Liquid Marble Based Digital Microfluidics

Author Kamalalayam Rajan, Sreejith

Published 2020

Thesis Type Thesis (PhD Doctorate)

School School of Eng & Built Env

DOI https://doi.org/10.25904/1912/3424

Downloaded from http://hdl.handle.net/10072/394318

Griffith Research Online https://research-repository.griffith.edu.au Liquid Marble Based Digital Microfluidics

Queensland Micro- and Nanotechnology Centre School of Engineering and Built Environment Griffith University. Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy By Mr. Sreejith Kamalalayam Rajan Bachelor of Technology (Electronics and Instrumentation Engineering) Master of Technology (Instrument Technology) February 2020

To my dear parents

Liquid marble based digital microfluidics

Sreejith Kamalalayam Rajan

Submitted to the School of Engineering and Built Environment on February 28th , 2020 in

partial fulfilment of the requirements for the degree of Doctor of Philosophy

Principal Supervisor: Prof. Nam-Trung Nguyen

Associate Supervisors: Assoc. Prof. Dzung Viet Dao

Dr. Chin Hong Ooi

Dr. Hongjie An

iii

Abstract

Sample handling with liquid marbles is a recent advancement in digital microfluidics.

Liquid marbles are liquid droplets coated with fine hydrophobic/oleophobic particles. The external particle coating isolates the liquid droplet from the surrounding ensuring a contamination-free environment for the droplet. Mobility manipulation of the liquid marble is easier and well developed compared to the droplet counterpart. These advantages have promoted the usage of liquid marbles as a microreactor for a range of biological applications.

Liquid marbles may help in reducing the non-reusable contaminated plastic waste generated in conventional biochemical reaction chambers such as plastic vials and microfluidic chips.

However, the use of liquid marble as a microreactor is limited to room-temperature applications as they are susceptible to the problems of evaporation at elevated temperatures. The development of a liquid marble based digital platform for biochemical applications at elevated temperature would broaden the scope of liquid marbles, reducing the generation of contaminated plastic waste. Polymerase Chain Reaction (PCR), a DNA amplification technique is a high-temperature process, which finds application in medical diagnosis, agriculture and forensics. Millions of PCRs are carried out in plastic vials and conventional microfluidic chips annually, contributing to the increasing problem of plastic waste. So developing a liquid marble based digital microfluidic platform tailored for PCR has an immense significance. This Ph.D. thesis focusses on the development of a liquid marble-based digital microfluidic platform particularly for carrying out PCR.

A basic PCR process consists of three different phases namely sample dispersion, thermal cycling, and output monitoring. Sample dispersion process should be precise and contamination-free. Manual intervention in sample dispersion should be minimised to avoid the contamination of the sample and to achieve the precise volume. We designed, developed

iv and tested an automated on-demand liquid marble generator. The instrument demonstrated a high precision over the volume of the marble and was highly repeatable.

Evaporation of the sample liquid is a major problem faced by liquid marbles operating at elevated temperature. Detailed knowledge of the evaporation dynamics of the liquid marble is essential for designing and developing a liquid-marble-based platform for PCR. We carried out experiments to study the evaporation dynamics of liquid marbles at elevated temperatures.

Studies revealed that the evaporation of liquid marble also obeys the “d-square law” at higher temperature. The lifetime of the liquid marbles is a function of its volume, temperature, the relative humidity of the surrounding environment, the number of liquid marbles and the distribution of liquid marbles. We demonstrated that the lifetime of a liquid marble can be increased by providing a vapour saturated ambient.

Next, we designed an experiment to demonstrate the use of a liquid marble as a microreactor for PCR. With the knowledge acquired from the previous study, a humidity- controlled environment was developed. Custom-built thermal cycler was integrated with the humidity-controlled chamber. Thermal cycling of the PCR mixture inside the liquid marble was carried out. Successful DNA amplification was observed, and the results were comparable with those obtained from the PCR in a conventional commercial machine. However, the sample volume used in this experiment was high and the marble was able to undergo only 9 thermal cycles.

Furthermore, we tested the synthesis of core-shell beads using composite liquid marble technology. A volume of 2 µL PCR sample was used as a core liquid where a photopolymer was used as the shell material. We successfully achieved the synthesis of core-shell beads.

Thirty thermal cycles were carried out without evaporation. Successful DNA amplification was

v verified. We achieved twenty-five times reduction in sample volume and 86.1% reduction in plastic waste.

The present thesis explains in details the various stages of developing a liquid marble based digital microfluidic platform for PCR. A little research on characterisation and optimisation of the methods proposed in this thesis might result in a commercially viable liquid marble-based platform for PCR.

Statement of Originality

This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

(Signed)______

Sreejith Kamalalayam Rajan

Date: 21/02/2020

vii Acknowledgements

I would like to express my special appreciation to my principal supervisor Prof. Nam-

Trung Nguyen and associate supervisors Associate Prof. Dzung Viet Dao, Dr. Chin Hong Ooi and Dr. Hongjie An for the tremendous support they had provided to me from the very beginning of my HDR candidature. It was a privilege to carry out research under the supervision of Prof. Nam-Trung Nguyen, who is a renowned and leading scholar in the fields of micro- and nanofluidics and nanoscience. His profound knowledge and meticulous work style have a significant impact on moulding my research skills and methods.

It was also very fruitful and helpful to discuss my research with Associate Prof. Dzung

Viet Dao. He was instrumental in providing suggestions in professionally organising my research and setting up my research objectives. Dr. Chin Hong Ooi and Dr. Hongjie An was kind enough to give valuable suggestions in my experiments, data analysis and paper writing.

Moreover, I would like to take this opportunity to thank Queensland Micro- and

Nanotechnology Centre and Griffith University for providing the research facilities and scholarships to support my PhD research. I sincerely thank the Australian Research Council for funding support through the grant DP170100277.

I would like to thank all the present and past members of Nguyen group especially Dr.

Raja, Jing Jin, Sharda and Lena for sharing their knowledge and helping me to carry out experiments. Sincere thanks to Peyush for helping and supporting me during tough times.

Special thanks to Lacey for all the administrative support.

I extend my sincere gratitude to my family members and my friends for their love and emotional support.

viii

I would like to thank my country, the republic of India for providing me high-quality education from primary school to Master’s degree course and financially supporting me with various scholarships time to time.

Publications during candidature

Peer reviewed journal articles

1. K R Sreejith, CH Ooi, J Jin, DV Dao, NT Nguyen, ‘Digital polymerase chain reaction technology–recent advances and future perspectives’, Lab on a Chip 18 (24), 3717- 3732

2. K R Sreejith, CH Ooi, DV Dao, NT Nguyen, ‘Evaporation dynamics of liquid marbles at elevated temperatures’,RSC Advances 8 (28), 15436-15443

3. K R Sreejith, CH Ooi, J Jin, DV Dao, NT Nguyen, ‘An automated on-demand liquid marble generator based on electrohydrodynamic pulling’, Review of Scientific Instruments 90 (5), 055102

4. K R Sreejith, L Gorgannezhad, J Jin, CH Ooi, H Stratton, DV Dao, NT Nguyen, ‘Liquid marbles as biochemical reactors for the polymerase chain reaction’, Lab on a Chip 19 (19), 3220-3227

5. Sreejith, K.R.; Gorgannezhad, L.; Jin, J.; Ooi, C.H.; Takei, T.; Hayase, G.; Stratton, H.; Lamb, K.; Shiddiky, M.; Dao, D.V.; Nguyen, N.-T. Core-Shell Beads Made by Composite Liquid Marble Technology as A Versatile Microreactor for Polymerase Chain Reaction. Micromachines 2020, 11, 242.

6. R Vadivelu, N Kashaninejad, K R Sreejith, R Bhattacharjee, I Cock, NT Nguyen, ‘Cryoprotectant-free freezing of cells using liquid marbles filled with hydrogel’, ACS applied materials & interfaces 10 (50), 43439-43449

7. J Jin, CH Ooi, K R Sreejith, DV Dao, NT Nguyen, ‘Dielectrophoretic trapping of a floating liquid marble’, Physical Review Applied 11 (4), 044059

8. CH Ooi, J Jin, K R Sreejith, AV Nguyen, GM Evans, NT Nguyen, ‘Manipulation of a floating liquid marble using dielectrophoresis’, Lab on a Chip 18 (24), 3770-3779

9. J Jin, CH Ooi, K R Sreejith, J Zhang, AV Nguyen, GM Evans, DV Dao, NT Nguyen, ‘Accurate dielectrophoretic positioning of a floating liquid marble with a two-electrode configuration’, Microfluidics and Nanofluidics 23 (7), 85

x 10. J Jin, K R Sreejith, CH Ooi, DV Dao, NT Nguyen, ‘Critical Trapping Conditions for Floating Liquid Marbles’, Physical Review Applied 13 (1), 014002

11. L Gorgannezhad, K R Sreejith, J Zhang, G Kijanka, M Christie, H Stratton, NT Nguyen, ‘Microfluidic Array Chip for Parallel Detection of Waterborne Bacteria’ , Micromachines 10 (12), 883

12. Adrian Teo, Fariba Malekpour-galogahi, Kamalalayam Rajan Sreejith, Takayuki Takei, and Nam-Trung Nguyen, ‘Surfactant-free, UV-curable Core-Shell Microcapsules in a Hydrophilic PDMS Microfluidic Device’, Applied Physics Letters(Submitted).

Conference oral presentations

1. K R Sreejith and Nam-Trung Nguyen , Liquid marbles as a bio reactor for Polymerase Chain Reaction, The 10th Australia and New Zealand Nano and Microfluidics Symposium, University of Wollongong, Australia. 1st -3rd July , 2019.

2. Jing Jin, Chin H. Ooi, Kamalalayam R. Sreejith,Dzung V. Dao,and Nam-Trung Nguyen, The 10th Australia and New Zealand Nano and Microfluidics Symposium, University of Wollongong, Australia. 30th June -3rd July , 2019.

Conference poster presentations

1. K R Sreejith, Lena Gorgannazhad and Nam-Trung Nguyen, ‘Liquid Marbles – A

versatile microfluidic platform for “green bio laboratory” practices’, Microfluidics &

Organ-on-a-Chip Asia 2019 conference, at Tokyo , Japan. 14th -15th November 2019.

Table of Contents Abstract ………………………………………………………………………………..iv Acknowledgements...………………………………………………………………...viii Publications during candidature …………………………………………………… x Table of contents …………………………………………………………………….. xii List of figures ………………………………………………………………………. xvii List of tables ………………………………………………………………………… xix

1 Introduction ...... 1

1.1 Background and motivation ...... 1

1.2 Aim of the research ...... 4

1.3 Objectives of the research ...... 4

1.4 Thesis framework ...... 5

1.5 Thesis structure as published journal articles ...... 7

References ...... 8

2 Digital polymerase chain reaction technology – recent advances and future

perspectives ...... 11

2.1 Introduction ...... 12

2.2 Sample dispersion techniques ...... 17

2.2.1 Droplet-based sample dispersion ...... 17

2.2.2 Microwell-based sample dispersion ...... 21

2.2.3 Channel-based sample dispersion ...... 24

2.2.4 Printing-based sample dispersion ...... 25

2.3 Thermal cycling methods ...... 26

xii

2.3.1 Joule heating ...... 28

2.3.2 Thermoelectric heating ...... 29

2.3.3 Acoustic heating ...... 30

2.3.4 Photonic heating ...... 31

2.3.5 Induction heating ...... 32

2.3.6 Microwave heating ...... 33

2.3.7 Solar heating ...... 35

2.4 Output monitoring techniques ...... 36

2.5 Conclusions and future perspectives ...... 40

Conflicts of interest ...... 42

Acknowledgements ...... 42

References ...... 43

3 An automated on-demand liquid marble generator based on electrohydrodynamic

pulling ...... 53

3.1 Introduction ...... 54

3.2 Principle of operation of the liquid marble generator ...... 55

3.2.1 Derivation of design parameters ...... 58

3.3 Fabrication of automated on-demand liquid marble generator ...... 63

3.3.1 Fabrication of droplet generation and deposition subsystem ...... 63

3.3.2 Fabrication of hydrophobic powder coating subsystem ...... 64

3.4 Experimental evaluation of the on-demand liquid marble generator ...... 66

3.5 Conclusion and future perspectives...... 67

xiii Acknowledgments ...... 68

References ...... 68

4 Evaporation dynamics of liquid marbles at elevated temperatures ...... 71

4.1 Introduction ...... 72

4.2 Theoretical background ...... 74

4.3 Materials and methods ...... 77

4.3.1 Preparation of the aluminium crucible as water reservoir ...... 77

4.3.2 Preparation of liquid marbles ...... 78

4.3.3 Experimental setup and procedures ...... 78

4.4 Results and discussion ...... 78

4.4.1 Temperature dependence of evaporation ...... 78

4.4.2 Evaporation of a group of liquid marbles ...... 82

4.4.3 Behaviour of liquid marbles with surrounding water well at elevated

temperature ...... 86

4.5 Conclusions ...... 87

Conflicts of interest ...... 88

Acknowledgements ...... 88

Appendix ...... 88

References ...... 88

5 Liquid marbles as biochemical reactors for the polymerase chain reaction ...... 93

5.1 Introduction ...... 94

5.2 Materials and methods ...... 96

xiv

5.2.1 Preparation of the polymerase chain reaction mixture ...... 96

5.2.2 Preparation of PCR mixture liquid marbles ...... 97

5.2.3 Design and fabrication of the thermal cycler ...... 98

5.2.4 Design and fabrication of the polymerase chain reaction chamber ...... 99

5.2.5 Design and fabrication of the fluorescence detection system ...... 100

5.3 Experimental procedure ...... 101

5.4 Results and discussion ...... 103

5.5 Conclusions ...... 105

Ethical approval ...... 108

Conflicts of interest ...... 108

Acknowledgements ...... 108

Appendix – Electronic supplementary material ...... 108

References ...... 109

6 Core-shell beads made by composite liquid marble technology as a versatile

microreactor for polymerase chain reaction ...... 114

6.1 Introduction ...... 115

6.2 Materials and Methods ...... 117

6.2.1 Preparation of photopolymer liquid ...... 117

6.2.2 Synthesis of composite liquid marbles and core-shell beads ...... 117

6.2.3 Design and fabrication of thermal cycler ...... 119

6.2.4 Preparation and optimization of the PCR mixture ...... 120

6.2.5 Design and fabrication of fluorescent detection system ...... 121

6.3 Experimental ...... 122

6.4 Results and discussion ...... 125

6.5 Conclusions ...... 128

Conflicts of interest ...... 131

Ethical approval ...... 131

Author contributions ...... 131

Acknowledgments ...... 131

References ...... 134

7 Conclusion and future perspectives ...... 138

7.1 Conclusions ...... 138

7.2 Future perspectives ...... 140

xvi

List of Figures

Fig.1. 1 Classification of techniques in microfluidics ...... 2

Fig.1. 2 Thesis structure as published papers ...... 7

Fig.2. 1 a) Various stages of polymerase chain reaction (PCR) ...... 13

Fig.2. 2 Concept and workflow of various PCR techniques ...... 16

Fig.2. 3 PCR sample dispersion techniques...... 18

Fig.2. 4 Concept diagram of slip-chip technology ...... 23

Fig.2. 5 Thermal cycling in digital PCR platform...... 27

Fig.2. 6 Fluorescence detection techniques of PCR ...... 38

Fig.3. 1 Schematic of pendant droplet under electric field...... 55

Fig.3. 2 Experimental setup of electro hydrodynamic pulling of liquid droplet ...... 58

Fig.3. 3 Graph a) droplet volumes vs applied voltage. b) Time of detachment(calculated vs obseved values) ...... 62

Fig.3. 4 The experimental setup of on demand liquid marble generator...... 65

Fig.3. 5 Performance characeterisation of the liquid marble generator...... 66

Fig.4. 1 (a) Model of a liquid marble; (b) Various arrangements of liquid marbles; (c)

Schematic of the experimental setup ...... 74

Fig.4. 2 Surface regression of 10 µl liquid marbles at various conditions...... 80

Fig.4. 3 Buckling time histogram of liquid marbles at various conditions...... 84

Fig.4. 4 Buckling time histogram of symmetrically and asymmetrically arranged liquid marbles ...... 86

xvii

Fig.5. 1 Experimental set up of liquid marble based PCR ...... 96

Fig.5. 2 Comparison of temperature between aluminium block and liquid marble...... 99

Fig.5. 3 Characterisation of humidity controlled chamber and thermal cycler...... 102

Fig.5. 4 Liquid marble based PCR results...... 104

Fig.5. 5 Characterisation of photobleaching of fluorophores...... 105

Fig.6. 1 Various stages of core-shell making process and structure and characterisation of the thermal cycler...... 118

Fig.6. 2 Temperature comparison between heater block and polymer bead ...... 122

Fig.6. 3 Gel electrophoresis results of DNA and primer optimization ...... 123

Fig.6. 4 Schematic of the experimental setup and results of core-shell bead PCR ...... 124

xviii

List of Tables

Table 2.1 Qualitative comparison of various thermal cycling techniques ...... 35

Table 4. 1 Comparison of surface regression rates k of liquid marbles at various physical conditions ...... 81

Table 4. 2 bBckling time of various set of liquid marbles heated at 95 0C ...... 85

xix 1 Introduction

1.1 Background and motivation

The broad field of microfluidics deals with the science, engineering, and applications of fluids in microscale. Microfluidics is considered as the road to Damascus in biomedical engineering, disease diagnosis, and life science research. Based on the way liquids are handled, methods and applications in microfluidics can be classified into continuous-flow microfluidics

(CFM) and digital microfluidics (DMF)[1].

Continuous-flow microfluidics mainly relies on microfluidic chips with microchannels and microchambers for the manipulation of working liquids. Continuous-flow microfluidic chips need external pumps and tubes to deliver and manipulate working liquids in the microchannels [2]. The bulky pumps and tubing required for the operation of continuous-flow microfluidic chips have made it less accessible for practical field applications [3]. Most of the continuous-flow microfluidic devices in operation are made of Polydimethylsiloxane (PDMS) or Polymethyl methacrylate (PMMA). Thus, the contaminated plastic waste generated by the single-use microfluidic chip is enormous.

Digital microfluidics, on the other hand, relies on droplets of the working liquid on a planar surface with volume ranging typically from nanolitre to microliter. The manipulation of a liquid droplet is usually achieved by means of an electric field [4-6] or a magnetic field [7].

Droplets on an open planar surface are more susceptible to contamination. Evaporation of the liquid droplet is another challenge of digital microfluidics. Surface interaction of the working liquid with the planar surface may hinder its mobility and even cause liquid loss while moving.

Liquid marble based technology is a recent advancement in digital microfluidics. Liquid marbles are liquid droplets covered with fine hydrophobic or oleophobic particles [8].

Conventional liquid marbles are made by depositing the working liquid droplet on a hydrophobic or oleophobic powder bed and subsequently rolling it on the powder bed to get a thin layer of coating on the droplet [9]. The exterior powder coating allows a liquid marble to hold its shape even under a relatively high mechanical stress [10-13]. Mobility manipulation

[14-18] of liquid marbles is relatively simple and well developed. The exterior powder coating provides a contamination-free environment for the interior liquid. There is no surface interaction between the liquid droplet and the operating surface, since a thin layer of powder and an air gap exist between them. These properties make a liquid marble the ideal candidate for digital microfluidic applications in cell biology, biomedicine, bioengineering, genetic analysis. Arbatan et al. used liquid marbles as a microreactor for rapid blood typing [19]. Sarvi et al. explored liquid marbles as bioreactors for cardiogenesis of embryonic stem cells [20].

Serrano et al. applied liquid marbles in cryo-preservation of mammalian cells [21]. Tian et al. utilised liquid marbles for the cultivation of microorganisms [22]. There are plenty of other

Fig.1. 1 Classification of techniques in microfluidics

2 Fig.1 1 Classification of techniques in microfluidics applications reported on liquid marble as a microreactor in life sciences. Fig.1.1 summarises the classification of methods in microfluidics, based on liquid handling methods.

Urbina et al. estimated that bio labs around the world could have produced approximately

5.5 million tons of plastic waste in 2014 [23]. Conventional plastic vials and microfluidic chips used to carry out biochemical reactions are contributing to this contaminated non-reusable plastic waste from bio labs. These alarming figures motivate us to develop alternative technologies, which could either completely eliminate or reduce the use of single-use plastic.

Liquid marbles could be used as an alternative packaging technology for liquid samples. The idea of liquid marbles as a microreactor can be explained as follows. Liquid marble is a microreactor platform, in which the working liquid is closely packed (covered) by an external thin powder coating instead of inserting the working liquid into an available container

(microfluidic chips or conventional plastic vials). In other words, the size and volume of the

“reaction chamber” could be easily tailored depending on the volume of the working droplet liquid. This versatility of liquid marbles could be effectively explored to reduce the amount of plastic waste generated from biochemical processes carried out in conventional platforms such as plastic microfluidic chips or vials.

However, like droplets, conventional liquid marbles also suffer from the problem of sample evaporation [9, 24, 25]. This disadvantage limits the use of liquid marbles in high- temperature microreactor applications. That is the reason, why there was no research ever reported in the literature before on using liquid marbles as a biochemical reactor for Polymerase

Chain Reaction (PCR). PCR is a DNA amplification technique invented by Karry Mullis in

1985 [26]. In PCR, the template DNA, the primers, the DNA polymerase deoxyribonucleotide phosphates (dNTPs) and buffer solution undergo a series of thermal cycles to generate millions of copies of the DNA. The thermal cycles usually consist of three different stages known as denaturation, annealing and extension, which are typically carried out at temperatures between

3 95 0C to 58 0C. A PCR mixture is most likely to be evaporated at these elevated temperatures.

However, the development of a liquid marble based digital microfluidic platform to carry out

PCR is expected to open up new “green laboratory practices”, reducing the use of non-reusable contaminated plastic waste and eliminating other practical difficulties of conventional microfluidic chip-based applications. The same is expected to accelerate the growth of liquid marble microfluidics in the future.

1.2 Aim of the research

Aim of the proposed research is to design and develop a liquid marble based digital microfluidic platform to carry out Polymerase Chain Reaction.

1.3 Objectives of the research

The following objectives are derived from the aim of the research discussed in the previous section:

1. To design, develop and test an automated on-demand liquid marble generation system

using electrohydrodynamic pulling with precise control over the volume of the liquid

marble. The volume of the liquid marbles generated can be controlled by controlling the

applied voltage.

2. To modify the existing “d-square law” of droplet evaporation and apply it to characterise

the evaporation dynamics of liquid marbles at elevated temperatures. “d-square law” of

droplet evaporation states that the square of the diameter of an evaporating droplet

decreases linearly with respect to time. The parameters of the modified “d-square law” can

be effectively tuned to reduce the premature evaporation of liquid samples of the liquid

marble at elevated temperature.

3. To design, develop and test a customised liquid marble based PCR device.

4. To apply the concept of composite liquid marble technology to synthesise core-shell beads

and use them as a microreactor to perform PCR without evaporation and with a

considerable reduction in single-use contaminated plastic waste.

1.4 Thesis framework

This thesis proposes novel methods to achieve the objectives stated in the previous section. The thesis chapters are arranged in order of achieving the objectives. The framework of the thesis is summarised below.

Chapter 1 introduces the background and motivation behind the project. Chapter 1 also explains the formulation of the project including research question, research hypotheses, aims and objectives of the research.

A thorough literature analysis which was necessary to study the existing techniques in microfluidics for polymerase chain reaction and to identify the research gaps in those techniques. Chapter 2 consists of a critical review of the existing microfluidic techniques for

PCR. The research gaps in the existing techniques and the possible solutions using liquid marble as a digital microfluidic platform to carry out PCR is extensively discussed in Chapter

Despite the advancements in liquid marble technology and its applications, there was no instrument available to automatically synthesise the liquid marbles on-demand with precise control over the volume. Chapter 3 presents the design, development, and characterisation of an on-demand liquid marble generator using the electrohydrodynamic pulling technique.

Evaporation of samples is a major concern in liquid marble based digital microfluidics.

PCR is a biochemical reaction performed at elevated temperatures. It is essential to understand the evaporation dynamics of liquid marbles to design and develop a liquid marble-based PCR system. Chapter 4 explains the evaporation dynamics of a liquid marbles at elevated

5 temperatures. The results obtained from the experiments summarised in Chapter 4 was helpful in designing a liquid marble-based PCR platform

Chapter 5 reports the design, development, and testing of a liquid marble-based PCR system. Polytetrafluoroethylene (PTFE) was used as a hydrophobic powder to make the PCR liquid marble.

Chapter 6 elaborates the design, development, and testing of a method to synthesis core- shell beads using composite liquid marble technology. The core-shell bead thus generated was used as a microreactor to carry out PCR. Premature evaporation of PCR mixture was the major problem with PTFE liquid marble based PCR. Core-shell based PCR effectively addressed this issue. Single-use contaminated plastic waste reduction was also achieved using this technology.

Chapter 7 summarises the results of the project and extensively discusses future perspectives.

1.5 Thesis structure as published journal articles

Fig.1.2 depicts the structure of the thesis as published journal articles and the same are arranged in the order of achieving the proposed objectives to meet the aim of the research.

Chapter 1: Introduction -Unpublished chapter

Chapter 2: Digital polymerase chain reaction technology – recent advances and future perspectives

Chapter 3: An automated on-demand liquid marble generator based on electrohydrodynamic pulling

Chapter 4: Evaporation dynamics of liquid marbles at elevated temperatures

Chapter 5: Liquid marbles as biochemical reactors for the polymerase chain reaction

Chapter 6: Core-shell beads made by composite liquid marble technology as a versatile microreactor for polymerase chain reaction

Chapter 7: Conclusion and future perspectives-Unpublished chapter

Fig.1. 2 Thesis structure as published papers

References

1. Nguyen, N.-T., et al., Recent Advances and Future Perspectives on Microfluidic Liquid Handling. Micromachines, 2017. 8(6): p. 186. 2. Freire, S.L.S., Perspectives on digital microfluidics. Sensors and Actuators A: Physical, 2016. 250: p. 15-28. 3. Sreejith, K.R., et al., Digital polymerase chain reaction technology – recent advances and future perspectives. Lab on a Chip, 2018. 18(24): p. 3717-3732. 4. Pollack, M.G., A.D. Shenderov, and R.B. Fair, Electrowetting-based actuation of droplets for integrated microfluidics. Lab on a Chip, 2002. 2(2): p. 96-101. 5. Geng, H., et al., Dielectrowetting manipulation for digital microfluidics: creating, transporting, splitting, and merging of droplets. Lab on a Chip, 2017. 17(6): p. 1060- 1068. 6. McHale, G., et al., Dielectrowetting Driven Spreading of Droplets. Physical Review Letters, 2011. 107(18): p. 186101. 7. Nguyen, N.-T., et al., Magnetowetting and Sliding Motion of a Sessile Ferrofluid Droplet in the Presence of a Permanent Magnet. Langmuir, 2010. 26(15): p. 12553-12559. 8. Aussillous, P. and D. Quéré, Liquid marbles. Nature, 2001. 411: p. 924. 9. Sreejith, K.R., et al., Evaporation dynamics of liquid marbles at elevated temperatures. RSC Advances, 2018. 8(28): p. 15436-15443. 10. Polwaththe-Gallage, H.-N., et al., The stress-strain relationship of liquid marbles under compression. Applied Physics Letters, 2019. 114(4): p. 043701. 11. Planchette, C., et al., Coalescence of armored interface under impact. Physics of Fluids, 2013. 25(4): p. 042104. 12. Draper, T.C., et al., Mapping outcomes of liquid marble collisions. Soft Matter, 2019. 15(17): p. 3541-3551. 13. Liu, Z., et al., Mechanical Compression to Characterize the Robustness of Liquid Marbles. Langmuir, 2015. 31(41): p. 11236-11242. 14. Mahadevan, L. and Y. Pomeau, Rolling droplets. Physics of Fluids, 1999. 11(9): p. 2449- 2453. 15. Bormashenko, E., et al., Composite non-stick droplets and their actuation with electric field. Applied Physics Letters, 2012. 100(15). 16. Xue, Y., et al., Magnetic liquid marbles: a "precise" miniature reactor. Adv Mater, 2010. 22(43): p. 4814-8. 17. Zhao, Y., et al., Magnetic liquid marbles, their manipulation and application in optical probing. Microfluidics and Nanofluidics, 2012. 13(4): p. 555-564. 18. Khaw, M.K., et al., Digital microfluidics with a magnetically actuated floating liquid marble. Lab Chip, 2016. 16(12): p. 2211-8.

8 19. Arbatan, T., et al., Liquid marbles as micro-bioreactors for rapid blood typing. Adv Healthc Mater, 2012. 1(1): p. 80-3. 20. Sarvi, F., et al., A novel technique for the formation of embryoid bodies inside liquid marbles. RSC Advances, 2013. 3(34). 21. Serrano, M.C., et al., Mammalian cell cryopreservation by using liquid marbles. ACS Appl Mater Interfaces, 2015. 7(6): p. 3854-60. 22. Tian, J., et al., Respirable liquid marble for the cultivation of microorganisms. Colloids Surf B Biointerfaces, 2013. 106: p. 187-90. 23. Urbina, M.A., A.J.R. Watts, and E.E. Reardon, Labs should cut plastic waste too. Nature, 2015. 528(7583): p. 479-479. 24. Dandan, M. and H.Y. Erbil, Evaporation rate of graphite liquid marbles: comparison with water droplets. Langmuir, 2009. 25(14): p. 8362-7. 25. Tosun, A. and H.Y. Erbil, Evaporation rate of PTFE liquid marbles. Applied Surface Science, 2009. 256(5): p. 1278-1283. 26. Mullis, K., et al., Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol, 1986. 51 Pt 1: p. 263-73.

2 Digital polymerase chain reaction technology – recent advances and future perspectives

Abstract

Digital polymerase chain reaction (dPCR) technology has remained a “hot topic” in the last two decades due to its potential applications in cell biology, genetic engineering, and medical diagnostics. Various advanced techniques have been reported on sample dispersion, thermal cycling and output monitoring of digital PCR. However, a fully automated, low-cost and handheld digital PCR platform has not been reported in the literature. This paper attempts to critically evaluate the recent developments in techniques for sample dispersion, thermal cycling and output evaluation for dPCR. The techniques are discussed in terms of hardware simplicity, portability, cost-effectiveness and suitability for automation. The present paper also discusses the research gaps observed in each step of dPCR and concludes with possible improvements toward portable, low-cost and automatic digital PCR systems.

The original version of this chapter has been published as

Sreejith, K. R., Ooi, C. H., Jin, J., Dao, D. V., & Nguyen, N.-T. (2018). Digital polymerase chain reaction technology – recent advances and future perspectives. Lab on a

Chip, 18(24), 3717-3732. doi:10.1039/C8LC00990B

11 2.1 Introduction

Polymerase chain reaction (PCR) is an artificial method for DNA amplification invented by Kary Mullis in 1985.[1] In a conventional PCR, template DNA, primers, DNA polymerase, deoxyribonucleotide triphosphates (dNTPs) and buffer solution are combined in a mixture to undergo a series of thermal cycles to generate millions of copies of a template DNA. The first stage of a thermal cycle begins with a process called initialization, also known as a hot start, to activate the polymerase. It is conducted at a temperature of from 94 °C to 96 °C for a duration of between 1 min and 10 min depending on the template DNA and the type of polymerase. It is followed by a denaturation step conducted typically at a temperature between 93 °C and 98

°C. The hydrogen bonds in double stranded DNA (dsDNA) are broken resulting in the formation of two single-stranded DNA (ssDNA) molecules from each dsDNA (denaturation step). The temperature is then lowered to a primer-specific annealing temperature in the range from 55 °C to 65 °C so that the primers are annealed to complementary sequences of the single- stranded DNA molecules (annealing step). Subsequently the PCR mixture is heated up to temperature between 72 °C and 80 °C based on the polymerase used. During this step the incomplete DNA sequence is extended by a polymerase in the presence of free deoxynucleotide triphosphates (dNTPs) synthesizing a new double-stranded DNA, a copy of the original DNA template (extension step). These last three steps (denaturation, annealing and extension) are repeated to produce multiple copies of the original DNA.[2-4] The process of polymerase chain reaction is depicted in Fig.2.1a. Right from its inception, PCR has found applications in various fields such as biotechnology, cell biology, genetic engineering, forensic science, medical science, drug discovery research etc.[5-11] Finely optimized methods have evolved over the

Fig.2. 1 a) Various stages of polymerase chain reaction (PCR) and b) different phases of a real- time polymerase chain reaction. Digital polymerase chain reaction is carried out up to the linear phase of the fluorescence and obtains either a positive or a negative fluorescence.

last three decades to perform PCR efficiently. These methods are generally categorized as end- point PCR, quantitative PCR (qPCR) and digital PCR (dPCR).[12] The outcome of end-point

PCR is analysed at the end of the entire reaction process, typically using gel or capillary electrophoresis to quantify the outcome of end-point PCR,[13, 14] such as the presence or absence of a target DNA.[15, 16] The end-point PCR technique also finds applications in gene editing,[17] genotyping[18] etc. In qPCR or real-time PCR, the products are continuously monitored throughout the reaction cycles using fluorescent dyes. There are four phases in the qPCR process: (i) initiation (baseline), (ii) exponential, (iii) linear and (iv) plateau.[19] During the first initiation phase, the number of DNA molecules doubles (ideally) during each PCR cycle, but the fluorescence induced by their concentration is below the threshold level of the fluorescence imaging (detection) system. Nevertheless, its amplitude is used for normalization of the PCR curve. Thus, the initiation phase is also called the baseline phase. The exponential phase follows next and doubles the number or DNA molecules during each PCR cycle, but the fluorescence induced by the DNA concentration is above the system detection limit

(exponential phase). Next, the amplification efficiency is reduced due to the limited availability of primers and dNTPs. Various phases of a qPCR process are depicted in Fig.2.1.b.

There are not enough materials to double the number of DNA molecules in each cycle.

Thus, the fluorescence does not increase exponentially anymore, but linearly. Finally, the

13 solution is totally depleted and the fluorescence does not increase anymore (plateau phase).

The cycle threshold (Ct) is an artificially set threshold to compare qPCR curves for templates with different known initial numbers of DNA copies and create the PCR standard curve. The initial quantity of the DNA template can be determined by comparing the fluorescence output curve of the qPCR with a standard curve. The qPCR technique is more accurate than end-point

PCR. qPCR finds application in gene expression profiling,[20] copy number variation detection,[21] molecular diagnosis[22] and genotyping.[23]

The term “digital PCR” was coined by Vogelstein and Kinzler in 1999.[24] Their seminal paper reported a novel approach for converting the exponential analog nature of PCR to a linear digital signal which is essential and most suitable for identification of genetic mutation and for a variety of other research and clinical applications. Experiments were carried out with stool specimens of patients with colorectal cancer on a commercially available 384 well plate. The experiments successfully demonstrated the presence of mutant c-Ki-Ras genes in the stool sample of patients suffering from colorectal cancer. In dPCR, the polymerase chain reaction mixture along with the necessary fluorophore is compartmentalized into small smaller units, and each unit undergoes the same thermal cycles as in the case of a conventional PCR. Each unit is expected to have a single template DNA and some units may not even contain a template

DNA. The units which have undergone a positive PCR reaction can be identified by the fluorescent dye. A positive fluorescence from a unit is regarded as ‘1’, while no fluorescence or negative fluorescence is considered a ‘0’, similar to signals in digital electronics.[25, 26]

The compartmentalization of a PCR mixture into smaller units increases the relative concentration of the target DNA sequence in each unit, reducing the “template competition effect”, which in turn helps in the detection of rare mutations among other wild-type sequences.[19] One of the key differences between qPCR and dPCR is that the fluorescence output is monitored throughout the qPCR process, while it is done only during the exponential

14 phase in dPCR. Also, qPCR requires the generation of standard fluorescence curve and Ct values with known number of nucleic acid copy numbers to compare the outcome of sample with unknown concentration of nucleic acids. On the other hand, dPCR technique does not need any standard for comparison. Poisson distribution is used for the quantification of the total number of target molecules within the sample. The target concentration C can be estimated as[27]

C = λ/V (1) where λ is defined as the mean copies per partition

λ = −ln(z/n) (2) and z is the number of units with negative fluorescence, n is the total number of units and V is the volume of PCR reagents in each unit. It should be noted that the Poisson statistics-based model for estimating the concentration of the target molecule assumes a uniform volume for all units present in a typical dPCR experiment. Any violations in this assumption can affect the quantification accuracy of the technique. Majumdar et al. suggested a “Poisson plus” model for effectively addressing this issue in 2017.[28]

Digital PCR is considered as the most precise PCR technique compared to qPCR and end-point PCR.[29, 30] The dPCR technique has demonstrated a comparable sensitivity with qPCR in several potential clinical applications.[31, 32] One of the major advantages of dPCR over qPCR is the reduced possibility of PCR inhibition.[33, 34] In addition, dPCR offers good repeatability and reproducibility for some applications.[35] With these advantages, digital PCR has gained popularity, and thus a number of significant advancements have been achieved in digital PCR technology over the last two decades. However, digital PCR technology still requires complex, bulky hardware, and a laboratory setup to carry out the desired operation.

The process of PCR is time-consuming, laborious and requires a significant amount of manual

15 interventions at various stages. Commercially available dPCR machines and various dPCR techniques reported in scientific journals also suffer the above-mentioned shortcomings. In addition, the cost of the PCR machines reported to date are not favourable to make it an affordable technology for most applications, especially point-of-care clinical applications. The development of a simple, compact, automatic and cost-effective digital PCR technology with reasonable throughput, sensitivity, precision, and inhibition resistance still remains as a research gap.

Fig.2.2 illustrates the conceptual diagram of the workflow of three different types of

PCR techniques. Digital PCR is fundamentally a three-step process, [25] namely (i) sample dispersion, (ii) PCR thermal cycling (amplification), and (iii) output monitoring. A systematic engineering approach towards each of these processes could possibly result in a simple, compact, automatic and cost-effective PCR platform. The present review concentrates on the critical evaluation of recent engineering advances of these processes in terms of operation simplicity, compactness, the suitability for automation and integration and cost-effectiveness.

Fig.2. 2 Concept and workflow of a) end-point PCR, b) qPCR or real time PCR and c) digital PCR(dPCR).

The review concludes by summarizing the research gaps in each of these processes and providing a few possible research perspectives to fill these gaps.

2.2 Sample dispersion techniques

Sample dispersion is the process in which the PCR mixture is compartmentalised into smaller units for undergoing thermal cycling. Each dPCR sample compartment is expected to possess a maximum of one nucleic acid template to warrant the high accuracy of the measurement. Even if the compartment consists of more than one nucleic acid template, it will give a positive fluorescence (digital ‘1’) during readout, leading to errors in the output quantification. Thus, it is desirable to reduce the sample volume in each compartment to the minimum so that the probability of multiple template DNAs in one compartment is less. A notable cycle threshold reduction is also possible in small volume droplets.[36] A major problem is the contamination of the sample. If liquid handling and compartmentalisation are not properly implemented, the PCR reagents may become contaminated, leading to no amplification of target nucleic acid or the amplification of undesirable nucleic acid molecules.

Undesirable coalescence of reagents in different compartments is another prospective issue of the sample dispersion process. The coalescence of reagents in different compartments may occur as a result of poor design of the compartmentalisation. Some popular methods for sample dispersion and compartmentalisation are discussed in the following subsections.

2.2.1 Droplet-based sample dispersion

The field of microfluidics has advanced in the last three decades.[37, 38] Droplet-based microfluidics is a subfield of microfluidics in which minute sample volumes (10−9 to 10−18 liters) generated as droplets are dispersed in another immiscible liquid.[38-40] Advancements in droplet-based microfluidics have revolutionized the PCR sample dispersion process.

Production and manipulation of droplets down to volumes on the order of attoliters have been

Fig.2. 3 PCR sample dispersion a) by droplets formed by a flow focusing device, b) by droplets formed by a T-junction microfluidic device, c) by droplets formed by a co-flow type microfluidic device, d) in microwells created on a base substrate, e) by channels created on a base substrate, and f) by printing the PCR mixture (step 2) on pre-deposited oil droplets (step 1) with the help of print heads. reported in the literature.[41, 42] The ability to produce droplets in very small volumes considerably reduced the possibility of contamination and decreased the probability of multiple template nucleic acids in a single droplet. Reduction in droplet volume also increases the number of simultaneous reactions and consequently the throughput of digital PCR. The high- throughput capability of microfluidic devices [43] also makes them a good choice for dPCR.

The most commonly used configurations to create droplets with a given volume are T-junction configuration,[44-46] flow-focusing configuration,[47-49] and co-flow configuration.[50, 51]

Active droplet generation methods include dielectrophoresis-based droplet generation [52-54] and electrowetting-based droplet generation.[55-57] Microfluidic droplet generation using T- junction, flow-focusing and co-flow configurations are the most commonly used methods in digital PCR platforms (Fig.2.3.a–c) as the requirement for instrumentation is relatively simple compared to other methods. In these techniques, the PCR mixture forms immiscible droplets in an oil phase and subsequently undergoes thermal cycling. The major advantage of this method is the protection of the PCR mixture from contamination. The oil phase also prevents the droplets of a PCR mixture from evaporation. The availability of various actuation methods

18 such as pneumatic,[58] electrostatic [59, 60] and magnetic [61] manipulation makes microfluidics-based sample dispersion more popular for PCR.

Beer et al. reported the design and development of a real-time on-chip droplet-based

PCR device in 2007. The PCR reaction was carried out in water-in-oil droplets with an average volume of 13 picolitres.[36] The smaller droplet size was achieved using an on-chip hydrophobic T-junction channel with a junction size of 60 μm. The channel size increases to

300 μm downstream of the junction to improve the field of view of the fluorescence monitoring system. Syringe pumps were used for delivering the liquids into the device. The PCR mixture was pumped at a flow rate of 2.3 mL h−1. The flow rate of the oil phase was 0.3 μL h−1. This type of pumps provides a better control over the flow rates and allows for their optimization to achieve monodisperse droplets with a desirable size. The frequency of droplet generation is also an important aspect of microfluidic droplet generation as it is directly related to the possibility of droplet coalescence. The frequency of droplet generation can be controlled by adjusting the flow rates. However, the lack of a feedback system for monitoring the droplet size and the frequency of its formation limits the reliability of such devices. Crawford [62] et al. reported a feedback control system that may be helpful to produce highly monodisperse

PCR reagent droplets with the desired frequency. The possibility of droplet coalescence and cross-contamination can thus be reduced. Automation of a syringe pump-based microfluidic system is relatively simple. The syringe pumps can be programmed to provide a closed-loop feedback for the droplet production process.

Most automated microfluidic droplet generation systems reported in the literature used a computer and commercial software (MATLAB, LabVIEW etc.) for their operation.[63, 64]

The use of third-party software increases the cost and complexity of the PCR device. Syringe pumps and associated tubing make the system bulky and difficult to use. Moreover, there is a high probability that the pumping system is contaminated after the first use, making it difficult

19 to use the same system for different samples. The ‘single use and throw’ type of devices may increase the overhead expenses if such systems are commercialized. Moreover, the manufacturing process of disposable microfluidic chips could be tedious and time-consuming.

Tanaka et al. (in 2015) came up with a hands-free method to prepare highly monodisperse water in droplets using PDMS microfluidic chips for droplet digital PCR.[65]

The design essentially consisted of a T-junction microfluidic chip with 50 μm width at the junction and 250 μm width at the upstream and downstream regions. The channels were connected to two inlet reservoirs and one outlet reservoir. The pumping mechanism of the device is designed by exploiting the gas porosity of PDMS. When a degassed PDMS chip is brought back into standard atmospheric pressure, atmospheric gas diffuses into the PDMS chip.

Thus, if the inlet reservoirs are filled with oil and PCR mixture, the liquids would automatically be pumped to the outlet reservoir through the channel, producing monodisperse PCR mixture droplets in oil. The group successfully demonstrated the tenability of the droplet size by varying the channel dimensions. For a typical experimental setup, 90.5% of the droplets have no DNA molecule, 9% a single DNA and 0.5% multiple DNA templates. This shows a better performance in terms of the undesired condition of multiple DNA in a single droplet as discussed previously. Another advantage of this device is that it does not require a pumping mechanism for droplet generation. However, the tedious and time-consuming process of degassing of the PDMS chip prior to use is necessary. Also, the frequency of droplet generation and droplet speed may reduce over time. The possibility of coalescence of droplets is high with the reduced speed of droplets through the channel. The absence of external pumping and tubing makes this device compact. However, manual intervention and tedious pre-processing steps of the chip make it less viable for a practical PCR device. Using the same concept, this team also designed a flow-focusing configuration for generating monodisperse PCR sample droplets in oil.

2.2.2 Microwell-based sample dispersion

The basic idea of microwell-based PCR systems is to make micro-/nano-sized reaction chambers in a base substrate and fill them with the PCR mixture for thermal cycling. Fig. 2.3.d shows a microwell configuration for dPCR. A top cover or oil was used to prevent evaporation of the reagents during thermal cycling. Microwell-based digital PCR platforms allow for high parallelism, robustness and sensitivity in measurement compared to droplet-based digital PCR platforms.[66] A physical boundary between the wells provides a better isolation between samples compared to the droplet based digital PCR method.

Lindstrom et al. were successful in demonstrating the PCR amplification and genetic analysis in a specially designed 672 microwell chip.[67] The team demonstrated the possibility of heterogeneous operations in a single microwell. The microwell had sloped walls, 650 × 650

μm2 bottom and 1360 × 1360 μm2 top and a volume of 500 nL.[68] The device was used to analyse two human cell lines, one wild type and one with a single-base mutation in the p53 gene. The method involved cell cultivation, cell lysis, PCR, the capture of PCR products on beads, denaturation, and mini-sequencing, all performed in individual wells on-chip. The possibility of heterogeneous operations with a single cell is an advantage of microwell-based

PCR compared to droplet-based PCR.[66] Conversely, multiple operations in droplet samples require complex system design and processes.[69] Another advantage of a microwell-based

PCR system is the easy and inexpensive integration of a heater and a fluorescence monitoring system.

One of the major problems faced during the PCR process is the difficulty in handling cells and DNA samples. Mostly, pre-processing is required to enrich the sample with target

DNA. The possibility of contamination is high during the pre-processing. Microwell-based

PCR systems can be engineered for the hassle-free handling of samples. Cai et al. had

21 successfully developed a point-of-care pathogen detection device with a multiplex PCR array and integrated dielectrophoresis.[70] The device consists of two hydrophobic glass plates; the bottom plate has five microchannels (800 μm wide, 15 μm deep) each with PCR reaction chambers (800 μm × 800 μm in size, 135 μm in depth). The top plates are integrated with electrodes to perform dielectrophoresis. The device successfully detects pathogens in complex samples such as human blood in a relatively short time. The major advantage of this device is that human blood can be directly fed into the microfluidic device without any pre-processing.

The target pathogens were separated inside the microfluidic device using dielectrophoresis.

The pathogens were then mixed with PCR reagents using slip-chip technology and subsequently underwent thermal cycling. Easy and contamination-free processing is possible since the pathogen extraction and PCR are carried out inside the device. Slipchip technology was introduced in 2009 by Du et al.[71] The device has two plates in contact with each other.

The bottom plate usually contains wells with preloaded reagents, and the top plate acts as a lid for the well with reagents. The device also has fluidic channels with ducts on the bottom plate and wells on the top plate, which are connected when the top and bottom plates are aligned in a particular configuration. The device does not require complex pumps or valves for its operation. The major advantage of slip-chip technology in PCR is its ability to conduct multiplexed reactions. It is easy to expose the same reagents to multiple nucleic acid templates or vice versa. Fig.2.4 illustrates the operation concept of the slip-chip technology.

Shen et al. used a slip-chip microfluidic device to conduct PCR and to quantify nucleic acids.[72, 73] The device utilised two plates with wells for PCR sample loading and circular reservoirs preloaded with oil to conduct PCR. A fluidic channel was integrated with the wells for sample loading. The wells were elongated and overlapped on sample loading. A simple relative slip of one plate will disconnect the elongated wells from the fluid channel and makes them overlap with the circular reservoir. Thermal cycling was carried out, and the reaction

22 outcome was monitored using fluorescent dyes. The device is composed of 1280 2.6 nL compartments.[72] A microfluidic real-time PCR rotational slip-chip device was also reported in 2011 for multiplexed quantification of nucleic acids.[74]

The throughput of microwell-based dPCR is limited by its design as compared to microfluidic droplet-based PCR. Droplet-based dPCR systems can handle smaller volumes.

However, modern fabrication technologies and highly optimised designs can remove these limitations. Dimov et al. were successful in performing multiplex single-cell RNA cytometry in a microwell array of 60 000 chambers with a volume as small as 20 pL.[75] 100-plex amplification of pneumonia pathogens was successfully demonstrated by Li et al. in 2011.[76]

Heyries et al. reported a megapixel digital PCR device which uses surface tension-based sample partitioning. The device has 1 000 000 reactor chambers with a volume of only a few picolitres. The device was reported to have a dynamic range of 107.[5] Sefrioui et al. developed a chip-based digital PCR platform to detect circulating DNA metastatic colorectal cancer.[77]

Witters et al. followed a complex fabrication procedure to develop a microwell array of 38 fL volume each. The device utilised magnetic beads for the detection of single DNA with a concentration as low as 10 aM.[78]

Fig.2. 4 Concept diagram of slip-chip technology

2.2.3 Channel-based sample dispersion

The fundamental building block of a channel-based digital PCR system is a microfluidic device with thin mutually independent microfluidic channels. Each channel is connected with a number of reaction chambers. The PCR sample is prepared outside of the microfluidic device and subsequently fed into the microchannels. The sample flows through the microchannel and fills the chamber where PCR thermal cycling is carried out. Each channel and the associated chambers are separated from each other by means of a thin layer of microfluidic valves. The separation between chambers helps in reducing cross-contamination and sample coalescence. Fig. 2.3.e shows the conceptual diagram of the channel-based sample dispersion method. High parallelism and the possibility of multiple cell processes are the advantages of this sample dispersion technique.

The high-throughput microfluidic single cell digital polymerase chain reaction platform reported by White et al.[79] is a good example of this category. The device performs parallel processing of single cells and is capable of executing all the processes such as cell capture, washing, lysis, reverse transcription, and digital PCR. The device is composed of 1020 25 pL chambers. The device is capable of running 204 000 digital PCR reactions for a device footprint of 10 cm2. Tian et al. successfully developed an integrated microfluidic system for bovine DNA purification and digital PCR detection.[80] The device integrates a nucleic acid extraction module and dPCR module in a single chip. The dPCR module is composed of 650 chambers of 200 μm diameter and 230 μm depth. In 2017, Fu et al. demonstrated a microfluidic chip with

10,000 0.785 nL compartments that are connected by narrow channels. The specially designed pre-degassed PDMS chip was able to load and compartmentalise the sample autonomously.

The authors successfully demonstrated dPCR on this device.[81]

Ramakrishnan et al. reported a microfluidic device for digital PCR.[82] The device was based on the commercialised digital PCR technology of Fluidigm Corporation. The Fluidigm device has 48 individual panels with 770 reaction chambers per panel for digital PCR. The reaction volume of each chamber is approximately 0.8 nL, and the reaction chambers are isolated from each other by pressure-controlled valves.[82]

In most of the channel-based PCR platforms, the micro-compartments are connected in series with microchannels. The reagents enter through an inlet at one end and fill the compartments serially. The remaining reagents would come out through the outlet. This process may cause sample loss and inaccuracy in detection.[79, 83] Zhu et al. reported a device with a novel scalable self-priming fractal branching microchannel network for dPCR in

2017.[84] The device design was inspired by a natural fractal-tree-based structure which enables even distribution and 100% compartmentalisation of the sample without any loss. The device rectified the need for precise control systems and complicated tubing connection. Also, the device used only oil to separate the compartments instead of the conventional valves.

2.2.4 Printing-based sample dispersion

Printing-based sample dispersion is a relatively simple technique compared to the other methods discussed above. The PCR mixture is deposited on top of a base substrate which is pre-deposited with an oil drop. The PCR mixture is deposited in such a way that it is covered by the oil drop. The oil prevents evaporation of the PCR mixture during thermal cycling. An automated inkjet printing technique can be used for depositing the oil and the PCR mixture.

Fig.2.3.f depicts the concept of printing-based PCR sample dispersion. This technique possesses a number of advantages over the other methods. (i) Unlike polymeric microfluidic devices, silicon, glass or other non-reactive materials are used as the base substrate, which can be reused after proper cleaning. This reduces the overhead cost of the PCR technique. (ii) The

25 deposition method can be precisely controlled using the necessary electronic control.[85] This reduces the possibility of cross contamination and opens the possibility of a completely automated PCR system. (iii) The oil droplet covering the PCR mixture reduces the possibility of contamination from the ambience. (iv) Integration of the heater with the substrate is relatively easy. (v) The fluorescence detection system can be integrated without much engineering complexity. (vi) Injection of multiple reagents is possible at any time.

Sun et al. proposed a novel double inkjet printing-based platform for parallel PCR in picolitre droplets.[86] The device utilises piezoelectric inkjet printing to deposit oil droplets on top of a hydrophobic and oleophobic substrate. PCR reagents are subsequently deposited on top of the oil using the same printing technology. Zhu et al. reported a two-dimensional array for reverse transcription PCR.[87] A sequential-operation droplet-array-based microfluidic robotic system was used to print PCR mixture droplets with 50 nL volume on an oil-filled microchip. Zhang et al. reported the use of inkjet printing technology to generate highly monodisperse droplets in an oil-filled glass capillary instead of a substrate to perform PCR in

2018.[88]

2.3 Thermal cycling methods

Thermal cycling is the process in which the dispersed PCR reagents undergo heating at various temperatures. Conventional PCR uses programmed thermal cyclers with highly accurate temperature control to perform thermal cycling. Being the key stage of any PCR process, the thermal cycling technique deserves an intense review in this context. The ability to maintain the accurate temperature for an exact duration of time determines the amplification efficiency of PCR. The ramping rate during the heating and especially the cooling period is another important parameter affecting the overall efficiency and specificity of PCR. The ramping rate determines the overall time required to complete the polymerase chain reaction.

The time required to complete the desired number of cycles of PCR would be higher with a 26

Fig.2. 5 Thermal cycling in digital PCR platform using a) Joule heating technique, b) TEC heating, c) surface acoustic wave technology, d) contact type photonic heating, e) non-contact type photonic heating, f) direct photonic heating with a thin layer of gold coating in a microchip, g) induction heating, h) microwave heating and i) solar heating. thermal cycling technique with a poor ramping rate. It should be noted that a lower ramping rate of heating may only delay the PCR cycles, while a poor cooling rate would sacrifice the amplification specificity. If the cooling time of thermal cycling is not optimised, the ssDNAs formed after denaturation may undergo hybridisation before reaching the annealing temperature, resulting in a reduced yield in each cycle. Hence, the thermal cycling subsystem plays an important role in developing a simple, compact and low-cost digital PCR system.

Various novel methods for performing thermal cycling in digital PCR devices have been reported in the literature. These methods can be classified as Joule heating, thermoelectric, photonic, acoustic, inductive, microwave-based and solar heating. Each of these methods and their use for thermal cycling in a simple, compact and low-cost digital PCR system are discussed in the subsequent subsections.

2.3.1 Joule heating

Joule heating relies on an electric current passing through an ohmic conductor, also called the resistive heater. The heat energy generated is proportional to the square of the electric current and the resistance of the ohmic conductor. Either a stand-alone or an integrated resistive heater can be used for thermal cycling of the PCR mixture. In stand-alone heaters, the heating element and the PCR reaction chamber are developed separately and then made to come in contact physically for thermal cycling. The heating element material can be deposited on a base substrate using various physical or chemical deposition strategies. The base substrate is subsequently attached to the PCR reaction chamber for thermal cycling.[89] Different microheaters can be integrated in the reaction chambers.[90] Micro-machined resistive heaters can also serve as a heating element for PCR.[91] In the case of an integrated device, the material of the resistive heater is directly deposited on the surface of the PCR chamber. Fig. 2.5a depicts the concept of a typical Joule heating-based thermal cycler.

El-Ali et al. developed an SU-8 based PCR chip with an integrated platinum heater and a temperature sensor in 2004.[92] The device was the first polymer-based PCR chamber with integrated heater and temperature sensor. The chip has a chamber volume of 20 μl with fast heating and cooling rates of 50 and 30 K s−1, respectively. The team successfully demonstrated the amplification of yeast gene ribosomal protein S3 and Campylobacter gene cadF. Liao et al. reported a PDMS-based real-time PCR chip with integrated platinum microheaters and a platinum temperature sensor.[93] The heating and cooling rates of the system were 20 and 10

K s−1, respectively. The system successfully amplified and detected dengue virus type 2 and enterovirus 71 (EV 71).

Neuzil et al. presented a low-cost lab-on-a-chip micro PCR device.[91] The device consisted of a microscope glass coverslip placed on top of a micromachined silicon chip. The

28 silicon chip has integrated heaters and temperature sensors. A single 1 μl PCR sample droplet was placed over the glass slip and covered with mineral oil to prevent evaporation. Mineral oil serves as the thermal contact between the PCR sample droplet on the glass coverslip and the micro-machined silicon chip. The device takes half a second to heat up from 72 °C to 94 °C and two seconds to cool from 94 °C to 55 °C. The glass slip on which the PCR sample is placed is disposable so that the device is highly efficient in eliminating cross-contamination of samples during the tests.

Joule heating-based thermal cycling has the following advantages: (i) easy to integrate with the PCR chamber; (ii) heat energy variation is directly proportional to the applied electric current, which makes precise temperature control relatively simple; (iii) the heater element and associated control elements can be miniaturised to eliminate the overall system complexity and size; (iv) multiple heating elements can be used on a substrate to provide multiple temperature regimes. This ability can be exploited for a PCR platform; (v) minimum cost. Non-uniform heating of the target is the major disadvantage of resistive heaters. An array of microheaters can eliminate this problem to a greater extent. However, the array may increase the complexity of the system. The ramping rates of heating and cooling are limited in thermal cyclers based on Joule heating. The cooling rate can be increased by a cooling fan or thermoelectric coolers.

2.3.2 Thermoelectric heating

When an electric current passes through the junction of two dissimilar metals, one junction experiences cooling while the other junction experiences heating. This effect is known as the Peltier effect, discovered by Jean Charles Athanase Peltier in 1834.[94] Peltier effect- based thermoelectric modules (TEC) in various specifications are commercially available. A number of thermal cyclers using thermoelectric heating have been implemented in end-point

PCR and qPCR technology. [95-97] Fig.2.5b shows the basic arrangement of thermal cycling

29 using a TEC. The major advantage of a TEC is its ability to reverse the thermal effect based on the polarity of the input bias voltage. This characteristic can be utilised to reduce the cooling time of the thermal cycle, which in turn reduces the overall cycling time. The major disadvantage of thermoelectric modules is the high thermal inertia. The thermal response of a system based on the Peltier effect can be improved by optimising the PCR chamber. Shigeo et al. sandwiched a copper PCR tube tip directly between the p-type and the n-type thermoelectric material. [96] This design demonstrated considerable improvements in thermal response of the overall system.

Thermoelectric modules and Joule heating modules can work together in a hybrid thermal cycler. The high ramping rate of the Joule heating module is beneficial to reduce the cycle time. The TEC can be used for forced cooling during the cooling cycle, which also reduces the overall cycle time.

2.3.3 Acoustic heating

Longitudinal waves traveling on the surface of a material is known as surface acoustic waves (SAWs). Due to the energy dissipation, the liquid over the SAW transmitting surface can be heated. The temperature of the liquid can be controlled by the applied voltage and the duty cycle of the SAW generator. [98, 99]Later in 2009, Kondoh et al. developed a temperature control system for liquid droplets using SAW.[100] The team successfully demonstrated the thermal cycling for PCR by controlling the duty cycle of the SAW generator.

Shilton et al. demonstrated controllable heating using SAWs in 2015.[101] The device consisted of a gold patterned lithium niobate (LN) substrate mounted on a TEC. The piezoelectric material LN was responsible for generating surface acoustic waves with excitation from a radio frequency (RF) source. The surface acoustic waves propagating along the substrate heat up the substrate. Water droplets of 1 μL volume were placed over the

30 substrate. The SAW-driven temperature changes in the droplets achieved the corresponding set value within 150 ms and the temperature overshoot in the microdroplets was within 12 K. The fastest heating rate in the experiments was reported to be 70 K s−1. A short settling time is essential for a better yield in each cycle of the reaction. Furthermore, the short settling time may help in protecting the nucleic acid template from degradation. A fast ramping rate is the key factor for reducing the overall time duration of the PCR. However, this method is not directly applicable to PCR as the liquid droplet is kept in an open environment and prone to contamination and evaporation. A suitable system should be designed in association with this thermal cycling method to prevent contamination of the reaction mixture. Using liquid marbles as a microreactor [102-104] for carrying out PCR is a good option as it needs little modification to prevent contamination.[105]

Ha et al. reported acoustic heating of a polydimethylsiloxane microfluidic system for

PCR.[106] Although the system could be categorised as a continuous-flow PCR system, the

PCR achieved a billion-fold amplification of a 134 amplicon DNA in less than 3 minutes. The heating mechanism was based on the damping of the acoustic wave in PDMS. The high- frequency waves were generated from a conventional SAW generator. Fig.2.5.c shows the conceptual diagram of a SAW-based thermal cycler for PCR.

2.3.4 Photonic heating

Photonic heating converts the energy of light into heat. Generally, laser or light- emitting diodes (LEDs) serve as the source of light. Roche et al. demonstrated a plasmonic thermocycler for the amplification of human androgen receptor DNA.[107] This is the first paper reporting the practical application of photonic thermal cycling. Gold nanoparticles with an average diameter of 60 nm were used to produce heat energy when activated with a 532 nm laser. A cooling fan was used to achieve rapid cooling. The heating and cooling rates are

31 reported to be 7.62 ± 0.81 K s−1 and 3.33 ± 0.24 K s−1, respectively. Thermal cycling was implemented in contact and non-contact setups. The gold nanoparticles were separated from the PCR reagents in the non-contact setup, while gold nanoparticles were dispersed in the PCR reagents in the contact type setup. The team successfully demonstrated PCR in both setups.

Fig.2.5.d and e depict the conceptual diagram of the experimental setup. However, non-contact plasmonic thermal cycling requires a large number of gold nanoparticles to heat up a small amount of PCR reagents. Moreover, the actual cost of this method should also include the high cost of the laser source, which makes this method impractical for desktop and portable use.

Son et al. reported in 2015 an ultrafast photonic PCR.[108] The device utilised plasmonic photothermal conversion using a photon–electron–phonon coupling. The plasmon- assisted optical absorption capacity of a thin gold film was utilised for thermal cycling. The thickness of the gold film was observed to be 120 nm for optimum results. Light-emitting diodes (LEDs) with a wavelength of 450 nm were used as the source of light. The use of LEDs significantly reduced the cost of the device. LEDs consume less power than conventional lasers. The heating and cooling rate of the device was reported to be 12.79 ± 0.93 K s−1 and 6.6

± 0.29 K s−1, respectively. This PCR platform can be considered as low-cost, ultrafast and compact.

2.3.5 Induction heating

Induction heating is the process wherein an electrically conductive metal is heated when it is placed in a varying magnetic field. The eddy currents produced by electromagnetic induction are responsible for the heating of metal. A coil powered with an AC power supply is used to generate the varying magnetic field. Fig.2.5.g depicts the concept of induction heating.

The heating efficiency of inductive heating is a function of the frequency of the AC source, the relative permeability and the resistivity of the target material.[109, 110] This heating method

32 has been used for biological applications[111, 112] including the thermal cycling of a PCR mixture. Pal and Venkataraman reported an induction-based portable, battery-operated thermal cycler.[113] The setup consists of two subsystems: (i) a magnetic field generator: a 10 mm long coil made by winding 2 mm enameled copper wire on a 10 mm diameter ferrite core. The coil has an inductance of 203 μH at 100 kHz; (ii) PCR chips fixed on a metal ring: the PCR chip is made by anodically bonding a 3 mm × 3 mm Pyrex glass slide to silicon. A 1 mm deep hole drilled in the glass served as the reaction chamber. The heating and cooling rates were 6.5 and 4.2 K s−1, respectively, for a 0.2 mm metal sheet. A simple K type thermocouple and the simple ON–OFF strategy were adopted for temperature control. The setup was powered by a

12 V battery. The power consumption of each chip was reported to be 2 mW.

Pal et al. used a similar induction heating technology to amplify a 350 bp segment from a plasmid and a human sex determining gene.[114] Although the reported technology was designed for end-point PCR, it also has the potential to be used in digital PCR. The lack of a complex control circuitry and the use of rechargeable battery make this method an attractive candidate for a portable and inexpensive digital PCR system. The major disadvantage is the complex fabrication procedure of the microchip. Similar to other microfluidic chips, the device also poses the risk of contamination after the first use. However, this heating method combined with a suitable sample handling technique may yield an effective dPCR platform. Bio

Molecular Systems has commercialised the world's first qPCR thermal cycler based on magnetic induction technology (https://biomolecularsystems.com).

2.3.6 Microwave heating

Microwave heating has been extensively used in lab-on-a-chip devices assisting biological processes and biochemical reactions. The major disadvantage of conventional thermal cycling methods including Joule heating is the thermal inertia associated with the

33 heating block. The heating block takes a substantial amount of time to reach the setpoint temperatures due to its high thermal inertia. The low thermal inertia and higher energy density of microwave heating make it suitable for rapid thermal cycling.[115] The absence of the wall effect[116] in microwave heating can accelerate the speed of PCR. Fermer et al. demonstrated the use of microwave for thermal cycling.[115] The team used a commercially available single mode microwave cavity (Microwell 10) to amplify a 220-base pair segment of the chromosomal 23S rRNA gene of thermophilic campylobacters. The conventional end-point

PCR successfully amplified the target DNAs using microwave heating. Orrling et al. successfully demonstrated PCR thermal cycling with external single cavity resonators in millilitre scale.[117] Garg et al. successfully developed a low-cost, low power, programmable

PCR thermal cycler for point-of-care devices.[118] A copper microstrip transmission line was used to provide 0.7 W power to a chamber with 4 μL capacity. The whole system was incorporated in a polycarbonate substrate. Temperature programmability was achieved by tuning the amplitude and frequency of the microwave. Fig.2.5.h shows the conceptual diagram of microwave heating on the microchip. A similar device was reported by Marchiarullo, who successfully conducted thermal cycling of 1 μL PCR mixture containing a fragment of the lambda-phage genome, proving the ability of microwave heating to be incorporated into a portable lab-on-a-chip device.[119]

Compared to conventional thermal cycling, microwave heating stands out with its higher ramping rate. However, proper impedance matching between the target PCR mixture and the microwave resonator is necessary for optimum heating efficiency. The possibility of variation in permittivity of the PCR mixture with rising temperature may degrade the efficiency of microwave heating.[119] Also, it would be difficult to design an efficient microwave heating-based thermal cycling system for PCR of a wide range of target volumes. In other words, the operation is limited to a narrow range of volumes and a typical microwave

Table 2.1 Qualitative comparison of various thermal cycling techniques

Techniques Heating form Ramping rate Portability Cost Drawbacks

Joule Conduction **** ***** Low -

Thermo electric Conduction *** ***** Low Higher thermal inertia

Acoustic Conduction *** *** High Complex

Photonic Radiation ***** ** Low-High Complex ,bulk and expensive

Inductive Radiation ***** *** Medium Complex

Microwave Radiation ***** ** High Complex and lacks universality

Solar Radiation ** ** Medium Lacks universality

frequency. In the near future, we may expect the development of a generic microwave thermal cycling technique with a wider range of target volumes, simple design, efficient temperature control and low cost for portable digital PCR devices.

2.3.7 Solar heating

Conventional PCR techniques require an electricity supply. Thus, it is difficult to use conventional PCR machines or techniques in areas where electric power is unavailable or not stable. The major part of the power is consumed by the thermal cycling subsystem of the PCR machine.[120-122] Recent studies showed promising results in using solar thermal energy as a source for PCR thermal cycling.[123-127] Jiang et al. reported the use of solar thermal energy in the detection of Kaposi's sarcoma herpesvirus (KSHV/HHV-8) using PCR in a couple of papers published in 2013 and 2014. The authors used a combination of collimating lens, aluminium foil mask, and a microfluidic device to achieve solar thermal cycling of a PCR mixture. The collimating lens focuses light on to the microfluidic device containing the PCR mixture through the aluminium foil mask. The mask contains a circular opening through which the passage of solar energy and the temperature at various regions of the microfluidic device are controlled. The distance between the lens and the aluminium mask can be varied to control

35 the temperature of each region. This research group used smartphone technology for monitoring the temperature of the thermal cycle. Fig.2.5i depicts the concept of solar thermal cycling.

The solar heating technique utilizes inexpensive hardware components such as a collimating lens and an aluminium foil mask. The temperature control was obtained by varying the relative position between the lens and the microfluidic chip. Thus, solar thermal cycling could be considered for designing a portable, cost-effective digital PCR device. However, solar thermal PCR systems reported to date are end-point PCR systems. No research has been reported on the use of solar thermal energy for digital PCR systems. One of the major engineering challenges of using solar thermal energy in a digital PCR system is the possibility of light interference and probability of noise in the fluorescence detection system by the sunlight. However, optimised systems could be designed to isolate the solar thermal cycling and fluorescence detection to avoid the possibility of errors in detection. Using microchannel geometry and dimension to control the period of thermal cycles sacrifice the broader range of applications. Table 2.1 provides the qualitative comparison of various aspects of the thermal cycling techniques discussed in this paper.

2.4 Output monitoring techniques

Successful thermal cycling of the PCR mixture yields amplification and fluorescence in each microreactor. Detection and quantification of the amplified DNAs is the final stage of a PCR process. Two techniques are widely used for the detection of the fluorescence output:

(i) using sensors such as a photodiode, phototransistor and photomultiplier tubes; (ii) using scanning devices such as charge-coupled devices (CCD) or complementary metal-oxide- semiconductor (CMOS) sensors.

The photomultiplier tube (PMT) captures fluorescent light emitted by fluorophores in the target and converts it into electrical signals corresponding to the intensity of the light. This method is well suited for fluorescence probing in droplet-based microfluidic devices.[128, 129]

Fig.2.6.a shows the concept of a PMT-based output monitoring system.

Mazutis et al. reported the kinetic analysis of an in vitro translated enzyme.[128] The work utilised photomultiplier tubebased fluorescence detection for laccase activity in the reaction mixture. Pekin et al. used the same setup to quantify the mutated oncogene in gDNA.[130] The experimental setup consisted of two laser beams for exciting the target droplets containing PCR products of a mutated gene, wild-type DNA and corresponding fluorophores. The amplified mutant gene emits green light while the wild type produces red light. The red and green lights were captured using two photomultiplier tubes. The voltage output of PMTs were used to quantify the mutant gene. LabVIEW software was used for data processing.

Zhong et al. demonstrated the use of photomultiplier tubes for multiplex PCR.[131]

The paper reported a novel method of multiplex digital PCR in droplets. The team verified the accurate multiplexed measurement of gene copy numbers for different targets and one reference. The instrumentation for fluorescence detection consisted of a 2× expanded, 20 mW,

480 nm laser source, which is focused onto the microchannel using the objective lens of a microscope. The fluorescence detected from the droplets was discriminated by two band-pass filters. Data processing was done by LabVIEW software. The colour and intensity of fluorescence were used to identify and quantify the different targets. Error due to coalescence and doublets were rectified by analysing the duration and amplitude of the fluorescence burst.

Quantification using a photomultiplier tube is advantageous for use with microfluidic devices. Errors due to false positives and doublets can be reduced by optimising the signal

37 conditioning and detection algorithms. The major disadvantage of PMT-based detection is the low throughput. PMT-based quantification is a sequential method wherein only one droplet can be evaluated at a time. This characteristic makes the method not suitable for simultaneous quantification of high throughput digital PCR platforms. Although the photomultiplier tubes and corresponding electronics are economical, the laser excitation source and software used for data processing are costly, resulting in a relatively expensive detection system.

Advancements achieved in the fields of flexible electronics and integrated sensors [132-135] are yet to be exploited for developing simple, inexpensive dPCR quantification methods. A less expensive light source could replace lasers as the excitation source. Inexpensive microcontrollers for data processing may make PMT-based quantification systems more compact and affordable.

Camera-based fluorescence detection is a three-step process: (i) fluorescent excitation of PCR microreactors with a light source; (ii) scanning the fluorescence using a CCD/CMOS camera; (iii) image/data processing of the scanned image and subsequent quantification.

Mercury vapour or laser lights are the conventional sources of light, which are bulky and expensive.[136] LED can considerably reduce the size and cost of the light source in a PCR fluorescence detection system. The camera-based fluorescence detection system is

Fig.2. 6 Fluorescence detection using a) a photomultiplier tube and b) a camera

38 advantageous, as it scans the entire reaction platform at once resulting in reduced detection time as compared to its PMT-based counterparts. Although the primary detection is quick, PCR quantification may take longer, depending on the speed and the efficiency of the image processing algorithm. Also, a camera-based system is capable of handling high-throughput digital PCR fluorescence output. Fig.2.6.b depicts the concept of camera-based fluorescence detection.

Hatch et al. reported a 1-million droplet array with wide field fluorescence imaging for digital PCR.[137] The device is a droplet-based microfluidic platform with fluorescence imaging. Wide-field images of the droplet array were captured by a 21-megapixel digital camera (Canon 5D Mark II) with a CMOS sensor and an f/2.8 USM macro lens. A 350 W

Omniprint-1000 portable light source with 3 mm fiber optic cable and a 25 mm filter kept at

5–8 inches above the microfluidic reaction chamber at an angle of 30–45° was used to illuminate the reaction chamber. The camera was positioned vertically above the reaction chamber. The setup was capable of imaging areas of 8.6 and 17.2 cm2 with 1× and 0.5× magnification, respectively. The device successfully captured 1 million 50 picolitre drops in an 11 cm2 viewing area with a resolution of 15–20 pixels per droplet. Image and data processing were carried out using ImageJ and MATLAB. The image quantification required 0.5–1.5 hours depending on droplet lattice uniformity, fluorescence uniformity, the degree of droplet overlap and the percentage of amplified droplets.

In summary, fluorescence detection systems used in most digital PCR platforms reported to date are not well optimised in terms of size and cost. Reduction of size and cost of the fluorescence detection system is important for the design and development of a simple, compact, fully automated and cost-effective digital PCR platform. Novak et al. observed that the integration of conventional fluorescence detection methods with lab-on-a-chip devices actually makes the system a “chip-in-lab”.[136] The authors developed a compact and

39 integrated fluorescence detection system using a LED as a source of excitation and a photodiode as a detector. The system incorporates all the necessary electronics and optics for efficient detection of fluorescence within a device dimension of 30 mm × 30 mm × 11 mm.

Peham et al. demonstrated in 2011 fluorescence detection of end-point PCR using a LED– photodiode sensor system.[138] Recent developments of the integration of conventional as well as organic LEDs and photodetectors on lab-on-a-chip devices indicate the high potential that they may be exploited for compact and inexpensive quantification strategies of digital

PCR.[139, 140]

2.5 Conclusions and future perspectives

The present paper reviews recent advances in the area of digital PCR. The review evaluates this technology in terms of simplicity of operation, compactness, possibility of automation and cost. The main processes involved in digital PCR are sample dispersion, thermal cycling, and output monitoring. Research advances reported in each of these processes were critically evaluated.

The scope of droplet-based sample dispersion, microwell based sample dispersion, and channel-based sample dispersion is limited due to their complexity in manufacturing and associated hardware requirements. Extensive overhead cost for these technologies once commercialised is also a drawback. Special arrangements and care are required to prevent evaporation and contamination. Printing-based sample dispersion is found to be better compared to other methods of dispersion. The possibility of automation and reuse of this platform makes it a relatively good choice of sample dispersion. However, the system requires an oil phase to prevent contamination and evaporation. Liquid marble is a different sample dispersion method that could meet all the requirements of a handheld dPCR device. Liquid marbles are formed by coating an inert hydrophobic substance on liquid droplets.[141]

Hydrophobic powder coating on liquids can be done by rolling the liquid droplets on a 40 hydrophobic powder bed. This technique can be used to make liquid marbles containing a PCR mixture. Systems for keeping these liquid marbles for thermal cycling are relatively simple.

Moreover, the inert hydrophobic powder does not react with the PCR mixture. The hydrophobic powder coating prevents contamination of the PCR mixture. Liquid marbles are extensively used as microreactors for biochemical reactions, cell biology, bioengineering, biomedicine, and genetic analysis.[102],[142-147] Various mobility manipulation techniques[102, 142],[148-152] are available for manipulating liquid marbles. The major challenge in using liquid marbles as a dPCR reactor is the possibility of evaporation of the PCR mixture during thermal cycling. However, our earlier studies showed promising results on the evaporation stability of liquid marbles at high temperatures.[105]

Joule heating is the simplest method for thermal cycling. It is easy for an engineer to control the heating time of Joule heating elements. However, the cooling time depends on the system thermal time constant as heat is removed only by natural convection. A combination of

Joule heating and active TEC could be an ideal method for thermal cycling of dPCR. This technology may reduce the unnecessary lag in completing one thermal cycle. Moreover, the optimum ramping rate of heating and cooling optimises the amplification efficiency. Many commercially available PCR machines use a combination of Joule heating and TEC to optimise the cycling time. Joule heating elements and TEC are relatively cheap and easy to integrate into a compact system. Photonic- and surface acoustic-based heating methods are costly and require complex and expensive setups. Microwave heating suffers from the drawback of hardware complexity. The efficiency of heating is limited to a narrow range of sample volumes for a typical microwave-based thermal cycling system. The solar heating technique is bulky and its current form lacks universality to be considered as a serious heating method for a simple, cost-effective and handheld dPCR system.

41 Output monitoring systems for dPCR need more optimisation in terms of hardware simplicity, cost, and compactness. LED-based illumination systems can meet almost all the requirements to be used in future dPCR systems. A low-cost digital camera can serve as the detector. The data processing module and the control of the entire device can be conducted by an inexpensive microcomputer board such as Raspberry Pi, Beagle Bone etc. The use of indigenously developed open source software for image processing and output quantification can considerably reduce the cost of the system. Smartphone-based fluorescence detection [153-

157] is also an option for reducing the hardware complexity and cost.

Ahrberg et al. reported in 2016 a handheld qPCR machine.[158] This portable real-time

PCR device is the first of its kind ever reported. Ahram Biosystems has developed a handheld

PCR device called Palm PCR. The device claims to be capable of amplifying almost all kinds of DNA within 25–30 minutes. Even though the system claims to be a miniature handheld

PCR, it should be noted that the system does not incorporate the monitoring part of the PCR.

It is just a battery-operated thermal cycler where the output should be quantified separately using gel electrophoreses. We conclude that extensive research is still needed to design and develop a simple, inexpensive and portable digital PCR system with a reasonable accuracy, precision, and throughput.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors acknowledge the Australian Research Council for funding support through grant

DP170100277.

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3 An automated on-demand liquid marble generator based on electrohydrodynamic pulling

Abstract

Liquid marble is a recently emerging digital microfluidic platform with a wide range of applications. Conventional liquid marbles are synthesized by coating liquid droplets with a thin layer of hydrophobic powder. Existing and emerging applications of liquid marbles require a contamination-free synthesis of liquid marbles with a high degree of reproducibility of their volume. Despite this requirement, the synthesis of liquid marbles has been still carried out manually. Manual production of liquid marbles leads to inconsistent volume and the possibility of contamination. The synthesis of liquid marbles with sub-microliter volume is difficult to achieve and prone to large errors. This paper discusses the design and development of the first automated on-demand liquid marble generator with sub-microliter capability. The device utilizes electro hydrodynamic pulling of liquid droplets on to a hydrophobic powder bed and subsequently coat them with the hydrophobic powder to synthesize liquid marbles of a desired volume.

The original version of this chapter has been published as

Sreejith, K.R., et al., An automated on-demand liquid marble generator based on electrohydrodynamic pulling. Review of Scientific Instruments, 2019. 90(5): p. 055102.

This paper was selected as an Editor’s pick.

53 3.1 Introduction

Liquid marbles are liquid droplets covered with thin coating of hydrophobic powder.

Aussillous and Quere first reported the production of liquid marbles by coating water droplet with lycopodium grains covered with fluorinated silanes in 2001.[1] Conventional liquid marbles are formed by rolling a small liquid droplet over a hydrophobic powder bed. However, some recent papers reported the synthesis of mono layer nano-particle covered liquid marbles using sol-gel technique.[2-5] The hydrophobic powder coating on the liquid marble physically isolates the liquid droplet from the surrounding, providing a contamination free environment to carry out sensitive chemical and biochemical reactions. Moreover, the hydrophobic coating helps the liquid droplet to keep its spherical shape even under relatively high mechanical stress.[6] Mobility manipulation[7-12] of liquid marbles is easier compared to liquid droplets without hydrophobic coating. Hence, liquid marbles find profound applications in biotechnology, biochemical reactions, cell biology, genetic research, bioengineering, drug discovery research etc.[9, 13-17] Research works have been advancing towards using liquid marbles as a digital microfluidic platform for DNA amplification techniques such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP).[18, 19]

Existing and emerging applications of liquid marbles in various fields of digital microfluidics demand high-throughput generation of liquid marbles with precise control and consistency over their volume. Despite this requirement, the production of liquid marbles is currently still accomplished manually by depositing the liquid droplet on a hydrophobic powder bed using a micropipette and subsequent rolling. Limited throughput, inconsistency in volume, lack of repeatability, the possibility of human error and contamination of liquid sample are some of the main disadvantages of this manual method. Moreover, the generation of liquid marbles with sub-microliter volume is laborious and prone to significant volume inconsistencies. To the best

54 of our knowledge, no other automated on-demand liquid marble generation system with sub- microliter volume capacity has been reported in the literature.

In this paper, we report the design and development of an automated on-demand liquid marble generation system with sub-microliter volume capacity using the concept of electrohydrodynamic pulling. The project utilizes electric field assisted deposition of liquid droplets on a hydrophobic powder bed and subsequent automated rolling for synthesizing liquid marbles of the desired volume. The proposed strategy is capable of synthesizing liquid marbles of sub-microliter volume. Also, this method avoids contamination of liquid samples due to manual interventions.

The paper is structured as follow. Section 3.2 explains the principle of operation of the proposed liquid marble generator and the design parameters of an on-demand liquid marble generator. Section 3.3 describes the fabrication of the automated on-demand liquid marble generator. Experimental evaluation of the developed on-demand liquid marble generator is explained in section 3.4. Section 3.5 concludes the paper.

3.2 Principle of operation of the liquid marble generator

The fundamental idea behind the proposed liquid marble generator can be summarized as a three-step process: (i) Generation of a liquid droplet using a mechanically actuated syringe;

Fig.3. 1 Schematic of pendant droplet under electric field. a) Without hydrophobic powder bed between anode and cathode and b) with hydrophobic powder bed between anode and cathode

(ii) Using an electric field to pull the liquid droplet of the desired volume formed at the tip of a syringe needle; (iii) Collecting the ejected droplet on a hydrophobic powder bed for subsequent automated rolling to synthesize liquid marbles. Knowledge of the physical parameters involved in these processes are essential to designing and optimizing the proposed liquid marble generator, and is explained as follow.

Fig.3.1.a shows the schematic of a pendant droplet formed at the tip of a syringe needle with a high DC voltage applied between the syringe needle and a metallic plate kept vertically below the needle. The force balance equation of a pendant drop at the tip of the syringe needle, when subjected to an electric field is given by:[20]

퐹푔푟푎푣𝑖푡푎푡𝑖표푛 + 퐹푒푙푒푐푡푟표푠푡푎푡𝑖푐 − 퐹푠푢푟푓푎푐푒 푡푒푛푠𝑖표푛 = 0 (1)

4 where, 퐹 = ( 휋푟3) 휌푔 (2) 푔푟푎푣𝑖푡푎푡𝑖표푛 3

1 퐹 = 휀 푆퐸2 (3) 푒푙푒푐푡푟표푠푡푎푡𝑖푐 2 0

and 퐹푠푢푟푓푎푐푒 푡푒푛푠𝑖표푛 = 2휋푅훾 (4)

푟 is the radius of the pendant droplet, 휌 is the density of the liquid, 푔 is the acceleration due to gravity, 휀0 is the permittivity of the medium between the droplet surface formed at the tip of the needle and the cathode plate, 푆 is the surface area of the droplet formed at the tip of the needle and 퐸 is the electric field strength between the droplet surface formed at the tip of the needle and the cathode plate, 푅 is the outer radius of the syringe needle, and 훾 is the surface tension constant of the liquid. The pendant droplet would detach from the needle tip if the total downward force (퐹푔푟푎푣𝑖푡푎푡𝑖표푛 + 퐹푒푙푒푐푡푟표푠푡푎푡𝑖푐) exceeds the surface tension force(퐹푠푢푟푓푎푐푒 푡푒푛푠𝑖표푛 ). It should be noted that the electric field strength 퐸 represented in equation (3) is a function of electrical properties (permittivity, polarizability etc.) of the liquid droplet.[21, 22] However, equations (1) to (4) indicate that for a particular pendant liquid

56 droplet with radius 푟, 퐹푔푟푎푣𝑖푡푎푡𝑖표푛 and 퐹푠푢푟푓푎푐푒 푡푒푛푠𝑖표푛 are constant, whereas 퐹푒푙푒푐푡푟표푠푡푎푡𝑖푐 is a function of the intensity electric field strength 퐸 between droplet surface formed at the tip of the needle and the cathode plate. Alternatively, we may conclude that the intensity of the electric field at the tip of the needle is the parameter that determines the radius of the droplet detaching from the needle tip by overcoming the surface tension force. The radius of the droplet detaching from the needle decreases with increasing electric field strength.

It has been reported in previous in the literature that the electric field strength 퐸 at the tip of the needle is affected by the droplet formation. The electric field intensity at the tip of the needle with a liquid droplet can be estimated as[20]:

√2푉 퐸 = 4퐷−2푟 (5) [퐾(푟−푅)+푅] ln 퐾(푟−푅)+푅

where 푉 is the applied voltage, 푟 is the radius of the droplet, 푅 is the radius of the needle tip, 퐷 is the distance between the cathode plate and the tip of the needle, and 퐾 = 1.2 is a correction factor.

The droplet ejected from the tip of the syringe needle can be collected on a hydrophobic powder bed by placing the bed between the needle tip and the cathode plate, Fig.3.1b. However, the introduction of a hydrophobic powder bed between the syringe needle tip and cathode may alter the electric field at the tip of the needle and so the electrostatic force. The hydrophobic powder bed may act as a dielectric undergoing polarization in an electric field. The effective electric field at the tip of the needle is now reduced to:

퐸퐸푓푓푒푐푡𝑖푣푒 = 퐸 − 퐸푃표푙푎푟𝑖푠푎푡𝑖표푛 (6)

where 퐸푃표푙푎푟𝑖푠푎푡𝑖표푛 is the electric field generated in the hydrophobic powder bed due to dielectric polarisation. The reduced electric field would decrease the electrostatic force acting on the droplet:

1 퐹 = 휀 푆(퐸 − 퐸 )2 (7) 푒푙푒푐푡푟표푠푡푎푡𝑖푐 2 0 푃표푙푎푟𝑖푠푎푡𝑖표푛

Hence, the volume of the droplet detached from the tip of the needle for a typical value of applied voltage is expected to be greater than that obtained in a setup without powder bed.

3.2.1 Derivation of design parameters

Fig.3. 2 Experimental setup of electro hydrodynamic pulling of liquid droplet a) without PTFE powder bed between anode and cathode and b) with PTFE powder bed between anode and cathode

The numerical or analytical modeling of electric field intensity between anode and cathode with a hydrophobic powder bed between them is complex and out of the scope of this paper. However, the electric field strength 퐸퐸푓푓푒푐푡𝑖푣푒 between the tip of the needle and the aluminium plate can be considered as constant for a typical value of applied voltage as long as

58 the mechanical arrangement of the setup and the ambient conditions remains unchanged. We can safely assume that the electric field 퐸퐸푓푓푒푐푡𝑖푣푒 at the tip of the needle and the electrostatic force 퐹푒푓푓푒푐푡𝑖푣푒 acting on the droplet at the tip of the needle is only a function of the applied voltage ‘푉’. Hence, the volume ‘푣’ of the droplet that can be pulled from the tip of the needle is also a function of the applied voltage. The relationship between the droplet volume ejected from the tip of the needle and the applied voltage can be determined experimentally. The details of the experiments are discussed as follows.

A primary experiment was carried out to find out the volume of droplets produced by various values of the applied voltage without the hydrophobic powder bed placed between the needle tip and the cathode plate. Fig.3.2a shows the schematic of the experimental setup. A vertically arranged 1-ml syringe with an inner diameter of 4.6 mm, attached with a blunt stainless-steel needle with an outer diameter 250 µm was used to produce liquid droplets of the desired volume. Deionized (DI) water (density =0.997 g/ml and surface tension = 71.99±0.05 mN.m-1 at 25 0C) was used as the working liquid. The syringe piston was pushed by a motorized micrometer stage (Zaber technologies) moving at a constant speed of 10.2 µm/s. The velocity of the piston should be carefully selected since a higher velocity of the piston would result in liquid jet instead of a pendant drop while a lower velocity of the piston would cause the evaporation of liquid at the tip of the needle before the droplet formation. Moreover, even in the suitable operating velocity range, the frequency of droplet generation also depends on the velocity of the piston. A higher frequency of droplet generation may cause constraints in the automation of the liquid marble synthesising process. The speed of the piston was selected by trial and error method. The syringe needle was connected to the positive terminal of a high voltage source (HVPS2, 20kV high voltage module, Eastern Voltage Research) while a thin aluminium plate (10 cm  10 cm) placed vertically below the needle and connected to the negative terminal of the voltage source. The distance between the tip of the needle and the

59 aluminium plate was 10 cm. A high voltage, varying from 0 to 3.5 kV was applied between the syringe needle tip and the aluminium plate, while the syringe piston was pushed at a speed of

10.2 µm/s. The formation and detachment of droplet from the needle at various voltages are recorded using a high-speed camera (Photron FASTCAM SA3, attached with Nikon AF Micro

Nikkor 60mm f/2.8D lens) at a frame rate of 1000 fps. The liquid droplet ejection was observed to be in the micro-dripping regime from 0-3.4 kV and exhibited the formation of Taylor–cone beyond 3.4kv. The micro dripping regime is suitable for the design of a highly reproduceable on demand liquid droplet production. Images of the droplets just before their detachment from the needle tip was captured for evaluation. These images were processed in OpenDrop software

[23] to get the droplet volume before detaching from the needle tip.

The experimental setup was subsequently rearranged by introducing a hydrophobic powder bed between the needle tip and the aluminium plate cathode. PTFE

(Polytetrafluoroethylene) powder (Sigma-Aldrich, 1 μm nominal diameter, ρ = 2.2 g cm−3) was used as the hydrophobic powder. A plastic rectangular spatula (35 mm  35 mm  8 mm) with negligible thickness, partially filled with PTFE powder forms the PTFE powder bed. The PTFE powder bed was arranged at a distance of 2 cm vertically below the syringe needle to collect the ejected droplet. The horizontal high-speed camera was replaced by a compact CMOS camera (ximea xiQ), attached with 0.5 telecentric lens (Edmund Optics, EO-63074) for hardware simplicity. A new vertical CMOS camera (Edmund Optics, E-05012C) was attached with 1 telecentric lens (Edmund Optics, EO-58430) was introduced in the setup to record the images of liquid droplet deposited on the PTFE bed and the subsequently synthesized liquid marble. Fig. 3.2b shows the experimental setup. High voltages varying from 0 to 3.4 kV were applied in steps and the droplets were collected on the PTFE powder bed. These droplets were manually rolled on the PTFE powder bed to coat the droplet with PTFE powder and form liquid marbles. The liquid droplet and the PTFE powder were kept under an ionizing air blower

(STATIC-ST101A) for a few seconds before starting the rolling process. This step ensures the charge neutralization of the PTFE powder and the droplet. Charge neutralization of PTFE powder and droplet allows for error-free rolling process and uniform coating of the powder over the liquid droplet. Images of the deposited droplet and the corresponding liquid marble were captured by the vertical camera. These images were subsequently processed using ImageJ software to evaluate their respective volume. The variation of the volume of the droplets with respect to the applied voltage in the first and second experiment is shown in Fig.3.3a. The droplet volume obtained in the first experiment (without PTFE powder bed between the anode and cathode) is smaller than the droplet volume observed in the second experiment for the applied voltage up to a threshold value, after which the volumes of both cases are approximately equal. The effect of reduced electric field intensity was observed to be significant at lower voltage resulting in relatively larger differences in droplet volumes between the first and the second experiment. The difference in volume is smaller at a higher voltage.

Curve fitting was performed with the data obtained from the experiments using MATLAB curve fitting tool. A generalized relationship between the volume of the droplet deposited on the PTFE powder bed and the applied voltage was obtained as,

푣 = 3.67 × sin(0.19 × 푉 + 1.90) (8) where 푣 is the volume of the droplet in µL and 푉is the applied voltage in kV. The sine function is evaluated in radians.

The volume of the droplet at the tip of the needle is a function of time, since the droplet formation is caused by the continuous uniform actuation of syringe piston. The rate of change of volume at the tip of the needle can be given as:

푑푣(푡) 푑ℎ = 휋푟2 (9) 푑푡 푠푦푟𝑖푛푔푒 푑푡

푑ℎ where 푟 is the inner diameter of the syringe cavity and is the downward 푠푦푟𝑖푛푔푒 푑푡 velocity of the syringe piston. The volume of the droplet on the needle tip at any instant of time

푡 can be obtained by solving equation (9), substituting the constant values of 푟푠푦푟𝑖푛푔푒 = 2.3

푑ℎ mm and = 10.23 µm/s. Volume of the droplet 푣(푡) at any instance 푡 is given as: 푑푡

푣(푡) = 0.17 × 푡 (10)

Equation (10) can be used to find out the time ‘푡’ at which a particular volume of droplet detaches from the needle tip. The calculated and observed values of 푡 for various volumes of liquid droplets deposited on the PTFE powder bed is shown in Fig.3.3b

Fig.3. 3 Variation of droplet volumes with applied voltage. b) Graph showing the calculated and observed values of time of detachment of liquid droplet for various values of droplet volume

3.3 Fabrication of automated on-demand liquid marble generator

A fully automated on-demand liquid marble generation system was designed and tested by considering the design parameters discussed in the previous section. The automated liquid marble generator consists of two main subsystems, namely (i) the droplet generation and deposition subsystem and (ii) the hydrophobic powder coating subsystem. The details of the fabrication process of each of these subsystems are explained as follow.

3.3.1 Fabrication of droplet generation and deposition subsystem

A vertically arranged 1-ml syringe with an inner diameter of 4.6 mm, attached with a metallic needle of outer diameter 250 µm was used to produce liquid droplets of a desired volume. The motorized micrometer stage moving at a velocity of 10.2 µm/s actuated the syringe piston. The micrometer stage was controlled by an Arduino UNO microcontroller board through a relay. The liquid droplet started forming at the tip of the syringe needle upon the actuation of micrometer stage by the microcontroller. The volume of the droplet at the tip of the syringe needle 푣(푡) follows equation (10).

The deposition of the droplet generated at the tip of the needle on PTFE powder bed is done by applying a suitable electric field between the syringe needle and an aluminium plate placed 10 cm vertically below the needle tip. The Arduino UNO microcontroller is programmed to receive the volume input through a computer interface. The microcontroller calculates the high voltage required to pull the specific volume of a liquid droplet using the relationship shown in equation (8). The microcontroller subsequently uses the calibration data of the high voltage module to determine the DC input voltage required for the high voltage module. A digital equivalent of the calculated DC input voltage is generated by the microcontroller and converted into an analogue DC voltage using the MCP 4725 digital to analogue converter. The analogue DC input voltage is applied to the high voltage DC module

63 through a buffer amplifier designed using LM 358 operational amplifier and TIP 120

Darlington power transistor to generate the high voltage required to electro-hydrodynamically pull the required volume of droplet. The buffer amplifier was biased at 12V, 1A DC using a programmable power supply (KEITHLY, 30V, 5A). The accuracy of the high voltage applied between the tip of the needle and aluminium plate determines the accuracy of the droplet volume deposited on the PTFE powder bed. Hence, feedback is provided from the high voltage module to the microcontroller using a 1000:1 voltage probe (40 kV HV probe, TENMA 72-

3040) and a PID control action with 5% tolerance were performed by the microcontroller to ensure the accuracy of the generated high voltage. It should be noted that the microcontroller actuates the syringe piston only after setting the suitable high voltage between the anode and cathode. The droplet detaches from the needle tip when its volume 푣 is equal to the value, which can be pulled by the applied voltage according to equation (8). The pulled liquid droplet would be deposited on to a PTFE powder bed placed 2 cm vertically below the syringe needle tip.

3.3.2 Fabrication of hydrophobic powder coating subsystem

The droplet deposited on the PTFE powder bed has to be coated with the PTFE powder to form a liquid marble. This was achieved by oscillating the PTFE powder bed 20 times between 0° and 40° with an angular velocity of 1.2 rad/s. The optimum value of the angular velocity was obtained by trial-and-error method. A servomotor (Tower Pro-MG 995) controlled by Arduino UNO microcontroller was used to oscillate the PTFE powder bed. It should be noted that the PTFE powder bed should start oscillating only after (i) the droplet is deposited on the powder bed and (ii) the powder bed and droplet being neutralized by the ionizing air blower. The time 푡 required to generate a specific volume of the droplet at the tip of the needle can be calculated using equation (10). An upper tolerance of 10% was allotted to the oscillation subsystem to address the errors due to the mechanical inertia of the motorized micrometre stage and the syringe piston. The ionising air blower was turned on subsequently by the micro controller for 8 seconds through a relay. The servo motor starts the oscillation

Fig.3. 4 Schematic of the experimental setup of on demand liquid marble generator. Photograph of the setup is shown as inset picture

process after the air blower is stopped. Fig.3.4 shows the schematic of the fully automated on demand liquid marble generator.

3.4 Experimental evaluation of the on-demand liquid marble generator

Fig.3. 5 Graph showing the expected and obtained volume of droplets using the automated on- demand liquid marble generator. The micrographs of droplets and corresponding liquid marbles are also depicted.

The on-demand liquid marble generator in section 3.3 was employed to generate liquid marbles of various volumes. Liquid marbles of volumes ranging from 700 nL to 5 µL were generated automatically with an average error of 8.6% in volume. Fig. 3.5 shows the liquid droplet volumes generated by the automated on-demand liquid marble generator against the expected volumes. The error obtained in the droplet volume compared to the expected value of droplet volume can be attributed to the errors of the high voltage module. This error can be reduced by bringing down the tolerance band of the PID control algorithm from 5% to a suitable value. Reduction in the tolerance band of the PID algorithm would generate the high voltage values precisely, which in turn increases the accuracy in droplet volumes. However, narrowing the PID tolerance band may increase the settling time of the PID controller so that the time required to generate the required precise voltage values would be increased. This may limit the throughput of the proposed liquid marble generator.

3.5 Conclusion and future perspectives

This paper reported the design and development of an on-demand liquid marble generator with sub-microliter volume capacity. The liquid marble generator utilized electric field to get control over the volume of liquid marbles. The volume of the liquid droplet was found to be a function of the applied electric field. The relationship between the droplet volume and the applied voltage was obtained experimentally. A simple theoretical model to estimate the time required to generate a specific volume of the droplet was also developed. These parameters were utilized to implement the on-demand liquid marble generator. Mechanical and electrical subsystems were designed and fabricated to automate the process of droplet deposition and coating. The on-demand liquid marble generator was successfully tested and liquid marbles of a desired volume were synthesized with an average error of 8.6%.

Future works are extended to be the development of a generalized theoretical model representing the relationship between the droplet volume and the applied voltage, with a hydrophobic powder bed between anode and cathode. An extensive study on the effect of physical properties of liquid like density, viscosity, surface tension etc can be conducted and included in the generalised theoretical model. The physical, chemical and electrical properties of the hydrophobic powder is also expected to play a significant role in the high throughput production, quality of coating and also the accuracy of the droplet volume. This revised theoretical model can be used in the already developed on demand liquid marble generator to produce liquid marbles with nano-pico liter volume capacities and better operating volume ranges. The optimization of the PID control algorithm can also be carried out to achieve better accuracy on the volume of the droplet without sacrificing the throughput capability. Though the fundamental electrohydrodynamic pulling strategy of liquid droplet offers a high throughput production of liquid marbles, the proposed single hydrophobic bed beneath the syringe needle limits the throughput capability to a certain extent. Multiple hydrophobic

67 powder bed along with suitable marble transfer mechanism would result in increasing the throughput capability of the proposed instrument. Any of the existing liquid marble mobility manipulation techniques can be suitably engineered to use as a marble transfer mechanism.

Acknowledgments

The authors acknowledge the Australian Research Council for funding support through grant DP170100277.

References

1. Aussillous, P. and D. Quéré, Liquid marbles. Nature, 2001. 411: p. 924. 2. XiaoguangLi, et al., Monolayer nanoparticle-covered liquid marbles derived from a sol- gel coating. Applied Physics Letters, 2017. 111(26): p. 261604. 3. XiaoguangLi, et al., Effective surface tension of liquid marbles using controllable nanoparticle monolayers. Applied Physics Letters, 2018. 113(10): p. 101602. 4. Li, X., et al., A capillary rise method for studying the effective surface tension of monolayer nanoparticle-covered liquid marbles. Soft Matter, 2018. 14(48): p. 9877- 9884. 5. Lin, K., et al., Transparent Bioreactors Based on Nanoparticle-Coated Liquid Marbles for in Situ Observation of Suspending Embryonic Body Formation and Differentiation. ACS Applied Materials & Interfaces, 2019. 11(9): p. 8789-8796. 6. Polwaththe-Gallage, H.-N., et al., The stress-strain relationship of liquid marbles under compression. Applied Physics Letters, 2019. 114(4): p. 043701. 7. Mahadevan, L. and Y. Pomeau, Rolling droplets. Physics of Fluids, 1999. 11(9): p. 2449- 2453. 8. Bormashenko, E., et al., Composite non-stick droplets and their actuation with electric field. Applied Physics Letters, 2012. 100(15). 9. Xue, Y., et al., Magnetic liquid marbles: a "precise" miniature reactor. Adv Mater, 2010. 22(43): p. 4814-8. 10. Khaw, M.K., et al., Digital microfluidics with a magnetically actuated floating liquid marble. Lab Chip, 2016. 16(12): p. 2211-8. 11. Zhang, L., D. Cha, and P. Wang, Remotely controllable liquid marbles. Adv Mater, 2012. 24(35): p. 4756-60. 12. Zhao, Y., et al., Magnetic liquid marbles, their manipulation and application in optical probing. Microfluidics and Nanofluidics, 2012. 13(4): p. 555-564. 13. Heyries, K.A., et al., Megapixel digital PCR. Nat Methods, 2011. 8(8): p. 649-51.

14. Zhang, H., et al., Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics. Anal Chem, 2012. 84(8): p. 3599-606. 15. Arbatan, T., et al., Liquid marbles as micro-bioreactors for rapid blood typing. Adv Healthc Mater, 2012. 1(1): p. 80-3. 16. Sarvi, F., et al., A novel technique for the formation of embryoid bodies inside liquid marbles. RSC Advances, 2013. 3(34). 17. Serrano, M.C., et al., Mammalian cell cryopreservation by using liquid marbles. ACS Appl Mater Interfaces, 2015. 7(6): p. 3854-60. 18. Sreejith, K.R., et al., Evaporation dynamics of liquid marbles at elevated temperatures. RSC Advances, 2018. 8(28): p. 15436-15443. 19. Sreejith, K.R., et al., Digital polymerase chain reaction technology – recent advances and future perspectives. Lab on a Chip, 2018. 18(24): p. 3717-3732. 20. Kim, Y.-J., et al., Numerical and Experimental Analysis of electrostatic Ejection of Liquid Droplets. Vol. 51. 2007. 21. Bormashenko, E., et al., Electrically Deformable Liquid Marbles. Journal of Adhesion Science and Technology, 2012. 25(12): p. 1371-1377. 22. Notz, P.K. and O.A. Basaran, Dynamics of Drop Formation in an Electric Field. Vol. 213. 1999. 218-237. 23. Berry, J.D., et al., Measurement of surface and interfacial tension using pendant drop tensiometry. Journal of Colloid and Interface Science, 2015. 454: p. 226-237.

4 Evaporation dynamics of liquid marbles at elevated temperatures

Abstract

Study of evaporation dynamics of liquid marbles at elevated temperature is essential to determine the feasibility of liquid marbles to be used as micro compartments for digital polymerase chain reaction (PCR). We have modified an existing theoretical model of evaporation of a liquid droplet and verified its applicability on the evaporation of liquid marbles. The evaporation dynamics of an individual and a group of liquid marbles are analysed.

This paper demonstrates that the evaporation dynamics of liquid marbles obeys the theoretical framework for elevated temperatures. The evaporation of a group of liquid marbles are observed as a coupled function of their diameter, their number in a group, the vapour density of the surrounding atmosphere and their spatial distribution.

The original version of this chapter has been published as

Sreejith, K.R., et al., Evaporation dynamics of liquid marbles at elevated temperatures.

RSC Advances, 2018. 8(28): p. 15436-15443.

71 4.1 Introduction

Liquid marbles are formed by coating liquid droplets with fine particles of hydrophobic materials. This coating enables the liquid droplet to hold its spherical shape even under a relatively high mechanical stress. Moreover, the hydrophobic particle coating helps to isolate the liquid droplet from the surrounding environment reducing the possible contamination of the sample inside the liquid marble. Being inert, the hydrophobic material coating does not react with the liquid sample, making it suitable to carry out chemical reactions. The manipulation of liquid marbles in terms of mobility can be effectively achieved by gravitational,[1] electrostatic[2, 3] or magnetic methods.[4-7] With these capability, liquid marbles find applications in gas/pollution sensing,[8, 9] micro-pumps,[10] biochemical reactions,[4, 5] genetic analysis and lab on chip devices.[11] Excellent reviews and articles have been published on the properties, applications and mobility manipulation techniques of liquid marbles.[12-18] Significant advancement in cell biology, bioengineering and biomedicine have been achieved recently using liquid marbles.[13, 18-21] Arbatan et al. successfully verified the use of liquid marbles as bioreactor for rapid blood typing.[22] Sarvi et al. demonstrated the usage of liquid marbles as a bioreactor for cardio genesis of embryonic stem cells.[23] Serrano et al. used liquid marbles as a cryopreservation tool for mammalian cells.[24] The porosity of liquid marble surface was successfully utilized by Tian et al. for the cultivation of microorganisms.[25]

Though much advancement has been achieved in exploring the possibilities of liquid marbles as a reactor for biological and chemical reactions, vast application areas are still unexplored, such as polymerase chain reaction (PCR). In terms of technology, there has been a leap in the field of PCR since its invention. Qualitative PCR (qPCR) and digital PCR are the two prominent techniques to perform DNA amplification.[26] In qPCR, the products are continuously monitored throughout the reaction cycles using fluorescent dyes. The quantity of

72 a target DNA can be found by comparing the fluorescent output curve with a standard curve.

In digital PCR, the reaction mixture is compartmentalised into many smaller units. Each unit undergoes the same thermal cycles as in the case of a conventional qPCR. Each unit is expected to have one or more template DNA and some unit may not even contain a template DNA. The units which had undergone a positive PCR can be identified by fluorescent dyes. A positive florescence from a unit is regarded as ‘1’ while the negative fluorescence (no fluorescence) is considered to be a ‘0’ similar to signals in digital electronics.[27, 28] Digital PCR techniques are gaining the attention of researchers and practitioners recently.[29-33] Better accuracy, and efficiency in preventing cross contamination are the main reasons behind the popularity of digital PCR techniques. Liquid sample containing PCR reagents along with DNA templates and fluorescent dyes can be coated with hydrophobic particles to form a liquid marble based digital PCR micro reactor. Liquid marbles can be an ideal candidate for being a micro-reactor to carry out PCR because of their self-sustainability, ability to prevent contamination, availability of various manipulation techniques [16] and ease of handling. The major constraint in using liquid marble as a micro-compartment for PCR is the possibility of evaporation of reactants through the semi permeable membrane of the liquid marble. Evaporation is expected as the polymerase chain reaction can be only achieved by a sequence of thermal cycling with temperature ranging from 55 °C to 95 °C.[34] In this context, it is essential to study the evaporation of liquid marble at elevated temperatures.

It is also essential to understand the evaporation behaviour of a group of liquid marbles at elevated temperatures. The present paper discusses the evaporation behaviour of individual and a group of liquid marbles at elevated temperatures. The paper also demonstrates a method to increase the lifetime of liquid marbles and makes it suitable for biochemical reactions at elevated temperatures such as PCR.

4.2 Theoretical background

A few studies have been carried out by various research groups on the evaporation of liquid marbles under different conditions. Most of these studies were carried out to analyse the evaporation dynamics of liquid marbles at room temperature and atmospheric pressure.[11, 35-

40], Moreover, not many researchers had attempted to evaluate the evaporation process of a group of liquid marbles. These studies do not provide useful information on the feasibility of liquid marbles for polymerase chain reaction. In this context, we are adapting an existing model of liquid droplet evaporation and modifying it to fit the physical characteristics of liquid marbles. Fig. 4.1(a) shows the model of a liquid marble. The evaporation mechanism of a liquid marble can be considered as a two-step process. The region close to the liquid marble surface

(region B) would be uniformly saturated with liquid vapour if the hydrophobic coating of the marble is thin and uniform.

The vapour molecules in region B would be topped up from liquid molecules in region

A (liquid state) as the diffusion from B to C progresses, maintaining a dynamic equilibrium of mass transport from A to B and subsequently to C. Therefore, the factor determining the

Fig.4. 1 Experimental study of evaporation dynamics of liquid marbles at elevated temperature: (a) Model of a liquid marble; (b) Various arrangements of liquid marbles; (c) Schematic of the experimental setup

74 evaporation rate of liquid marble is the diffusion rate from region B to C. We hypothesize that the hydrophobic coating of liquid marble offers a constant resistance to the evaporation process as compared to an ordinary liquid droplet. At any given instance, the mass flux at the radial location x (represented to an arbitrary point c′ in Fig.4.1.a) around a liquid marble can be described as[41]

푑푤 푚̈ (푥) = 푚̈ (푥)푤 - 휌 퐷 퐴 (1) 퐴 푣푎푝 푊퐴 푑푥

where 푚̈ (푥) is the mass flux of liquid vapour, 푤퐴 is the mass fraction of liquid vapour at corresponding point, 휌푣푎푝 is the density of liquid vapour phase, 퐷푊퐴 is the binary diffusivity

푑푤 of liquid in the air and 퐴 is the mass fraction gradient in space. The total mass of a liquid 푑푥 marble (푚퐿푀) is the combined mass of liquid droplet (푚퐿) and mass of the hydrophobic coating

(푚퐻).

푚퐿푀 = 푚퐿 + 푚퐻 (2)

Since the mass reduction in liquid marble during evaporation is only due to the evaporation of water, the change in mass of hydrophobic coating, 푚퐻 over time is zero. Thus the rate of change mass can be represented as

2 푚̇ 퐿푀 = 푚̇ 퐿 = 4휋푟 × 푚̈ (푥) (3)

Substituting 푚퐿푀̇ in the expression for 푚̈ (푥) and rearranging result in

푑푤퐴 −푚̇ 퐿푀 (1−푤퐴 ) = 2 (4) 푑푥 휌푣푎푝퐷푊퐴 4휋푟

and

푚̇ (1−푤 ) exp (− 퐿푀⁄ ) 퐴푋 4휋 휌푣푎푝퐷푊퐴 푥 푤퐴 (푥) = 1 − 푚̇ (5) exp (− 퐿푀⁄ ) 4휋 휌푣푎푝퐷푊퐴 r

Let the mass fraction of liquid vapour in the region close to the PTFE coating (푥 → 푟) of the liquid marble is 푤퐴푆 and that at infinity (푥 → ∞) is 푤퐴∞. Mass fraction of liquid vapour in the bulk atmosphere is taken as 푤퐴∞ in all practical cases. Applying these boundary conditions and solving for mass flow rate of liquid marble yield:

1−푤퐴∞ 푚̇ 퐿푀 = 4휋r 휌푣푎푝퐷푊퐴 푙푛 ⌊ ⌋ (6) 1−푤퐴푆

Since the mass reduction is due to the evaporation of the liquid inside the marble, the rate of change of mass can be approximated as

3 푑 4휋푑 1−푤퐴∞ 푚̇ 퐿푀 = − ( ) 휌푤푎푡푒푟 = 4휋푟 휌푣푎푝퐷푊퐴 ln ⌊ ⌋ (7) 푑푡 24 1−푤퐴푆

where 푑 is the diameter of the liquid marble. The expression can be rearranged to:

푑 2 휌푣푎푝 1−푤퐴∞ (푑 ) = −8 퐷푊퐴 푙푛 ⌊ ⌋ (8) 푑푡 휌푤푎푡푒푟 1−푤퐴푆

and

2 2 휌푣푎푝 1−푤퐴∞ 푑 (푡) = 푑 (0) − 8 퐷푊퐴 푙푛 ⌊ ⌋ × 푡 (9) 휌푤푎푡푒푟 1−푤퐴푆

This is similar to the 푑2 law of droplet evaporation. It shows that the liquid marble obeys 푑2 law of evaporation and the rate of surface regression depends on the initial diameter of the marble and the mass fraction of vapour at infinity. But this theoretical model has not considered the possible attenuation caused by the thin hydrophobic layer covering the liquid droplet. For a uniform and thin hydrophobic coating, we can assume a constant attenuation.

We can then rewrite equation (9) as

2 2 휌푣푎푝 1−푤퐴∞ 푑 (푡) = 푑 (0) − 8 휑퐷푊퐴 푙푛 ⌊ ⌋ × 푡 (10) 휌푤푎푡푒푟 1−푤퐴푆

where 휑 is the attenuation factor to evaporation. Bhosale et al. proposed 푑2 law with a correction factor for the evaporation dynamics of liquid marbles.[35] Laborie et al. adopted the theoretical framework of drying of granular packing to model the evaporation of liquid marbles. According to this framework, a liquid marble can be considered as a liquid droplet coated with very fine granules of hydrophobic particles which acts as a porous membrane. The authors have introduced a similar correction factor which is a function of porosity and

휌푣푎푝 1−푤퐴∞ tortuosity of the hydrophobic membrane [36]. The term 8 휑퐷푊퐴 푙푛 ⌊ ⌋ is the 휌푤푎푡푒푟 1−푤퐴푆 surface and known as the regression coefficient (푘) that has a dimension of 푚2푠−1.

4.3 Materials and methods

4.3.1 Preparation of the aluminium crucible as water reservoir

An aluminium crucible with customized size and shape was made for efficient heat transfer and to ensure repeatability of the experiments. A concentric well-shaped mould was designed in 123D software (Autodesk Inc) and 3D printed using Nobel 1.0 advanced SL printer

(XYZ Printing). Heavy duty aluminium foil (Alcan foil Products) of 0.024 mm thickness was pressed over the mould to make aluminium crucible for the experiment. The aluminium crucible thus obtained was fixed on to a 75 × 50 × 2 mm glass plate to avoid accidental physical deformation during the experiments. The central cavity of the crucible was designed to keep the liquids marbles while other surrounding cavities were designed to carry water for later experiments, Fig. 4.1.b. The diameter of the central cavity was 14 mm. The concentric well shaped structure provides controllability on the amount of water around the liquid marbles.

4.3.2 Preparation of liquid marbles

Liquid marbles were formed by dispensing and subsequent rolling of deionised water droplets on a bed of polytetrafluoroethylene (PTFE) powder (Sigma-Aldrich, 1 μm nominal diameter, ρ = 2.2 g cm−3). A micropipette (model CH 02607 by Thermo Electron Corporation) was used for droplet dispensing as it provides good controllability on the volume and the diameter of the droplet. Liquid marbles with 2 μl, 5 μl and 10 μl volumes were prepared for the experiments.

4.3.3 Experimental setup and procedures

The required number of liquid marbles for each experiment was transferred to the aluminium crucible. The crucible was subsequently transferred to a hotplate with a stabilised temperature value. The top view of the liquid marbles was recorded with a CMOS USB camera and a telecentric (0.5× and 0.3×) lenses (model: 63074, Edmund Optics). A motorized stage

(Zaber technologies T-LS 28M) was used for fine focusing throughout the experiments. Fig.

4.1.c shows the schematic of the experimental setup. The diameter of liquid marbles was measured by comparing the liquid marble image with a calibrated scale using ImageJ software.

All the experiments were carried out in the air-conditioned laboratory environment (24 ± 1 °C temperature, 50 ± 2% relative humidity and atmospheric pressure) until there was a visible buckling on the liquid marbles. The time at which buckling is visible on the liquid marble is termed as buckling time (tb). We consider tb as the lifetime of liquid marble in this paper.

4.4 Results and discussion

4.4.1 Temperature dependence of evaporation

4.4.1.1 Behaviour of liquid marbles at room temperature.

We carried out the first set of experiments to verify the theoretical model of d2 law on a liquid marble and compared the result with that of a water droplet of similar volume at room temperature. A liquid marble of 10 μl volume was kept under the microscope till we observed visible buckling. The buckling time was noted to be 27 minutes. A 10 μl water droplet was then dispensed on a thin layer of PTFE powder. Thin PTFE layer makes the surface hydrophobic and allows the water droplet to get a similar shape and contact angle with the surface as a liquid marble does. The thickness of the PTFE layer is minimised to avoid the possible permeation of liquid into the PTFE bed and subsequent liquid marble effect reported by Kumar et al.[42]

No visible permeation effect and liquid marble effect were observed in water droplet on thin layer of PTFE bed. The water droplet was observed for 27 minutes for the change in diameter.

Fig. 4.2.a shows the change of the normalised diameter of a water droplet and a liquid marble. The behaviour of water droplet was in accordance with the d2 law, whereas the liquid marble exhibited a nonlinear behaviour. The dotted lines represent the least square fit of liquid marble surface regression. The R2 value of the fitted line was 0.9968 for water droplet and 0.98 for liquid marble. Average surface regression rate (k) observed for the liquid marble is 1.2 times higher than the corresponding water droplet. This indicates that the liquid marble evaporates faster compared to uncoated water droplet of similar volume. A few research groups have reported a longer lifetime for liquid marbles compared to pure droplets of similar volume.

[11, 38] They concluded that the hydrophobic coating is offering a barrier effect for the evaporation of water in the liquid marble which in turn reduces the rate of evaporation.

Conversely, Laborie et al. reported a higher evaporation rates for liquid marbles with thin hydrophobic coating compared to similar water droplets. They had pointed out two effects that

2 2 Fig.4. 2 Surface regression(D /D 0) of 10 µl liquid marbles: (a) Single water droplet and liquid marble at room temperature; (b) Single liquid marble at elevated temperatures; (c) A group of 10 liquid marbles; (d) A group of 10 liquid marbles with surrounding water well. The images of the marbles in the initial and final time of the experiment are included in each graph. cause faster drying: (i) water loss rate through a layer of dense micro particles is almost the same as the one from bare interface; (ii) the solid particles lead to incompressibility of the interface whose area remains constant over the drying process. Thus, liquid marbles covered with monolayer particles exhibit a larger interface than liquid droplets and dry faster.[36] From these studies, we conclude that at room temperature the rate of evaporation of a liquid marble compared to a corresponding bare liquid droplet is related to the thickness of the hydrophobic coating. Liquid marbles with a thick hydrophobic coating evaporate slower while that with a thin or monolayer of hydrophobic coating evaporates faster compared to a corresponding uncoated liquid droplet. Our experiment showed a higher surface regression rate for liquid marbles compared to its uncoated counterpart at room temperature.

4.4.1.2 Evaporation of a single liquid marble at elevated temperatures.

Table 4. 1 Comparison of surface regression rates k of liquid marbles at various physical conditions Condition Number of Temperature(C) k Marbles x 10-9 m2s-1

10 µl Water droplet 1 25 1.4

25 1.7

55 4.5 10 µl Liquid marble 1 65 6.6

75 8.1

85 8.7

95 11.6

10 95 5.0±0.7

10 µl Liquid marble with surrounding water 10 95 2.1±0.4 well

The study of evaporation characteristics of liquid marbles at elevated temperatures is essential to determine their suitability for being used as bioreactors. A 10 μl liquid marble was heated at various elevated temperatures from 55 °C to 95 °C. From the experiment, we observed that the liquid marble is deviating from d2 law at lower temperatures up to 55 °C. Fig.4.2.b shows the surface regression of a single 10 μl liquid marble heated at elevated temperatures.

The R2 value of the fitted curve for the liquid marble at 55 °C was 0.9852. The liquid marble started obeying d2 law from 65 °C to 95 °C. The surface regression rates of liquid marbles were found to increase with temperature. The d2 law of liquid marble evaporation discussed in the previous section is modelled by assuming a uniform and thin coating of hydrophobic material over the liquid droplet, which is expected to allow water in the liquid marble to evaporate homogeneously through the coating.

A heterogeneous hydrophobic coating of liquid marble may lead to non-homogeneous evaporation of water through the coating, in turn causing the marble to deviate from d2 law. At

81 higher temperature, water molecules on the surface will get sufficient kinetic energy to evaporate through the hydrophobic coating, almost uniformly through the hydrophobic surface.

This may be the reason for liquid marbles to exhibit constant surface regression rate at higher temperatures. The surface regression rate k of liquid marbles at various temperatures is shown in Table 4.1. Liquid marbles with 2 μl and 5 μl volume was also tested at 95 °C to ensure the behaviour at elevated temperature. The results exhibited good alliance with d2 law. The buckling time for 2 μl, 5 μl and 10 μl was 2 minutes, 3 minutes and 3.5 minutes (see ESI, Movie

S1†) respectively at 95 °C.

4.4.2 Evaporation of a group of liquid marbles

Evaporation behaviour of a group of liquid marbles is vital as the PCR application is a high throughput process. Ten liquid marbles with volumes of 5 μl and 10 μl respectively were heated at 95 °C to analyse their group evaporation dynamics. The change in diameter was noted until visible buckling was observed on any one of the liquid marbles in the group. The average change in the square of the diameter is plotted against time, Fig.4.2.c. The results showed good agreement with the d2 law model. The average surface regression rate was lesser compared to a single liquid marble with similar volume leading to an extended average buckling time (tavg).

The decrease in surface regression rate and rise in average buckling time of grouped liquid marbles compared to their single counterpart can be attributed to the general evaporation dynamics discussed in the theory section. The transport of water molecules from region A to

B is governed by the diffusion of water vapour from B to C, Fig.4.1a. In a group setting, region

B of the marble group becomes saturated rapidly with very little evaporation from individual liquid marbles whereas the diffusion from B to C remains almost constant. Thus, the rate of water loss from individual liquid marble is lower, leading to lower surface regression rate and extended the average buckling time.

The above hypothesis can be extended with three further hypotheses: (i) the average buckling time (life time) of a group of liquid marbles can be increased by increasing the number of liquid marbles in the group; (ii) the liquid marbles situated at the edges in a group are expected to buckle faster compared to those in the centre since the diffusion of water vapour from region B to C through their outward facing surface might be higher. This may decrease the average buckling time of the group; (iii) the average buckling time can be extended if the liquid marbles are kept in a vapour saturated environment.

To verify the first hypothesis, a group of 5, 10 and 20 liquid marbles of 5 μl and 10 μl volumes were heated at a constant temperature of 95 °C (see ESI, Movie S2†). The marbles were continuously monitored for visible buckling. Average buckling time was calculated after observing visible buckling on each marble in the group. Average buckling time of the group is calculated as the weighted average time:

∑ 푡 푛 푡 = 푖 푖 (11) 푎푣푔 푁

83 where 푛𝑖 is the number of liquid marbles buckling at a specific time 푡𝑖 and 푁 is the total number of liquid marbles in the group. Table 4.2 shows the comparison of average buckling time of different liquid marble groups. Generally, we observed that the average buckling time of grouped liquid marbles are extended. But, it is interesting to observe that the average buckling time of 5 μl liquid marbles exhibited an anomalous behaviour between the groups of 5 and 10 liquid marbles. This anomalous behaviour can be explained by plotting the buckling time histogram of liquid marbles. Buckling time histogram is a plot of number of liquid marbles buckled at a specific time interval.

Fig.4. 3 Buckling time histogram of (a) 5 x 5 µl; (b) 5 x 10 µl; (c) 10 x 5 µl ; (d)10 x 10 µl; (e) 20 x 5 µl; and (f) ) 20 x 10 µl liquid marbles.

Fig.4.3.a–c show the buckling time histogram of different group of liquid marbles. The histogram shows that 4 out of 5 marbles in the group had buckled between 4.5 and 5 minutes resulting the average buckling time of the group to be 4.9 minutes. On the other hand, 8 out of

10 liquid marbles buckled between 4 and 4.5 minutes leading the weighted average to be 4.4 minutes. We observed that the 8 marbles that buckled during 4–4.5 minutes were relatively distant from the central marble. This observation gives a primary confirmation about the second hypothesis. It can be speculated that the average life time of group of liquid marbles depend on the spatial distribution of liquid marbles.

Table 4. 2 Comparison of buckling time of various set of liquid marbles heated at 95 0C

Condition Volume Number In a Buckling Time, tb group (Minutes)

1 3

5 µl 5 4.9±0.42 Random arrangement 10 4.4±0.39

20 6.5±1.43

1 3.5 10 µl 5 7.5±0.96

10 8.3±1.76

20 8.8±1.55

Radial symmetry arrangement 10 µl 9 8.8±1.17 water well 10 µl 10 25.2±5.18

We had conducted another experiment to verify the effect of distribution of liquid marbles on their average buckling time and to verify the second hypothesis. Nine liquid marbles of 10 μl each were arranged in the crucible in radial symmetry and heated at a constant temperature of 95 °C (see ESI, Movie S3†). We observed that the rate of change of diameter of the liquid marble at the centre is lower compared to those situated at the edges. The central liquid marble was the last one to buckle. This confirms the second hypothesis. Fig.4.4.a shows

85 the buckling histogram of 9 symmetrically arranged liquid marbles. The average buckling time was noted as 8.8 minutes. The average buckling time of 9 symmetrically arranged liquid marbles is equal to that of 20 randomly distributed marbles and greater than 10 randomly distributed liquid marbles of similar volume. We can conclude that the average buckling time of a group of liquid marbles at elevated temperature is a coupled function of number of liquid marbles and their spatial distribution. It is evident from the experiments that the decrease in average buckling time of a group liquid marbles is due to relative fast evaporation of the liquid marbles on the edge.

Fig.4. 4 Buckling time histogram of (a) 9 symmetrically arranged liquid marbles and (b) 10 liquid marbles with surrounding water well. Photos of liquid marbles at initial and final time of the experiment are included

4.4.3 Behaviour of liquid marbles with surrounding water well at elevated temperature

According to the third hypothesis, the average buckling time of the group can be improved by increasing the vapour density in the region around liquid marbles situating at the edges. We conducted the following experiments to verify the effect of increased vapour density on the life time of the liquid marbles.

The concentric grooves on the aluminium crucible were filled with water. A single 10

μl liquid marble was kept in the central cavity and the crucible is heated at 95 °C till visible buckling is observed on the marble (see ESI, Movie S4†). The buckling time was 9 minutes whereas the same without surrounding water well was 3.5 minutes. The experiment was repeated with 10 numbers of liquid marbles each with 10 μl volume. The average buckling time of the group was observed as 25.2 minutes whereas it was 8.3 minutes for marbles without surrounding water well. Fig.4.4 shows the buckling histogram of the experiment. The average life time of the group was extended three times compared to its counterpart without surrounding water wells. It can also be noted that the buckling of the first liquid marble occurred after 17 minutes while it occurred after 5.5 minutes for the corresponding group without water well.

This indicates that the individual buckling time of liquid marbles situating at the edges in group can be extended by providing a vapour saturated environment around it. The extended buckling time of liquid marbles at the edges would extend the average buckling time of the group. We can summarise that the average lifetime of a group of liquid marbles can be extended by heating them in a vapour saturated environment.

4.5 Conclusions

The present study shows that the evaporation of liquid marbles obeys d2 law at elevated temperatures. The non-homogeneous evaporation of water through the porous hydrophobic coating of the marble might be the reason for liquid marbles to deviate from d2 law at lower temperatures. The d2 law was obeyed for individual as well as grouped liquid marbles from 65

°C to 95 °C. Buckling time of liquid marbles was observed to depend on the number of marbles in the group, vapour density in the surrounding atmosphere and their spatial distribution of liquid marbles in the group. The average buckling time of a group of liquid marbles can be extended by providing a vapour saturated environment around the group of liquid marbles. We conclude that the average life time of a group of liquid marbles and their evaporation behaviour

87 can be accurately predicted with d2 law for liquid marbles with the same diameter and homogeneous spatial distribution under a controlled environment. The present study also reveals the possibility of liquid marbles to survive catastrophic evaporation during the thermal cycles of PCR. More extensive studies on the prediction of life time of grouped liquid marbles and their evaporation dynamics under real PCR thermal cycles would be carried out in future works.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The author acknowledges the Australian Research Council for funding support through the grant DP170100277.

Appendix

The supplementary movie S1-S4 mentioned in this paper can be watched respectively on the following links.

http://www.rsc.org/suppdata/c8/ra/c8ra02265h/c8ra02265h1.mp4

http://www.rsc.org/suppdata/c8/ra/c8ra02265h/c8ra02265h2.mp4

http://www.rsc.org/suppdata/c8/ra/c8ra02265h/c8ra02265h3.mp4

http://www.rsc.org/suppdata/c8/ra/c8ra02265h/c8ra02265h4.mp4

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5 Liquid marbles as biochemical reactors for the polymerase chain reaction

Abstract

The polymerase chain reaction (PCR) is a popular and well-established DNA amplification technique. Technological and engineering advancements in the field of microfluidics have fuelled the progress of polymerase chain reaction (PCR) technology in the last three decades. Advances in microfluidics-based PCR technology have significantly reduced the sample volume and thermal cycling time. Further advances led to novel and accurate techniques such as the digital PCR. However, contamination of PCR samples, lack of reusability of the microfluidic PCR platforms, complexity in instrumentation and operation remain as some of the significant drawbacks of conventional microfluidic PCR platforms.

Liquid marbles, the recently emerging microfluidic platform, could potentially resolve these drawbacks. This paper reports the first liquid marble based polymerase chain reaction. We demonstrated an experimental setup for the liquid-marble based PCR with a humidity- controlled chamber and an embedded thermal cycler. A concentrated salt solution was used to control the humidity of the PCR chamber which in turn reduces the evaporation rate of the liquid marble. The successful PCR of microbial source tracking markers for faecal contamination was achieved with the system, indicating potential application in water quality monitoring.

The original version of this chapter has been published as

Sreejith, K.R., et al., Liquid marbles as biochemical reactors for the polymerase chain reaction. Lab on a Chip, 2019. 19(19): p. 3220-3227.

93 5.1 Introduction

The polymerase chain reaction (PCR) has been emerging as a popular and powerful amplification technique of deoxyribonucleic acids (DNAs) since its invention in 1985.[1] In a

PCR, a mixture containing template DNAs, primers, DNA polymerase, deoxyribonucleotide triphosphates (dNTPs) and buffer solution is subjected to a series of thermal cycles to yield millions of copies of template DNA.[2-5] End-point PCR, quantitative PCR (qPCR) and digital

PCR (dPCR) are the three well established, optimised and widely used PCR techniques. The outcome of an end-point PCR is evaluated at the end of the reaction using a separate gel electrophoresis device, while the outcome of a qPCR and dPCR is evaluated in real time using the fluorescence signal emitted from the reaction mixture upon successful DNA amplification.

These PCR techniques have their own advantages, disadvantages and specific applications.[5]

However, PCR generally finds applications in biotechnology, genetic engineering, cell biology, forensic science, water research, drug discovery research, food micro-biology, etc.[6-12]

Advances in microfluidics technology in the last three decades have revolutionised the implementation of PCR technology.[5, 13-16] Many research papers were published and demonstrated PCR carried out in various microfluidic platforms including droplet,[17, 18] micro well,[19-21] micro channels,[22, 23]etc. However, these methods are limited by the vulnerability to contamination, lack of reusability of the platform and the requirement for sophisticated instrumentation.

Despite the advanced microfluidics-based PCR technologies, the use of liquid marbles as biochemical reactors for PCR has not been reported in the literature to the best of our knowledge. Liquid marbles are liquid droplets covered by a thin layer of hydrophobic particles.[24] Conventional liquid marbles are generated by placing and rolling a liquid droplet over a hydrophobic powder bed. The hydrophobic powder coating avoids physical interaction between the liquid and the ambient atmosphere, preventing the possibility of contamination. 94

The production [25] and manipulation [26-37] of liquid marbles are recently relatively well developed. Liquid marbles remain mechanically robust even under relatively high mechanical stress.[38-41] One of the major concerns needing attention is the non-recyclable plastic waste produced by bioscience laboratories across the world. Approximately 5.5 million tons of plastic waste was estimated to be generated from laboratories around the world in 2014.[42] Liquid marbles utilise only a thin hydrophobic powder to encapsulate the liquid droplet, thus eliminating the usage of disposable plastic consumables for carrying out biochemical reactions.

The potential of liquid marbles for “green bio-laboratory” practices are still to be explored.

These advantages make liquid marbles an ideal candidate to be used as bioreactors to carry out biological and biochemical reactions.

Arbatan et al. demonstrated rapid blood typing in a liquid marble.[43] Sarvi et al. used liquid marbles for cardiogenesis of embryonic stem cells.[44] Ledda et al. successfully demonstrated in vitro maturation of sheep oocytes inside a liquid marble.[45] Vadivelu et al. successfully used liquid marbles as bioreactors for cryopreservation of cells.[46] Despite the vast usage of liquid marbles as bioreactors for various biological and biochemical reactions, the potential of liquid marbles as bioreactors for the polymerase chain reaction has never been explored. To the best of our knowledge, no research paper has reported the usage of liquid marbles for PCR.

The fundamental difficulty in using liquid marbles as practical reactors for PCR is the evaporation of the reagent mixture during thermal cycling.[47] The evaporation process of different liquid marble types under various physical conditions was extensively studied and reported in the literature.[48-51] Our previous studies had provided insights into the different parameters affecting the evaporation rate of liquid marbles.[47] In the present work, a customised experimental setup was developed to minimize the evaporation of liquid marbles

Fig.5. 1 Liquid marble based PCR: a) Experimental setup; b) Exploded view of the PCR chamber and thermal cycler assembly; c) Photograph of the experimental setup during the PCR experiments. The present paper reports the first successful PCR using a liquid marble as a biochemical reactor.

5.2 Materials and methods

Fig.5.1.a shows the schematic of the experimental setup. Fig.5.1.b demonstrates the exploded view of the custom-made liquid marble PCR chamber with an embedded thermal cycler. Fig.5.1c is the photograph of the experimental setup. Detailed description of the fabrication of the liquid marble PCR chamber and thermal cycler is provided in the following subsections.

5.2.1 Preparation of the polymerase chain reaction mixture

Aiming at application in water quality monitoring, DNA was extracted from fecal samples of a healthy individual using a QIAamp DNA Stool Mini Kit (Qiagen) according to the manufacturer's protocol and was stored at −20 °C. A primary amplification of the extracted

DNA was carried out in a conventional PCR machine using the GoTaq Green master mix

(Promega). Reverse primer sequence: 5′CGTTACCCCGCCTACTATCTAATG-3′ and forward primer sequence: 5′-TGAGTTCACATGTCCGCATGA-3′ were used for the detection of the human specific faecal DNA marker (BacHuman). The reaction mixture (20 μl) contained

10 μl of GoTaq Green master mix, 2.5 μl (10 μM) of each forward and reverse primer, and 2.5

μl of the template DNA and 2.5 μl of DNA free water. The PCR was performed under the following conditions: initial annealing at 50 °C for 2 min, 95 °C for 10 min, followed by 39 cycles at 95 °C for 15 second and 60 °C for 1 min. Then, the resulting template DNA was used for DNA and primer optimization (provided in the supplementary material). Serial dilutions of the template DNA (25–400 ng μl−1) were carried out and used in the subsequent experiments.

Five 50 μl PCR mixture samples were prepared in duplicate by mixing the template

DNA, forward and reverse primer sequences and SsoFast EvaGreen Supermix (Bio-Rad). The sample set contained 400 ng μl−1, 200 ng μl−1, 100 ng μl−1, 50 ng μl−1, and 25 ng μl−1 template

DNA concentrations. One sample set was subsequently used for liquid marble PCR experiments, and the other sample set was used to carry out the conventional qPCR. A negative control for BacHuman was prepared using S. aureus DNA.

5.2.2 Preparation of PCR mixture liquid marbles

Liquid marbles made of the PCR mixture were formed by dispensing the PCR mixture liquid droplets on a bed of polytetrafluoroethylene (PTFE) powder (Sigma-Aldrich, 1 μm nominal diameter, ρ = 2.2 g cm−3) and subsequent rolling. A micropipette (model CH 02607 by Thermo Electron Corporation) was used for droplet dispensing as it provides good controllability on the volume and the diameter of the droplet. The PTFE powder used for the experiment was sterilised for 20 minutes in an ultraviolet irradiation chamber. Liquid marbles with 50 μl volume were prepared and used in the experiments.

5.2.3 Design and fabrication of the thermal cycler

A customised thermal cycler was developed to carry out the polymerase chain reaction in a liquid marble coated with PTFE powder. A 20 × 20 × 15 mm3 aluminium block embedded with a 30 W cylindrical cartridge heater (Core Electronics) was used as the heating platform of liquid marbles. An LM 35 semiconductor temperature sensor was mounted on the aluminium heater block for measuring the temperature. The heating platform was mounted on a 40 × 40 ×

3.5 mm3 thermoelectric Peltier cooler module (TEC-12706 AUS Electronics) using a heat conductive adhesive (Stars-922 heat sink plaster). The Peltier module was subsequently mounted on an aluminium heat sink (85.4 × 68.3 × 41.5 mm3) of a computer CPU cooler fan

(12 V, 3300 rpm, 70 × 70 × 25 mm3) using a heat conductive adhesive. The cartridge heater and the Peltier cooler modules were controlled by a PID control algorithm implemented in an

Arduino Mega microcontroller board. The proportional, integral and derivative gains were optimized to obtain faster heating and cooling with minimum overshooting and steady-state error. The smaller size of the heating platform also helped to reduce thermal inertia and achieve a faster thermal response. The temperature ramping rate of our customised thermal cycler was

0.68 K s−1 during heating and 1 K s−1 during cooling.

A dummy experiment was carried out to check the thermal response of the liquid marble placed on the thermal cycler. The Arduino microcontroller was programmed to heat the aluminium block to 75 °C. A 50 μl water liquid marble was placed on the aluminium heating block. A calibrated negative temperature coefficient (NTC) thermistor (Build Circuit,

Australia) was vertically inserted into the liquid marble using a precision positioning stage.

Fig.5. 2 Comparison of aluminium block temperature and liquid marble temperature. Inset picture shows the photograph of the liquid marble inserted with temperature sensor.

The temperature of the liquid marble during ramping and steady state was recorded in real time.

Fig. 5.2 compares the temperatures of the aluminium heater block and of the liquid marble.

The inset shows a liquid marble with an NTC thermistor. The liquid marble temperature closely followed the heater block temperature with an average difference of only 1.24 ± 0.62 K. This experiment confirms reasonably good heat transfer from the heater block to the liquid marble.

5.2.4 Design and fabrication of the polymerase chain reaction chamber

The primary difficulty in using liquid marbles as a microreactor to carry out the PCR is the evaporation of the PCR mixture through the porous PTFE coating. We already reported in a previous study that high ambient humidity reduces the rate of evaporation of a liquid marble at elevated temperatures.[47] Hence, a humidity controlled chamber was developed to carry out the liquid marble based polymerase chain reaction. A 100 × 100 × 60 mm3 rectangular airtight plastic container was used to make the humidity-controlled PCR chamber. The heating

99 platform of the thermal cycler subsystem was inserted into the container through a specially made rectangular opening at the bottom of the container. Proper sealing at the bottom of the container ensured airtightness. A rectangular opening was made on the top cover of the plastic container at a position vertically above the heating platform. A clear glass plate was used to close this opening and proper sealing was ensured for air isolation. The clear glass helps to obtain better visibility of the liquid marble lying on the heating platform.

An additional cartridge heater module was affixed on the outer side of the clear glass using a heat conductive adhesive. This cartridge heater could be turned on to avoid vapour condensation on the glass plate during the thermal cycling process of the liquid marble. The condensation would reduce the visibility of the liquid marble. An 8 × 5 mm2 rectangular openable window was made at the side of the plastic reaction chamber. This window is used to place the liquid marble on the heating platform before starting the thermal cycling process.

A well-known method [52] with a saturated salt solution was used to control the relative humidity inside the reaction chamber. A saturated solution of potassium sulfate was prepared and subsequently kept inside the reaction chamber in 6 separate rectangular plastic dishes (35

× 35 × 8 mm3). The reaction chamber was kept closed for 16 hours for humidity stabilisation.

The humidity inside the chamber was continuously monitored using a humidity sensor

(Senonics Minnow) positioned inside the reaction chamber. The average relative humidity of the reaction chamber over the duration of the experiment was 96.3 ± 0.7%.

5.2.5 Design and fabrication of the fluorescence detection system

The fluorescence excitation wavelength of the proposed PCR mixture is between 450 and 490 nm (blue light) and it is expected to provide an emission spectrum between 520 and

560 nm (green) upon successful DNA amplification. The intensity of the emitted green light is the indicator of amplification efficiency. A blue light emitting diode (LED) (450–490 nm)

100 served as the source of fluorescence excitation. An LED lighting system used in this experiment was custom-built with a concentric circular arrangement of 30 blue LEDs (1500 mCd, Jaycar,

Australia). The green fluorescent light emitted from the liquid marble PCR microreactor was captured using a vertically mounted CMOS camera (Edmund Optic EO-5012C) attached with a 0.5× telecentric lens (Edmund Optics-63074). The light emitted from the liquid marble was filtered using a green optical filter (520–560 nm) before being captured using the camera to improve the signal to noise ratio.

5.3 Experimental procedure

The synthesized PCR mixture liquid marble (sections 5.2.1 and 5.2.2) was transferred to the heating platform with a steel spatula through an openable window made on the sidewalls of the PCR chamber. The window was closed after successful placement of the liquid marble.

The liquid marble was placed vertically below the camera so that a clear image can be captured.

The heater mounted on the top of the glass window was turned on and the glass window was heated to prevent possible vapor condensation during the thermal cycling process of the liquid marble. Heating of the glass window ensured better visualisation of the liquid marble during the thermal cycling process.

The thermal cycling subsystem was subsequently turned on. The thermal cycler was programmed in such a way that the liquid marbles underwent a onetime initiation process at 95

°C for 15 seconds. The liquid marble was subsequently subjected to denaturation at 95 °C for

15 seconds and a combined annealing and extension process at 60 °C for 40 seconds. The thermal cycle was repeated for 15 minutes. Fig.5.3.a shows the temperature characteristics of the thermal cycler. Fig.5.3.b shows the relative humidity of the PCR chamber during the period of experiment. The thermal cycler exhibited a maximum overshoot of +8 K from the upper temperature setpoint (95 °C) during the first thermal cycle and stabilised to steady state in

101

Fig.5. 3 Parameters of PCR using a liquid marble: a) Thermal cycles of the PCR machine. b) Variation of humidity inside the PCR chamber during the experiment.

subsequent cycles. The temperature of the block read 95 ± 0.7 °C and 60 ± 1.5 °C during the

steady-state thermal cycles. The recorded video of the liquid marble during the thermal cycling process was captured throughout. A volume of 50 μl PCR mixture with DNA concentrations varying from 25 ng μl−1 to 400 ng μl−1 was used for the experiments. Experiments were repeated three times.

Thermal cycling of the 50 μl PCR mixture (five samples with DNA concentrations varying from 25 ng μl−1 to 400 ng μl−1) was carried out in a conventional qRT-PCR machine

(Bio-Rad CFX connect) for 15 minutes with similar thermal cycling conditions explained earlier.

102

5.4 Results and discussion

Thermal cycling of the liquid marbles containing the PCR reaction mixture was carried out as explained above in section 5.2.6. Fig.5.4.a shows the representative fluorescence intensity emitted from the liquid marbles with various DNA concentrations. The numerical equivalent values of fluorescence intensities of the liquid marbles at various time intervals were evaluated using ImageJ[53] open source software. The fluorescence intensity values of liquid marbles were offset corrected and normalised subsequently. Offset correction and normalisation of fluorescence intensities were done as follows:

퐼푠푡_푁표푟푚푎푙𝑖푠푒푑 = (퐼푠푡 − 퐼푠0)⁄퐼푀푎푥 (1)

where 퐼푠푡 is the fluorescence intensity of a sample measured at a given time, 퐼푠0 is the fluorescence intensity of that sample at the beginning of thermal cycling and 퐼푀푎푥 is the maximum fluorescence intensity recorded among all the samples in the experiment. Fig.5.4.b shows the change of the normalised fluorescence intensities over time of liquid marbles with different DNA concentrations. The results indicate that the fluorescence intensity of liquid marbles containing the PCR mixture with different concentrations increased over time up to a certain time point and subsequently decreased. The increase of fluorescence intensity of the

PCR mixture liquid marbles indicates the positive polymerase chain reaction inside the liquid marbles. The liquid marble containing the negative sample of the PCR mixture was not giving any significant fluorescence on thermal cycling. This proves the specificity of the PCR inside the liquid marble. Maximum fluorescence was observed during a period of 11–12 minutes

(corresponding to 9 thermal cycles) for all the liquid marbles. We observed that the fluorescence intensity of the liquid marbles reduced after reaching a maximum value.

103

Fig.5. 4 PCR results: a) Micrographs showing the fluorescence from PCR mixture liquid marbles with different DNA concentrations at various intervals of thermal cycling. b) Variation of fluorescent intensity of PCR mixture liquid marbles with respect to time during thermal cycling. c) Comparison of normalised peak fluorescent intensities of liquid marble PCR and conventional qRT-PCR at the time of maximum fluorescence emission from corresponding liquid marble PCR sample.

Fig.5.4.c shows the comparison of normalised fluorescence intensities of reactions carried out in a conventional qRT-PCR machine (Biorad CFX Connect) and that of a real-time liquid-marble PCR. We observed that the trends in maximum fluorescence intensities emitted by the samples in both cases are similar. The optimum DNA concentration for maximum fluorescence intensity was observed to be 100 ng μl−1 in both experiments. The fluorescence intensity variation with respect to the DNA concentration follows a similar pattern in both experiments. These results confirm the successful polymerase chain reaction and amplification of template DNA inside a liquid marble.

We hypothesize that the reason for the deterioration of fluorescence is photo-bleaching of fluorescent dye due to constant illumination. The fluid medium of the PCR mixture was observed to be completely evaporated after 12 minutes of thermal cycling, resulting in no further DNA amplification. Continuous illumination of the sample with blue light may cause photo-bleaching and can result in deterioration of fluorescence intensity over time.

104

Fig.5. 5 Deterioration of fluorescent intensity of liquid marble PCR sample on continuous illumination

A separate experiment was carried out to test the possibility of photo-bleaching. A volume of 50 μl of PCR sample mixture with 100 ng μl−1 template DNA underwent 20 thermal cycles in a conventional PCR machine (Biorad CFX Connect). A liquid marble is made out of this DNA amplified sample and kept under the fluorescence detection system described in section 5.2.5. A continuous video of the fluorescence emitted from the liquid marble is captured for 15 minutes. The experiment was repeated three times. The fluorescence intensity of the liquid marble at regular time intervals was evaluated using ImageJ software. An average deterioration of 22.5% in fluorescence intensity was observed over a period of 15 minutes of continuous illumination. Fig.5.5 shows the decrease in magnitude of fluorescence intensity with respect to time. This decrease in intensity verifies that the fluorescence deterioration of the liquid marble PCR after reaching a maximum fluorescence intensity is due to photobleaching of the dye under continuous illumination.

5.5 Conclusions

We designed and developed a customised thermal cycler to conduct the liquid marble- based polymerase chain reaction. A humidity-controlled chamber was developed to reduce the

105 rate of evaporation of the PCR mixture from the liquid marble during the thermal cycling process. Successful amplification of DNA extracted from fecal samples of a healthy person was carried out by the real-time polymerase chain reaction using the PTFE liquid marble as a bioreactor.

The fluorescence emitted from the liquid marble was captured in real time using a camera. The fluorescence intensity emitted from liquid marbles containing various DNA concentrations was plotted as a function of time. The optimum DNA concentration for efficient amplification was 100 ng μl−1. This optimum DNA concentration for efficient amplification obtained from the liquid marble-based PCR was confirmed with a conventional qRT-PCR experiment conducted in a commercial machine (Biorad CFX Connect). The trend in variation of fluorescence intensities with template DNA concentration was also observed to be similar in both the liquid marble PCR and conventional qRT-PCR.

Evaporation of the PCR mixture through the porous walls of the liquid marbles limits the duration of thermal cycling and hence the amplification efficiency of the proposed method.

Deterioration of fluorescence was observed in the liquid marble-based PCR after a certain number of thermal cycles. Photobleaching of the fluorescent dye was found to be the reason for the deterioration of fluorescence.

The optimum detection sensitivity achieved with our present work was 25 ng μl−1. Highly optimised conventional qPCR machines demonstrate detection sensitivity on the order of pg

μl−1. The relatively lower detection sensitivity of the present work can be attributed to three major reasons: (i) transparency of the hydrophobic powder used in the experiment; (ii) premature evaporation of the liquid marbles and (iii) sensitivity and resolution of the optical detection system. Our work utilised PTFE powder as the hydrophobic coating. The optical transparency of the PTFE coating is worse compared to other transparent hydrophobic powders

106 reported in some studies.[54, 55] Utilising a truly transparent coating may greatly enhance the optical detection characteristics of liquid marble-based PCR technology. Transparent liquid marbles will improve the detection limit of PCR amplification. The presented liquid marble- based polymerase chain reaction could only undergo 9 thermal cycles before getting completely evaporated. An optimised liquid marble technology with a lower rate of evaporation of the sample would significantly increase the amplification efficiency and detection limit. A hydrophobic powder material with controllable crosslinking could potentially solve the problem of evaporation during the thermal cycling process. Furthermore, our experiments utilised a commercial off-the-shelf CMOS camera for the detection of fluorescence signals.

This camera is inferior to highly sensitive and optimised fluorescence detection systems

(photodiodes, photomultiplier tubes, charge coupled devices, etc.) of conventional qPCR machines. In a nutshell, the detection limit of liquid marble-based PCR technology can be optimised with optically more transparent coating powder, evaporation prevention and highly sensitive fluorescence detection.

In conclusion, this paper reported the successful PCR of template DNA using a PTFE liquid marble as a biochemical reactor. The detection sensitivity of the present work is relatively lower compared to conventional commercial qPCR methods due to limited transparency of the hydrophobic coating, evaporation of the liquid marble and the low sensitivity of the camera used in our experiments. All these issues can be addressed in future studies. The optimized liquid-marble-based PCR is expected to open a new era for the real- time PCR as well as the digital PCR. Future studies will also be extended to explore the potential of liquid marbles as a bioreactor for the loop mediated isothermal amplification

(LAMP) technique, which requires a lower working temperature than that of the PCR. The isothermal low-temperature LAMP process is expected to reduce the evaporation rate of the

107 liquid marble, thereby could provide a comparable performance with existing conventional

LAMP techniques.

Ethical approval

All experiments were performed in compliance with relevant laws or guidelines. All experiments followed the guidelines of Griffith University. Ethical approval for the study on the effectiveness of molecular assays in detecting human faecal pollution and the effect of freezer storage on human faecal samples was done by the ethics committee through Griffith

University Office of Research, ref no. BPS/01/13/HREC. No human subject has been used in the experiment and informed consent was obtained.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors acknowledge the Australian Research Council for funding support through the grant DP170100277. This work was performed in part at the Queensland node of the

Australian National Fabrication Facility, a company established under the National

Collaborative Research Infrastructure Strategy to provide nano- and micro-fabrication facilities for Australia's researchers.

Appendix – Electronic supplementary material

The following is the electronic supplementary material (DNA and Primer optimisation) mentioned in this chapter.

To optimize the amount of PCR reaction reagents, DNA and primer optimization were carried out. The reaction mixture (20 μl) for DNA optimization contained 10µl GoTaq Green master mix, 1 μl (10µM) of each forward and reverse primer, and a range of the template DNA

108 including 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 µl (100ng/µl). The reaction mixture (20 μl) for primer optimization consisted of 10µl GoTaq Green master mix, 2.5 μl (100ng/µl) of template DNA, and a range of each forward and reverse primer (0.25, 0.5, 1, 1.25, 1.5, 2, 2.25, 2.5 µl). The

DNA Optimisation Primer Optimisation

S1: DNA and Primer optimisation results demonstrate that the amount of 2.5 µl for DNA and primers is the optimum amount for

PCR in this experiment. Photograph of the gel electrophoresis is given below.

References

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113

6 Core-shell beads made by composite liquid marble technology as a versatile microreactor for polymerase chain reaction

Abstract

In the last three decades, polymerase chain reaction (PCR), a DNA amplification technique, has been optimized and well developed in terms of protocol. However, no significant improvements have been made in terms of sample dispersion for this technique.

This paper reports the synthesis of a core-shell bead microreactor for PCR using photopolymerization of a composite liquid marble. We also demonstrated successful PCR on this platform. The core-shell bead serves as a simple and effective sample dispersion platform for PCR that can significantly reduce plastic waste generated from conventional PCR processes. The platform improves the throughput capability, enhancing the performance and portability of the thermal cycler. The platform also allows for the contamination-free storage of sample after thermal cycling.

The original version of this chapter has been published as

Sreejith, K.R.; Gorgannezhad, L.; Jin, J.; Ooi, C.H.; Takei, T.; Hayase, G.; Stratton, H.;

Lamb, K.; Shiddiky, M.; Dao, D.V.; Nguyen, N.-T. Core-Shell Beads Made by Composite

Liquid Marble Technology as A Versatile Microreactor for Polymerase Chain Reaction.

Micromachines 2020, 11, 242.

114 6.1 Introduction

Since the invention of polymerase chain reaction (PCR) in 1985,[1] this DNA amplification technique has gained extensive adaptation in various fields including biotechnology, cell biology, genetic engineering, forensic science, medical science, and drug discovery [2-8]. Various advancements and optimizations of PCR techniques such as quantitative reverse transcription PCR (qRT-PCR) and digital PCR have enabled scientific advancements in fundamental and applied molecular biology. Moreover, advances in microfluidics and optics have greatly improved the efficiency and detection limit of PCR techniques. However, very few updates have been reported in sample dispersion platforms for

PCR. This lack of innovation means that sample dispersion platforms are limited to either conventional PCR tubes or conventional microfluidic chips based on polymers such as polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA).

Urbina et al. estimated that bio labs around the world may have produced around 5.5 million tons of plastic waste in 2014 [9]. In light of the environmental situations all over the world, this alarming figure motivates the development of alternative technologies to address the problem of plastic waste. Technological innovations should either eliminate or significantly reduce single-use plastic laboratory consumables. Biochemical reactions and disease diagnostic methods like PCR contribute a significant share to the non-reusable contaminated plastic wastes. A single 0.2 mL PCR tube weighs ~0.14 g and a 100 µL pipette tip weighs ~0.3 g. Millions of conventional PCR is being carried out in laboratories around the world annually, contributing towards the plastic wastes.

Conventional microfluidic chips for PCR poses the same problem of plastic waste. Lack of reusability of conventional microfluidic chips is the main disadvantage. Moreover, the design and development of conventional microfluidic devices are complex. Bulky external

115 support systems like pumps and tubing are required to deliver the samples into the microfluidic device, making many of them not suitable for practical field applications [10]. In this context, a suitable sample dispersion platform that has comparable performance to conventional PCR tubes and microfluidic devices but eliminating other disadvantages is of great interest.

Liquid marble, a liquid droplet coated with hydrophobic/oleophobic powder [11], is an ideal candidate for this purpose. The powder coating on the surface of the droplet isolates it from the surrounding, eliminating the possibility of contamination. Conventional liquid marbles possess reasonably good mechanical strength [12-15]. Moreover, the generation [16,

17] and manipulation [18-26] of liquid marbles have been relatively well developed for other applications. In fact, significant advances in cell biology, bio-engineering, and bio-medicine have been made using liquid marbles as bio-reactors [27-31]. However, to our best knowledge, no significant advances in using liquid marbles for DNA amplification have been reported in the literature. The major issue of using a conventional liquid marble for polymerase chain reaction is the evaporation of the PCR mixture at elevated temperatures. We have previously developed a simple method to reduce evaporation of the PCR mixture in liquid marbles and successfully conducted PCR in a liquid marble coated with polytetrafluoroethylene (PTFE) powder [32]. However, the sample volume used in the experiment was relatively large (50 µl) and the PCR mixture could only last for 9 to 10 thermal cycles due to evaporation of PTFE- coated liquid marbles [33].

In this paper, we make use of core-shell beads synthesized from a composite liquid marble as a microreactor for carrying out PCR. The composite liquid marble consists of two immiscible liquid droplets forming a concentric spherical geometry and a coating of hydrophobic/oleophobic powder. Composite liquid marbles possess all the advantages of a conventional liquid marble, but also have to increased protection from external contamination of the core droplet by the shell liquid as well as the powder coating. Moreover, the shell liquid

116 can transform through polymerization into a solid, converting the liquid marble into a core- shell bead. This versatility allows for convenient manipulation and storage of the liquid sample as a solid particle. We propose that the shell liquid in its polymerised state (solid-state) may also prevent the evaporation of interior liquid droplet at elevated temperatures, as the exterior solid polymer acts as a closed chamber. Here, we used the PCR mixture as the core liquid and a photopolymer as the shell liquid for manufacturing the composite liquid marble. The close packing of the exterior photopolymer shell liquid on the interior PCR mixture will considerably reduce the amount of plastic waste compared to the conventional PCR.

6.2 Materials and Methods

6.2.1 Preparation of photopolymer liquid

The photopolymer liquid is prepared by dissolving 0.05 g of camphorquinone and 0.06 g of ethyl-4-(dimethylamino) benzoate in 10 g of Trimethylolpropane trimethacrylate (TRIM) using a magnetic stirrer at 600 rpm for 2 minutes. The photopolymer liquid was stored in an opaque container for subsequent use. Trimethylolpropane trimethacrylate[34] is a crosslinking monomer which finds its applications in the field of dentistry and for the development of bone cement [35, 36]. There is no cytotoxicity reported for the compound to the best of our knowledge.

6.2.2 Synthesis of composite liquid marbles and core-shell beads

A super amphiphobic silicon monolith called “marshmallow-like gel” (MG) [37] was powdered using a mortar and collected in a plastic weighing container (35 mm x 35 mm x 10 mm), forming a super amphiphobic powder bed. A volume of 20 µl of the photopolymer prepared previously was deposited on to the powder bed using a micropipette as shown in Fig.

6.1a, step 1. A volume of 2 µl of the PCR mixture was subsequently injected into the

117 photopolymer (Fig 6.1a, step 2). The PCR mixture droplet was observed to be partially immersed in the polymer as shown in Fig 6.1a, step 3. Photopolymerization of the shell liquid at this stage would cause a part of the core droplet to expose to the ambient atmosphere, causing evaporation of the PCR mixture during the thermal cycling process. The composite liquid droplet obtained after step 3 was gently rolled on the powder bed to coat the powder on the liquid droplet, forming the composite liquid marble (Fig 6.1a, step 4). We observed that the powder coating on the liquid droplet had pushed the PCR mixture droplet into the shell polymer liquid due to the highly “phobic” nature of the PCR mixture to the powder as compared to the polymer liquid.

Fig.6. 1 (a) (1) Deposition of photopolymer droplet on super amphiphobic powder bed. (2) Inserting PCR mixture into the predeposited drop. (3) Inner droplet in a partially immersed state. (4) Amphiphobic powder-coated composite liquid marble. (5) Photopolymerization of composite liquid marble rotating at 140 rpm in a motorized drum. (6) Core-shell bead embedded with PCR mixture droplet. (b) Exploded view of the custom-built thermal cycler. (c) (1) Photograph of core-shell bead generated. 2) Photographs of the core-shell bead after at various cycles of thermal cycling. (d) Thermal cycles of the custom-built thermal cycler.

118

Photopolymerization of the shell liquid at this stage would result in a core-shell bead with the PCR mixture encapsulated by the hardened polymer shell. However, the thickness of the hardened coating over the PCR mixture was relatively thin and could break due to thermal stress during the thermal cycling process. Hence, the composite liquid marble obtained after step 4 was transferred to a motorized cylindrical drum. The drum was rotated at 140 rpm. The rotating composite liquid marble inside the drum was illuminated with blue light and kept at a distance of 5 cm above the liquid marble for photopolymerization (Fig.6.1a, step 5). Takei et al. reported that the rotary motion of composite liquid marble would push the inner liquid droplet towards the center of the outer droplet.[38] The process of rotation and simultaneous photopolymerization was carried out for 5 minutes. A hardened core-shell bead with powder coating was obtained after 5 minutes. The powder on the surface of the bead was washed using de-ionized (DI) water to obtain a transparent core-shell bead (Fig.6.1a, step 6). Photographs of each of these steps and the core-shell bead generating set up is provided in the supplementary material S1 (Fig. a and b).

6.2.3 Design and fabrication of thermal cycler

As a commercial thermal cycler for liquid marbles or core-shell beads does not exist, we developed a custom-built thermal cycler for the PCR experiment. A 20 mm x 20 mm x 15 mm aluminum block with embedded cartridge heater (5-mm diameter and 15-mm length, Core electronics) served as the heating platform. The aluminum heater block was attached to a 40 mm x 40 mm x 3.5 mm Peltier thermoelectric cooler (TEC-12706. AUS Electronics) using a heat conductive adhesive (Stars -922 heat sink plaster). The entire assembly was subsequently mounted on an aluminium heat sink (85.6 mm x 68.3 mm x 41.5 mm) of a computer CPU cooler fan (12 V, 3300 rpm, 70 mm x 70 mm x 25 mm) using heat conductive adhesive. An

Arduino Mega microcontroller board was programmed to run a PID (proportional-integral-

119 derivative) algorithm to control the temperature setpoints of the thermal cycle. Fig.6.1b shows the exploded view of the thermal cycler assembly. Photograph of the custom-built thermal cycler is provided in the supplementary material S1 (Fig. c).

A dummy experiment was carried out to test the characteristics of the thermal cycler and the performance of the core-shell bead. Core-shell bead containing a volume of 2 µl deionized water added with green fluorescent dye was prepared as per the methods explained in section

6.2.2. The side view image of the core-shell bead was taken with a CMOS camera (Edmund

Optic EO-5012C) attached to a 1x telecentric lens (Edmund Optics), Fig.6.1c (1). The core- shell bead was placed on the heater platform of the thermal cycler. A thin layer of heat sink paste was affixed between the heater platform and the core-shell bead to ensure efficient heat transfer. The thermal cycler was operated for 30 cycles each cycle having the following conditions: 95 0C for 15 seconds and 60 0 c for 45 seconds. The to-view image of the core-shell bead was taken with a CMOS camera (Edmund Optic EO-5012C) attached to a 0.5X telecentric lens (Edmund Optics), Fig.6.1c (2). The recorded image shows that the droplet inside the hardened bead was stable over the thermal cycles. The experiment was repeated in 3 core-shell beads to ensure repeatability.

The custom-built thermal cycler demonstrated a ramping rate of 0.68 K/s during heating and 1 K/s during cooling. A maximum overshoot of 8 K was shown in the first thermal cycle and stabilized during the subsequent thermal cycles. The steady-state temperature of the thermal cycle was observed to be 95 ± 0.7 °C and 60 ± 1.5 °C. Fig.6.1d depicts the performance of the thermal cycler.

6.2.4 Preparation and optimization of the PCR mixture

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DNA was extracted from the fecal sample of a healthy individual using the QIAmp DNA stool mini kit (Qiagen). This template DNA was used to synthesize artificial copies using a conventional PCR machine. Reverse primer sequence: 5ʹ

CGTTACCCCGCCTACTATCTAATG-3ʹ and forward primer sequence: 5ʹ -

TGAGTTCACATGTCCGCATGA-3ʹ were used for the detection of human-specific fecal

DNA marker (BacHuman). The reaction was performed using GoTaq green master mix

(Promega) under the following conditions: initial annealing at 50 °C for 2 min, 95 °C for 10 min, followed by 39 cycles at 95 °C for 15 seconds and 60 °C for 1 min. The artificial template

DNAs were serially diluted to 100 ng/µl, 150 ng/µl and 200 ng/µl concentrations for the subsequent experiments. Samples were prepared in duplicate. One set of samples would be used to carry out core-shell based PCR while the other set would be used for qPCR control experiments in a conventional commercial qPCR machine.

6.2.5 Design and fabrication of fluorescent detection system

The excitation and emission wavelengths of the PCR mixture used in the proposed experiments are 450-490 nm (Blue) and 520-560 nm (Green) respectively. Intensity of green fluorescence emitted from the sample is proportional to the amplification efficiency of PCR.

An LED lighting system was custom-built by arranging 30 blue LEDs (1500mCd, Jaycar,

Australia) in concentric circles. This lighting source was used as the source of illumination in the proposed experiment. A CMOS camera (Edmund Optic EO-5012C) attached with a 0.5X telecentric lens (Edmund Optics-63074) mounted vertically was used to capture the green fluorescent light emitted from the core-shell bead. The green light emitted by the PCR mixture in the core-shell bead was optically filtered using a green optical filter (520-560 nm) for better signal to noise ratio.

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6.3 Experimental

The thermal transferring characteristics of the core-shell bead is important for the efficient PCR inside the bead. A dummy experiment was carried out to test the thermal transfer characteristics of the core-shell bead. 50 µl photopolymer liquid is deposited on the oleophobic powder bed. A calibrated negative temperature coefficient (NTC) thermistor (Build Circuit,

Australia) was vertically inserted into the photopolymer liquid droplet using a precision positioning stage. The photopolymer droplet was kept under the blue light source for 5 minutes for hardening. After minutes, a hardened bead embedded with the thermistor was obtained. The hardened bead along with the thermistor was kept on the aluminium heater block. The Arduino microcontroller of the thermal cycler was programmed to heat the aluminium block to 95 °C for 30 seconds and 60 °C thereafter forever. One heating and cooling cycle are observed.

Fig.6.2 compares the steady-state temperature difference between the aluminium block and the polymerized bead. The inset shows the polymer bead with the NTC. The average temperature difference between the block and the bead was observed to be 2.12k at upper temperature and

1.31k at the lower temperature.

Fig.6. 2 Comparison of heater block temperature with the polymer bead temperature. The inset picture shows the polymer bead with the thermistor.

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Fig.6. 3 Gel electrophoresis results of (a) DNA optimization and (b) Primer optimization

DNA and primer optimization were carried out to determine the right amount of PCR reagents. The reaction mixture (20 μl) for DNA optimization contained 10 µl GoTaq Green master mix, 1 μl (10 µM) of each forward and reverse primer, and a range of the template DNA including 1, 1.5, 2, 2.5, 3, 3.5, and 4 µl (100 ng/µl). The reaction mixture (20 μl) for primer optimization consisted of 10 µl GoTaq Green master mix, 2.5 μl (100 ng/µl) of template DNA, and a range of each forward and reverse primer (0.5, 1, 1.25, 1.5, 2, 2.25, 2.5 µl). The results demonstrate that the amount of 2.5 µl for DNA and primers is the optimum amount for the

PCR in our experiment. Fig.6.3 shows the gel electrophoresis results of DNA and primer optimization.

A core-shell bead was prepared by using 2-µl PCR mixture as the core liquid and 20-µl polymer liquid as the shell liquid as described in Section 6.2.2. The core-shell bead containing the PCR mixture was kept on the heating platform of the thermal cycler. A thin layer of heat conductive paste was applied between the heating platform and the core-shell bead to ensure efficient heat transfer. The thermal cycler was subsequently turned on to run 95 °C for 15 seconds as one-time initiation and 30 thermal cycles having the following cycling conditions:

95 °C for 15 seconds and 60 °C for 45 seconds. The experiment was conducted in a dark laboratory environment at 23 °C and 50 % relative humidity. Blue light from an LED light source was aimed at the composite bead, the light emitted from the bead was filtered using a

123

Fig.6. 4 (a) Schematic of the experimental setup. (b)Variation of normalized mean fluorescent intensity of the PCR mixture in the core-shell bead with respect to thermal cycles. Sample micrographs of the core-shell bead with colour intensity scale are given as inset. Comparison of normalized peak fluorescent intensity of core-shell bead PCR with conventional qRT-PCR after c) 20 cycles., d) 30 cycles green optical filter and video captured using a vertically mounted CMOS camera (Edmund

Optic EO-5012C) attached with a 0.5x telecentric lens (Edmund Optics-63074). The blue light source was manually activated to illuminate the bead in intervals of 5 cycles. Switching off the excitation light reduces the possibility of photobleaching of fluorophores under continuous illumination. Experiments were repeated three times each for PCR mixture samples with 100 ng/µl, 150 ng/µl and 200 ng/µl DNA concentrations. Negative control for BacHuman was also prepared using S. aureus DNA. Experiments were repeated for negative control with similar thermal cycling conditions. Fig.6.4.a illustrates schematically the experimental setup.

The average pixel density of the circular region containing the core-shell bead in the images was identified using ImageJ software. The values represent the numerical equivalent of the fluorescent intensities emitted by the bead at respective times. The numerical equivalent values of fluorescent intensities were offset corrected and normalized as:

∗ 퐼푠푐 = (퐼푠푐 − 퐼푠0)⁄퐼푚푎푥 (1)

124

Where 퐼푠푐 is the fluorescent intensity of a sample measured at a given cycle, 퐼푠0 is the fluorescent intensity of that sample at the beginning of thermal cycling and 퐼푚푎푥 is the maximum fluorescent intensity recorded among all the samples in the experiment. The standard error of the measurements in fluorescent intensity is also calculated using the equation:

푆. 퐸 = σ⁄√푛 (2)

and

∑(퐼∗ − 퐼∗ )2 √ 푠푐 푠푐_푚푒푎푛) ⁄ 휎 = (푛 − 1) (3)

∗ Where 퐼푠푐_푚푒푎푛 is the average of normalised numerical equivalent values of fluorescent intensities measured for various samples of PCR mixture with a particular DNA concentration and 푛(=3) is the total number of samples tested.

Weighing of the core-shell bead was performed subsequently. The weight of the 5-core shell bead containing the PCR mixture was measured individually using an electronic weighing balance (Entris 124I-1s, Sartorius Lab Instruments). Weights of five 0.2mL conventional PCR tubes were also measured to compare the plastic waste reduction. Percentage reduction in non- re-usable contaminated plastic waste was calculated as

Δw = (푤푡 − 푤푏)⁄푤푡 × 100 (4)

Where 푤푡 is the weight of the conventional PCR tube (0.2ml) and 푤푏 is the weight of the core- shell bead.

6.4 Results and discussion

The experimental results, Fig. 6.4b ,6.4c, and 6.4d show that there is a steady increase in fluorescent intensity as the thermal cycling process progresses and that the fluorescent results are comparable to, though not the same as, a conventional qRT-PCT method. Maximum

125 fluorescent emission was detected from a sample with 100 ng/µl DNA concentration after 30 cycles. The peak fluorescent intensity of the samples was observed to decrease with increasing

DNA concentration. The results confirm the amplification of DNA inside the core-shell bead.

The negative samples did not show any significant fluorescence signal even after 30 thermal cycles. The fluorescent intensity values of samples were observed to be ~20% of the maximum fluorescent intensity throughout the first 20 thermal cycles, where the intensity then showed an exponential increase afterward up to a total of 30 cycles. Fig.6.4.c and 6.4.d depict the comparison of normalized peak fluorescent intensities of core-shell bead PCR and a conventional qRT-PCR. The result of fluorescent intensities from the conventional qRT-PCR shows that the optimum DNA concentration for optimum amplification efficiency is 100 ng/µl, matching that of core-shell bead PCR. The trend of the amplification efficiency with respect to the DNA concentration also matches with the observation derived from core-shell bead PCR within a reasonable band of error. It should be noted that the results depicted in Fig. 6.4 c and

6.4.d are the comparisons of trends in DNA amplification efficiency with respect to the DNA concentrations. Fluorescent intensity values of core-shell bead PCR and conventional qRT-

PCR cannot be compared in an absolute sense due to the following reasons. A) The commercial machine uses a highly sensitive optical instrumentation system and algorithms to evaluate the fluorescence. B) The method of core-shell bead synthesis is not completely optimized to ensure repeatability of the geometric location of the core PCR mixture inside the core-shell. C) Minor variations in fluorescence will have a magnified effect on the off the shelf optical detection method used in this experiment. D) Variations in size and shape of the core-shell bead, as well as the core PCR mixture, are also expected to make a difference in the fluorescent intensity.

These results confirm that, despite this method being non-optimized and using off-the- shelf equipment, the core-shell dispersion method is a potential replacement for other dispersion methods used in PCR techniques. This method of manufacturing core-shell

126 structures using composite liquid marble methods for sample dispersion and containment is able to protect the sample during thermal cycling and achieve fluorescence results comparable to the conventional method.

In addition, for the 10 experiments presented here, the conventional dispersion method produced approximately ~1.41 g of contaminated plastic waste, while the core-shell method produced ~0.21 g of contaminated plastic waste. The percentage reduction in contaminated plastic waste is ~85.1 %. Sample pictures of the weight measurement are given in supplementary material (S1.d).

Although the increase in fluorescent intensity with respect to increasing thermal cycles is evidence of successful polymerase chain reaction, there are a number of interfering inputs that may affect the fluorescent intensity at any instant of time during the experiment. The volume of the PCR sample inside the core-shell bead, sensitivity and resolution of the camera- based detection system, transparency of the core-shell bead, geometrical location of the PCR sample inside the core-shell bead, uniformity in size, and shape of the core-shell bead are some of the interfering factors which can affect the fluorescent intensity measured during the experiment. It should be noted that the sample volume used in this experiment is only 2 µl. The sensitivity and resolution of the off the shelf camera-based fluorescent detection system used in this experiment is not good enough to distinguish the fine variations in fluorescence, which may be the reason for the “roughly equal” trend of fluorescent intensity curves until approximately 20 thermal cycles.

This study has shown that this core-shell bead techniques can reduce plastic waste and improving the stability of samples in thermal cycling, however, further improvements optimization to this method are still required. The variations in the geometrical position of the

PCR sample inside the core-shell bead and uniformity in size and shape of the core-shell bead

127 are two important factors affecting the quantification of fluorescent intensity. Though the method of core-shell bead production proposed in this paper successfully synthesized the core- shell beads, extensive characterization and optimization of the method, including the development of ideal compatible photopolymers with similar densities to the PCR mixture, has not been completed. The proposed core-shell bead preparation method in its optimized state should enable the synthesis of highly uniform core-shell beads that will reduce plastic waste produced during PCR techniques, as well as improve the storage ability of samples.

6.5 Conclusions

In this paper, a core-shell polymer bead was synthesized using composite liquid marble technology and subsequent photopolymerization aimed to reduce the contaminated plastic wastes and improve PCR methods of sample dispersion. A volume of 2 µl of PCR mixture with three different concentrations was successfully embedded in the core-shell beads. A customized thermal cycler was designed and fabricated to perform thermal cycling of the core- shell bead, and thermal cycling of the PCR mixture core-shell bead was performed and the first successful DNA amplification inside a core-shell bead using composite liquid marble technology was presented. It was observed that the core-shell bead prevents the evaporation of

PCR liquid and allows an increased number of thermal cycles compared to previously presented PTFE liquid marbles. The mechanical handling of these hardened polymer core-shell beads is also easier compared to conventional liquid marbles.

The method of generation of the core-shell bead using composite liquid marble technology is simpler compared to the conventional PDMS/PMMA based microfluidic chips.

The proposed method was shown to reduce the plastic wastes generated in the conventional

PCR sample dispersion methods by up to 85.1 %. As the laboratory bench surface area required to conduct PCR of a 2-µl liquid marble is considerably less than the conventional method, this method can improve the throughput capability of the PCR substantially. The core-shell sample 128 containment also offers contamination-free storage of the PCR sample after thermal cycling in an easy and space-efficient way. Though there is a considerable amount of plastic waste reduction in the proposed method, there is still a small amount of chemical waste generated in the proposed method. The amphiphobic marshmallow like gel powder used to generate composite liquid marbles (prior to photopolymerization) is the chemical waste generated in the process. However, the amount of powder required to make one core shell bead is negligibly small as it only forms a thin layer of coating over the droplet.

The core-shell beads based on liquid marble technology has potential application in both qRT-PCR and in digital PCR. However, the technology still has room to improve. Customized photopolymer with a similar density that of a PCR mixture should further reduce the time and complexity of processes involved in the synthesis of the beads. An optimized optical probing and fluorescence detection system may significantly enhance the detection limit of the proposed method.

Though generates plastic waste and needs complex auxiliary setups, droplet-based PCR in conventional microfluidic chips promises very high throughput and the method is well developed[10]. The throughput capability of the core-shell bead PCR presented here is limited as the method of core-shell bead synthesis involves a significant amount of mechanical processes. The same could be improved by developing an automated core-shell bead generation system. Since it is a matter of photopolymerizing the exterior polymer liquid, conventional microfluidic droplet generation chips using PDMS or PMMA can be developed to make core- shell beads in applications where the issue of plastic waste and complexity due to pumps and tubing are insignificant compared to the required throughput capability. Conventional microfluidic droplet generation techniques are so advanced that it can generate droplets of picolitre(pL) volume[39]. Those techniques could be explored to synthesize core-shell beads with much lower volumes.

129

The present paper reports the successful PCR of 2-µl PCR mixture in 20-µl polymer core-shell bead. Variations in the volume ratios between interior and exterior liquids are expected to affect the performance and efficiency of the core-shell bead-making process as well as the efficiency of PCR. A significantly lower volume of PCR liquid compared to the exterior polymer liquid might affect the efficacy of thermal cycling. Sensitivity and resolution of the fluorescence detection system must be on the higher side to detect the fluorescence from a smaller volume of PCR liquid. On the other hand, a significantly higher volume of core liquid than the polymer liquid is expected to make it difficult to embed the interior liquid inside the exterior core-shell. Though it is embedded, the exterior shell would become delicate and there is a higher possibility for the delicate shell to break at the higher temperatures of thermal cycles.

Characterization and optimization of the core-shell bead generation system for various core liquid to shell liquid volume ratios are necessary to establish a complete protocol for the proposed method. Characterization and optimization of the core-shell bead generation system are out of the scope of this paper; however, future studies should address these issues.

Sample retrieval after the thermal cycling is not possible in the proposed method as the shell material is hard in the polymerized state. Sample retrieval after the thermal cycling may be made possible by using a suitable reversible polymer as the shell material. Reversible polymers are those, which can be depolymerized back to original monomers under suitable conditions [40-43]. It should be noted that the proposed method was not optimized for the fluorescent intensity characterization of the PCR. Errors due to size and shape of the synthesized core-shell bead, errors due to the variation in geometrical location of the PCR sample inside the core-shell bead, inability of sample retrieval from the core-shell bead are the major hindrances towards the fluorescent intensity characterization and performance benchmarking of the proposed method. Sample retrieval from the micro reactor by using a suitable reversible polymer as shell material along with the development of a highly repeatable

130 and optimized core-shell bead generation system might significantly advance the output characterization and calibration of the proposed core-shell bead PCR. Further researches should be carried out in this direction.

Conflicts of interest

There are no conflicts to declare.

Ethical approval

Ethical approval for the study of the effectiveness of molecular assays in detecting human faecal pollution, and the effect of freezer storage on human faecal samples was done by the ethics committee through Griffith University office of research. Ref No. BPS/01/13/HREC.

Author contributions

KRS and NTN established the concept of technology. TT and GH contributed to the formulation of the polymerizable liquid. LG and HS optimized the PCR samples and primers.

JJ, CHO, KL and MS contributed to the experimental setup and evaluation of the results. KRS carried out the experiments. KRS, KL, MS and NTN wrote the final manuscript.

Acknowledgments

The authors acknowledge the Australian Research Council for funding support through the grant DP170100277.

Appendix – Electronic supplementary material

131

The following are the supplementary materials specified in this chapter.

Fig. S1.a. Photographs of various stages of core-shell bead production. (1) Deposition of photo polymer droplet on super amphiphobic powder bed. (2) Inserting fluorescent dye mixed water into the predeposited drop. (3) Inner droplet in partially immersed state. (4) Amphiphobic powder coated composite liquid marble. (5) Photo polymerization of composite liquid marble rotating at 140 rpm in a motorized drum. (6) Core-shell bead embedded with water droplet.

132

Fig.S1. b) Photograph of the core-shell bead generating setup. c) Photograph of the custom-built thermal cycler

Fig.S1. d) Photograph of weighing conventional PCR tube and core-shell bead

133

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137 7 Conclusion and future perspectives

7.1 Conclusions

The present thesis focuses on the development of a simple, portable and cost-effective liquid marble-based platform for polymerase chain reaction (PCR). The Ph.D. research was successful in meeting the planned objectives and developing a state-of-the-art liquid marble technology-based platform for PCR.

An extensive literature survey was conducted to understand the state-of-the-art techniques of digital PCR technology and to identify the research gap. The review was focused on three major phases of digital PCR process namely sample dispersion, thermal cycling, and output monitoring. Potential research gaps were identified in the existing technologies for each of the three phases. Complexity in design and development, need of bulk, complex tubing and pumps and possibility of contamination were identified as the potential disadvantages of existing sample dispersion methods. All existing microfluidic techniques create contaminated plastic waste in a large scale. Thermal cycling techniques except Joule heating were complex, bulk, and/or expensive. Some of the techniques were not universal in terms of application.

Existing output monitoring techniques of PCR were complex and /or expensive due to the use of complex hardware and expensive proprietary software. The potential of the development of a liquid marble based technology for polymerase chain reaction was evident from the literature study. The subsequent part of the research was directed in developing liquid marble based state of the art techniques and address the disadvantages of the existing methods.

Precise and contamination-free sample dispersion is essential for carrying out polymerase chain reaction. It is always advantageous to minimize manual intervention in sample dispersion to avoid the possibility of contamination. Manual sample dispersion can cause error in sample volume and lacks repeatability. So it is always better to have an

138 automated sample dispersion technique. We identified that there was no automated on-demand liquid marble generation system with precise control over the volume of the marble. We developed the first automated on-demand liquid marble generator based on electrohydrodynamic pulling. The device could generate liquid marbles of a desired volume and with a precise control over the volume and with reasonable repeatability.

Evaporation was the potential bottle neck for carrying out PCR in conventional liquid marbles. An understanding of the evaporation dynamics of liquid marbles was essential for designing and developing a liquid marble based PCR technology. A detailed study of the evaporation dynamics of liquid marbles was carried out. The results revealed that a liquid marbles do not obey the d2 law of evaporation at lower temperatures. However, a liquid marble was observed to obey the d2 law of evaporation at elevated temperatures. We proved that the lifetime of liquid marbles at elevated temperature is a function of the volume of the liquid marble, temperature, number of liquid marbles, spatial arrangement of liquid marbles and the relative humidity of the surrounding atmosphere. We also proved that the rate of evaporation of liquid marbles could be considerably reduced by providing a vapour saturated environment around them. The study revealed the possibility of liquid marbles to survive the catastrophic evaporation during the thermal cycles of PCR.

A customised set up was developed to use liquid marble as a microreactor to carry out

PCR. With prior knowledge in the evaporation dynamics of liquid marbles at elevated temperature, a humidity-controlled chamber was developed to reduce the premature evaporation of PCR mixture. The well-known saturated salt solution method was used to achieve 96% relative humidity inside the chamber. A thermal cycler was custom developed and integrated into the humidity-controlled chamber. Thermal cycling for 50 µl PCR mixture was carried out. Nine thermal cycles were carried out prior to the evaporation of the sample.

Successful DNA amplification was observed. The result analysis was carried out in ImageJ

139 open software instead of expensive proprietary software. The results of PCR were comparable to the results obtained from a conventional commercial PCR machine. Though successful PCR was demonstrated in liquid marbles, evaporation of the sample was still limiting the total number of thermal cycles. Sample volume used was also high.

Optimisation of liquid marble PCR in terms of number of thermal cycles and sample volume was subsequently carried out. Core-shell bead was generated using composite liquid marble technology. A volume of 2 µl PCR mixture formed the core of the bead while 20 µl photopolymer served as the shell material. An amphiphobic marshmallow-like gel powder was used to make the composite liquid marble. Photopolymerisation of this composite liquid marble resulted in the synthesis of the core-shell bead. 30 thermal cycles were run on the core-shell bead without evaporation of sample. Successful DNA amplification was observed. 86.1% plastic waste reduction was achieved compared to the conventional PCR technology.

7.2 Future perspectives

Though the present research successfully explored the possibility of using liquid marble technology for polymerase chain reaction, there is plenty of room for future developments in terms of characterisation, optimisation, automation, and commercialisation. The automated on- demand liquid marble generator could be characterised for various liquids such as PCR mixture. The range of volume of liquid marble can be improved to meet the requirements of various applications. Engineering optimisations are possible to make the system more accurate and portable.

The evaporation dynamics of liquid marbles could be carried out at various relative humidity and temperature for various volumes and study the effect of each of these parameters on the lifetime of the liquid marble. This might help to develop an intelligent system, which

140 optimises the physical parameters to prevent the premature evaporation of samples in applications involving conventional liquid marbles.

Various materials can be tested for the shell material of the core-shell beads. Plenty of characterisation and optimisations are also necessary for the proposed method of core-shell bead synthesis.

Integration of the liquid marble generation system with proposed liquid marble based

PCR systems is another possibility of future research. The fluorescence detection system used in our liquid marble PCR technology can be integrated with the open-source image processing software (ImageJ) to automate the output monitoring of PCR. Characterisation and calibration of the fluorescence output is also essential. Commercialisation of liquid marble based digital microfluidic platform for PCR may be possible with further optimisation of the methods reported in this thesis.

Future works can be also extended to use liquid marbles as a microreactor for digital polymerase chain reaction. Digital PCR requires the sample to be compartmentalised into many small units of picolitre to nanolitre volume and before the thermal cycles. Liquid marbles down to 3 mm (~ 15fL) diameter has been reported so far. This enables the possibility of a highly efficient sampling dispersion technique for dPCR, that means, each reaction chamber may contain either one nucleic acid template or none. The problem of premature evaporation of liquid marble has to be effectively addressed in such a case as the volume of each liquid marble will be on the order of picolitres to femtolitres. However, development of such a technology may lead to a more efficient dPCR with reduced the device complexity and single-use plastic waste.

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