KAIST

Understanding biological phenomena, including protein-protein interactions and enzyme-substrate reactions occurring in microseconds to milliseconds, is essential for comprehending life processes and advancing drug development. KAIST researchers have developed a method for freezing and analyzing biochemical reaction changes within a span of just a few milliseconds, a critical step towards better understanding these complex biological reactions.

< Photo. (From left) Professor Jin Young Kang and Haerang Hwang of the Integrated Master's and Doctoral Program of the Department of Chemistry along with Professor Wonhee Lee of the Department of Physics >

KAIST (represented by President Kwang Hyung Lee) announced on the 24th of March that a joint research team led by Professor Jin Young Kang from the Department of Chemistry and Professor Wonhee Lee from the Department of Physics has developed a parylene-based thin-film microfluidic mixing-and-spraying device for ultra-fast biochemical reaction studies.

*Parylene: A key material for microfluidic devices used to observe protein reactions at ultra-high speeds. It can be fabricated into thin films, just a few micrometers thick, which can be used in making spray nozzles.

This research overcomes the limitations of the existing time-resolved cryo-electron microscopy (TRCEM) method by reducing sample consumption to one-third of the conventional amount while improving the minimum analyzable reaction time by several orders of magnitude—down to just six milliseconds (6 ms).

TRCEM is a technique that rapidly freezes protein complexes during intermediate reaction stages under cryogenic conditions, which allows researchers to analyze their structures. This approach has gained significant attention recently for its potential to capture fleeting biochemical events.

< Figure 1. Time-resolved cryo-EM (TRCEM) technique using microfluidic channels. In order to capture the intermediate structure of biomolecules during a biochemical reaction over time, biomolecules and reaction substrates are mixed in a microfluidic channel, and then sprayed on a grid after a certain reaction time and frozen in liquid ethane to prepare a cryo-EM sample. This can then be analyzed by cryo-EM to observe the structural changes of proteins over time. >

Traditional cryo-electron microscopy has struggled to capture transient intermediate states due to their extremely short lifespans. Although several TRCEM techniques have been developed to address this issue, previous methods were hindered by high sample consumption and limited time resolution. To overcome these challenges, the KAIST team developed a new mixing-and-spraying device using ultra-thin parylene films. The integrated design of the device further enhanced the precision and reproducibility of experiments.

< Figure 2. TRCEM grid fabrication setup using a parylene-based thin-film microfluidic device and actual appearance of the device. You can see that a thin-film parylene channel is inserted into the injection nozzle. The integration of the reaction channel and the injection nozzle allowed the residence time in the device to be reduced to at least 0.5 ms. >

“This research makes TRCEM more practical and paves the way for diverse applications of the parylene thin-film device in structural biology, drug development, enzyme reaction studies, and biosensor research.” Professor Jin Young Kang explained, emphasizing the significance of the study.

Professor Wonhee Lee added, “The team aims to continue this research, focusing on further performance enhancements  of the device and the application for various biochemical reaction analysis.”

< Figure 3. Comparison of the spraying patterns of the parylene mixing-jet device and the conventional mixing-jet device and the filament length in the resulting RecA-ssDNA filament formation reaction. It was shown that the thin film spray nozzle structure affects the uniformity and accuracy of the final reaction time. >

The research findings, with Haerang Hwang (a graduate student in the integrated master's and Ph.D. program in the Department of Chemistry) as the first author, were published online on January 28, 2025, in the international journal Advanced Functional Materials. (Paper Title: “Integrated Parylene-Based Thin-Film Microfluidic Device for Time-Resolved Cryo-Electron Microscopy”, DOI: doi.org/10.1002/adfm.202418224)

This research was supported by the National Research Foundation of Korea (NRF), the Samsung Future Technology Development Program, and the CELINE consortium.