Periodic Reporting for period 1 - ENERGSYNTISSUE (Energy-generating synthetic tissues) Reporting period: 2018-07-01 to 2020-06-30 Summary of the context and overall objectives of the project Bridging the gap between biology and electronics has never been so crucial. Given the rapid progress in miniaturized electronics and the recent greater understanding in brain science, the seamless integration of electronics with living systems is poised to contribute to basic biology as well as to medical diagnostics and therapeutics. Despite the name 'bioelectronics', most devices in this area are built of rigid, dry electronic components and lack biological features. Therefore, the differences between biological tissues and man-made electronics lead to formidable obstacles in the deployment of bioelectronic devices. Important distinctions between biological tissues and man-made electronics are mechanical stiffness and the nature of the electricity generated by the two divergent systems. Native tissues consist of cells within an extracellular matrix (ECM). The nature of the cells and the features of the ECM differ between organs, producing the diversity of stiffness, topography and complexity. In addition, man-made electronics rely on electrons as carriers of information, while bioelectronic activities in the human body use ions as carriers. Therefore, bioelectronics must adopt the principles of biology so that devices can work synergistically with living tissues and organs. Synthetic tissues can be made from droplet networks comprising aqueous compartments separated by lipid bilayers. Among all synthetic materials, droplet networks offer the closest approximation to natural tissues, notably with comparable mechanical properties. Each droplet represents a simplified cell. Information can be exchanged between these compartments internally, and with the external environment. For example, by encapsulating enzymes within the compartments, synthetic cells can receive and process biological cues. In this project, we aim to combine the advances in synthetic cells based on droplet networks, and create tissue-like energy-generating electronics, in which electrical circuits and electronic functions are established within biological components. We seek to:I. construct stable droplet networks that allow long-term energy productionII. integrate enzymatic reactions in different compartments producing electrical output functionsIII. demonstrate the capability of tissue-like energy-generating electronics for biological applications Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The work conducted has concentrated on the following:I. the construction of energy-generating droplet networks II. the systematic optimization of the factors that impact energy generationIII. the replacement of the oil surrounding droplet networks by aqueous solutionsWe have constructed a tissue-like energy-generating droplet network. To attain this goal, we screened a library of lipids and polymers and achieved stable droplet networks with 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DphPC). A model system was constructed with an anode droplet and a cathode droplet. The oxidation of nicotinamide adenine dinucleotide (NADH) by NADH dehydrogenase in the anode droplet generated electrons, which were shuttled to a gold electrode by electron mediators, 9,10-Anthraquinone-2,6-disulfonic acid (AQDS). The electrons subsequently moved to the cathode droplet via the electrode to reduce oxygen. Meanwhile, to balance the charges, monovalent cations were transported across the lipid bilayer through gramicidin A (gA) channels. We have confirmed and assessed the generation of electricity using an amplifier. Unlike the 'bioelectronic' devices composed of non-natural components (e.g. elastomers), our energy-generating droplet networks are built of delicate biological molecules. Initially, electrical outputs that were high enough to rupture the lipid bilayer in 3 mins were achieved. In control experiments, we observed droplet coalescence under either a high voltage (> 0.73 V) generated between the gold electrodes, or a high density of charges accumulated on the sides of lipid bilayer as shown by the high capacitance current. To sustain a stable generation of electricity over a long period of time, we systematically varied the components and optimized the electrical output along with the droplet stability. In particular, we included the electron mediators, AQDS and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), in both anode and cathode droplets, respectively, to moderate the electrical potential. We found that the current was powered solely by the enzymatic oxidization of NADH in the anode droplet, which drove the oxygen reduction in the absence of a catalyst in the cathode droplet. This configuration resulted in a more stable droplet network for functional electrical output by preventing a charge overload on the lipid monolayers prior to the formation of droplet interface bilayers (DIBs). To move droplet networks formed in oil to aqueous solutions is challenging. Our preliminary results showed that the droplet-droplet interactions are not sufficiently stable to allow this transition. We, therefore, used the agarose hydrogel to stabilize the droplets from inside. The hydrogel-supported droplet networks preserved the intact structure during the transition from oil to water. This paved the way for further applications of energy-generating droplet networks under physiological conditions at the interface with living cells.The conclusions of the action:1. Tissue-like energy-generating droplet networks have been constructed.2. The components inside the droplet battery for sustainable electrical output and sufficient stability particularly have been optimized.3. Transferring droplet network from oil to aqueous solution has been achieved. 4. The electrical potential built from the droplet battery was capable to drive the directional movement of monovalent cations and membrane-permeable pyronin Y across the droplet network. 5. The possibility of regulating cellular behavior by droplet battery was investigated preliminarily. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Bioelectronics have brought new perspectives into the diagnosis and the treatment of conditions such as cardiovascular disease, neurodegenerative disease, blindness, cancer, diabetes and asthma. The energy-generating droplet network constructed in the project can produce both electrical and ionic currents, and thereby bridge classical electronics and bioelectric devices. Moreover, the key components in the system also play core roles in cellular metabolism. Their cellular levels differ between cell types and decline with the age of patients. The activity of enzymes is also affected by the temperature, the pH, and the ionic concentration. Therefore, the application of our platform will allow the sensing of changes in biological environments. Figure. The energy-generating droplet networks built of biological elements.
