Droplet-based microfluidic platforms for protein investigations

Martino, Chiara (2013) Droplet-based microfluidic platforms for protein investigations. PhD thesis, University of Glasgow.

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Abstract

In the last two decades, the integration of life science and micro-engineering has developed systems which are able to perform laboratory functions on devices only 10-100 μm in size. These microfluidic systems, called lab-on-chip (LOC), show promising capabilities in reducing both the time and the cost of a wide range of chemical and biological processes. More recently, the creation of microfluidic systems which are able to form and control sub-nanolitre droplets, comprising two phase emulsions, have been developed to deliver new experimental platforms. Such systems, also known microdroplets or segmented flow platforms, consist of stable liquid droplets, suspended in a second immiscible phase, with volumes on the nanolitre to the femtolitre scale. The potential of these systems within chemical and biological sciences has already been clearly demonstrated in the literature and commercial platforms are now becoming available. Some of the appealing features that these systems allow are the compartmentalization, the ultra-high throughput experimentation, the imitation of cellular conditions in terms of volumes and chemical composition. The aim of this research project is to exploit the droplet-based LOC systems using the two phase segmented flow to create platforms where proteins can be investigated and even expressed within the droplet chassis. Initial work used a single emulsion strategy (i.e. droplet of water in oil) to selectively capture cellular proteins from a cell suspension, which was directly processed on chip. It was observed that proteins remain active in these systems. In addition, a complexity of conventional laboratory procedures for protein quantification assays was reduced, both in terms of investigation times and amounts of valuable biological samples used. The obtained results demonstrated that this system has the potential to provide the same level of quantitative information obtained using standard biological techniques (i.e. Western blot) at a lower cost. The research has been moving over the development of artificial cell models, nanolitre sized watery droplets comprising a membrane separating the inner from the external environment. Within these systems, realised using a double emulsion strategy (i.e. droplet of water with a droplet of oil or other immiscible phase surrounded by another watery phase), proteins have been expressed using cell-free protein expression systems. The technology adopted lies broadly within the field of synthetic biology involving the transformation of microorganism’s DNA for the production of the desired proteins. Fluorescent proteins were designed and expressed within the artificial cell and fluorescence assays, implemented within the microfluidic format, confirmed not only the functionality of the expressed protein which behaved like in vivo, but also the possibility of its controlled release. Protein release was possible through the use of polymers, within the double emulsion format, which represent a good class of material for the production of nanometre thickness shells. Future developments in this research will aim to (i) expand the capabilities of the single emulsion system for the capture of multiple proteins from a cell lysate and (ii) use different class of polymers to enrich the artificial cell membrane with membrane proteins aiming towards the natural cell mimicking. Emulsions capable to mimic some aspects of the living cell, like the protein synthesis, represent a great opportunity to be used as tools for protein investigations.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: microfluidics, microdroplet, emulsion, artificial cell, synthetic biology, DNA, cell-free protein expression
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Science and Engineering > School of Engineering > Biomedical Engineering
Funder's Name: UNSPECIFIED
Supervisor's Name: Jonathan, Professor Cooper
Date of Award: 2013
Depositing User: Miss Chiara Martino
Unique ID: glathesis:2013-4005
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Feb 2013 09:52
Last Modified: 19 Feb 2013 09:55
URI: http://theses.gla.ac.uk/id/eprint/4005

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