Rapid affinity purification based on ion concentration shock waves and porous polymer monoliths
- 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 m^2. 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. 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 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.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Stanford University, Department of Mechanical Engineering.
|Santiago, Juan G
|Santiago, Juan G
|Goodson, Kenneth E, 1967-
|Mani, Ali, (Professor of mechanical engineering)
|Goodson, Kenneth E, 1967-
|Statement of responsibility
|Submitted to the Department of Mechanical Engineering.
|Thesis (Ph.D.)--Stanford University, 2015.
- © 2015 by Viktor Shkolnikov
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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