Enabling multivariate investigation of single-molecule dynamics in solution by counteracting Brownian motion
Abstract/Contents
- Abstract
- Although single-molecule fluorescence spectroscopy has developed into a mature toolkit that allows one to follow the dynamical behaviors of individual biomolecules in exquisite detail, its application in the solution phase is often limited by Brownian diffusion of the nanometer-sized biomolecules to only a 1-millisecond observation window. To remove this limitation while avoiding potential perturbation via immobilization, a device known as the Anti-Brownian ELectrokinetic (ABEL) trap has been developed to counteract Brownian motion that combines position-sensitive fluorescence detection and closed-loop feedback based on electrokinetic forces in a microfluidic geometry. This dissertation describes my work on recent methodology developments and new applications of the ABEL trap technology. First, we describe an optimal redesign of the feedback control strategy that operates close to the ultimate physical limits imposed by diffraction, shot-noise, photobleaching and information bandwidth provided by fluorescence emission. This advanced system greatly improves the capability to hold individual proteins and oligonucleotides in buffer solution and enables multivariate interrogation of single-molecule dynamics for as long as 30 seconds in a non-perturbative aqueous environment. Next, we combine multimodal fluorescence detection with the ABEL trap and illustrate its power using single organic fluorophores and a photosynthetic protein as examples. Direct observation of the synchronized dynamics of different fluorescence parameters reveals the internal states of a single nano-emitter and the interconversions between these states on a 1-second timescale in solution. Last, we show that by applying machine-learning techniques to the ABEL trap's photon data stream, a single molecule's diffusive and electric-field-induced motion parameters can be extracted in real-time during its residence in the trap. We demonstrate how this new measurement paradigm enables molecule-by-molecule sensing of size and charge heterogeneity and their fluctuations at the nanoscale. Two biophysical applications are illustrated: resolving the heterogeneous molecular mixture along a multimeric protein dissociation pathway and real-time visualization of the binding/unbinding interactions between single DNA strands.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2014 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Wang, Quan |
|---|---|
| Associated with | Stanford University, Department of Electrical Engineering. |
| Primary advisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Fan, Shanhui, 1972- |
| Thesis advisor | Solgaard, Olav |
| Advisor | Fan, Shanhui, 1972- |
| Advisor | Solgaard, Olav |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Quan Wang. |
|---|---|
| Note | Submitted to the Department of Electrical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2014. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2014 by Quan Wang
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Also listed in
Loading usage metrics...