Integrated Parylene‐Based Thin‐Film Microfluidic Device for Time‐Resolved Cryo‐Electron Microscopy

The observation of transient protein complex structures facilitates a deeper understanding of the underlying biochemical reactions. However, conventional cryo‐electron microscopy (cryo‐EM) methods require tens of seconds for sample preparation, impeding the visualization of intermediate structures in short timescales. To address this limitation, time‐resolved cryo‐EM (TRCEM) is developed as a rapid sample preparation method that can mix and vitrify samples in a sub‐second timescale. However, polydimethylsiloxane (PDMS)‐based microfluidic devices, commonly employed in TRCEM methods, suffer from large sample consumption and high time heterogeneity, limiting their widespread implementation. Here, a parylene‐based microfluidic mixing‐spraying device is presented, which features a thin‐film structure containing a mixer and a spray nozzle in a single layer. The high bonding strength of the device enables it to withstand high channel pressure, facilitating rapid mixing at a wider range of flow rates. Furthermore, the spray nozzle, comprising an ultra‐thin parylene nozzle tip, generates a stable jet at low flow rates, thereby reducing sample consumption and time heterogeneity. The monolithic mixing‐spraying device design minimizes the internal volume, achieving an in‐chip reaction time of