Spatiotemporal Control of Chemical Reaction Networks using Droplet Microfluidics

A number of cellular organisms, such as yeast, bacteria and slime moulds, exhibit dynamic behaviour, in particular switching and rhythms that are controlled by feedback mechanisms in enzyme-catalysed reactions. The mechanisms of these processes are well understood, and recently there has been a focus on generating similar reactions in synthetic biocatalytic systems to establish bioinspired analogues for applications in materials and medicine. In this context, compartmentalisation of biochemical reactions within synthetic cell models such as micelles, vesicles, and W/O/W-based double emulsions is attracting growing attention for applications in the field of therapeutics. In this respect, it is necessary to adopt easier-to-use stimuli-responsive (react to pH, temperature or light) biochemical reactions, to apply artificial cell models to the biomedical context, and regulate artificial cell communication in a spatiotemporal controlled way. As a first step, it is crucial to control the output of a chemical reaction that maybe exploited for applications in the field of programmable materials and biomedicine. Droplet emulsion and synthetic vesicle systems have been widely employed as bioinspired micro- or nanoreactors for production of materials such as hydrogels and ceramic particles. They also provide test platform for biomimetic cell like behaviour.

To address this, we have developed and fine-tuned a platform with synthetic bottom-up chemistry that has enabled us to systematically and thoroughly investigate the effects of entrapment on a feedback-driven enzymatic reaction. As a result of this process, we have revealed a system that is more intricate than originally thought. Firstly, taking advantage from pressure driven droplet microfluidics, we developed a system of enzyme-encapsulated (urea-urease) double emulsion (W/O/W) droplets to obtain a localised pH pulse, with a controllable induction time to program material properties. The urease-catalysed hydrolysis of urea (urea-urea reaction), has a feedback through the production of the base (NH3). This leads to a change from an acidic to a basic pH after an induction time (Tind), resulting in an environment with auto-changing pH conditions. Reaction was initiated by addition of urea and a pulse in base (ammonia) was observed in the droplets after a time lag of the order of minutes. The pH-time profile can be manipulated by the diffusion timescale of urea and ammonia through the oil layer, resulting in localised pH changes not accessible in bulk solutions.

Secondly, we performed a computational investigation of the nonlinear reaction chemistry (urea-urease) within the designed platform of the W/O/W-based reactor. A radially distributed reaction diffusion model is presented for a layered sphere mimicking a double emulsion. Here we have combined the experiments with simulations (shell-core model) to demonstrate the influence of urea transport triggered by the shell, the core and the external solution surrounding the cell model (µ-reactor) on the induction time/period (Tind) of urea-urease reaction.

Third, inspired from natural cellular systems (e.g. bacterial quorum sensing), we focus on the use of urea-urease reaction confined to double emulsions to investigate chemical communications. We observed a system that resulted in a system of microreactors acting as individual units with distinct induction periods (Tind) for the first time. We show that in contrast to other systems, the release of ammonia can accelerate the reaction in all the droplets but there is no evident synchronisation of activity characterised by a wide distribution of induction times across the population of micro-reactors. However, the investigation of behaviour of population/group of µ-reactors as a function of substrate urea concentration and the density of µ-reactors highlights the possibility of transitions to collective behaviours.

Finally, we aimed to use the double emulsion template for potential biomedical and therapeutic applications using the autocatalytic urea-urease reaction. We used the platform to produce thiol-acrylate gels in the form of double emulsion loaded gel films and spherical microcapsules for potential drug delivery applications. In addition, we employed the encapsulated double emulsion platform of the enzyme urease to study the inhibition of the enzyme itself; which is important in the development of anti-microbials for ureolytic bacteria.

By building this platform, we have not only learned how to control the kinetic output of the reaction (urea-urease), but have also demonstrated its potential in future applications.