Microfluidic Preparation and Transport of Long DNA using Pillar Arrays

• This thesis presents a set of microfluidic tools and experimental studies for preparing (> 20 kbp) for genetic analysisas 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. Ifsize-selective separation is to be achieved, it is also expensive. Long-DNA preparation is essential for detectinggenetic sequences that ranges above kilobase pairs such as large scale structural variations. These can in turn beimportant for diagnosing genetic diseases.Deterministic lateral displacement (DLD) has been used to prepare the long DNA. DLD is a continuous microfluidicseparation 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 meanflow velocity of approximately 34 mm/s. This increases the separation throughput immensely (one to five orders ofmagnitude in throughput compared to other microfluidic techniques) which makes it possible to collect enoughseparated sample after a few minutes to hours, depending on the post-separation analysis method. We explore theeffect of high concentration and show that long-DNA separation can both be enhanced and lessened as aconsequence of concentration-based effects. We also integrate long-DNA isolation in DLD with subsequent surfacestretching of the isolated DNA molecules. Combining the analysis on-chip after the separation eliminates anyproblematic sample transfer steps and allows the analysis to work with dilute samples of only a few hundredmolecules.Novel elastic flow phenomena have been discovered. Large-scale ordered regular DNA waves have been observed toemerge in pillar arrays when trying to increase the throughput of DNA separation in DLD. It is possible that thesewaves could either improve separation or worsen it and thus set the limits for it. A large part of the presented workaimed to understand the emergence and character of these waves. The peaks of these waves consist of high localDNA concentration with the DNA strands stretched and oriented with the wave fronts. These have been found tooccur at high flow velocity, u, and high concentration to overlap concentration ratio (C/C*). We have explored thewave onset in C/C* and u by changing the polymer length, concentration and ionic strength of the buffer. Thesewaves arise together with periodic cycles of growth and shedding of masses of DNA that collect in the pillar gaps inthe flow direction.We also show that the macroscopic and microscopic DNA flow patterns in micro pillar arrays depend highly on thepillar distribution and the pillar shape. By changing the pillar array distribution to hexagonal instead of quadratic, largescalechaotic zig-zag patterns are observed. By changing the distribution to a disordered one, no large-scale flowpattern is observed. We speculate that the induction or avoiding of a large-scale flow pattern could be useful fordifferent degrees of mixing. By changing the pillar shape from circular cross-section to a triangular one, we form largewaves of only one orientation instead of two. The large waves appear in a different orientation depending on the flowdirection. In addition, the microscopic vortex behavior emerges at different flow velocities for the two directions as wellas with different flow resistances. This could be exploited in microfluidic components such as one-way valves orpumps.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biofysik (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biophysics (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Nanoteknik -- Nanoteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Nano-technology -- Nano-technology (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Annan medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Other Medical Biotechnology (hsv//eng)

Nyckelord

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

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