New detection modes for droplet microfluidics

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry


This project is based on the idea that the classical test tube in experimental biology can be replaced by miniature water-in-oil droplets. The droplet is the equivalent of an ultra-small test tube. Droplet volumes are typically in the femto- to nanolitre range. Furthermore, up to 10e10 droplet reactors fit into a millilitre-sized tube. Importantly, an equivalent number of experiments can be carried out simultaneously. The key idea is that the droplet compartment combines the functional molecule with information on its identity and a readout of its function. Thus the droplet contains everything needed to assess and decode a particular experiment or profile a library member. This system is ideally suited for directed evolution, because genotype and phenotype are combined in the droplet. We have recently performed directed protein evolution in this format (Kintses, B. et al & Hollfelder, F. Chemistry & Biology 2012, 19(8):1001-9) and now propose to add another detection mode for assays that are based on generation of a chromophore, to allow a wider range of biological functions to be screened.

Technical Summary

The toolbox of unit operations for droplet microfluidics consists of modules can be integrated like jigsaw pieces to suit the requirements of specific biological experiments. A large number of these unit operations have been established in the Cambridge group and elsewhere, but one detection mode familiar in the large scale lab has not been miniaturized. The objective of this proposal is to add an alternative module for absorbance detection to this toolkit (in addition to the current detector that requires a fluorescent signal). Integration with a droplet sorter will allow directed evolution based on many more assays than currently possible. This is important as many reaction for which catalysts are sought do not involve fluorophores. Therefore, optimization is carried out in a way that may bioas the outcome of directed evolution away from the desired target reaction according to the adage "you get what you select for". For example, by creating space for the bulky fluorogenic group that may not be required or even detrimental for turnover of the desired substrate directed evolution is not quite as directed as one would like it to be. The availability of a droplet evolution system based on absorbance will significantly expand the possibilities for a wider range of assays in directed evolution.

Planned Impact

In the area of high throughput screening, microdroplet technology has the potential to improve on existing microtiter plate-based approaches by up to six orders of magnitude: smaller (from ml to pl), cheaper (from 1$ per assay to <0.01$) and with less sample (from 1000 cells to a single cell). The beneficiaries of the proposed research will be individuals and organisations interested in a transformational platform in directed protein evolution (but also, by extension, for experimental high-throughput science in general).
Description We have built an analytical interface that monitors processes in extremely miniaturised reaction vessels (droplets with picolitre volumes) by absorbance changes. We showed the utility of this device by performing selections for enzymes with improved activities. In this way large collections of enzyme variants (up to 10e6 per day) can be analysed. This facility will be used in protein engineering by 'directed evolution', a process in which randomised enzyme mutants are improved in iterative Darwinian selections.
Exploitation Route The ability to perform absorbance detection in microdroplets enables many new assays of enzymes for use in biotechnology. We are collaborating with two industrial partners to apply this technology to directed evolution of enzymes for green manufacturing.
Sectors Chemicals,Energy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Darwinian evolution of enzyme relies on the ability to test millions of enzyme variants for their ability to make useful products. One of the most efficient ways of testing many variants is to compartmentalise them in picolitre droplets. We have established a new way of product detection in microdroplets (by absorbance) and are now using this new mode of product detection with two industrial partners to improvement of enzymes for 'green' and efficient manufacturing of pharmaceutical reagents.
Sector Chemicals,Energy,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Title DropBase: repository of device designs for handling moicrofluidic droplets 
Description DropBase is a collection of microfluidic droplet device designs that are free to download and use. We are making these designs freely available as a service to the microdroplet research community. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Positive direct feedback from user community 
Description Collaborative project 
Organisation Johnson Matthey
Country United Kingdom 
Sector Private 
PI Contribution The absorbance detector developed in this grant was used for ultrahogh-throughput screening of enzymes.
Collaborator Contribution Exertise, reagents, molecular modelling.
Impact Multi-disciplinary: biocatalysis (new enzymes), microengineering (a microfluidic sorting module), technology (assay development)
Start Year 2015
Description The invention relates to a microfluidic system for generating droplets, the controlled merging of two or more droplets, and the analysis of droplets. 
IP Reference WO2004087308 
Protection Patent application published
Year Protection Granted 2014
Licensed Yes
Impact A droplet-on-demand platform was commercialised via Drop-Tech and Dolomite (marketed as the Mitos Dropix -