Title: Manipulation of magnetic microparticles in droplet microfluidics
Other Titles: Manipulatie van magnetische micropartikels in druppelgebaseerde microfluidics
Authors: Verbruggen, Bert; S0105759
Issue Date: 27-May-2014
Abstract: Lab-on-a-chips and micro total analysis systems have been around for more than three decades. They find applications in medical, environmental and food diagnostics and appear in many different configurations and complexities. They all have in common that fluids are transported in small volumes through micrometer sized structures. A promising and growing subcategory of these microfluidic systems are the multiphase, droplet or segmented flow microfluidics. Discrete volumes of reagents are transported inside microfluidic channels, surrounded with an immiscible fluid or a gas, which prevents cross-contamination between the plugs or droplets. These droplets with a typical volume between 1 microliter and 1 picoliter can be used as reaction vessels for (bio)chemical reactions or for bioassays such as ELISA and PCR. They can be generated at high frequencies, up to 10000 per second, which is especially promising for high-throughput applications.Many commonly applied bioassays (e.g., ELISA) use a solid support to anchor the antibodies or capture probes, allowing the separation of targets from the complex biological matrices. Magnetic nano- or microparticles are perfectly suited for this task due to their high surface-to-volume ratio and the potential to magnetically separate them from the sample matrix. In that context droplet-based segmented flow microfluidics (DBSF) and on‑chip manipulation of magnetic microparticles appears a logic and attractive combination. However, integrating both technologies requires a good understanding of micro-engineering, microfluidics and bioassay development to come to a successful diagnostic device. The objective of this PhD research was to study the integration of magnetic microparticle manipulations in a DBSF microfluidic chip.The first part of this work consisted mainly of capacity building. First, existing fabrication protocols for polydimethylsiloxane (PDMS) microfluidic chips were adjusted to match the available equipment and the design requirements. Next, DBSF was implemented in the microchannels. In order to minimize the absorption and migration of biomolecules, specific fluorocarbon was introduced, replacing traditional hydrocarbon-based oil. This in turn required corresponding surfactants and surface coating and made it necessary to repeat much previously reported work. The basic unit operations such as droplet generation, mixing and splitting were studied before novel concepts were attempted.In the second part, a novel microfluidic concept was developed to actively control the splitting ratio of droplets. A computational fluid dynamics model was composed, validated and implemented to simulate the effect of different designs and flow parameters. With the final design, droplet splitting ratios between 50/50 and 95/5 were achieved solely by controlling the operational parameters. This considerably reduces the time necessary to match the splitting ratio with potential (bio)assays in future work. The dynamic control especially accelerated the experimental phase of microparticle separation in the follow-up research.In the third part, the magnetic configuration to separate the microparticles from the sample matrix was studied using mathematical simulations and experimental tests. A detailed three-dimensional model of the magnetic field in proximity of a cubic permanent magnet was applied to accurately calculate the magnetic force on superparamagnetic particles. Using these results, the conditions for particle aggregation, attraction and immobilization were studied and good separation conditions were determined. In the improved setup, up to 90% of the droplet volume was removed from the microparticles while the non-separated particle fraction remained below 5%. Only when more than 95% of the original sample volume was removed in a single purification step, 10% of the microparticles were not correctly separated.Finally, in a last part, a selective DNA extraction assay with microparticles was studied in the DBSF microfluidic system to evaluate the overall performance of the novel design. It was demonstrated that the hybridization and capture efficiency of the biofunctionalized particles was identical for off‑chip and on‑chip methods. Also, the effect of the particle separation efficiency on the extraction efficiency was tested for different splitting regimes. Finally, the impact of separation at a higher splitting ratio for the repeated washing of the particles was discussed. The successful implementation of selective DNA extraction is new to the field of DBSF microfluidics and is very promising for future applications.
Table of Contents: Table of contents
Preface i
Abstract iii
Beknopte samenvatting vii
List of abbreviations xi
List of symbols xv
Table of contents xvii
Chapter 1 General introduction 1
1.1 An introduction to microfluidic systems 1
1.2 Dividing the flow 3
1.3 Problem statement 6
1.4 Objectives and outline of the thesis 8
1.5 References 11
Chapter 2 Fabrication of a microfluidic system in PDMS 17
2.1 Introduction 17
2.2 Objective and outline of the chapter 18
2.3 Design of the microfluidic channels 19
2.4 Fabrication of the SU 8 mold 22
2.4.1 Surface treatment of the silicon wafer 23
2.4.2 Spin-coating of the SU 8 film 25
2.4.3 Patterning of the SU 8 film 30
2.4.4 Development of the SU 8 structures 32
2.4.5 Post-treatment of the mold 34
2.4.6 Complete fabrication protocol of the SU 8 mold 35
2.5 Fabrication of the PDMS microfluidic chip 38
2.5.1 Molding the PDMS chip 39
2.5.2 Sealing the PDMS chip 40
2.5.3 Post-treatment of the microfluidic chip 45
2.5.4 Complete fabrication protocol of the PDMS microfluidic chip 46
2.6 Conclusions 47
2.7 References 49
Chapter 3 Unit operations in droplet-based segmented flow 53
3.1 Introduction 53
3.2 Objective and outline of the chapter 55
Materials and methods 56
3.2.1 Reagents 56
3.2.2 Microfluidic design 56
3.2.3 Operational parameters 57
3.2.4 Microfluidic setup 57
3.2.5 Imaging setup 58
3.3 Results and discussion 59
3.3.1 Droplet analysis 59
3.3.2 General design strategy 61
3.3.3 Droplet formation 63
3.3.4 Droplet volume 69
3.3.5 Mixing of reagents 70
3.3.6 Splitting of droplets 74
3.3.7 Droplet collection 81
3.4 Conclusions 84
3.5 References 86
Chapter 4 Design of a flow controlled asymmetric droplet splitter using computational fluid dynamics 89
4.1 Introduction 89
4.2 Objective and outline of the chapter 90
4.3 Materials and methods 91
4.3.1 Microfluidic design 91
4.3.2 Data analysis 92
4.4 The multiphase model 92
4.4.1 Numerical simulation 92
4.4.2 Numerical solution procedure 94
4.4.3 Mesh sensitivity analysis 95
4.4.4 Experimental validation using droplet formation 96
4.5 Results and discussion 99
4.5.1 Design of T junction split 99
4.5.2 Design of the extra inlet 101
4.5.3 Simulating and validating the extra flow 102
4.6 Conclusions 105
4.7 References 107
Chapter 5 Separation of magnetic micro-particles in segmented flow using asymmetric splitting regimes 109
5.1 Introduction 109
5.2 Objective and outline of the chapter 112
5.3 Materials and methods 112
5.3.1 Reagents and materials 112
5.3.2 Microfluidic system 113
5.3.3 Microfluidic design 113
5.3.4 Microfluidic experiments 115
5.4 The magnetic model 115
5.4.1 Numerical simulation of magnetic field density 115
5.4.2 Calculation of the magnetic forces 117
5.5 Results and discussion 119
5.5.1 Forces on the superparamagnetic microparticles 119
5.5.2 Particle behavior in a magnetic field: continuous flow 122
5.5.3 Particle behavior in a magnetic field: segmented flow 126
5.5.4 Magnet position and orientation at the split 135
5.6 Conclusions 140
5.7 References 142
Chapter 6 Selective DNA extraction with microparticles in segmented flow 147
6.1 Introduction 147
6.2 Objective and outline of the chapter 148
6.3 Materials and methods 148
6.3.1 Reagents and buffer solutions 148
6.3.2 Microfluidic system 150
6.3.3 Microfluidic design 150
6.3.4 Capture probe immobilization on magnetic particles 152
6.3.5 Target capture efficiency of the functionalized microparticles 152
6.3.6 Microparticle separation efficiency of the microfluidic system 153
6.3.7 Quantification of DNA 154
6.3.8 Kd of the hybridization reaction 154
6.4 Results and discussion 156
6.4.1 Immobilization of the capture probes 156
6.4.2 qPCR standard curve 157
6.4.3 Temperature and hybridization rate 158
6.4.4 On chip hybridization 160
6.4.5 Capture efficiency and saturation 162
6.4.6 Microparticle separation and total DNA extraction efficiency 165
6.5 Conclusions 167
References 169
Chapter 7 General conclusions and perspectives 170
7.1 General conclusions 170
7.2 Future research and perspectives 174
7.2.1 Coupling of separation modules 175
7.2.2 Difficult sample matrices 176
7.2.3 Quantification with ELISA 177
7.2.4 Digital quantification 177
7.2.5 Digital PCR 178
7.2.6 Complex microfluidic systems 179
7.3 References 180
List of Publications 181
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Division of Mechatronics, Biostatistics and Sensors (MeBioS)

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