Microscale Surface-based Bioanalytical Applications Using Microfluidic Probe Technologies
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2017-05
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Doctoral Thesis
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Abstract
Compartmentalization is central to the study of heterogeneity in biological substrates in
several areas of research in the life-sciences. For this, it is important to localize chemical
reactions at the micrometer length-scale in immersed environments with conservative use
of reagents and samples. Such controlled localization of chemicals on surfaces has been
demonstrated in microfluidic systems wherein the small scale provides unique advantages
in terms of transport and reaction kinetics resulting in lower reagent and sample
consumption as well as reduced time for testing. Most current microfluidic systems are
“closed”, i.e. they are sealed within four walls. This limits their interactivity, versatility
and precision in use with a diverse set of biological samples such as tissue sections and
adherent cells. To address this large diversity of samples, and enable interaction with
standard biological substrates (e.g. microscope slide, Petri dishes, microtiter plates), the
Microfluidic Probe (MFP) was developed.
In this thesis, technological advancements in the instrumentation, including the design
and microfabrication of new MFP heads along with numerical and analytical models of
the MFP and their applications to selected problems in the bioanalytical sciences are
developed. The MFP is a scanning, non-contact microfluidic technology, which enables
local (bio)chemical reactions on surfaces in the “open-space” by hydrodynamically
confining nanoliter volumes of liquids in a wet environment (water, buffer solutions,
culture media).
Within the framework of this dissertation, a compact MFP (cMFP) system that can be
used on standard inverted microscopes, that assists in local processing of tissue sections
and biological specimens was developed. The cMFP has a footprint of 175 × 100 × 140
mm3 and can scan an area of 45 × 45 mm2 on a surface with an accuracy of ±15 μm. The
applicability of the cMFP to tissue staining, exemplified by melanoma cell blocks was
shown using hematoxylin to create contours, lines, spots and gradients.
Further, a new methodology was introduced for efficient and high-quality patterning of
biological reagents for surface-based biological assays. The interplay between diffusion,
advection, and surface chemistry is studied. As compared to standard deposition
techniques, a 2- to 5-fold increase in deposition rate is demonstrated together with a 10-
vi
fold reduction in analyte consumption with unprecedented level of deposition
homogeneity.
In order to expand the applicability of the MFP to a larger set of bioassays, a new concept
was developed termed Hydrodynamic Thermal Confinement (HTC), that can be generally
applicable, along with its implementation, for the creation of microscale dynamic thermochemical
microenvironments on biological surfaces. HTC is based on an MFP and
operates under physiological conditions. The temperature can be regulated between 30°
and 80° C with ±2° C precision and temperature ramps of 5° C/s over a footprint of ~50
μm × 80 μm in a volume of ~50 × 80 × 15 μm3.
Finally, the concept of Tissue Lithography (TL), which enables retrospective studies on
formalin-fixed paraffin-embedded tissue sections was described and implemented. TL
uses a microfluidic probe to remove microscale areas of the paraffin on formalin-fixed
paraffin-embedded biopsy samples. Stain patterns as small as 100 μm × 50 μm as well as
multiplexed immunostaining within a single tissue microarray core are achieved with a
20-fold time reduction for local dewaxing as compared to standard protocols.
It is very likely that such a bioanalytical tool and the associated analytical models that we
developed in this thesis will be a facilitator towards the development of quantitative
surface-based biological assays and will bring a new level of versatility in biological
sample processing and budgeting.
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Examiner : Nelson, Bradley
Examiner : Gadegaard, Nikolaj
Examiner : Kaigala, Govind
Examiner : Maerkl, Sebastian
Examiner : Moch, Holger
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ETH Zurich
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03627 - Nelson, Bradley J. / Nelson, Bradley J.