Wu, Jiamin
(2013)
DNA SEPARATION AT A STRETCH AND MULTISTAGE MAGNETIC SEPARATION OF MICROSPHERES.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
This thesis consists of two parts. The first part focuses on development of a novel DNA separation technology by tethering DNA strands to a solid surface and then stretching the DNA with an electric field. The anchor is such designed that the critical force to detach a DNA is independent of its size. Because the stretching force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based removal of the DNA from the surface and thus DNA separation. This strategy may provide a convenient, low-cost, and high-speed alternative to existing methods for DNA separation, because sieving matrices are not required, separated DNA can be readily recovered, and in principle, there is no upper limit on the length of DNA that can be separated. Using this method, we have demonstrated (i) efficient separation of lambda double-stranded DNA (dsDNA) (48,502 bp) from human genomic dsDNA (>100 kbp) in a dc electric field applied between two parallel plates, (ii) separation of short single-stranded DNA (ssDNA) with less than 100 nucleotides (nt) at 10-nt resolution by tethering and stretching DNA in microfluidic channels filled with a low conductivity buffer, and (iii) separation of short ssDNA by taking the advantage of the strong yet evolving non-uniform electric field near the charged Au surface in contact with an electrolyte.
The second part of my thesis focuses on development of a multistage separation technology to circumvent the challenge caused by non-specific interactions in current single-stage magnetic separation techniques. The key idea is to allow the magnetic particles (MNPs) to reversibly capture and release the targets by manipulating the hydrophobic interaction between the MNPs and the targets. This will be enabled by attaching temperature-responsive polymers to both the MNPs and the targets. Through temperature cycling, which triggers the reversible hydrophilic-to-hydrophobic phase transition of the polymers, the targets can be reversibly captured and released by the MNPs (due to hydrophobic interaction) at a higher efficiency than the non-targets which may also be captured and released by the MNPs due to non-specific interactions. The difference in the capture-and-release efficiencies of targets versus non-targets in a single cycle will be amplified by multiple separation stages, following a similar concept to the distillation process. As a proof-of-concept demonstration, we have demonstrated efficient separation of poly(N-isopropylacrylamide) (PNIPAM, a temperature responsive polymer)-functionalized polystyrene (PS) microspheres from bare PS microspheres by using PNIPAM-functionalized MNPs. The overall enrichment factor is observed to significantly increase with the number of separation stages, and reaches as high as 1.87 E+5 after 5 stages.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
31 January 2013 |
| Date Type: |
Publication |
| Defense Date: |
7 November 2012 |
| Approval Date: |
31 January 2013 |
| Submission Date: |
13 November 2012 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
117 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Swanson School of Engineering > Chemical Engineering |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
DNA separation; electric field; nucleic acids; microfluidics; electrical double layer; magnetic separation; multistage separation; temperature-responsive polymers; nonspecific interaction; magnetic nanoparticles |
| Date Deposited: |
31 Jan 2013 21:29 |
| Last Modified: |
15 Nov 2016 14:07 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/16402 |
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