Magnetic bead actuated microfluidic immunoassay platform for

147 Pages·2014·4.26 MB·English

Magnetic bead actuated microfluidic
immunoassay platform for sensitive
detection of biomarkers and pathogens

Von der Fakultät für Elektrotechnik und Informationstechnik
der Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades einer
Doktorin der Ingenieurwissenschaften
genehmigte Dissertation

vorgelegt von
Master of Science Biomedical Engineering
Raiah Gottheil (geb. Bhullar)
aus Chandigarh, Indien

Berichter: Universitätsprofessor Dr. rer. nat. Wilfried Mokwa
Universitätsprofessor Dr. rer. nat. Gerald A. Urban

Tag der mündlichen Prüfung: 23.06.2014

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

The research reported in this thesis was conducted at the Natural and Medical Sciences
Institute (NMI) in Reutlingen under the supervision of Dr. Martin Stelzle and Prof. Dr.
Wilfried Mokwa. The funding for this project was supported through grants from the
Federal Ministry for Research and Education 16V5111 (BMBF-Bundesministerium für
Bildung und Forchung) and the Spitzencluster microTEC Südwest.

Acknowledgements
First and foremost, I would like to express much appreciation to all my advisors
startly with Dr. Martin Stelzle, group leader BioMEMS and Sensors Group, Natural and
Medical Sciences Institute (NMI), Reutlingen. I would like to thank him for giving me the
opportunity to work in his group and supporting me throughout my thesis. I have
learned how to look at things from a very different perspective under his guidance,
during these past three years.
Professor Dr. rer. nat. Wilfried Mokwa, Institute of Materials in Electrical
Engineering , RWTH Aachen University for agreeing to be my first advisor and for
following my progress during the thesis time. I appreciate the time he took to write back
to me and organize meetings when I needed his support.
Professor Dr. rer. nat. Gerald A. Urban, Department of Microsystems engineering,
University Freiburg for agreeing to being my second advisor on such short notice. I am
thankful that he undertook the task of reading, correcting and advising me on the
written report. His input was greatly appreciated and helped improve the presentation
of the dissertation.
My gratitude also goes to the scientists in the Department of Biochemistry at the
NMI, Nadja Baur, Dr. Nicole Schneiderhahn Marra and Dr. Oliver Pötz. They were always
reachable and prepared to answer my numerous queries regarding the assay
development. It was an immense learning experience to work with scientists in a
completely different discipline.
Many thanks are due to the project collaborators from Mediagnost GmbH,
microfluidic ChipShop GmbH and MultiChannelSystem GmbH, for working with me
towards our common goal in this project. Our numerous discussions and exchanges
helped me in understanding the commercial aspects of this project apart from all I
gleaned from their expertise.
I also want to acknowledge the support provided Simon Werner and Gorden (Steve)
Link, from the BioMEMS and Sensors group, during the hardware and software
development of the experimental set-up. The many challenges encountered during the
establishment of the set-up were solved through their help, especially when there was a
predicament.
The three students Sabrina Ott, Dimitri Maier and Andreas Geiger who helped with
the many experiments needed during this thesis work. They made it possible to validate
the platform thoroughly. Furthermore, supervising them taught me how to co-ordinate
and create enthusiasm within ones teammates.
My colleagues and peers in the BioMEMS and Sensors group whose experiences and
knowledge I drew on for numerous tasks, their aid has in no way been trivial to me. I
appreciate the assistance they provided me when faced with unfamiliar equipment and
methods, no matter how engaged they were. Not to mention all the barbeques, cake and
get-to-togethers, that were organized and made working at the NMI fun.
A most special thanks goes to my best friend and partner, my husband, and our
parents and family, who gave me unconditional support, loads of motivation and love
throughout this endeavor. Lastly, my son, who brings me joy and laughing with him
gives me the strength to move mountains.

Contents

1. Introduction ..................................................................................................... 13
1.1. Diagnostics and healthcare ..................................................................................................... 13
1.1.1. In-vitro diagnostic market .....................................................................................................15
1.1.2. Microfluidics in Point-of-Care Testing ..............................................................................16
1.2. Objectives ....................................................................................................................................... 21
1.3. Strategy ........................................................................................................................................... 23
2. State of the Art ................................................................................................. 24
2.1. Magnetic beads for mixing ...................................................................................................... 25
2.2. Magnetic beads for reagent exchange ................................................................................. 26
3. Theory ................................................................................................................ 29
3.1. Microfluidics and fluid behavior ........................................................................................... 29
3.1.1. Interfacial physics ......................................................................................................................29
3.1.2. Passive fluid control ..................................................................................................................32
3.2. Magnetic bead technology ....................................................................................................... 36
3.2.1. Magnetism and magnetic materials ..................................................................................36
3.2.2. Magnetic Beads ...........................................................................................................................40
3.2.3. Magnetophoretic forces ..........................................................................................................41
3.3. Immunoassay concepts ............................................................................................................ 43
3.3.1. Immunometric assay design .................................................................................................43
3.3.2. Kinetics of antibody-antigen interactions .......................................................................44
3.3.3. Assay sensitivity ..........................................................................................................................46
3.4. Fluorescence spectroscopy ..................................................................................................... 48
3.4.1. Fluorescence and fluorophores ............................................................................................48
3.4.2. Fluorescence techniques .........................................................................................................50
4. The Microfluidic Cartridge .......................................................................... 53
4.1. Contact angle ................................................................................................................................ 55
4.2. Cartridge design .......................................................................................................................... 57
4.2.1. Fluid control: Phaseguides and Valves .............................................................................59
4.2.2. Phaseguide: Numerical Simulation ....................................................................................61
4.3. Performance .................................................................................................................................. 62
4.3.1. Filling Scheme .............................................................................................................................63
4.3.2. Pressure Profile ...........................................................................................................................64
4.4. Discussion ...................................................................................................................................... 67
5. The Platform .................................................................................................... 71
5.1. Reconfiguring sandwich assays ............................................................................................. 72
5.2. Experimental set-up ................................................................................................................... 73
5.3. Magnetic actuation ..................................................................................................................... 75
5.3.1. Mixing and active incubation ...............................................................................................75
5.3.2. Aggregation and transport ...................................................................................................80
5.4. Image acquisition ........................................................................................................................ 82
5.5. Discussion ...................................................................................................................................... 84
6. Interleukin-8 Assay ....................................................................................... 87
6.1. Luminex bead based assays .................................................................................................... 89
6.1.1. The Luminex technology .........................................................................................................89
6.2. Interleukin-8 assay design ...................................................................................................... 90
6.2.1. The Luminex technology: Interleukin-8 assay ...............................................................91
6.3. Interleukin-8 assay on cartridge ........................................................................................... 94
6.3.1. Preparation of assay reagents .............................................................................................94
6.3.2. Assay scheme on cartridge .....................................................................................................96