The use of droplet-digital (D2) microfluidics to study protein-protein interaction in a cell-free system

Chiara Leal Alves^a, Kenza Samlali^a, Alaa Selim^b, Fatemeh Ahmadi^a, Steve Shih^a^,^b

^aDepartment of Electrical and Computer Engineering, Concordia University, De Maisonneuve Blvd. W., Montreal, Canada

^bDepartment of Biology, Concordia University, Rue Sherbrooke Ouest, Montréal, Canada

Proceedings of Emerging Investigators in Microfluidics Conference (EIMC)

Online, Spain, 2021 July 20th - October 6th

Organizers: Adrian Nightingale, Darius Rackus and Claire Stanley

Oral, Chiara Leal Alves, presentation 009
DOI: https://doi.org/10.29363/nanoge.eimc.2021.009
Publication date: 5th July 2021

Synthetic biology aims to engineer new organisms using engineering principles by modifying their genetic makeup consisting of DNA, RNA, proteins and metabolites. Proteins are of significant interest since they are the machinery of life. Their interactions induce control over the general behaviour of the cells and organisms. Thus, the study of protein-protein interaction (PPI) has broad applications in the pharmaceutical, food and agriculture industries and in the development of environmentally friendly renewables. There are many methods to study PPIs, some of which are yeast two-hybrid systems and affinity purification coupled to mass spectrometry. These are generally coupled with other systems – e.g., NMR, isothermal titration, and colorimetric methodologies to study their kinetics and interactions. Yet, these techniques are slow, which are not ideal for studying weaker interactions. Furthermore, studying PPIs in a cell environment increases the complexity given the requirement to culture cells and to provide maintenance to ensure their high viability. In our work, we present the first cell-free system to study PPIs in a microfluidic device. We use a D² (droplet-digital) microfluidic device since microfluidics provide conditions that are close to the native state of the protein and use a droplet-digital system since it provides the advantages of control and throughput. Our device consists of several components – (1) on demand electrofusion of droplets of different proteins and inhibitors, (2) on demand picoinjection of different concentrations of fluorescence substrate, (3) kinetics analysis of enzymes interactions using fluorescence readout. As proof of concept, we are studying the interaction of different cellulases enzymes (mainly cellobiohydrolases and betaglucosidases) from different thermophilic organisms in droplets using cell free system. Our goal is to test different combinations of those enzymes to improve cellulose breakdown and study how those combinations could affect the hydrolysis under ionic liquid environment. We anticipate that this device could be used for other applications related to PPI – e.g., studying how new drugs could affect target proteins related to neurodegenerative diseases (e.g., Alzheimer), develop novel biocontrol agents (e.g. amylase inhibitors for fungal control in plants) and discovering new metabolic pathways to produce biofuels.

References:

[1] Ahmadi, F., Samlali, K., Vo, P. Q., & Shih, S. C. (2019). An integrated droplet-digital microfluidic system for on-demand droplet creation, mixing, incubation, and sorting. Lab on a Chip, 19(3), 524–535. https://doi.org/10.1039/c8lc01170b

[2] Samlali, K., Ahmadi, F., Quach, A. B., Soffer, G., & Shih, S. C. (2020). Single Cell Isolation: One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation (Small 34/2020). Small, 16(34), 2070190. https://doi.org/10.1002/smll.202070190

[3] Giss, D., Kemmerling, S., Dandey, V., Stahlberg, H., & Braun, T. (2014). Exploring the Interactome: Microfluidic Isolation of Proteins and Interacting Partners for Quantitative Analysis by Electron Microscopy. Analytical Chemistry, 86(10), 4680–4687. https://doi.org/10.1021/ac4027803

[4] Arola, S., Linder, M. Binding of cellulose binding modules reveal differences between cellulose substrates. Sci Rep 6, 35358 (2016). https://doi.org/10.1038/srep35358

Acknowledgements:

We thank the Natural Sciences and Engineering Research Council (NSERC), the Fonds de Recherche Natureet technologies (FRQNT), and the Canadian Foundation of Innovation (CFI) for funding. CLA thanks Concordia University department of Electrical and Computer Engineering for FRS Funding, NSERC CREATE SynBioApps program for graduate funding and the Biology Department for academic resources. KS thanks FRQNT B2Xs & NSERC CREATE SynBioApps program for graduate funding. AS thanks the Biology Department for academic resources and funding, and the NSERC CREATE SynBioApps program for graduate funding. FA FRQNT PBEEE and Concordia University department of Electrical and Computer Engineering for FRS Funding. SCCS thanks Concordia for a University Research Chair.

nanoGe is a prestigious brand of successful science conferences that are developed along the year in different areas of the world since 2009. Our worldwide conferences cover cutting-edge materials topics like perovskite solar cells, photovoltaics, optoelectronics, solar fuel conversion, surface science, catalysis and two-dimensional materials, among many others.

MATSUS

Previously nanoGe Spring Meeting (NSM) and nanoGe Fall Meeting (NFM), MATSUS is a multiple symposia conference focused on a broad set of topics of advanced materials preparation, their fundamental properties, and their applications, in fields such as renewable energy, photovoltaics, lighting, semiconductor quantum dots, 2-D materials synthesis, charge carriers dynamics, microscopy and spectroscopy semiconductors fundamentals, etc.

International Conference on Hybrid and Organic Photovoltaics

International Conference on Hybrid and Organic Photovoltaics (HOPV) is celebrated yearly in May. The main topics are the development, function and modeling of materials and devices for hybrid and organic solar cells. The field is now dominated by perovskite solar cells but also other hybrid technologies, as organic solar cells, quantum dot solar cells, and dye-sensitized solar cells and their integration into devices for photoelectrochemical solar fuel production.

Asia-Pacific International Conference on Perovskite, Organic Photovoltaics and Optoelectronics

The main topics of the Asia-Pacific International Conference on Perovskite, Organic Photovoltaics and Optoelectronics (IPEROP) are discussed every year in Asia-Pacific for gathering the recent advances in the fields of material preparation, modeling and fabrication of perovskite and hybrid and organic materials. Photovoltaic devices are analyzed from fundamental physics and materials properties to a broad set of applications. The conference also covers the developments of perovskite optoelectronics, including light-emitting diodes, lasers, optical devices, nanophotonics, nonlinear optical properties, colloidal nanostructures, photophysics and light-matter coupling.

International Conference on Perovskite Thin Film Photovoltaics Perovskite Photonics and Optoelectronics

The International Conference on Perovskite Thin Film Photovoltaics Perovskite Photonics and Optoelectronics (NIPHO) is the best place to hear the latest developments in perovskite solar cells as well as on recent advances in the fields of perovskite light-emitting diodes, lasers, optical devices, nanophotonics, nonlinear optical properties, colloidal nanostructures, photophysics and light-matter coupling.