Microfluidic Microdroplet Reactors

Lead Research Organisation: University of Cambridge
Department Name: Chemistry

Abstract

We plan to generate a novel platform technology for experimental science, and demonstrate its utility by applying it to the identification of novel biological catalysts for chemical reactions. The key features of the technology will be its speed, scale and general utility. Individual reactions will take place inside microdroplets of water carried in a fluorocarbon continuous phase within microfluidic channels. These microreactors will be load up with the components for a reaction. Additional compounds can then be added by fusing a droplet with a second droplet. Once a reaction has occurred in a droplet the presence of product will be determined either spectroscopically or by mass spectrometry. Mass spectrometry has the advantage that it can provide high resolution structural information. However interfacing it with microdroplets is an unexplored and very challenging part of the proposal. In this way new catalysts will be identified which can then be improved by taking them through several rounds of selection.A modular device will be assembled that load, fuse, incubate sort and split droplets and present then to either a spectroscopic or mass spectrometric screen. The fabrication of this device, and especially the integration of the various components is a significant challenge, and will be the main focus of the first part of the project.We plan to use this modular device for the discovery of novel biological catalysts. These will either be enzymes that incorporate new functionality, or enzymes from unusual organisms. This is an example of an important project that will become more accessible using the system we develop than using than with existing approaches. We expect to get an efficiency enhancement of over 104. The project is multidisciplinary and at the forefront of several areas: the use of microdroplets, the use of microfluidics for lab-on-a-chip applications, the use of mass spectrometry, and the directed evolution of enzymes. It will be possible to customise the devices we generate to tackle a broad range of problems in biological, chemical and materials science.

Publications


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Casadevall I Solvas X (2011) Fluorescence detection methods for microfluidic droplet platforms. in Journal of visualized experiments : JoVE
Casadevall I Solvas X (2011) Droplet microfluidics: recent developments and future applications. in Chemical communications (Cambridge, England)
Casadevall I Solvas X (2011) High-throughput age synchronisation of Caenorhabditis elegans. in Chemical communications (Cambridge, England)
Casadevall I Solvas X (2012) Microfluidic evaporator for on-chip sample concentration. in Lab on a chip
 
Description The Basic Technology funded complementary research into microdroplets technology at Cambridge and Imperial. At Imperial a focus was to create and define sophisticated optical techniques for droplet interrogation, create functional components for droplet manipulation and to integrate these activities with a range of biological programmes at Cambridge. At Cambridge there was additionally a major effort to integrate microfluidics with mass spectrometry.

We have successfully fabricated microfluidic devices to manipulate individual droplets on-chip. Devices have been fabricated that create droplets with frequencies up to 20 kHz, with volumes ranging from 20 fL to several nL. These droplets can be fused passively using surface chemistry, or actively with strong electric fields generated by microfabricated electrodes. Fluorescence intensity detection is used to study the contents of droplets at high frequencies, allow droplet-based enzyme assays and high-throughput screening. Sub-nM sensitivity is achieved routinely, and single molecule sensitivity has been demonstrated. Electric fields have also been used to sort droplets at kHz rates, where the

fields are triggered by fluorescence signals generated via laser-induced fluorescence.

We have demonstrated DNA amplification, in vitro transcription translation (IVTT) followed by a fluorescent assay of the expressed enzyme, developing all the components required for protein evolution experiments. However our achievements in biology have gone much further as we have become increasingly aware of the potential of microdroplets for cell-based studies. The key achievements (by area) include:

FABRICATION We have pioneered numerous innovations in microdroplet device design including: novel methods to controllably merge droplets based on device architecture (pillar arrays) or surface modification; and the development of a sorter/de-emulsifier that feeds droplets contents into any analytical device. We have devised novel approaches for droplet-based dilution for high-throughput screening applications that are now being implemented into an automated instrument. We have also developed emulsion formulations that stop (99%) leaking from droplets.

DETECTION We were the first to demonstrate direct mass spectrometry of individual droplets. Other firsts include: demonstration of fluorescence lifetime imaging, enhanced Raman spectroscopy, and FTIR of microdroplet flows; demonstration of refractive index variation, and single molecule detection in picolitre-volume droplets. We have combined the analytical capability with sorting to develop droplet-based microfluidics as a high efficiency tool in 2D proteomics.

SCREENING We have defined multiple formats for cell-based and enzymatic assays in droplet arrays and in movingdroplets analysed in flow. We showed that continuous microdroplet flows can be used to perform high-throughput DNA binding assays and to probe protein-protein interactions. We were the first to demonstrate that picolitre-volume droplets can be used for single cell experiments, and developed a method for rapid compartmentalisation of eukaryotic cells. We developed a device to follow up to 4000 cell based assays simultaneously over several days, constantly monitoring

several different parameters.

BIOLOGY We demonstrated droplet-based continuous flow PCR with an efficiency similar to benchtop PCR, and droplet IVTT from a single copy of a gene. Subsequently we carried out DNA amplification followed by IVTT in the same droplet. A protocol for directed evolution of a hydrolase that consisted of cell lysis at droplet formation, incubation with a substrate and sorting after optical interrogation for product appearance was developed and its utility shown by determination of the enrichment in a full evolution cycle. We have initiated studies to use microdroplets to study quorum sensing in bacteria, and for algal strain selection for biofuel development.
Exploitation Route The research has applications in all aspects of experimental science, most clearly those relating to molecular and cell biology. The will be of interest to companies involved in biopharmaceuticals, antibody production, assay development, and diagnostics. This research provides the basis for a new platform technology for experimental science with applications across biology, cell biology, biochemistry and molecular biology, into chemistry and material science. Several patents have been filed and the ground work done for setting up for potentially establishing a spinout company.

UPDATE In 2011 a spin out was created Sphere Fluidics
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
 
Description They stimulated further research which subsequently led to several spinouts
First Year Of Impact 2010
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description BBSRC Studentship
Amount £70,000 (GBP)
Funding ID BB/I016589/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2011 
End 09/2015
 
Description ERC Advanced Grant
Amount £1,890,000 (GBP)
Funding ID 246812 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 04/2010 
End 03/2015
 
Description Follow-on-fund (from Impact Acceleration Account)
Amount £59,814 (GBP)
Funding ID EP/K503757/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2013 
End 10/2014
 
Description Follow-on-fund (from Impact Acceleration Account)
Amount £36,486 (GBP)
Funding ID EP/K503757/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2013 
End 10/2013
 
Description MC-IIF - International Incoming Fellowships (IIF)
Amount € 174,240 (EUR)
Funding ID 255500 
Organisation European Commission (EC) 
Sector Public
Country European Union (EU)
Start 06/2010 
End 05/2012
 
Description Partnership Development Award (from Impact Acceleration Account)
Amount £49,963 (GBP)
Funding ID EP/K503757/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 06/2014 
End 06/2015
 
Description Standard Research
Amount £505,226 (GBP)
Funding ID EP/I013342/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2011 
End 09/2015
 
Description Standard Research
Amount £1,067,243 (GBP)
Funding ID EP/H046593/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2010 
End 04/2014
 
Description Wellcome Trust, The
Amount £1,508,112 (GBP)
Organisation The Wellcome Trust Ltd 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2011 
End 03/2014
 
Title IONISATION MASS SPECTROMETRY 
Description Systems that employ microdroplets are used in embodiments for Microdroplet Electrospray Ionisation Mass Spectrometry (ESI MS). Thus, a method of detecting an analyte includes providing an oil composition comprising oil and an aqueous microdroplet comprising the analyte, the oil composition comprising a surfactant to stabilise the aqueous microdroplet in the oil composition; and performing ionisation mass spectrometry analysis of the oil composition. 
IP Reference US2013187040 
Protection Patent application published
Year Protection Granted 2010
Licensed Yes
Impact None
 
Title MICROFLUIDIC DEVICES 
Description We describe a method of layer-by-layer deposition of a plurality of layers of material onto the wall or walls of a channel of a microfluidic device, the method comprising: loading a tube with a series of segments of solution, a said segment of solution bearing a material to be deposited; coupling said tube to said microfluidic device; and injecting said segments of solution into said microfluidic device such that said segments of solution pass, in turn, through said channel depositing successive layers of material to perform said layer-by-layer deposition onto said wall or walls of said channel. Embodiments of the methods are particularly useful for automated surface modification of plastic, for example PDMS (Poly(dimethylsiloxane)), microchannels. We also describe methods and apparatus for forming double-emulsions. 
IP Reference US2012168010 
Protection Patent application published
Year Protection Granted 2009
Licensed Yes
Impact None
 
Title MICROFLUIDIC SYSTEMS 
Description This invention relates to microfluidic systems and more particularly to methods and apparatus for accessing the contents of micro droplets (114) in an emulsion stream. A method of accessing the contents of a droplet (114) of an emulsion in a microfluidic system, the method comprising: flowing the emulsion alongside a continuous, non- emulsive stream of second fluid (118) to provide an interface (120) between said emulsion and said stream of second fluid (118); and in embodiments applying one or both of an electric (112a, 112b) and magnetic field across said interface (120) to alter a trajectory of a said droplet (114) of said emulsion to cause said droplet to coalesce with said stream of second fluid (118); and accessing said contents of said droplet (114) in said second stream (118). 
IP Reference US2012091004 
Protection Patent application published
Year Protection Granted 2007
Licensed Yes
Impact None
 
Title Microfluidic device for removing oil from oil separated aqueous sample droplets 
Description The device comprises a sample channel 11 with an oil outlet channel and an aqueous flow outlet channel. The dimension of the oil outlet channel opening is such as to prevent water from passing through it. The micro-fluidic device also includes an encapsulation device 1 for separating portions of a pre- separated aqueous analyte stream 4 passing along a conduit using oil 2. The oil separated droplets 12 being fed into the aforementioned device, with the aqueous material 14 being fed into a further separation device 21. The analyte 4 is preferably peptide (protein), nucleic acid (DNA, RNA), amino acid or cells. Preferred pre- and post-separation devices 21 include high pressure liquid chromatography (HPLC), liquid chromatography (LC), isoelectric focussing, capillary electrophoresis (CE), capillary gel electrophoresis (CGE), isotachoporesis or micellar electrokenetic chromatography, coupled with ultraviolet (UV), fluorescence, phosphorescence, staining, radioactivity, refractive index or vibrational infrared (IR) spectroscopy detector. 
IP Reference GB2474228 
Protection Patent application published
Year Protection Granted 2009
Licensed No
Impact None
 
Company Name Sphere Fluidics Limited 
Description Sphere Fluidics Limited is an established Life Sciences company which has developed unique products for use in single cell analysis and characterisation and provides collaborative Research and Development services in this area. 
Year Established 2010 
Impact Sphere Fluidics is one of the very few teams in the world with expertise in single cell analysis and pico droplet technology. The company has leading-edge, patented technology and proprietary know-how enables analysis and detection of single cells and their biomolecules. Sphere Fluidics has extensive management experience in successfully commercialising innovative science and engineering. Its expertise in new science with key applications has particular impact for novel biopharmaceutical and biosimilar discovery. Sphere Fluidics' novel process for single cell analysis and characterisation provides significant saving of time, resource and money compared to conventional workflows.
Website http://www.spherefluidics.eu/index.php
 
Description Droplet Reactors with Catalytic Interfaces: An Active Fluorous Phase for Segmented-Flow Microfluidic Reactions 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Shared research output with other research scientists in the field

Interest in our research at the Microdroplets group in the Univesity of Cambridge, UK
Year(s) Of Engagement Activity 2009