Smart Molecular Nanosheets for Advanced Preparation of Biological Samples in Electron Cryo-Microscopy

Abstract

Transmission electron cryo-microscopy (cryo­EM) of vitrified biological specimens is a powerful tool for structural biology. Current preparation of vitrified biological samples starts off with sample isolation and purification, followed by the fixation in a freestanding layer of amorphous ice. Here, we demonstrate that ultrathin (∼10 nm) smart molecular nanosheets having specific biorecognition sites embedded in a biorepulsive layer covalently bound to a mechanically stable carbon nanomembrane allow for a much simpler isolation and structural analysis. We characterize in detail the engineering of these nanosheets and their biorecognition properties employing complementary methods such as X-ray photoelectron and infrared spectroscopy, atomic force microscopy as well as surface plasmon resonance measurements. The desired functionality of the developed nanosheets is demonstrated by in situ selection of a His-tagged protein from a mixture and its subsequent structural analysis by cryoEM.

Elastic Properties of Poly(ethylene glycol) Nanomembranes and Respective Implications

“…The bioinert and ultraflexible character of the PEG nanomembranes makes them suitable for different applications ranging from extremely sensitive sensing elements in microelectromechanical systems [ 26 , 29 , 44 , 45 ] to biomolecule-friendly supports for biological samples in high-resolution transmission electron microscopy [ 19 , 46 , 47 ].…”

Synthesis and Nanoscale Characterization of Hierarchically Assembled Molecular Nanosheets

“…The S 2p XP spectrum presented in Figure S7 (Supporting Information) shows thiolates as well as disulfides or thiols formed upon the electron irradiation. [25,48] Spectroscopic details including full width at half maximum (FWHM) values of the individual peaks and relative intensity ratios of their components are presented in Table S1 (Supporting Information).…”

“…[20,21] In this respect carbon nanomembranes (CNMs)-molecular nanosheets with ≈1 nm thickness, deliver a universal platform for the hierarchical assembly of 2D materials. [22][23][24][25] CNMs can be synthesized by electron irradiation induced cross-linking of aromatic selfassembled monolayers (SAMs) on a large scale, [23] with tunable thickness [24] and porosity [24,26] and allow chemical functionalization [27,28] as well as gas and ion permeation, [29,30] etc. Examples for the employment of CNMs are the hierarchical assembly of 2D sheets for bio-recognition [31] and energy-funneling [27] applications as well as for implementation Chemical functionalization of molecular 2D materials toward the assembly of hierarchical functional nanostructures is of great importance for nanotechnology including areas like artificial photocatalytic systems, nanobiosensors, or ultrafiltration.…”

Synthesis and nanoscale characterization of hierarchically assembled molecular nanosheets

“…The S 2p XP spectrum presented in Figure S7 shows thiolates as well as disulfides or thiols formed upon the electron irradiation. [25,48] Spectroscopic details including full width at half maximum (FWHM) values of the individual peaks and relative intensity ratios of their components are presented in Table S1 of the SI.…”

“…[20,21] In this respect carbon nanomembranes (CNM) -molecular nanosheets with 1 nm thickness, deliver a universal platform for the hierarchical assembly of 2D materials. [22][23][24][25] CNMs can be synthesized by electron irradiation induced cross-linking of aromatic self-assembled monolayers (SAMs) on a large scale, [23] with tunable thickness [24] and porosity [24,26] and allow chemical functionalization [27,28] as well as gas and ion permeation [29,30] , etc. Examples for the employment of CNMs are the hierarchical assembly of 2D sheets for bio-recognition [31] and energy-funneling [27] applications as well as for implementation of organic-inorganic hybrids [32] .…”