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Microfluidic Preparation and Transport of Long DNA using Pillar Arrays

Ström, Oskar LU orcid (2023)
Abstract
This thesis presents a set of microfluidic tools and experimental studies for preparing (> 20 kbp) for genetic analysis
as well as the transport of high-concentration, long-DNA solutions in pillar arrays.
Long-DNA sample preparation with conventional gel-based techniques is slow (tens of hours to days) and laborious. If
size-selective separation is to be achieved, it is also expensive. Long-DNA preparation is essential for detecting
genetic sequences that ranges above kilobase pairs such as large scale structural variations. These can in turn be
important for diagnosing genetic diseases.
Deterministic lateral displacement (DLD) has been used to prepare the long DNA. DLD is a continuous microfluidic
separation... (More)
This thesis presents a set of microfluidic tools and experimental studies for preparing (> 20 kbp) for genetic analysis
as well as the transport of high-concentration, long-DNA solutions in pillar arrays.
Long-DNA sample preparation with conventional gel-based techniques is slow (tens of hours to days) and laborious. If
size-selective separation is to be achieved, it is also expensive. Long-DNA preparation is essential for detecting
genetic sequences that ranges above kilobase pairs such as large scale structural variations. These can in turn be
important for diagnosing genetic diseases.
Deterministic lateral displacement (DLD) has been used to prepare the long DNA. DLD is a continuous microfluidic
separation method. Long-DNA separation in DLD has previously been thought to be limited to very low flow velocities
(up to 40 μm/s) and thus low throughput. In this work, we show that it is possible to displace long DNA up to a mean
flow velocity of approximately 34 mm/s. This increases the separation throughput immensely (one to five orders of
magnitude in throughput compared to other microfluidic techniques) which makes it possible to collect enough
separated sample after a few minutes to hours, depending on the post-separation analysis method. We explore the
effect of high concentration and show that long-DNA separation can both be enhanced and lessened as a
consequence of concentration-based effects. We also integrate long-DNA isolation in DLD with subsequent surface
stretching of the isolated DNA molecules. Combining the analysis on-chip after the separation eliminates any
problematic sample transfer steps and allows the analysis to work with dilute samples of only a few hundred
molecules.
Novel elastic flow phenomena have been discovered. Large-scale ordered regular DNA waves have been observed to
emerge in pillar arrays when trying to increase the throughput of DNA separation in DLD. It is possible that these
waves could either improve separation or worsen it and thus set the limits for it. A large part of the presented work
aimed to understand the emergence and character of these waves. The peaks of these waves consist of high local
DNA concentration with the DNA strands stretched and oriented with the wave fronts. These have been found to
occur at high flow velocity, u, and high concentration to overlap concentration ratio (C/C*). We have explored the
wave onset in C/C* and u by changing the polymer length, concentration and ionic strength of the buffer. These
waves arise together with periodic cycles of growth and shedding of masses of DNA that collect in the pillar gaps in
the flow direction.
We also show that the macroscopic and microscopic DNA flow patterns in micro pillar arrays depend highly on the
pillar distribution and the pillar shape. By changing the pillar array distribution to hexagonal instead of quadratic, largescale
chaotic zig-zag patterns are observed. By changing the distribution to a disordered one, no large-scale flow
pattern is observed. We speculate that the induction or avoiding of a large-scale flow pattern could be useful for
different degrees of mixing. By changing the pillar shape from circular cross-section to a triangular one, we form large
waves of only one orientation instead of two. The large waves appear in a different orientation depending on the flow
direction. In addition, the microscopic vortex behavior emerges at different flow velocities for the two directions as well
as with different flow resistances. This could be exploited in microfluidic components such as one-way valves or
pumps. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Prof. Dorfman, Kevin, University of Minnesota, USA.
organization
alternative title
Mikrofluidisk provförberedning och transport av lång DNA med hjälp av mikropelare
publishing date
type
Thesis
publication status
published
subject
keywords
Microfluidics, Deterministic Lateral Displacement, DNA Separation, Elastic Turbulence, Micro-pillar arrays, DNA Sample Preparation, Lab-on-a chip, elastic waves, Sample Preparation, DNA Isolation, bumper array, Fysicumarkivet A:2023:Ström
pages
272 pages
publisher
Solid State Physics, Lund University
defense location
Lecture Hall Rydbergsalen, Department of Physics, Sölvegatan 14, Faculty of Engineering LTH, Lund University, Lund. The dissertation will be live streamed.
defense date
2023-02-10 09:15:00
ISBN
978-91-8039-390-4
978-91-8039-389-8
project
Microfluidic Preparation and Transport of Long DNA using Pillar Arrays
language
English
LU publication?
yes
id
1621b3a0-4f58-4d04-bbcd-beddbc42796d
date added to LUP
2022-08-31 11:35:16
date last changed
2023-05-31 10:23:33
@phdthesis{1621b3a0-4f58-4d04-bbcd-beddbc42796d,
  abstract     = {{This thesis presents a set of microfluidic tools and experimental studies for preparing (&gt; 20 kbp) for genetic analysis<br/>as well as the transport of high-concentration, long-DNA solutions in pillar arrays.<br/>Long-DNA sample preparation with conventional gel-based techniques is slow (tens of hours to days) and laborious. If<br/>size-selective separation is to be achieved, it is also expensive. Long-DNA preparation is essential for detecting<br/>genetic sequences that ranges above kilobase pairs such as large scale structural variations. These can in turn be<br/>important for diagnosing genetic diseases.<br/>Deterministic lateral displacement (DLD) has been used to prepare the long DNA. DLD is a continuous microfluidic<br/>separation method. Long-DNA separation in DLD has previously been thought to be limited to very low flow velocities<br/>(up to 40 μm/s) and thus low throughput. In this work, we show that it is possible to displace long DNA up to a mean<br/>flow velocity of approximately 34 mm/s. This increases the separation throughput immensely (one to five orders of<br/>magnitude in throughput compared to other microfluidic techniques) which makes it possible to collect enough<br/>separated sample after a few minutes to hours, depending on the post-separation analysis method. We explore the<br/>effect of high concentration and show that long-DNA separation can both be enhanced and lessened as a<br/>consequence of concentration-based effects. We also integrate long-DNA isolation in DLD with subsequent surface<br/>stretching of the isolated DNA molecules. Combining the analysis on-chip after the separation eliminates any<br/>problematic sample transfer steps and allows the analysis to work with dilute samples of only a few hundred<br/>molecules.<br/>Novel elastic flow phenomena have been discovered. Large-scale ordered regular DNA waves have been observed to<br/>emerge in pillar arrays when trying to increase the throughput of DNA separation in DLD. It is possible that these<br/>waves could either improve separation or worsen it and thus set the limits for it. A large part of the presented work<br/>aimed to understand the emergence and character of these waves. The peaks of these waves consist of high local<br/>DNA concentration with the DNA strands stretched and oriented with the wave fronts. These have been found to<br/>occur at high flow velocity, u, and high concentration to overlap concentration ratio (C/C*). We have explored the<br/>wave onset in C/C* and u by changing the polymer length, concentration and ionic strength of the buffer. These<br/>waves arise together with periodic cycles of growth and shedding of masses of DNA that collect in the pillar gaps in<br/>the flow direction.<br/>We also show that the macroscopic and microscopic DNA flow patterns in micro pillar arrays depend highly on the<br/>pillar distribution and the pillar shape. By changing the pillar array distribution to hexagonal instead of quadratic, largescale<br/>chaotic zig-zag patterns are observed. By changing the distribution to a disordered one, no large-scale flow<br/>pattern is observed. We speculate that the induction or avoiding of a large-scale flow pattern could be useful for<br/>different degrees of mixing. By changing the pillar shape from circular cross-section to a triangular one, we form large<br/>waves of only one orientation instead of two. The large waves appear in a different orientation depending on the flow<br/>direction. In addition, the microscopic vortex behavior emerges at different flow velocities for the two directions as well<br/>as with different flow resistances. This could be exploited in microfluidic components such as one-way valves or<br/>pumps.}},
  author       = {{Ström, Oskar}},
  isbn         = {{978-91-8039-390-4}},
  keywords     = {{Microfluidics; Deterministic Lateral Displacement; DNA Separation; Elastic Turbulence; Micro-pillar arrays; DNA Sample Preparation; Lab-on-a chip; elastic waves; Sample Preparation; DNA Isolation; bumper array; Fysicumarkivet A:2023:Ström}},
  language     = {{eng}},
  month        = {{01}},
  publisher    = {{Solid State Physics, Lund University}},
  school       = {{Lund University}},
  title        = {{Microfluidic Preparation and Transport of Long DNA using Pillar Arrays}},
  url          = {{https://lup.lub.lu.se/search/files/136526224/phdthesis_oskar_strom.pdf}},
  year         = {{2023}},
}