Droplet-based microfluidics for single cell-omics

Lead Research Organisation: University of Glasgow
Department Name: School of Engineering

Abstract

Water will readily form droplets in oil, providing miniature enclosed environments, in which the aqueous (water phase) is separate from the oil phase. If these droplets are created in very small channels (the width of a human hair, for instance), then the size of the droplets can be comparable to that of individual single cells. By using the techniques of microfabrication, first established within the semi-conductor industry, we are able to create appropriate microchannel architectures that can create reproducible droplets that contain cells (the droplets behave like very small test tubes). However, to date, attempts to make such droplets have proved difficult. Currently they are unstable and will coalesce and disappear as rapidly as they form. Using a new technology, we propose to use the same two phase (oil and water) systems to create droplets with a structured coat, so that once the cells have been placed within the droplet, the micro-environment is stable and long lived. The techniques used for making such droplets will enable us to change the nature of the coating so that they can be considered 'intelligent'. In other words, by engineering the chemical composition of the coating, we can decide which molecules enter the micro-droplet. Intriguingly, we can also introduce nanometre scale sensors within the droplet wall that can report upon the metabolic state of the cell. Finally, we will create arrays of such structured droplets so that many single cells can be stored and analysed readily, and ultimately used in the process of finding and testing new medicines.

Technical Summary

Many methods have previously been used to create coated droplets on both a macro- and micro-scale, including polymer gelation at the surface of water droplets and the deposition of charged particles at water oil interfaces. In contrast to these existing methods, we propose to use the generation of a water-in-oil emulsion followed by the adsorption of colloidal particles at the droplet interface, to provide a novel method by which stabilised droplets, named 'colloidosomes', can be created. To date these capsules have not been produced within a microfluidic lab-on-a-chip device, nor have they been used to manipulate the environment around cells. We propose to create families of different capsule 'shells' that will be biocompatible (i.e. not only non-toxic to cells, but able to modulate cell behaviour). We also wish to be able to control the both gas and analyte permeability, enabling us to control the delivery of nutrients, ions, gases and drugs from the bulk media. This will provide the ability to stabilise the cell's physiology with the possibility of influencing its metabolism, through drug intervention, for example. Finally we wish to create microdroplet membrane interfaces containing optically active nanostructures. This will enable quantitative and extremely sensitive molecular fingerprinting of metabolites profiles of single cells using surface enhance Raman spectrsocopies (SERS). We will develop a platform that enables long-term and sensitive drug, proteomic and metabolic measurements of single cells, on-chip. There is also the potential for investigating cell-cell interactions. The colloidsomes will be sedimented on chemically or physically modified chips with the potential to create single cell microarrays for performing long-term assays in microfluidic systems. The inert and stable nature of colloidosomes also makes them ideal for a range of other future applications relevant to the biotechnology and bio-pharma industries.

Publications

10 25 50

publication icon
Zagnoni M (2010) Hysteresis in multiphase microfluidics at a T-junction. in Langmuir : the ACS journal of surfaces and colloids

publication icon
Zagnoni M (2009) Electrically initiated upstream coalescence cascade of droplets in a microfluidic flow. in Physical review. E, Statistical, nonlinear, and soft matter physics

publication icon
Zagnoni M (2010) Electrocoalescence mechanisms of microdroplets using localized electric fields in microfluidic channels. in Langmuir : the ACS journal of surfaces and colloids

publication icon
Zagnoni M (2010) A microdroplet-based shift register. in Lab on a chip

 
Description Microfluidic device structures were designed, fabricated and tested to exploit novel analytical procedures using stable
water-in-oil droplets on-a-chip. These techniques were successfully used to create bioreactors in which chemical and
biological assays were performed:

(i) A detailed experimental analysis was carried out to understand and use droplet electrocoalescence as a means to
perform dilution and mixing of substances within emulsions (leading to the publication of 3 peer-reviewed journal papers in Langmuir, Lab Chip and Phys Rev E).
(ii) A novel strategy was developed to carry out a microfluidic, droplet-based immunoassays for the analysis of intracellular
proteins using HEK-293 cells and MCF-7 cells. Cells were introduced into the device in suspension and were electrically
lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water
in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently, quantifying the level of two intracellular proteins (HRas-mCitrine and GFP-actin). This work was carried out following
collaboration with the group of Dr Andrew Pitt at Glasgow (The work led to the publication of a peer-reviewed journal paper in Analytical Chemistry).
(iii) Encapsulation of single cells into droplets was also successfully achieved. Experiments with cell-loaded colloids,
encapsulated into droplets, for SERS and SERRS analysis was also performed. However, preliminary experiments
produced insufficient results for publication, as the emulsion provided large background spectra. This work is currently
being continued by Dr Christopher Syme, working on a Basic Technology grant.
(iv) Formation of double emulsion to create cell-like compartments was performed and we obtained encouraging results, which have been submitted to a high impact journal. By tailoring the membrane structure, and performing cell free
expression of a protein within the structure, we have shown we can assemble functional membrane proteins at the droplet
interface. We term this a "protocell" providing a first step towards a synthetic cell.
(v) We have further developed the protocell concept by creating polymerosomes, improving the functionality of the
membrane in the droplet based cell structure. This has enabled us to include glycosolated polymers into the membrane which we will use to anchor proteins involved in cellular microstructure.

In summary:
Synthetic biology is an emerging field of science that offers the prospect of the design and construction of new biological systems that do not exist in nature. In one aspect of this field, there is a need to create artificial cell surrogates, or
"protocells", which enable biochemical pathways to be reconstructed, to perform complex processing steps. We have
shown that by manipulating oil and water in tiny channels, a subject known as microfluidics, we can create emulsions of
regular microdrops, which look and behave like cells. These emulsions are not dissimilar to mayonnaise as they comprise a
mixture of water dispersed in oil.
Next, we took this basic technological development and by using more complex networks of channels, we made more
complicated dispersions of droplets. Importantly, we showed we can make structures which were not dissimilar from cells
(we called them protocells). The protocells are made by controlling the flow rates of the flow of the water (W) relative to an oil (O). As the aqueous flow moves through the chip, it is segmented by a cross flow of oil which encases it within an
aqueous environment (giving a micordrop or emulsion, described as W/O/W). The thin oil layer behaves as a membrane that separates the inner aqueous phase and the outer aqueous phase, just like a natural cell.
In the next phase of the work, we introduced specific sequences of DNA and precursor biochemicals into the aqueous input channel. These components become entrapped within the oil coated droplets. Within moments of the protocell being
formed, the DNA starts to synthesise (or "express") a green coloured protein encoded in its sequence. This demonstrated
one of the fundamental characteristics of living cells, within our artificial cells, namely protein expression.
We then introduced a second sequence of DNA within the same protocells, which produces a red coloured protein. We
showed that in these artificial cells this red protein accumulates in the oily membrane, whereas the green protein stays in the watery middle. This is as would be expected from their behaviour in nature.
Although, on one level, the creation of artificial life may appear a little ominous, in future, this technology may provide
cheaper chemicals, cleaner energy and new medicines. It will almost certainly reduce the burden on animals in the process
of testing new medicines and cosmetics. For those concerned by the nature of the technology, it is helpful to remember
that these protocells are not dissimilar in their structure from mayonnaise.
Exploitation Route See above, and Unilever have shown interest in funding the microdroplets work and we anticipate some level of support in the near future.
We are in an ongoing discussion with Malvern instruments about developing new methods to measure the viscoelastic
properties of biological membranes and they are interested in using our 'bottom up' approach for producing model 'cells'
(protocells) as part of this collaboration.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Unilever have shown interest in funding the microdroplets work and we anticipate some level of support in the near future. We are in an ongoing discussion with Malvern instruments about developing new methods to measure the viscoelastic properties of biological membranes and they are interested in using our 'bottom up' approach for producing model 'cells' (protocells) as part of this collaboration. The PI has been involved in setting up an undergraduate degree in Glasgow, in Biomedical Engineering (the first in Scotland). As such he has made extensive visits to many Schools, primarily in the greater Glasgow area, but extending into Strathclyde region. In these visits he has discussed the topic of controlling microdrops in areas as diverse as the food industry and synthetic biology, the former being something familiar to a general audience and the latter being been central to much of the work performed in this grant. Together, these illustrate the links between engineering, biochemical assays and biology in a manner that is readily accessible. This link is of course, central to the subject of Biomedical Engineering and in interesting school children to take a subject that crosses disciplines. Similar presentations have been made at open days and at one public understanding of science festival, in Edinburgh (2010).
First Year Of Impact 2010
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description EPSRC Frontiers Engineering grant
Amount £5,100,000 (GBP)
Funding ID EP/K038885/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2018
 
Description Engagement associated with BB/F005024/1 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The PI has been involved in setting up an undergraduate degree in Glasgow, in Biomedical Engineering (the first in
Scotland). As such he has made extensive visits to many Schools, primarily in the greater Glasgow area, but extending into
Strathclyde region. In these visits he has discussed the topic of controlling microdrops in areas as diverse as the food
industry and synthetic biology, the former being something familiar to a general audience and the latter being been central
to much of the work performed in this grant. Together, these illustrate the links between engineering, biochemical assays
and biology in a manner that is readily accessible. This link is of course, central to the subject of Biomedical Engineering
and in interesting school children to take a subject that crosses disciplines. Similar presentations have been made at open
days and at one public understanding of science festival, in Edinburgh (2010).
Year(s) Of Engagement Activity 2010,2011,2012