石墨烯载网在冷冻电镜样品制备中的应用研究
宋佶岭，张晨辉，付韵桥，王宏伟*，刘 楠*
（1.清华大学 生命科学学院，北京生物结构前沿中心教育部蛋白质科学重点实验室，北京 100084；2.香港大学 生物科学学院，香港 999077；3. 北京石墨烯研究院有限公司，北京 100095）
摘 要 在冷冻电镜结构解析过程中，样品制备是关键限速步骤之一，经常受到气液界面等问题困扰。石墨烯支撑膜能够富集生物样品颗粒、降低电子束照射导致的样品漂移和充电效应，并且具有较低的背景噪音，已经被应用于冷冻样品的制备，缓解气液界面等问题造成的影响，提高样品制备成功率。为了更广泛地将石墨烯载网用以冷冻电镜样品制备，本文介绍了石墨烯载网的表征办法和对比了不同亲水化处理条件对石墨烯支撑膜产生的影响，着重针对使用石墨烯载网制备冷冻样品时出现的一些异常冰层现象进行了研究分析，并讨论了冷冻样品制备过程中溶液的扩散过程。该研究加深了对石墨烯表面液体分布和扩散过程的理解，为优化使用石墨烯载网用以冷冻电镜样品制备提供了基础。
关键词 冷冻电镜；样品制备；石墨烯；生物大分子
中图分类号：Q617；Q336 文献标识码：BDoi:10.3969/j.issn.1000-6281.2025.01.012
The application of graphene grids in Cryo-EM sample preparation
SONG Jiling1, ZHANG Chenhui2, FU Yunqiao3, WANG Hongwei1*, LIU Nan2*
(1. MOE Key Laboratory of Protein Sciences, Beijing Frontier Research Center for Biological Structures, School of Life Sciences, Tsinghua University, Beijing 100084；2. School of Biological Sciences, The University of Hong Kong, Hong Kong 999077, China；3. Beijing Graphene Institute Co., Ltd., Beijing 100095, China)
Abstract In the process of Cryo-electron microscopy (Cryo-EM) structural analysis of biological macromolecules, sample preparation is one of the key rate-limiting steps, often hindered by the air-water interface issue. Graphene offers advantages such as particle enrichment, reduction of beam-induced motion and charging effects, and minimal background noise. Consequently, graphene grids have been utilized in Cryo-EM to improve sample preparation success rates and address challenges like preferred orientation and structural damage caused by the air-water interface. To further expand the applications of graphene grids in Cryo-EM sample preparation, this study characterizes and optimizes usage conditions for graphene grids. We further investigated and analyzed anomalous ice layer phenomena encountered when utilizing graphene grids for Cryo-sample preparation, as well as examining the diffusion process of solutions during sample preparation. This research contributes to a deeper understanding of the utilization of graphene grids in Cryo-EM sample preparation.
Keywords Cryo-EM；sample preparation；graphene；macromolecule
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