Dielectrophoretic single bead-droplet reactor : an approach towards high-fidelity solid-phase enzymatic DNA synthesis
Abstract/Contents
- Abstract
- Synthetic DNA is indispensible for research in synthetic biology with widespread applications spanning healthcare, environment, agriculture, energy, nanomaterials, and data storage. To fuel its fast-expanding frontiers a rapid augmentation in the accurate and low-cost synthesis capability of arbitrarily long strands of oligonucleotides (single stranded DNA) is necessary. However, after decades of advancement and optimization state-of-the-art column and microarray-based platforms are limited to the synthesis of oligonucleotides that are 300 bases long. Accumulated reaction errors over multiple synthesis cycles curtail the yield of longer oligonucleotide sequences. In microarrays, the misalignment of reagent drops or optical beams to the synthesis spots leads to substitution or deletion errors. In synthesis columns with many beads packed together, reaction fidelities are fundamentally limited by bead-bead stacking that leads to suboptimal bead surface-to-reagent ratio. Hence, drastically new physical approaches to implement solid-phase synthesis are required to overcome the shortcomings of current systems and open a robust avenue for the high-purity synthesis of ultra-long strands of oligonucleotides. My thesis work developed Single Bead-Droplet Reactor (SBDR) as a novel physical approach to synthesize on individual microbeads by dielectrophoretically encapsulating and ejecting them from reagent microdroplets. Dielectrophoretic force overcomes the interfacial tension of the droplet-medium interface to manipulate the microbead across it. Reactions on isolated beads can circumvent bead-bead stacking to provide an enhanced bead surface-to-reagent ratio for higher fidelity of reactions with reduced errors. I will begin the thesis by discussing about the current state-of-the-art solid-phase DNA synthesis approaches and their limitations. I will introduce SBDR as a potential solution to these limitations and highlight its novelties. I will describe the physical principle underlying the encapsulation and ejection process using electric field driven fluid flow analysis through a coupled solution of the Navier Stokes equation and electric charge conservation equation. A more intuitive explanation of the process is described in terms of the supply voltage driven change in the electrocapillary potential energy of the bead-droplet system in the silicone oil suspension medium. Subsequently, I will discuss the detailed fabrication of the silicon-on-glass microfluidic platform used to implement SBDR. I will provide insight into the choice of materials, device dimensions and fabrication process subject to the device design considerations. Then I will discuss the detailed experimental setup consisting of the sample mount, fluidic, electrical, and optical subsystems designed to appropriately control the device and record experimental observations. Following this, I will discuss the detailed sample preparation to ensure experimental repeatability. Thereafter, I will discuss the experimental demonstration of the encapsulation and ejection of individual beads from reagent droplets using the fabricated device and the experimental setup. I will highlight the dielectrophoretic trapping of bead, droplet generation and droplet trapping leading up to the encapsulation and ejection process. Using this process, I will demonstrate the enzymatic coupling of fluorescently labelled bases to the 3' end of the initiator strands bound to the microbead. I will emphasize the control experiment used to eliminate the contribution of non-specific binding of the fluorescently labelled base to the observed fluorescence emanating from the bead. Furthermore, using fluorescence intensity measurements, I will highlight the higher fidelity of the coupling reaction implemented using SBDR compared to benchtop setups with many beads packed together. This will open up a robust route for the high-purity, low-cost synthesis of ultra-long (> > 300 bases) strands of oligonucleotides. Finally, I will conclude my thesis by highlighting the major achievements of my work. I will also discuss the follow-up work needed to extend this proof-of-concept demonstration of SBDR into a robust, accurate, low-cost, massively parallel, and high-throughput on-chip solid-phase synthesis platform for the synthesis of arbitrarily long strands of oligos. I will also highlight the alternate research avenues that can be pursued using the basic demonstration of SBDR.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2022; ©2022 |
| Publication date | 2022; 2022 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Padhy, Punnag |
|---|---|
| Degree supervisor | Hesselink, Lambertus |
| Thesis advisor | Hesselink, Lambertus |
| Thesis advisor | Howe, Roger Thomas |
| Thesis advisor | Soh, H. Tom |
| Degree committee member | Howe, Roger Thomas |
| Degree committee member | Soh, H. Tom |
| Associated with | Stanford University, Department of Electrical Engineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Punnag Padhy. |
|---|---|
| Note | Submitted to the Department of Electrical Engineering. |
| Thesis | Thesis Ph.D. Stanford University 2022. |
| Location | https://purl.stanford.edu/hc160gz7899 |
Access conditions
- Copyright
- © 2022 by Punnag Padhy
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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