Coupling isotachophoresis to reaction and separation assays
Abstract/Contents
- Abstract
- Molecular diagnostics are rapidly growing with applications in disease detection, genetic profiling, forensic investigation, and various research purposes. These techniques include PCR, sequencing, antibodies, and hybridization assays, and offer exquisite sensitivity and multiplexity. However, widespread adoption of molecular diagnostics is constrained by a number of key challenges. Assay times often take 16 hours or more, due to slow second order hybridization kinetics as well as time-consuming sample preparation methods. Furthermore, sample preparation itself is laborious, requiring manual intervention by highly-skilled personnel at several steps in the workflow. This dissertation discusses approaches that leverage isotachophoresis (ITP) and its coupling to at least one other assay step with an aim to significantly reduce analysis times and complexity of molecular detection assays. ITP is an electrokinetic technique which uses a heterogeneous buffer system to preconcentrate and separate ions based on their electrophoretic mobilities. In the first part of this dissertation, we present a simple analytical model to describe accumulation and reaction rates in ITP. This model is useful in the design of ITP experiments, and enables a user to make informed decisions regarding optimal sample placement in ITP assay design. In the second part of this dissertation, we describe a novel approach that leverages ITP to accelerate chemical reactions and also use an ionic spacer to separate reaction products. We first demonstrate this approach using synthetic DNA targets, and show high-sensitivity detection in a 10 min assay. We then extend this technique and use high-mobility probes to recruit typically non-focusing species (such as proteins) into ITP. We demonstrate this assay using C-reactive Protein (CRP) in buffer as well as in diluted serum. We also present a numerical and analytical model to describe ITP assays that involve non-focusing targets recruited into ITP. The last part of this dissertation shows how ITP purification can simplify sample preparation protocols and integrate with downstream analysis methods. We first show size-based RNA fractionation using ITP in a 10 min assay. We use a commercial electrophoresis system to analyze resulting sample fractions. Finally, we show how ITP purification can extend the applicability of recombinase polymerase amplification (RPA) to whole blood samples. We demonstrate lysis, purification, and detection of inactivated Listeria Monocytogenes cells from whole blood using ITP and RPA.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Eid, Charbel |
|---|---|
| Associated with | Stanford University, Department of Mechanical Engineering. |
| Primary advisor | Santiago, Juan G |
| Thesis advisor | Santiago, Juan G |
| Thesis advisor | Marshall, Lewis A, 1987- |
| Thesis advisor | Pruitt, Beth |
| Advisor | Marshall, Lewis A, 1987- |
| Advisor | Pruitt, Beth |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Charbel Eid. |
|---|---|
| Note | Submitted to the Department of Mechanical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Charbel Said Eid
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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