Electric field driven microfluidics for acceleration of CRISPR enzyme kinetics and its application in molecular diagnostics
Abstract/Contents
- Abstract
- Rapid and early-stage screening is crucial, especially during pandemics, for early identification of infected persons and control of disease spread. CRISPR biology offers new and effective methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps — factors which prolong the process and impede use in low-resource settings. Moreover, the sensitivity and assay time for CRISPR-based approaches are fundamentally limited by the enzyme kinetic rates. In this experimental and theoretical work, we combine microfluidics, on-chip electric field control, and CRISPR to directly address limitations of current CRISPR diagnostic methods. We find that electric field gradients can be used to control and accelerate CRISPR reactions by co-focusing Cas--gRNA, reporters, and target nucleic acids within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. In this thesis, we first develop and validate a model based on Michaelis-Menten enzyme kinetics theory and tailor it to regimes common in conventional CRISPR diagnostics. We map out an estimate of the limits of detection and achievable assay speeds of existing CRISPR-based diagnostics given current known and consistent (i.e., physically possible) kinetics rate data. We then develop a kinetics model which couples ITP physics with CRISPR reactions. We experimentally validate the model and identify key regimes for product formation and reaction acceleration in ITP. We then apply our ITP-CRISPR method to the rapid (30 min) detection of the RNA of SARS-CoV-2, the virus that causes COVID-19, starting from raw nasopharyngeal swab samples. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw samples. This electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays.
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 | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Ramachandran, Ashwin |
|---|---|
| Degree supervisor | Santiago, Juan G |
| Thesis advisor | Santiago, Juan G |
| Thesis advisor | Fordyce, Polly |
| Thesis advisor | Lele, Sanjiva K. (Sanjiva Keshava), 1958- |
| Degree committee member | Fordyce, Polly |
| Degree committee member | Lele, Sanjiva K. (Sanjiva Keshava), 1958- |
| Associated with | Stanford University, Department of Aeronautics and Astronautics |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Ashwin Ramachandran. |
|---|---|
| Note | Submitted to the Department of Aeronautics and Astronautics. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/kx045dc2211 |
Access conditions
- Copyright
- © 2021 by Ashwin Ramachandran
- License
- This work is licensed under a Creative Commons Attribution 3.0 Unported license (CC BY).
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