Nanofluidics for Pioneering Synthetic Biology of Bottom-Up Cell-Free Molecular Systems

Synthetic biology via bottom-up assembly is transitioning from stochastic, extract-based cell-free systems toward reconstituted, molecularly defined cell-free molecular systems. Transitioning to molecularly defined systems provides a path to quantitative design; however, the active assembly of these molecular building blocks into ordered spatiotemporal architectures remains a formidable challenge in synthetic biology. In this perspective, we propose nanofluidics as a transformative platform to bridge this gap. By leveraging nanoconfinement effects and precision mass transport, nanofluidics facilitates the active assembly of molecular building blocks into functionally integrated spatiotemporal structures, thereby pioneering the synthetic biology of bottom-up cell-free molecular systems. Specifically, we discuss how nanofluidics enables precise control over fluid dynamics and single-molecule behavior within nanochannels and facilitates molecular active-assembly and tunable interactions of molecular components by engineering design of nanofluidic devices. Furthermore, we highlight key challenges and opportunities using nanofluidics to build next-generation cell-free molecular systems with single-molecule resolution. This perspective provides a strategic roadmap for the synthetic biology of bottom-up cell-free molecular systems.