinds of electrodes are very easy to fabricate and the fabrication process is flexible in terms of precision. The fabrication precision also depends on the photoresist and the printing quality of the mask. S-1813 is a positive photoresist which allows achieving higher aspect ratios and resolutions. Using S1813 in the proposed design, 50 µm features were fabricated without the cleanroom and with excellent repeatability. 2.1.2  Mask Design  The mask is one of the most important components of the photolithography process because it defines the precision and accuracy of the ultimate patterns on the mask. The mask is used to transfer the designed pattern on top of the chip (Cu or Au coated) by photolithography. The mask is designed using K-Layout that is commercial software used for the mask design. In order to design a good mask, there are some important parameters which need to be taken into special consideration, such as limitations of size; etching procedure and types of photoresist. Limitations of Size: Microscopic glass slide has a dimension of 76 mm x 25 mm which is commonly used in micro-fabrication. So, limited space is available due to the dimensional constraint. This may cause the limitations of gap between the adjacent square electrodes. Etching Procedure: Wet etching method is the commonly used method. Unfortunately, there are various problems associated with the wet etching. Among them two are the most critical and tend to offset the successful fabrication phenomenon. First, it is difficult to control the etching rate. Second problem associated with the wet etching is under cutting. Due to presence of sharp edges and uncontrolled etch rate, sometimes etchant undercuts the designed pattern and changes the gap size in between the adjacent electrodes. 28  Types of Photoresist: Two types of photoresists are available in the market, i) positive and ii) negative. Mostly, micro-fabrication process in digital microfluidics uses positive photoresist to avoid the uncertainties occurred due to negative photoresist. Working principle of positive photoresist is shown in the Figure 2.2.  Exposure Mask  Developed state  Positive photoresist Substrate Copper coated layer UV light  Figure 2.2: Effect of positive photoresist As mentioned earlier, pre-coated copper or gold microscopic slides have been used as the experimental substrate throughout the study. The connecting electrodes are placed at the corner of the glass slide as shown in Figure 2.3. The main working electrodes or the control electrodes are in the middle where high electric voltage is applied. The connection electrode pads are placed in the corner of the substrate to ease the hassle of electric wiring. The connection electrode pads were placed in the corner to avoid damage of the working electrode or the control electrode. The size of the connecting electrode pads are approximately 4.0 times than the normal electrode or 4 mm width. The top plate is an ITO coated glass plate. The top plate is placed only above of the working electrodes. The spacer should be provided in between the top and bottom electrode to prevent short circuit or electric damage within the chip. Commercially available 3M scotch tape is used as spacer. Usually, 3M scotch tape is 40 µm in thickness. As per the resolution and accuracy of the mask pattern it can be printed either on the glass or transparency. The proposed designed mask was printed on transparency because the feature of the proposed cross-scale digital microfluidics design was greater than or equal to 50 µm. Printing the mask on transparency was cost-effective. If the feature of the designed pattern is less than 10 µm then 29  it is recommended to print the mask in metal or quartz or fused silica with accurate pattern generator. Pattern generated mask is also effective and efficient for sputtering. 2.1.3  Features of the Proposed Mask  The main features of the proposed device include one or more reservoirs, a linear array of square electrodes forming the channel for liquid droplets, a separation site consisting of multiple strip electrodes or high density electrode or strip electrode, and an open loop controller to implement droplet operations such as actuation, splitting and particles manipulation in the proposed cross-scale digital microfluidics prototype. Figure 2.3 shows the proposed design of mask for this study that is divided into three major regions, i) working area, ii) connecting wires and iii) connection area. In the proposed design, the working area including the control electrodes is designed as 1450 µm (square shaped). The connection wire is designed as 150-200 µm width. The proposed design contains an electrode which is divided into a series of strip electrodes. This region of strip electrodes is known as the high density electrode or the strip electrode. The strip electrodes in the high density electrode or the strip electrode region are designed as 50 µm width and 100 µm the inter electrode gap. The heights of the strip electrodes or high density electrodes are same as the square electrodes of 1450 µm. The connection wire is initially designed as 50 µm for the high density electrode or strip electrode region and at the end it is again matches 150-200 µm with the connection electrodes. The entire connection electrode pad is designed as 4000 µm or 4 mm. All the working electrodes are identical in size, including the high density electrode or the strip electrode region as a single square electrode. On the other hand, the reservoir electrode is much larger than the other working electrodes. The reservoir is 2.5 mm in size, to accommodate larger droplet or multiple droplets.  30  Figure 2.3 Schematics of the designed mask for the proposed cross-scale digital microfluidics prototype The reservoir electrodes, typically much larger than those used for transport or actuation, contain the initial liquid samples mixed with different biological species. Smaller droplets from each of the reservoirs are generated by applying electro-wetting-on-dielectric (EWOD) forces and transported or towards the separation site or the high density electrode or the strip electrode. The separation site or high density electrode region is defined by a number of fine stripped electrodes in a cell of the same size and shape as a typical actuation electrode. These stripped electrodes can act together as a transport electrode when activated at the same time. The high density electrode or strip electrode region helps the droplet actuation with better positing and accuracy. Droplet splitting is also tried on the proposed cross scale digital microfluidics prototype where the middle electrode is used as grounded or floating electrode in times of droplet splitting. So, the working principle will be same as the three electrode design. Figure 2.4 shows the detailed of the proposed mask.  a) Proposed design of the cross-scale digital microfluidics (DMF) prototype 31  b) Working or control electrodes in the proposed cross-scale DMF design  c) On chip separation site or the high density electrode or the strip electrode in the proposed cross-scale DMF design  Figure 2.4: Schematic of the proposed mask a) full mask, b) working or control electrodes and c) on chip separation or the high density electrode region The high density electrode or strip electrode region in the proposed DMF prototype is designed to accommodate the particles movements and separation analysis. The particles movements are analyzed with the help of travelling wave dielectrophoresis (tw-DEP). Particles trapping are also tried in the proposed cross-scale DMF prototype. When each of them in the high density electrode or strip electrode region is connected to an AC voltage having a phase difference of 90o with an adjacent one, a travelling wave of electric field is established. Thus, a travelling-wave dielectrophoretic (tw-DEP) effect can be introduced to the specific biological molecule of interest owing to the difference in permittivity's of the molecule and liquid medium. By repeating this process of applying tw-DEP for a certain period, it is possible to concentrate those molecules in a smaller region within the droplet. Subsequent splitting (i.e., division) of the droplet into two sister droplets will result in one with 32  significantly higher concentration of the desired molecule. A tw-DEP signal is also applied to the strip electrodes of the proposed prototype to observe the motion the particles.  2.2  Micro-Fabrication through Wet Etching  Although there are various types of fabrication methods, photolithography is commonly used as a cost effective micro-fabrication process for the digital microfluidics systems. Thus, photolithography is the second step after mask preparation or development. Fabrication at micron scale is a highly precise process which depends upon various parameters including spinning speed, spinning time, types of photoresist, developer solution concentration and etchant concentration. The optimal recipe for successful fabrication of the proposed cross-scale DMF prototype is described in Section 2.3. Photolithography is the commonly used process in micro-fabrication to pattern the glass or silicon substrate with specific designed pattern. Figure 2.5 shows the pictorial representation of the process of photolithography step by step. 33  Figure 2.5: Photolithography process for micro-fabrication Photolithography uses the mask and photoresist coated Cu or Au glass slide or the substrate. Afterwards, both the mask and photoresist coated (glass or silicon) slide are exposed underneath the UV-light source to pattern the desired design of the mask on top of the substrate or the glass or silicon slide. Two processes can be used for UV-exposure i) the UV-light cabinet and ii) Mask aligner. The UV-light cabinet uses a normal UV-light source which needs to go through a trial and error process to get the optimal time for perfect exposure. The mask aligner is more precise compare to the UV-light cabinet. The optimal exposure time for every photoresist is specified by the manufacturer for the mask aligner. 2.2.1  Wet Etching Process  After UV-light exposure the substrate is gone through several stages such as development stage, etching stage and photoresist removal stage. All these process can be classified as the wet etching procedure or process. This section will briefly describe the development, etching and resist removal stage. Development stage: The exposed substrate is developed using the developer solution. Developer washes all the unnecessary photoresist away from substrate except where the designed pattern should be printed or patterned. The concentrated developer solution MF-319 is used for the development stage. Etching stage: Etching is the process of removing the unnecessary material from the coated chip. There are two methods for etching: i) dry etching and ii) wet etching. Wet etching method is the 34  commonly used method in the micro-fabrication process. But, dry etching method as example deep reactive ion etching (DRIE) can be used to achieve higher resolution and better accuracy. Dry etching methods are expensive and they need some extra setup of elements. Wet etching is less expensive compared to dry etching method. Undercutting is a well-known problem for wet etching method which affects the resolution and accuracy of desired gap sizes and electrode sizes. Wet etching is also a faster process compared to dry etching method. When the gap size between the electrodes or the feature in the design is greater than 50 to 60 micron, undercutting has minimal effect. If the feature size is less than 50 micron then undercutting effect can affect desired resolution and accuracy. Ferric Chloride (Fe2Cl3) was used as copper etchant. The concentration ratio of 1:12 of Ferric Chloride and DI water is used in the current research work. Photoresist Removal: Usually, Acetone is used as the photoresist thinner in micro-fabrication labs. Acetone cannot remove 100% of the left over photoresist. So, photoresist remover can be used to remove all photoresist from the substrate surface. Microposit_remover-1165 solution is used as the photoresist remover. 2.2.2  Coating of Materials on top of the Patterned Substrate  Coating can be classified into two segments: i) dielectric layer coating and ii) hydrophobic layer coating. Electro-wetting on dielectric (EWOD) is used as the basis of experimental analysis of the proposed cross-scale digital microfluidics prototype. Dielectric layer is the coating of dielectric material which protects the metal coated surface from the application of high voltage. There are numerous dielectric materials available in the market. Examples of commercially available dielectric materials are: Polydimethylsiloxane (PDMS), CYTOP, Teflon, Siliconedioxide (Si02), Silicone Nitrite, Barium strontatnium titanate (BST), Cyanoethayl Pulullan (CEP) etc. Each dielectric material has its own dielectric constant. Hydrophobic layer is coated to achieve high contact angle and also helps the droplet not to wet the surfaces of the substrate. Dielectric Layer Coating: Typically, digital microfluidics (DMF) system uses Parylene C as a dielectric coating layer. As spin coater is available in our existing facilities. In order to satisfy the requirement, focus was only on the advantages and disadvantages of those dielectric materials which can be spin coated. According to Abdelgawad et al. Polydimethaylsiloxane (PDMS) is used as dielectric and hydrophobic layer (Abdelgawad and Wheeler, 2007, Abdelgawad and Wheeler, 2008). For using PDMS, it is obvious to use silicone elastomer cure agent and silicone elastomer base agent. The mixing ratio for both of this is 1:10. Main advantages of PDMS include i) spin coated, ii) no costly setup and iii) easy to fabricate. Though PDMS provides an easy fabrication, it has also some disadvantages. Main disadvantages of PDMS include i) high threshold voltage i.e., approximately 200 35  V, ii) prone to contamination being a sticky polymer and iii) low dielectric constant i.e., approximately 2.5. Bhattacharjee and Najjaran (2010) have suggested Cyanoethayl Pullulan (CEP) as a dielectric material which has a low threshold voltage of approximately 20 V. The dielectric constant of CEP is reported as 20. According to Bhattacharjee and Najjaran, the advantages of using CEP are - i) spin coated, ii) high dielectric constant, and iii) low threshold voltage. Granular CEP is bought from Biddle Sawyer Corporation, USA. CEP is dissolved in N, N-Dimethylformamide to produce a 20% (wt. /wt.) solution. After spin coating of CEP, the substrate was hard baked in a micro-oven at 95OC for approximately 12-13 hrs. Hydrophobic Layer Coating: Hydrophobic layer is used in DMF to achieve high contact which reduced the driving voltage requirement to move the droplet from one electrode to another. EWOD is used as our experimental process where droplet is sandwiched between the top and the bottom plates. Hydrophobic layer also provides an easier motion of droplet. Teflon is used as the hydrophobic layer. According to literature and our observations, Teflon provides a high contact angle of 115-117 degrees. Teflon has a smaller dielectric constant of 2.5 approximately. 1-3 % wt. concentration of Teflon is used in the proposed cross-scale digital microfluidics as hydrophobic layer solution.  2.2.3  Limitations of Micro-Fabrication through Wet Etching  Micro-fabrication is a very precise process. The most important parameter is the UV-light exposure time before the addition of mask aligner. The UV-light cabinet is a source of exposure for the photolithography process. The UV-light cabinet is known as the Ultra Violet Fluorescence Analysis Cabinet. The wavelength for such a setup is limited to 350-450 nm. This cabinet is used as the exposure source during fabrication for prototyping this proposed DMF chip successfully. When the UV-light cabinet is used for exposure, special attention should be taken into the exposure time. Exposure time in UV-light cabinet has to be precise and accurate. In the beginning of fabrication, the system has gone through trial and error process for finding the optimal time for exposure. During the process of exposure, some difficulties were raised. The substrate was getting under-exposed and over-exposed which was the main problem to achieve the desired pattern during fabrication. Figure 2.6 illustrates the phenomenon of under and over exposure during micro-  36  fabrication.  Figure 2.6: Difficulties faced during fabrication (a) over exposure and (b) under exposure Under exposure can pattern the desired design with presence of unwanted copper on top of substrate. Over exposure removes the thin lines and the connection wires from the substrate. To minimize the above mentioned problem, exposure time must be exact and accurate. The optimal time was derived through the trial and error process. It has been proven through the trial and error process that the optimal time can be  2 sec from the exact timing. Manufacturer specifies the exposure time of every photoresist for the mask aligner. So, the use of mask aligner easily resolves the above mentioned problems.  2.3  Optimal Fabrication Recipe for the Proposed Cross-Scale DMF Prototype  Fabrication process for the proposed cross-scale digital microfluidic system is illustrated step by step in the Figure 2.7 and the optimal fabrication recipe for every steps are summarised in Table 2.1. Table 2.1 Optimal fabrication recipe for the proposed cross-scale DMF prototype Optimal Fabrication Recipe Step  Description  Features  No 1  Photoresist Coating: S1813 positive photoresist  37  Spin coat photoresist  4500 (rpm), 500 (rpm/s), 60 (s)  Heating the substrate in a hot plate to harden the 105o (Celsius), 1-2 (min) photoresist 2  Photoresist Patterning: i) UV-light Cabinet (light intensity 350 nm to 450 nm) and ii) Mask aligner i)  Expose the substrate in UV-light Cabinet  10 cm (distance), 5 (min)  i)  Expose the substrate in UV-light Cabinet  1.5-2 cm (distance), 2 (min) 45 (s)  ii) Expose the substrate to the Mask aligner 3  Photoresist Development : MF-319 Developer solution Submerge the substrate in developer solution  4  7-10 (s) for S1813  45 (s) to 55 (s)  Copper Etching: Ferric Chloride diluted into DI water, Dilution ratio: 1:12 Submerge the substrate into the copper etchant  15 (s) to 25 (s)  Optimal Fabrication Recipe Step  Description  Features  No 5  6  Gold Etching: i) Gold Etchant and ii) Chromium Etchant Submerge the substrate in gold etchant  25 (s) to 40 (s)  Submerge the substrate in chromium etchant  45 (s) to 55 (s)  Photoresist Removal: i) Acetone and ii) Microposit_remover-1165  38  i)  Dip into the Acetone solution  ii) Dip into the Microposit_remover-1165 7  20 (s) to 25 (s) 10 (s) to 12 (s)  Dielectric Layer Coating: i) Cyanoethayl Pullulan (CEP) 20 % weight solution and ii) S1813 Photoresist  8  a) CEP coating  1500 (rpm), 500 (rpm/s), 30 (s)  b) Hard baking  90o to 95o (Celsius), 12-13 (hrs)  c) Photoresist coating  4500 (rpm), 500 (rpm/s), 45(s)  d) Hard baking  100o (Celsius), 12 (min) to 13 (min)  Hydrophobic Layer Coating: Teflon AF 1600 1%-3% weight solution i)  Teflon coating  ii) Hard baking  1000 (rpm), 500 (rpm/s), 30 (s) 150o to 160o (Celsius), 30 min to 35 (min)  39  Figure 2.7: Fabrication process step by step  40  The entire cross-scale digital microfluidics lab-on-a-chip prototype has been fabricated using the optimal recipe provided in Table 2.1. Figure 2.8 shows the successfully patterned chip.  Figure 2.8: Digital microfluidic chip shows (a) full schematics of the chip with all connectors and working electrodes, (b) only working electrodes and (c) on chip separation or particles manipulation site or the high density electrode region  41  CHAPTER 3 EXPERIMENTAL SETUP AND RESULTS In this chapter, the experimental analysis of droplet actuation, droplet splitting and manipulation of particles in a cross-scale digital microfluidics (DMF) prototype and the experimental results are described. First, droplet actuation is demonstrated by moving a droplet from one electrode to the other electrode of the proposed DMF prototype. Second, the experimental results of droplet splitting and manipulation of particles in a cross-scale electro wetting on dielectric (EWOD) based digital microfluidics lab-on-a-chip (DMF-LOC) are explained. Finally, concluding remarks are provided to highlight the limitations and the sources of error in the experiments.  3.1  Experimental Setup  The bottom and the top plates of the proposed DMF LOC were fabricated using the optimal fabrication recipe mentioned in Chapter 2. The fabricated bottom plate of the digital microfluidics (DMF) prototype containing the patterned electrode was cautiously placed on top of the upward facing light source. There was not sufficient amount of light needed to record the images properly. It was a high speed image frame recording using HiSpec 5 camera. So, the sufficient amount of light is needed to record the image properly. The light source was used to provide the proper amount of light during the recording of the experimental results. The specifications of the camera are 532 frames per second, resolution: 1696 X 1710, and pixel: 8 µm × 8 µm. Images can be recorded at a higher rate of frames per second. To achieve higher rates, sufficient amount of light as well as higher resolutions are necessary. The fabricated top plate was gently placed on top of the droplet presents in the bottom plate. Experimental setup is shown in Figure 3.1.  42  Figure 3.1: Full experimental setup The upward facing light source in the Figure 3.1 was unable to provide sufficient light to clarify the particles in the droplet. To resolve the problem, the light source was replaced by ring light and white back light source. The ring light was useful for the experimental purpose. But even for higher rates, it was not also a reliable source for the proper amount of light. The enlarged picture of the proposed digital microfluidics (DMF) prototype is shown in Figure 3.2. HiSpec 5 camera was attached with a Navitar 12X (0.58-7X) zoom lens through a 1.33X adapter. The camera and lens both was adjusted at maximum focus and resolution. The zooming level was also adjusted at its pick. Everything was adjusted at its maximum to achieve the maximum level of accuracy in image recording. But, still the images were under-exposed. The droplet areas were not completely identified. It can be happened due to the manual assembly of top plate. During the time of the top plate assembly the droplet meniscus was moved a bit to the left or right side of the electrode. In some cases, this slight movement causes blurry pictures. The camera was mounted vertically. It was focused through a focusing ring in front of the lens of the camera. The images were acquired through HiSpec software in a laptop computer and analyzed manually.  43  During the particles manipulation experiment the 12X Navitar lens was not sufficient to see the particles because of the smaller sizes of the particles (5 µm). A 2X lens was placed on top of the 12X lens to increase the magnification. The addition of this 2X lens increases the magnification to 24X. At this magnification the particles were visible. But, the higher magnification makes the system more sensitive. Thus, a smaller deviation or movement can result blurry or unclear images of the experiments.  Electrical connector pads  Working electrodes  Top plate  Bottom plate  Figure 3.2: Enlarge view of the DMF platform The gap between the lower and the upper plate is a very important factor for droplet splitting. The lower the gap size required voltage will be lower and vice versa. Commercial scotch tape (40 µm) was used to maintain the gap size between the top and the bottom plates. Required gap size was 80 µm for  44  the droplet splitting experiment. The gap size was created by placing two layers of scotch tape of 40 µm. The volume of droplet was less than 430 nl. For dispensing this small amount of liquid and due to the hydrophobicity of the surface, a micro-pipette was not possible to use. A nano liter droplet dispenser PipeJetTM P9 was used to resolve the dispensing problem. The PipeJet TM P9 is a product of Biofluidix Inc. The PipeJetTM P9 can dispense a droplet of 2 nl to 60 nl. The PipeJetTM P9 uses a piezoelectric actuator to dispense the droplet. The stroke of the piezoelectric actuator can be adjusted in the software provided by the manufacturer. The stroke of the piezoelectric actuator determines the volume of the droplet. The PipeJetTM P9 cannot dispense droplets more than 60 nl. So, multiple droplets can be dispensed and accumulated together to achieve the required droplet volume. The rate of the droplet dispense was 50 Hz. Though there were several factors which affected the final volume of the droplet, the accuracy of the dispenser was high enough. Several factors can affect the accuracy of the dispenser. They are i) landing accuracy- because of the surface, where the droplet lands, is hydrophobic. The droplet jumping was observed during the time of droplet landing on the surfaces of the substrate from the tip of the dispenser, and ii) disintegration of droplets at the time of detachment from the tip. These problems were resolved as follows: i) Landing problem was resolved by decreasing the distance between the tip and the substrate, ii) Disintegration only hampers the final volume. To achieve the desired final volume perfectly a little amount of tolerance was negotiated. The droplet was dispensed just above the target electrode. With the help of the top plate, droplet was brought on the center of electrode (it was possible only because of the hydrophobic surface). It was aligned manually as smooth as possible. Moreover, the top plate was placed very gently (by hand) on top of the droplet so that it does not make the droplet to be displaced or deformed. As the droplet was deformed by the top plate, a new droplet was dispensed to recover the effect of deformation and both plates were assembled gently to achieve the high accuracy. The actuation voltages were generated by the signal generator from the Tektronix AFG 3022B. Each signal generator has two identical channels. The applied voltage was amplified to the desired level through an amplifier of the Tabor Electronics. For the droplet splitting experiment, sinusoidal voltage signal was applied at 10-20 kHz. But, in case of particles manipulation experiment the applied frequencies were both sinusoidal and square wave. In both cases, the applied signal was in between 100-200 kHz.  45  3.2  Experimental Results  In this section, the experimental results are described by commenting on the achievements, limitations and the sources of error. The experimental results are shown for three fluidic operations including droplet actuation (transport), splitting and particles manipulation using a cross scale digital microfluidic lab-on-a-chip (LOC) prototype. 3.2.1 Droplet Actuation Effective droplet transport is the prominent operation in digital microfluidics system, so it can be used as a criterion to verify the workability of a digital microfluidic system. In EWOD-based DMF systems, droplet actuation is the result of the application of sufficient electric voltage to the patterned electrodes underneath the droplet. The EWOD-based LOC systems consist of a linear array of the actuation electrodes coated by a dielectric material. In this thesis, the actuation electrodes were patterned on a glass substrate using the wet etching process and coated by CEP as a strong dielectric material to reduce the minimum threshold voltage required for droplet actuation. In our experiments, the minimum voltage required for continuous transport of a droplet from one side of electrode to another side was 60 V at 1 kHz. The sequence of images in Figures 3.3 and Figure 3.4 illustrate droplet transport in the proposed cross-scale LOC prototype.  46  Figure 3.3: Droplet movement from middle strip electrode to the right side electrode Figure 3.3 illustrates the movement of droplet from the origin to the right electrode. Figure 3.4 shows the droplet movement to the left electrode. Droplet actuation in both cases has been shown from the strip electrodes. Initial droplet position is not energized. So, it can be done in any electrode. The strip electrodes were designed to experiment particles manipulation. Although the minimum voltage required for moving a droplet was 60 V, the droplet actuation was also demonstrated at 110 V.  47  Figure 3.4: Droplet movement from middle electrode to its left side electrode The gap size between the two plates is also changed during the droplet actuation process. The higher gap size required higher voltage to actuate a droplet. Table 3.1 shows the droplet actuation voltage and time for different gasp sizes. The droplet actuation is observed as the preliminary investigation of the droplet splitting and particles manipulation. Commercial scotch tape (40 µm) used as the gasket to determine the gap size. So, the gap sizes increased with the increasing number of the 40 µm layers. Table 3.1 Droplet actuation voltage for different gap sizes between two plates Actuation Test  Droplet  No.  Actuation Voltage (V)  Gap Sizes (µm)  Applied Frequency (KHz)  1  60  80  1  2  80  120  1  48  Actuation Test  Droplet  No.  Actuation Voltage (V)  Gap Sizes (µm)  Applied Frequency (KHz)  3  95  160  10  4  110  200  10  3.2.2 Droplet Splitting Droplet splitting is a basic phenomenon of digital microfluidics. All the researchers attempted to explain the splitting by their own theoretical understanding and experimental findings. In this thesis, droplet splitting is divided into two segments partial splitting, total droplet splitting or droplet splitting. Partial Splitting: The partial splitting is an intermediate phenomenon from a droplet's initial position to the phase of droplet splitting. In this thesis, the partial splitting has been shown to clarify the neck creation and also showed how the neck changes to make a droplet splitting process successful or unsuccessful. In the experiment, sometimes applied voltage to the right and the left electrodes also determines the neck occurring. If the applied voltage in right electrode is higher, the droplet will move towards the right electrode and vice versa. Basically, the partial splitting term is to describe the neck creation in between the right and the left electrodes. If the neck occurs in the process of droplet actuation and the applied voltage satisfies the proper conditions then the droplet must split. The neck creation in the middle electrode is as important as the applied voltage. Figure 3.5 below shows the partial splitting phenomenon. The experimental conditions in partial splitting were: electrode size: 1.45 mm × 1.45 mm, gap size: 80 µm, applied voltage: 105 V, and applied frequency 10 KHz. Table 3.2 shows the conditions of the partial splitting including the amount applied voltage and the effect of the applied voltage.  49  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  Figure 3.5: Partial splitting: a) Droplet original position, b) Droplet partial splitting and c) Splitting boundary Table 3.2 Conditions of the partial splitting including the amount of applied voltage and the effect of the applied voltage Partial  Left  Right  Splitting  Electrode  Electrode  Test No.  Voltage (V)  Voltage (V)  1  100  100  Gap Size (µm)  80  Effect of the applied voltages to the curvature of the droplet  Partially neck was created. Droplet was more on the right side electrode  2  105  105  80  Neck was created properly. But, droplet was not split because of insufficient voltage  Droplet (total) Splitting: Experimental investigation of the droplet splitting is used to find the exact voltage at which the droplet can be split successfully. In the procedure of manipulation of the  50  particles, droplet splitting was a part for checking the volume concentration of the two sides of the droplet in a cross-scale DMF prototype. In this thesis, splitting of a DI water droplet and the manipulation of particles (how the particles behave when it is subjected to high frequency and high voltages) are demonstrated. In the splitting process, one of the main goals was to determine the minimum voltage for the splitting. To determine this, we used low voltage in the beginning of the experiment and the applied voltage was increased to accelerate the process. We have kept increasing the voltage until the splitting was visible. The optimal voltage for splitting was also determined. The total volume in the center electrode was adjusted in each experimental iteration. Most of our experimental instruments and apparatus were organized or placed manually. So, there were lots of uncertainties associated with each of them. We were unable to align the droplet perfectly because of the manual positioning of the dispenser tip and the upper plate. Due to the faulty dispense of a droplet from the dispenser tip, it was always difficult to control the exact volume. Sometimes the droplet jumps in the hydrophobic surfaces which also creates difficulty to control droplet alignment and volume. Due to the manual assembly of the upper plate on top of the droplet creates difficulty to align the droplet position properly. Overall, beyond all the factors associated and the uncertainties, we were able to split the droplet successfully into two sister droplets. The main goal is to split the droplet into two sister droplets by applying voltages in the patterned electrodes. The typical duration of droplet splitting is in milliseconds which are less than a second. The first few milliseconds are very important during the droplet splitting because the maximum acceleration of the droplet splitting occurs in this duration. In the analysis of droplet (total) splitting exact timing is more important for applying the electric potential in the electrodes (left and right). If there is any timing differences occurs in the process of applying high voltage in the left or the right electrode, the droplet will move towards the higher voltage electrode.  51  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  Figure 3.6: Unsuccessful attempts of droplet splitting during initial investigation The minimum voltage needed to split a droplet successfully, as well as the response and the changes in the dynamics of droplet splitting were investigated. The figure 3.6 has shown the trials of unsuccessful attempts of droplet (total) splitting. Bhattacharjee and Najjaran (2012) reported about the droplet splitting both experimentally and simulation. The main idea of applied voltage has been taken from their results. The applied voltages in left electrode and the right electrode were 100 V p-p and 105 Vp-p, respectively. For ensuring the successful splitting, the applied voltages were increased in both side of the electrode. The increment was 5 V per attempts. The first successful droplet (total) splitting was revealed at the left electrode voltage 115 Vp-p and the right electrode at 120 Vp-p. Figure 3.7 and Figure 3.8 illustrate the successful droplet (total) splitting. Figure 3.7 illustrates the very first droplet (total) splitting with higher electrolysis on top of the electrode surface. Splitting time was 125 ms.  52  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  Figure 3.7: First successful droplet splitting Electrolysis occurs due to the presence of dust particles on top of the surfaces which creates nonuniformity in the surfaces. It can be contaminated with dust from breathing or open exposure in times of experimental analysis. Figure 3.8 shows images of successful droplet (total) splitting with less electrolysis or less contamination. The splitting process takes 130 ms to complete.  53  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  Figure 3.8: Droplet splitting-less electrolysis on the surface  54  Droplet splitting was observed successfully in this proposed cross-scale DMF prototype. Table 3.3 provides droplet splitting voltages and time for several iterations. Table 3.3 Droplet splitting experiment: applied voltage, splitting time, voltage ratio and gap size  Splitting Test  Left  Right  Voltage  Splitting time  Gap Size  No.  Electrode  Electrode  Ratio  Voltage (V)  Voltage (V)  (ms)  (µm)  1  110  115  1.05  125  80  2  115  125  1.1  130  80  3  120  135  1.125  110  80  3.2.3 Major Factors: Affect Droplet Splitting There are several factors associated with droplet splitting which can lead the process unsuccessful. There can also be also some other minor factors. But, the major factors are described because of their potential effect can lead the process to an unsuccessful attempts of splitting. In the experimental process, the effect of electric field distribution has been determined by the variations in electrode size, electrolysis due to the non-uniformity in the surfaces. Electric Field Distribution Due to Electrode Size Variations: Electric field distribution has greater affect in any kind of droplet movement because the electric voltage is the main driving force for the droplet movement analysis. For droplet actuation an electro-wetting on dielectric (EWOD) methodology is used. For better and proper distribution of electric field all the electrodes in our design was kept in same size and shape, except one electrode. The electrodes are the main working areas of any digital microfluidics experiments. The distribution of electric field depends on the size of the electrodes. The larger electrode in digital microfluidics platform has more driving force in it. It has also more strength than the other electrodes. In the experimental platform, we have fixed all the electrodes size. The shape of electrode in our design is square and rectangular in the high density or strip electrode region. For investigation of 55  electric field affect, only one electrode in our platform was designed as larger than the other electrodes. The droplet was placed in between the larger electrode and the regular electrode. The electric voltage was applied to investigate the droplet splitting process as well the distribution of the electric field. Sequence of images is shown this effect in Figure 3.9. From the Figure 3.9, it can be concluded that the neck was created. But the neck did not pinch off totally due to the difference of electric forces in two electrodes. Initially, it provides an impression that droplet is going to split successfully. Finally, it revealed the effect of electric force by moving the droplet to the larger electrode. Same voltages were applied in two electrodes. The amplitude of the applied voltage was 110 Vp-p. Though the same voltages were applied in both electrodes, the effect was not the same. The droplet totally moved on the larger electrode because of stronger electric force. Table 3.3 provides electric field distribution stages for electrode size variations. Table 3.4: Electric field distribution stages for electrode size variations  Applied Voltage to Left and Right Electrode (V)  Time  Effect  (ms)  110  30  Neck was created to initiate droplet actuation  110  50  70% droplet moved to the larger electrode  110  130  95% of the droplet moved to the larger electrode  110  280  Droplet completely moved on the larger electrode  110  310  Droplet at the larger electrode and electrolysis has started  56  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  Figure 3.9: Effect of the electric field due to electrode size variations  57  Electrolysis: The electrolysis is also a basic problem which affects the droplet movement or the droplet splitting experiments. The electrolysis occurs due to the non-uniformity in the surfaces. All the processes of deposition and curing are manual. Due to the manual assembly on every process, some non-uniformity occurs in the substrate surfaces. These non-uniformity at last causes the electrolysis during the experimental analysis. Effects are shown in a sequence of images in Figure 3.10. The applied voltage was 105 V at 10 KHz.  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  - 4.5 µm/pixels  Figure 3.10: Electrolysis – spreading in the whole electrode The electrolysis can burn the whole chip (substrate). Sequences of images in Figure 3.11 show how the electrolysis burns the whole chip when it finds a little bit of non-uniformity in the surfaces. Due to resolve this effect, use of clean room facilities is important for the micro-fabrication. The applied electric potential was 110 V at 10 KHz for the experimental analysis images in Figure 3.11.  58  Droplet–bubbles in right electrode shows electrolysis  Droplet–no electrolysis  - 4.5 µm/pixels  - 4.5 µm/pixels  Droplet –electrolysis is increasing  - 4.5 µm/pixels  Electrolysis burns the whole electrode  - 4.5 µm/pixels  Figure 3.11: Electrolysis burns the whole chip  59  3.2.4 Manipulation of Particles or Separation in a Cross-Scale DMF Polystyrene particles solution was used as the working fluid instead of Deionized (DI) water in this cross-scale DMF prototype for particles manipulation. The particles were negatively charged. The particles in the solution were the carboxyl modified latex (-COOH group). The particles were 5 micron in diameter. The particles solution contains 2.5% of solid. The main purpose was to observe the movement of particles (present in the solution) in the strip electrode or the high density electrode of the proposed design. Basically, the strip electrode or the high density electrode in the design was introduced to apply different phases of electric voltage into each strip. Different phases of applied voltages create a non-uniform electric filed in the DMF-LOC prototype. The non-uniform electric field of different phases is known as travelling wave dielectrophoresis (tw-DEP). Cho et al. (2003) reported about the tw-DEP to separate two kinds of particles. The design was too complex. The high density electrode or the strip electrode as well as the linear array of square electrodes have been introduced to remove the complexity associated with the proposed method in Cho et al. (2003). The tw-DEP is applied to move the particles presents in the droplet. The particles movement was created on top of the regular electrode. But the high density or strip electrode can energize the small area of the droplet which helps to move the particle from one strip electrode to another one. This results the particles movement in one side of the droplet and separation will occur by splitting this droplet into two sister droplets. Particles motion was created in the proposed cross-scale DMF prototype to understand the physics associated with the particles movement. The particles in the solution were moved due to the application of high frequency and high voltages. The high frequency created the non-uniformity in the electric field distribution which helps the particles to move within the droplet. The particles were also trapped in between the gap of the electrode of the high density electrode or the strip electrode region. The strip electrodes were energized one by one. So, the electric force helps the particles to move. But, the particles were reached in a state where this electric field was not sufficient to move them towards another electrode. The particles trapping was observed due to the above described reason. The particle trapping opens a new path to analyze and detect the particles on chip. This provides a way to adopt on chip detection method. Movement of Particles in the cross-scale DMF Prototype: The working fluid is replaced by polystyrene particles solution. The carboxyl modified polystyrene particles were negatively charged. The particles were concentrated in times of purchase. The particles were diluted in the laboratory, 1µl particles diluted in 30 ml of DI-water. But, this dilution is not used in experimental analysis. The  60  more diluted solution of particles (5 ml and 20 ml DI-water) is used in experimental analysis. This makes the solution less dense and presence of particles was sufficient for the experimental analysis. The droplet size used for particle movement observation was larger in diameter. It was 0.8-1 µl. The micro-pipette was used to dispense the droplet into the cross-scale DMF prototype. Figure 3.12 illustrated the droplet with particles.  - 1.64 µm/pixels Figure 3.12: 5 µm particles in the droplet To observe the motion of the particles both the sine and the square waves were applied. The frequency was an important parameter for the particles movement in the cross-scale DMF device. The applied frequencies in both cases of the sine and the square waves were high. It was about 100-200 KHz. The amplitude of the applied voltage was also high. The applied voltage was about 100-130 V. Figure 3.13 shows the particle movement. The movement of the particles were too fast when the sine wave was applied in the system. The particles motion or movement was pretty slower than the sine wave, when the square wave was applied. Table 3.4 summarises the condition of the particles movement within the droplet for the proposed cross-scale DMF prototype  61  - 1.64 µm/pixels  - 1.64 µm/pixels  Figure 3.13: Movements of micro-particles in a droplet Table 3.5 Conditions applied for particles movement Experiment No  Applied  KHz  Frequency 1  Sine Wave  Applied  Effect  Voltage (V) 130  100-130  Particles movement were faster  2  Square Wave  150  100-130  Particles movement were slower  Particles Trapping: Particles trapping is a way to observe the motion of the particle and to detect particles type and its characteristics. Particles’ trapping is an effective phenomenon when two or more types of particles are presented in the droplet. Only one type of particles is used in our system, to show the particles trapping phenomenon. Particles trapping can be done in many ways- some position can be fixed to trap the particles, sometimes different size particles can be trapped into different position of the electrode. The particles’ trapping is basically done to detect the particles presence in the system. The characteristics of the particles can also be analyzed. It can also detect whether the particles is  62  appropriate for the system or not. It can be widely used in the clinical diagnostics or the medical related experiments. Figure 3.14 shows the particle trapping in between the electrodes. The applied conditions were: applied voltage: 100-125 V, energized electrode: only one (among 10), applied frequency: 100-150 KHz and droplet size: 0.2 µl. Particles were not aligned properly in the region of low voltage because application of sine wave caused dielectrophoresis and as well as electrophoresis with in the same droplet.  - 2.144 pixels/µm  - 2.144 pixels/µm Figure 3.14: Particles trapping- in between electrodes  Separation of Particles in One Side: The particles were introduced to move them in one side of the electrode. Figure 3.16 shows the particles movement into the one side of the electrode. The applied voltages were 100-120 V and frequency was 200-250 KHz. In Figure 3.15 the first two strip electrodes were energized. From the initial position to the final, it is visible that particles were collected in this two electrode region. Other electrode regions were almost free from particles.  63  Figure 3.15: Separation of Particles in one side of the droplet  3.3 Limitations and the Sources of Error There were a few variations and non-uniformity in the experimental results. The variations and the non-uniformity in the results were occurred by the several sources of error. Among the sources of error, the micro-fabrication may have provided the major contributions. The micro-fabrication was a process optimized by the trial and error. In micro-fabrication, copper and gold coated chips were used as substrate. Due to the presence of dust particles on top of the surface of the substrate, these dust particles worked as the dielectric breakdown, imperfection in coating the layers of chemicals, sources of the electrolysis. The dust particles were in micron size. It was not visible in normal eyesight.. In the etching section, it was mentioned that the wet etching affects the dimension of the design by under cutting. One more thing needs to be mentioned that some variations can also be occurred in times of the development state during patterning the substrate in the developer solution. The electrodes were designed as 1450 µm. Images recorded by the HiSpec camera provided less than 1450 64  µm. So, there were some variations in the actual experimental environment. The normal interelectrode gap size was designed as 100 µm and gap size in the strip electrodes were designed as 50 µm. From the image analysis, it can be also concluded that actual gap size was not exactly matched with the design. These variations can occur due to the under cutting during etching or during the development process or due to the misalignment of the mask or due to the over exposure during the exposure time. The wider the gap size, the required voltage would be higher in the droplet actuation. The connection electrodes may affect the droplet actuation. It can be the dimension of the connection electrodes or the position of connection electrodes. Applied high voltages can bias the results in both cases of the droplet actuation and the droplet splitting. The connections were given by the crocodile pads which was a manual connection. This can be a cause for variations in results. The droplet actuation and the droplet splitting were also unsuccessful due to the bad connection. The crocodile pads removed copper or gold from the connection electrodes because of its rough and sharp pins on the edges. So, perfect and more accurate design, and effective and automatic connection pad can minimize the above mentioned problem. Dielectric layer can also be a source of error. CEP was coated on the chip because not only it is a strong dielectric material but also it can be spin coated in a preliminary fabrication facility with a spin coater. During the spin coating, non-uniformity in the thickness of dielectric layer can occur. Any non-uniformity in the surface can bias the results of the experiments. The non-uniformity in the surface of the electrode can also cause electrolysis. Also, surface non-uniformity can lower the dielectric break down voltage. Electrolysis can also be a part of the process due to non-uniformity in copper or gold layer. Though CEP was completely de-aired during solution preparation, the presence of air bubble in the solution can result in non-uniform thicknesses or micron size holes in the dielectric layer which can in turn exasperate electrolysis. The imperfect functioning of the dispenser was also a source of error in the experiment. The variation in the droplet volume occurred due to the dispenser. The stroke rate, droplet volume, and droplet velocity were set in the software of the dispenser. But variation can occur due to the jump of the droplet on top of the hydrophobic surface. Due to manual assembly of top plate, droplet can be slightly deformed or moved from the origin. Multiple droplets were generated to make up the volume. This can also be a source of error. The top plate was positioned on top of the bottom plate manually. Clearly, manual assembly can displace the droplet. Sometimes the droplet moved more towards one electrode. Scotch tape (40 µm) was used as spacer in between the two plates. So, elasticity or improper alignment of tape can create non parallelism in between the top and the bottom plate. Moreover, manual removal of the 65  hydrophobic layer on top of the connection electrode damages the copper or gold frequently. Overall, above described all factors can produce biased results. As well as, droplet can be non-sensitive upon the application of high voltage. According to the discussion on limitations and sources of error, there were several factors which created measurement uncertainties during the experimental analysis. Table 3.6 describes the measurement uncertainties for this experimental study. Table 3.6 List of sources for measurement uncertainties Source No.  Name of the Sources  Accuracy  1  Micrometer  ± 3 µm  2  Tip of dispenser  ± 10 µl  3  Thickness of dielectric  ± 0.5 µm  layer 4  Thickness of  ± 0.5 µm  hydrophobic layer 5  UV exposure time  ± 3 sec  66  CHAPTER 4 CONCLUSIONS AND FUTURE WORK 4.1  Conclusions  The purpose of this thesis is to implement droplet actuation, droplet splitting and particles manipulation in a droplet using an electro wetting on dielectric (EWOD)-based cross-scale digital microfluidics lab-o-a-chip (DMF-LOC) device or prototype. The first and foremost concern was to design a novel and cost-effective cross-scale digital microfluidics prototype. The main focus of this cross-scale DMF prototype was to integrate various fluidic functions which establish this platform as a commercial lab-on-a-chip technology. So, to satisfy the above mentioned objectives, all the different fluidic operations including droplet actuation, droplet splitting and particles manipulation were performed to verify the diversity of this cross-scale DMF prototype or device. This section of the thesis describes the findings of this experimental analysis in a concise way. This study was initiated by investigating the methodology of EWOD-based digital microfluidics systems which includes the actuation or manipulation process and the droplet dynamics analysis. The geometric dimension and material attributes were also investigated during the process of preliminary investigation. The proposed cross-scale digital microfluidics prototype was designed in a linear array of square and rectangular electrodes which results an easy and smooth fabrication. This proposed design also consists of a high density electrode or strip electrode region. Based on literature, the proposed cross-scale DMF prototype was coated with unusual or new insulation or dielectric materials which enhanced the fluidic functions under continuous application of electric potential. The higher dielectric constant of the insulation materials enhanced the droplet actuation by lowering the actuation voltage and also protects the surface of the DMF chip from early breakdown. Experimental device or prototype was designed and fabricated to implement couple of fluidic operations in this cross-scale DMF prototype such as: droplet actuation, droplet spitting and particles manipulation. Before moving to the experimental analysis, all the underlying factors associated with the above mentioned fluidic operations were analyzed based on the literature. Droplet actuation was the first fluidic operation which was demonstrated to verify the performance of this proposed cross-scale DMF prototype. The droplet was initially dispensed on an electrode of the proposed DMF prototype and the electric potential was applied. The droplet moved to its adjacent electrode from where it was initially dispensed. Droplet actuation was performed to get an idea about the minimum voltage required to actuate a droplet. Higher electric potential was also applied to find 67  out the maximum range of the applied voltage. The minimum and maximum voltages applied in this cross-scale DMF prototype were 60 V and 130 V, respectively. The characteristics of the proposed DMF prototype were: electrode size: 1.45 mm X 1.45 mm, gap sizes: 80 µm, 120 µm, 160 µm, and 200 µm, dielectric layer thickness: 1.25 µm, and applied frequency: 1 KHz and 10 KHz. Droplet splitting was shown in the proposed cross-scale digital microfluidics prototype. Deionized water (DI water) was used as the liquid droplet for the splitting experiment. In the process of splitting, two different stages were observed which provide the detailed information and reasoning of the successful and unsuccessful attempts of droplet splitting. These two stages are: partial splitting and total droplet splitting or droplet splitting. The flow rate of the liquid droplet was observed as a faster process in the beginning of the applied electric potential. Then, the rate of flow decreases exponentially and finally, the droplet splits into two sister droplets. In addition, the maximum acceleration of the droplet was observed in the few milliseconds of the applied electric potential. Some important outcome of this experimental analysis can be expressed as follows:   Partial splitting process is an intermediate state of droplet splitting. Partial splitting basically revealed the neck creation or formation due to the application of electric potential during the droplet splitting process. The neck boundary was also shown in this stage. The radius of curvature of the deformed droplet and as well as the radius of newly formatted two sister droplets were also observed at this stage.    Droplet splitting was observed at 43.5 Vrms (120 V  peak to peak).  left and the right electrodes was approximately 1.05.  The voltage ratio between the  The characteristics of the DMF  prototype were: electrode size: 1.45 mm X 1.45 mm, gap size: 80 µm applied frequency: 10 KHz and dielectric layer thickness: 1.25 µm.   It was possible to determine the amount of applied electric potential and viscous shear requirement from the analysis of the droplet deformation pattern and rate. The deformation rate was directly proportional to the applied electric potential. The higher value of the deformation rate and higher electric potential help to obtain a faster droplet splitting.    Three successful droplet splitting iterations have been demonstrated on the proposed crossscale digital microfluidics platform. The ratio of the applied electric potential were 1.05, 1.1and 1.125, respectively.  Manipulation of particles in the droplet was done by using dielectrophoresis (DEP) in the proposed cross-scale DMF prototype. The high density electrode or the strip electrode in the current cross-scale 68  digital microfluidics prototype was used to manipulate particles in this region. Particles motions were observed in the current cross-scale DMF prototype. The used particles were polystyrene particles of 5 µm diameter. The particles solution contains 2.5% solid particles. Particles motions were observed by attraction and repulsion forces. Sine wave and square wave were used to observe particles motion. The applied voltage and frequency for particles movement were 100-130V and 100-200 KHz, respectively. Particles’ trapping was implemented in the proposed cross-scale DMF prototype. The high voltage and high frequency were applied in the high density electrode region; only two strip electrodes were energized. Particles were trapped in the gap between electrodes. The applied high voltage and high frequency were not sufficient to move the particles towards the energized electrodes during particles trapping. Particles trapping can move the focus of the research for on-chip detection implementation.  4.2  Future Work  Further investigations are required to modify the experimental results. In the experimental device, few components were assembled manually which affected the results. So, research can be done to make these parameters more automatic and precise. Due to the presence of dust in the environment and the limitations of spin coating process, thickness of hydrophobic and dielectric material coating was not uniform throughout the substrate surface. So, electrolysis was not possible to remove totally. Further investigation on these issues can remove the electrolysis. A simulation model can be designed to verify the experimental results. Overall, considering all the factors associated with the experimental analysis the following modifications can be done to improve the results:  69    Design of the proposed cross-scale digital microfluidics prototype can be improved by lowering the inter-electrode gap sizes of the electrodes. Interlocking electrode can also be implemented in this prototype to enhance the performance of the droplet manipulation.    Fabrication can be developed in more classified clean room environment which will minimize the possibility of dust particles presence on top of the coated substrate.    The thickness of the dielectric and the hydrophobic layer was not uniform throughout the proposed prototype. Alternative deposition process can be adopted to make a uniform layer of the dielectric and hydrophobic coating.    The experimental analysis in this proposed platform is focused only on the successful implementation of the fluidic operations. Droplet volume is not considered as a factor which can affect the analysis results. Proper relationship can be developed between the droplet volume and the applied electric potential.    DI water can be replaced by different conductive and viscous liquid droplets as the working fluid.    Particles can be manipulated with improved methodology which allows the system to define the particles motion and manipulate the particles with higher accuracy.    Particles purification and more enhanced separation method can be implemented.    Particles trapping region can be defined to trap the particles in a certain region within the DMF chip and a single particle trapping method can also be developed to gain more control over the particles characteristics.    Two types of particles can be used for particles manipulation, separation and purification in an improved cross-scale DMF prototype.  70  BIBLIOGRAPHY Abdelgawad, M., and Wheeler, A.R. (2007). Rapid prototyping in copper substrates for digitalmicrfluidics. Advance mater, 19, 133-137. 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