rapid affinity purification based on ion concentration shock waves and porous polymer monoliths

283 Pages·2015·5.13 MB·English

RAPID AFFINITY PURIFICATION BASED ON ION
CONCENTRATION SHOCK WAVES AND POROUS
POLYMER MONOLITHS

A DISSERTATION
SUBMITTED TO THE DEPARTMENT
OF MECHANICAL ENGINEERING
AND THE COMMITTEE OF GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

Viktor Shkolnikov
June 2015
© 2015 by Viktor Shkolnikov. All Rights Reserved.

Re-distributed by Stanford University under license with the author.

This work is licensed under a Creative Commons Attribution-

Noncommercial 3.0 United States License.
http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/yt510fd5490

ii
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Juan Santiago, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Kenneth Goodson
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Ali Mani
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost for Graduate Education
This signature page was generated electronically upon submission of this dissertation in
electronic format. An original signed hard copy of the signature page is on file in
University Archives.
iii
© 2015 by Viktor Shkolnikov. All Rights Reserved.

Re-distributed by Stanford University under license with the author.

This work is licensed under a Creative Commons Attribution-

Noncommercial 3.0 United States License.
http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/yt510fd5490

ii
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Juan Santiago, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Kenneth Goodson
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Ali Mani
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost for Graduate Education
This signature page was generated electronically upon submission of this dissertation in
electronic format. An original signed hard copy of the signature page is on file in
University Archives.
iii
Abstract
Micro total analysis systems have the potential to drastically improve the fields of
healthcare, agriculture, and environmental monitoring by rapidly providing decision
influencing answers directly in the field. Sample preparation represents on the remaining
challenges in achieving in achieving such systems. This dissertation focuses on developing
a rapid sample preparation method based affinity purification and preconcentration that is
compatible with integration into a micro total analysis system. Our method couples ion-
concentration shockwaves with porous polymer monolith based micro affinity columns to
achieve rapid affinity purification, minimizing purification time and maximizing affinity
substrate utilization.
We begin with describing methodology for synthesis of porous polymer monoliths
and control of their porous properties. We leverage the flexibility of structure, chemical
composition, and surface chemistry (including wettability) of porous polymer monoliths to
design, fabricate, and characterize hydrophilic porous monoliths, with the aim of achieving
high permeability wick materials. We show that variations in monomer concentration and
porogen composition can affect mode pore diameters ranging from 6.3 to 10.1 µm and
permeabilities ranging from 0.73 x 10-12 to 1.9 x 10-12 m2. In addition, we identify a rough
dependence of monolith permeability on porosity times the square of mode pore diameter
and discuss key figures of merit characterizing capillary transport. As an example
application, we then detail a custom injection molding procedure, where we in situ
polymerize ~150 µm thick wicks conformally onto the surface of metal channels of a
polymer electrolyte fuel cell cathode.
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We then present a novel technique where we couple ion-concentration shock waves
(isotachophoresis) with affinity chromatography to achieve rapid, selective purification
with high column utilization. We use isotachophoresis to simultaneously preconcentrate
analytes and purify them based on differences in mobility of sample components, excluding
species that may foul or compete with the target at the affinity substrate. Isotachophoretic
preconcentration accelerates the affinity reaction, reducing assay time, improving column
utilization and allowing for capture of targets with higher dissociation constants.
Furthermore, our method separates the target and contaminants into non-diffusing zones,
thus achieving high resolution in a short distance and time. We present an analytical model
for spatiotemporal dynamics of our method. We identify and explore the effect of key
process parameters including target distribution width and height, isotachophoresis zone
velocity (shock velocity), forward and reverse reaction constants, and probe concentration
on necessary affinity region length, assay time, and capture efficiency. Our analytical
approach shows collapse of these variables to three non-dimensional parameters. The
analysis yields simple analytical relations for capture length and capture time in relevant
regimes, and demonstrates how isotachophoresis greatly reduces assay time and improves
column utilization in affinity choromatography.
Lastly, we present an experimental study of coupling of isotachophoresis and
affinity chromatography to effect rapid, selective purification with high column utilization
and high resolution. We provide a detailed protocol for performing this method and
describe the design of a buffer system to perform sequence specific separation of nucleic
acids. We describe the synthesis and functionalization of our affinity substrate,
poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith. This
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substrate allows easy immobilization of affinity probes, is non-sieving (even to
macromolecules), and exhibits negligible non-specific binding. We demonstrate our
method with 25 nt, Cy5 labeled DNA target and a DNA probe and study the spatiotemporal
dynamics using epifluorescence imaging. We make qualitative and quantitative
comparisons between these data and the analytical model we developed for our method.
We vary the target concentration from 1 to 100 pg µl-1 and ITP velocity over the range of
10 to 50 µm s-1, and thereby explore over 4 orders of magnitude of scaled target amount.
We observe very good agreement between predictions and experimental data for the
spatiotemporal behavior of the coupled isotachophoresis and affinity process, and for key
figures of merit including scaled capture length and maximum capture efficiency. Finally,
we demonstrate that the resolution of our method increases linearly with time and purify
25 nt target DNA from 10,000-fold higher abundance background (contaminating)
genomic fish sperm DNA. We perform this capture from 200 µl of sample in under 1 mm
column length and in less than 10 min.

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Table of Contents
1 Introduction ................................................................................................................. 1
1.1 Ion concentration shockwaves (isotachophoresis) ............................................... 2
1.1.1 Peak-mode vs. plateau mode ..........................................................................3
1.1.2 Finite vs. semi-infinite injection ....................................................................4
1.2 Synthesis of porous polymer monoliths (PPM) ................................................... 6
1.2.1 Background and applications of PPM............................................................6
1.2.2 PPM synthesis theory .....................................................................................9
1.2.3 Discussion ....................................................................................................14
1.2.4 Summary of PPM synthesis .........................................................................23
1.3 Functionalization of pore surfaces in GMA-co-EDMA PPM ............................ 24
1.3.1 Functionalization of GMA-co-EDMA PPM surface with small molecules 24
1.3.2 Functionalization of GMA-co-EDMA PPM surface with nucleic acids .....27
1.3.3 Functionalization of GMA-co-EDMA PPM surface with proteins .............30
1.4 Scope of thesis .................................................................................................... 32
1.5 Supplementary Information................................................................................ 34
2 Design and Fabrication of Porous Polymer Wicks ................................................... 41
2.1 Introduction ........................................................................................................ 41
2.2 Materials and Methods ....................................................................................... 43
2.2.1 Materials ......................................................................................................43
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2.2.2 Polymer chemistry .......................................................................................44
2.2.3 Fabrication of samples for ex-situ permeability and pore size
measurements .............................................................................................................45
2.2.4 Measurement of permeability ......................................................................45
2.3 Results ................................................................................................................ 46
2.3.1 Review of general mechanisms and trends in monolith polymerization .....47
2.3.2 Control and optimization of wick porosity and pore structure ....................48
2.3.3 Designing high performance porous polymer wick material .......................52
2.3.4 Fabrication of resin negative mold ..............................................................61
2.3.5 Injection molding of wicks ..........................................................................61
2.3.6 Removal and cleaning ..................................................................................63
2.4 Conclusions and Recommendations................................................................... 64
2.5 Supplementary Information................................................................................ 65
2.5.1 Notes regarding permeability measurement setup .......................................67
3 Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective
Purification with High Column Utilization: Theory ......................................................... 68
3.1 Introduction ........................................................................................................ 68
3.2 Concept and theory............................................................................................. 72
3.2.1 Method concept ............................................................................................72
3.2.2 Transport and focusing of trace analytes in isotachophoresis .....................74
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