New detection modes for droplet microfluidics

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.

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.

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).