Fabrication of ultrathin suspended polymer membranes as supports for serial protein crystallography

Abstract

We present a process sequence for the fabrication of ultrathin perforated membranes suspended in polymer frames. Nanoimprint lithography was used to imprint high-density pillar arrays into a double layer consisting of 300 nm of water-soluble PVP and 2–3 μm COC sequentially spin coated onto a silicon wafer. After an RIE step to remove the residual layer and 3D-printing of PLA frames onto the structured COC film, the assemblies were released from the wafer by dissolving the sacrificial PVP bottom layer in water. The suspended membranes were developed as supports for serial protein crystallography, where small amounts of protein microcrystals are deposited onto a membrane and separated from most of their mother liquor through the sieving function of the membranes (blotting). After plunge freezing in liquid nitrogen crystallographic data are collected sequentially from the individual crystals, using X-rays from a synchrotron or femtosecond X-ray pulses of an X-ray free electron laser. Preliminary experiments confirmed that the supports allow for collection of good quality crystallographic data at high resolution, taking advantage of adequate liquid blotting, high stability and low X-ray interaction.

Keywords

• Free-standing membrane
• Nanoimprint lithography
• Additive manufacturing
• Serial crystallography
• Protein structure determination