Droplet-based separation tools for multidimensional biological separations

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Title: Droplet-based separation tools for multidimensional biological separations
Authors: Thamsumet, Nuchutha
Item Type: Thesis or dissertation
Abstract: Proteins have been extensively studied over the last decade as comprehensive understanding of the proteome can definitely lead to the discovery of novel biomarkers, early-stage disease diagnoses and the development of diagnostic tools and novel drug therapies. One of the crucial and fundamental processes in protein analysis is protein separation, which is usually performed as multidimensional separations to achieve high resolution and high peak capacity. However, high performance analyses are difficult to achieve due to the challenges involved in efficiently integrating different dimensions. In this work, we present the development of a microfluidic device for the effective transfer of protein droplets into the second separation dimension. Consequently, the device provides a stable, reproducible, easy to operate, portable and flexible system to connect a first dimension separation to the downstream second dimension analysis via droplets. The droplets act to preserve the resolution during transfer between separation techniques. In summary, a fluorescently labeled protein ladder serving as a representative of proteins separated from the first dimension is compartmentalized into droplets using the robotic droplet generator. These protein droplets are then transferred via the interfacing microdevice into the second dimension where the released proteins are further separated using capillary gel electrophoresis. Herein, several designs of interfacing microdevices were evaluated for the successful transfer of droplet contents (droplet injection) into the second dimension. The buffer for capillary gel electrophoresis was developed to achieve high-speed and high-resolution separations of proteins in droplet-based injection format. Several fluorescent dyes were also examined for protein labeling to achieve high fluorescent intensities necessary when using this droplet format. Successful droplet-based separation of proteins necessitates the seamless integration of all the developed components. This has been demonstrated here. This interface automates the oil depletion process, minimizes dead volume, prevents dispersion of analyte bands and reduces sample loss at the interface between separation dimensions. Furthermore, optimization of the entire system used in conjunction with the interfacing microdevice provided for ease of operation and more efficient droplet injections. Moreover, droplet injection into parallel separation channels was achieved, highlighting the interfaces capacity for high-throughput analyses.
Content Version: Open Access
Issue Date: Oct-2015
Date Awarded: Jun-2016
URI: http://hdl.handle.net/10044/1/33779
DOI: https://doi.org/10.25560/33779
Supervisor: deMello, Prof. Andrew
deMello, Prof. John
Sponsor/Funder: Thailand
Department: Chemistry
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Chemistry PhD theses



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