CN111470500B

CN111470500B - Bioactive ligand functionalized graphene film and preparation method thereof

Info

Publication number: CN111470500B
Application number: CN201910062667.7A
Authority: CN
Inventors: 王宏伟; 彭海琳; 陈亚楠; 张金灿; 刘楠
Original assignee: Tsinghua University; Peking University
Current assignee: Tsinghua University; Peking University
Priority date: 2019-01-23
Filing date: 2019-01-23
Publication date: 2021-10-08
Anticipated expiration: 2039-01-23
Also published as: CN111470500A

Abstract

The invention discloses a bioactive ligand functionalized graphene film and a preparation method thereof. The bioactive ligand is connected to the graphene film and is a Ni-nitrilotriacetic acid ligand; the preparation method of the bioactive ligand functionalized graphene film comprises the following steps: transferring the graphene film to an electron microscope carrier net; treating the graphene film by adopting a mixed aqueous solution of potassium permanganate and an alkaline compound; and (3) activating the graphene film by adopting nitrilotriacetic acid, and then reacting with nickel salt to obtain the graphene film. According to the invention, the functional modification of graphene can solve the problem of hydrophobicity of graphene, and high-density bioactive ligand is introduced by utilizing chemical linkage, so that selective specific combination of a support membrane and biomolecules can be realized, and protein distribution can be accurately controlled. It is contemplated that the bioactive ligand functionalized graphene thin films of the present invention may combine biomolecule purification and cryoelectron microscope sample preparation into one step by directly localizing or capturing the target biomolecule in the cell lysate in future optimization.

Description

Bioactive ligand functionalized graphene film and preparation method thereof

Technical Field

The invention relates to a bioactive ligand functionalized graphene film and a preparation method thereof, belonging to the technical field of biological cryoelectron microscopy.

Background

The cryoelectron microscope has become the most important structural biological research means at present through long-term development since the appearance of the last 70 th century, especially through scientific and technical breakthroughs in recent years. The water-containing biological sample under physiological condition is quickly frozen into amorphous glassy ice by a freezing electron microscope, then the sample under the frozen state is observed by using a transmission electron microscope, and the biomacromolecule structure is analyzed by combining an image processing technology, so that the method has unique advantages which are not possessed by other means. The refrigeration electron microscope technology has revolutionary breakthrough in the aspects of instrument hardware and structural analysis software in recent years, greatly expands the application range of the technology, and profoundly changes the research content and the pattern of structural biology. In the past two decades, the research on the structure biology in China has been rapidly developed and becomes a large country for the research on the structure biology, but the research results on the structure biology method based on the independent intellectual property and the related scientific and technical are very few.

Although the field of frozen electron microscopy has been developed vigorously, it is still not a mature technology, and its broader development and application are limited by scientific and technological bottlenecks. Frozen sample preparation, as a key step in frozen electron microscopy, has been a technical difficulty in this field. Until now, the preparation principle, preparation materials and assembly process of the sample for the cryoelectron microscope are not obviously changed compared with the preparation principle and the assembly process of the sample for the cryoelectron microscope before 20 years, the sample preparation and search stage is still performed by each experimenter according to different sample states, the repeatability is poor, and the efficiency of structural analysis of the cryoelectron microscope is greatly influenced. With the rapid development of electronic microscope hardware equipment and software technology, the bottleneck effect of sample preparation technology is more and more prominent. How to prepare a frozen sample with proper thickness, uniform sample distribution and good molecular property is a core problem of sample preparation. Therefore, designing a novel material for supporting the membrane of the cryoelectron microscope is the key point for obtaining an excellent frozen sample and is the core point for breaking through the technological bottleneck of frozen sample preparation.

The support membrane material of the currently and generally adopted cryoelectron microscope is a micro-sieve pore carbon membrane, and the material has a series of defects in the application field of cryoelectron microscopes, including uneven surface properties, inconsistent thickness of edges of micro-sieve pores, poor conductivity of an amorphous carbon framework at the temperature of liquid nitrogen, easy breakage due to poor mechanical rigidity and ductility and the like, and the gas-liquid interface of a solution film suspended in a micro-sieve pore has adverse effects on the structural stability, particle orientation and distribution uniformity of biomacromolecules. An ultrathin carbon film (the thickness is 3-5 nm) supported by a micromesh is also a commonly used freezing sample preparation supporting material, the influence of a solution gas-liquid interface on biomolecules is reduced to a certain extent, but the background noise of an amorphous carbon film is large, and the high-resolution structural research on a small molecular structure is limited. The above problems lead to a great reduction in reproducibility, universality, success rate, uniformity of sample quality, etc. of frozen samples. In the past few years, a plurality of groups of subjects are searching for frozen sample carrying nets prepared by new materials, including silicon nitride micromesh, gold micromesh, graphene oxide films and the like, but the frozen sample carrying nets are not widely applied so far, and the main reasons are that the materials are not ideal carrying net supporting film materials, the problems of poor conductivity, poor rigidity at liquid nitrogen temperature, easy cracking and the like exist, and a repeatable preparation process is not mature in practice. Therefore, it is necessary to provide an ideal novel material for supporting the grid of the cryoelectron microscope.

Disclosure of Invention

The invention aims to provide a bioactive ligand functionalized graphene film, which utilizes the unique properties (transparency, conductivity and mechanical property) of graphene to grow and prepare graphene, transfers the graphene to a micro-grid carrier mesh of a cryoelectron microscope to obtain a graphene supporting film, further performs chemical functionalization on the surface of the graphene film, and introduces high-density bioactive ligand (Ni-NTA) by utilizing chemical linkage, thereby fully improving the binding performance of the graphene and a biomacromolecule sample; the functional modification of the graphene can solve the problem of hydrophobicity of the graphene, and the high-density bioactive ligand is introduced by utilizing chemical linkage, so that the selective specific combination of a support membrane and biomolecules can be realized, and the protein distribution can be accurately controlled.

The graphene related by the invention can be large-area single crystal graphene, polycrystalline graphene, few-layer graphene and the like, and can be prepared by methods in the prior art, such as a CVD method, a mechanical stripping method, a liquid phase stripping method, a graphite oxidation reduction method and the like.

The bioactive ligand functionalized graphene film provided by the invention is formed by connecting a bioactive ligand to a graphene film, wherein the bioactive ligand is a nickel-nitrilotriacetic acid ligand.

In the functionalized graphene film with the bioactive ligand, the bioactive ligand is connected to the graphene film through a covalent bond.

The invention also provides a preparation method of the bioactive ligand functionalized graphene film, which comprises the following steps:

1) transferring the graphene film to an electron microscope carrier net;

2) treating the graphene film by adopting a mixed aqueous solution of potassium permanganate and an alkaline compound;

3) and activating the graphene film by adopting nitrilotriacetic acid, and then reacting with nickel salt to obtain the bioactive ligand functionalized graphene film.

In the above preparation method, in step 1), the electron microscope mesh may be a micro-grid mesh or other types of electron microscope meshes.

In the above preparation method, in step 1), the Graphene film may be transferred by a method of the prior art, such as a glue-free direct Transfer method (j.c. zhang et al, Clean Transfer of Large Graphene Single Crystals for High-integrity Suspended Membranes and Liquid cells, adv.mater.29,1700639 (2017); regan et al, a direct transfer of layer-area graphene, appl. phys.lett.96,113102 (2010); matkovic, u.ralevic, m.khikara, m.m.jakovljevic, d.jovanovic, g.bratia, r.gajic, j.appl.phys.2013,114, 093505; w.h.lin, t.h.chen, j.k.chang, j.i.taur, y.y.lo, w.l.lee, c.s.chang, w.b.su, c.i.wu, ACS Nano2014,8,1784), polymer assisted Transfer method (j.w.suk et al, Transfer of CVD-growth monomer Graphene on to array substrate, ACS Nano 5, 6916-ion 6924 (2011); l. Lin et al, Surface Engineering of coater files for Growing center-Sized Single-Crystalline graphene. acs Nano 10, 2922-; y.c. lin, c.c. lu, c.h.yeh, c.h.jin, k.suenaga, p.w.chiu, Nano lett.2012,12,414.; e.ledwosinska, p.gaskell, a.guermoune, m.siaj, t.szkopek, appl.phys.lett.2012,101, 033104; regan, n.alem, b.aleman, b.s.geng, c.girit, l.maserati, f.wang, m.crommie, a.zettl, appl.phys.lett.2010,96,113102.) and the like.

In the above preparation method, in step 2), the alkaline compound may be sodium hydroxide or potassium hydroxide;

in the step 2), in the mixed aqueous solution, the concentration of the potassium permanganate can be 0-0.4M but is not zero, and the concentration of the alkaline compound can be 0-0.2M but is not zero;

the treatment time may be 1-60 min, such as 50 min.

Processing the graphene film in the following way:

dropwise adding the mixed aqueous solution onto the graphene film; after standing, the mixed aqueous solution can be sucked off by using filter paper, and the graphene film is fully cleaned by using a sodium bisulfite aqueous solution (0-1M, but not zero) until no residual treatment solution is left on the surface of the graphene film.

And 2) applying to the surface of the graphene oxide film.

In the above preparation method, before the step 3), the method further comprises the following modification steps:

and treating the graphene film by using a mixed aqueous solution of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride, N-hydroxy-sulfosuccinimide and 2- (N-morpholino) ethanesulfonic acid.

In the above preparation method, the concentration of the 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) in the mixed aqueous solution may be 1 to 6mM, specifically 5mM, the concentration of the N-hydroxy-sulfosuccinimide (sulfo-NHS) may be 1 to 6mM, specifically 5mM, and the concentration of the 2- (N-morpholino) ethanesulfonic acid (MES) may be 10 to 200mM, specifically 100 mM.

In the preparation method, the pH value of the mixed aqueous solution is 4.0-6.0, and specifically can be 5;

the treatment time is 1-60 minutes.

In the preparation method, in the step 3), the graphene film is placed in a TBE buffer solution containing the nitrilotriacetic acid to perform the activation step;

the activation time can be 0.1-4 hours, specifically 2 hours;

the concentration of the nitrilotriacetic acid can be 1.0-15 mM, and specifically can be 11.3 mM;

the pH value of the TBE buffer solution is 7-9, and specifically can be 8.5.

In the above preparation method, in step 3), the nickel salt may be nickel sulfate, nickel chloride or nickel nitrate;

dropwise adding the aqueous solution of the nickel salt to the graphene film;

the reaction time can be 0.1-1.5 hours, specifically 1 hour;

the concentration of the nickel salt aqueous solution is 1.0-15 mM, and specifically 11.3 mM;

after the reaction is finished, the graphene film can be washed by deionized water.

In the specific embodiment of the invention, an atomic force microscope is adopted to test the flatness change of the functionalized graphene; determining the crystallinity change of the functionalized graphene by using Raman spectrum; and the integrity, stability and irradiation resistance of the graphene are detected by adopting a transmission electron microscope.

The bioactive ligand functionalized graphene film prepared by the method has little or no influence on graphene crystal lattices, and maintains the superior performance of single-layer single-crystal graphene, so that the background noise in the original cryoelectron microscope photomicrograph is effectively reduced compared with an amorphous carbon film. The bioactive ligand functionalized graphene film has the specificity of capturing protein with a His label, and is suitable for serving as a support material for three-dimensional reconstruction of a freezing electron microscope with atomic resolution.

When using bioactive ligand functionalized graphene thin films, making frozen samples with an optimal ice thickness of 20-30 nm is more controllable, and this ice thickness is considered to be an ideal choice for embedding most proteins and does not introduce additional background noise.

The bioactive ligand functionalized graphene film has little or no influence on graphene crystal lattices, and maintains the superior performance of single-layer single-crystal graphene, so that the background noise in the original cryoelectron microscope photomicrograph is effectively reduced compared with an amorphous carbon film. Compared with an amorphous carbon supported net, the bioactive ligand functionalized graphene film is naturally hydrophilic and is more friendly to biological molecules. Furthermore, it is capable of capturing the protein of interest, preventing the adsorption of protein molecules to the air-water interface. The bioactive ligand functionalized graphene thin films show high specificity in selecting a target protein and reducing the amount of protein used for sample preparation, thus simplifying protein purification and improving protein stability. These have opened up new prospects for the graphene materials to be used for cryoelectron microscopy. The outstanding advantages of the bioactive ligand functionalized graphene film enable the preparation of a vitrified cryoelectron microscope sample with uniform and appropriate ice thickness to be more controllable, and data processing to be more reliably carried out, especially in contrast transfer function estimation, for high resolution cryoelectron microscope structure determination. It is contemplated that the bioactive ligand functionalized graphene thin film of the present invention may combine biomolecule purification and cryoelectron microscope sample preparation into one step by directly localizing or capturing the target biomolecule in the cell lysate in future optimization.

Drawings

FIG. 1 is a representation of nickel atom modified suspended monolayer graphene; wherein, fig. 1(a) is a suspended graphene film covered on a freezing electron microscope Au grid; inset shows the corresponding pristine graphene grown on copper foil; fig. 1(b) is an atomic force microscope representation of a suspended graphene thin film; fig. 1(c) is a high resolution transmission electron microscope image of a graphene thin film; inset shows a magnified transmission electron microscope image of the graphene film; fig. 1(D) is a 2D atomic force microscope image of a bioactive ligand functionalized graphene membrane; fig. 1(e) is a high-power atomic force microscope 3D image of the corresponding bioactive ligand functionalized graphene film in the square region labeled in fig. 1 (D); fig. 1(f) is a statistical histogram of the height distribution of modified ligands on the graphene surface; fig. 1(g) is a scanning transmission electron microscope image of a Ni-NTA modified graphene film and the corresponding Ni atom energy dispersive X-ray spectroscopy elemental distribution, revealing a uniform distribution of nickel atoms on the graphene surface; fig. 1(h) is a high resolution scanning transmission electron microscope image showing potential individual nickel atoms on the graphene surface, marked by green circles; fig. 1(i) is a high resolution Ni 2p, N1 s XPS spectrum of Ni-NTA modified graphene.

FIG. 2 is a typical transmission electron microscope image of a transferred graphene film; wherein fig. 2(a) is a low magnification transmission electron microscope image of a transfer graphene film in which five pores are observed; fig. 2(b) is a typical transmission electron microscope image of a single hole covered by a suspended graphene film; fig. 2(c) is a higher magnification transmission electron microscope image of a graphene film suspended in a hole.

FIG. 3 is a suspended graphene film characterized by an atomic force microscope at different magnifications and modes; wherein fig. 3(a) and 3(b) are 2D atomic force microscope images of graphene films with 16 pores and a single pore, respectively; fig. 3(c) is a corresponding high power atomic force microscope 3D image of the graphene film in the square region marked in fig. 3 (b).

Fig. 4(a) is an atomic force microscope characterization of suspended bioactive ligand functionalized graphene films; FIG. 4(b) is a height profile of the dashed white line marked in FIG. 4 (a); highly consistent with the size of these bioactive molecules used to functionalize graphene membranes.

Fig. 5 is an X-ray photoelectron spectroscopy characterization of the original graphene (fig. 5(a)) and Ni-NTA modified graphene (fig. 5(b)) grids.

FIG. 6 is a representation of the hydrophilicity and strength of a bioactive ligand functionalized graphene membrane; wherein, fig. 6(a) is the hydrophilicity of graphene on Cu before (upper) and after (lower) the functionalization treatment, the water contact angles being 88 ° and 53 °, respectively; fig. 6(b) shows the hydrophilicity of suspended graphene on the carrier web before (top) and after (bottom) the functionalization treatment, with water contact angles of 56 ° and 29 °, respectively; fig. 6(c) is a raman spectrum of suspended graphene before (red) and after (blue) functionalization treatment; FIGS. 6(d) and 6(e) are Selected Area Electron Diffraction (SAED) patterns for graphene film and Ni-NTA functionalized graphene film (bioactive ligand functionalized graphene film), respectively, at a dose rate of 86e/A²The dashed rectangle shows the corresponding intensity; fig. 6(f) is a ratio of third-order integrated bragg intensity (I) to first-order integrated bragg intensity (Imax) as a function of the dose of the graphene film and the bioactive ligand functionalized graphene film, respectively.

FIG. 7 is a characterization of the binding specificity of bioactive ligand functionalized graphene membranes by fluorescence and negative staining electron microscopy; wherein, fig. 7(a) is that the incubation of His-tagged red fluorescent protein with graphene membrane as a bioactive ligand but in the absence of Ni ions, no red signal was detected under a fluorescence microscope; fig. 7(b) is a high density red signal with Ni ion binding to bioactive ligand functionalized graphene membrane; FIG. 7(c) shows that the red signal was washed off with 300mM imidazole buffer. Negative staining electron microscopy characterization of His-tagged PNPase binding affinity from fig. 7(d) to fig. 7 (i). FIGS. 7(d) and 7(g) show almost no protein particles attached to the graphene membrane carrier network as bioactive ligands but no Ni ions; FIGS. 7(e) and 7(h) representative photomicrographs of His-tagged PNPases supported by biologically active ligand functionalized graphene membrane networks, where protein particles are visibly identified, some of which are indicated by red arrows; FIGS. 7(f) and 7(i) show the washing away of protein particles with 300mM imidazole buffer; fig. 7(g), 7(h) and 7(i) are electron micrographs obtained with a higher magnification of fig. 7(d), 7(e) and 7(f), respectively.

Fig. 8 is a negative-stain electron microscope micrograph of a blank bioactive ligand-functionalized graphene membrane (fig. 8(a)) and a His-tagged PNPase-loaded bioactive ligand-functionalized graphene membrane (fig. 8(b)), in which monodisperse His-tagged PNPase particles can be clearly seen in fig. 8 (b).

FIG. 9 is an application of a bioactive ligand functionalized graphene film in the preparation of a frozen electron microscope sample; wherein fig. 9(a) is a representative micrograph of His-tagged 20S proteasome (arrowed) and 60S ribosomal precursor (circled) mixed proteins on amorphous carbon membrane, with the inset being the average class produced by Relion in the 2D classification, with the 60S ribosomal precursor in the upper part and the 20S proteasome in the lower part; fig. 9(b) is a representative micrograph of mixed proteins on a NTA bioactive ligand functionalized graphene thin film carrier network in the absence of Ni ions; fig. 9(c) is a representative micrograph of mixed proteins on a Ni-NTA modified bioactive ligand functionalized graphene membrane carrier network; fig. 9(d) is a statistical analysis of the change in protein ratio, with the ratio of His-tagged 20S proteasome to ribosome being about 30% on NTA-modified graphene membranes but 9-fold to 270% increase on Ni-NTA-modified bioactive ligand functionalized graphene membranes in the absence of Ni-ion NTA graphene membranes.

FIG. 10 is a cryo-electron microscopy texture determination of a composite prepared on a bioactive ligand functionalized graphene film mesh; wherein FIG. 10(a) is a cryo-electron tomography showing 20S particles localized in a porous carbon-coated carrier web, each dot representing a 20S particle; FIG. 10(b) lists three different slices from the Z-axis of the frozen electron tomography reconstruction in FIG. 10(a) and their relative positions are indicated by arrows, the thickness of the frozen specimen is estimated to be 50nm, and the scale bar in these slices represents 50 nm; FIG. 10(c) shows the localization of 20S particles in a Ni-NTA bioactive ligand functionalized graphene membrane network for cryo-tomography, each spot representing one 20S particle, the particles being distributed predominantly in the same layer; fig. 10(d) lists the three representative layers from the Z-axis of the frozen electron tomography reconstruction in fig. 10(c) and their relative positions are indicated by arrows. The thickness of the frozen specimen was estimated to be-25 nm, with a scale bar representing 50 nm; fig. 10(e) is a three-dimensional reconstruction of the 20S proteasome using particle images collected from Ni-NTA bioactive ligand functionalized graphene membrane samples; FIG. 10(f) is two different views of the alpha-and beta-subunit densities extracted from a three-dimensional reconstruction (including corresponding atomic model fits); FIG. 10(g) is an interaction alpha-helix density (reticular) atomic model segmented by alpha-and beta-subunit interfaces (purple bands, pdb code: 3J9I), and the density of some large side chains is clearly identified.

FIG. 11(a) is a representative frozen electron microscope micrograph of a porous carbon supported, network-supported 20S proteasome; 11(b) -11 (d) are different layers of the Z-axis extracted from the tomographic image reconstruction in FIG. 10 (a); layers (b) and (d) correspond to two air-water interfaces, which are also the regions of major protein particle distribution; the thickness of the frozen sample (-50 nm) can be estimated by calculating the distance between layer (b) and layer (d), layer (c) being a selected layer in the vitrified frozen sample, where some sparsely dispersed particles can be identified.

FIG. 12(a) is a representative cryoelectron microscope micrograph of the bioactive ligand functionalized graphene membrane supported 20S proteasome; fig. 12(b) -12 (d) are different layers of the Z-axis extracted by the tomographic image reconstruction in fig. 10(c), and it is found that almost all protein particles are distributed in the same layer (c); layer (b) was 4-5nm from layer (c) and some ice contamination (marked with arrows) was detectable, indicating that layer (b) was hardly located outside the frozen sample boundary; layer (d) and layer (c) were about 20nm in a direction different from (b), and the frozen sample thickness was estimated to be-25 nm by calculating the distance between layers (b) and (d).

Fig. 13(a) is an FSC curve of a 3D reconstruction of the 20S proteasome supported on Continuous carbon film (continuos carbon film), porous carbon film (Holey carbon film) or bioactive ligand functionalized graphene Film (FGM), all three reconstructions from the exact same number (6,095) of particles, the horizontal dashed line representing the FSC-0.143 resolution estimation criterion, and the corresponding resolution of each reconstruction is labeled; fig. 13(b) euler angle distributions of 20S proteasome particle orientation on continuous carbon, porous carbon and bioactive ligand functionalized graphene films, respectively, the diameters of these spots represent relative portions of the particles, and some of the major orientations of the structures are shown aside. D7 symmetry was applied in 20S proteasome reconstruction, so the profile covered only 1/14 of the 3D sphere. In addition to the side view (θ ═ 0 °) on the continuous carbon film and the porous carbon film, a part of the particles had other orientations. Such other orientations are hardly found on bioactive ligand functionalized graphene films (e.g., θ ═ 50 ° on continuous carbon films or θ ═ 90 ° on porous carbon films).

FIG. 14(a) is a 3D density plot of the 20S proteasome; FIG. 14(b) shows the α - α subunit interactions in 20S protease in vivo; FIG. 14(c) shows the in vivo beta-beta subunit interactions of 20S protease.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Graphene films used in the following examples were prepared by CVD and transferred to a cryoelectron microscope micro-grid Au carrier web by a gel-free direct Transfer method (j.c. zhang et al, Clean Transfer of Large Graphene Single Crystals for High-interaction Suspended Membranes and Liquid cells. adv.mater.29, 1709 (2017)).

The transferred graphene film is characterized:

FIG. 1(a) is a suspended graphene film covered on a freezing electron microscope Au grid; inset shows the corresponding pristine graphene grown on copper foil; fig. 1(b) is an atomic force microscope representation of a suspended graphene thin film; fig. 1(c) is a high resolution transmission electron microscope image of a graphene thin film; inset shows a magnified transmission electron microscope image of the graphene film.

Example 1 preparation of bioactive ligand functionalized graphene thin films

1. Preparation of

And (3) dropwise adding 7 mu L of mixed solution drops of 0.40M potassium permanganate and 0.20M sodium hydroxide on the surface of the transferred graphene film, standing for a period of time (about 50 minutes), sucking the mixed solution by using filter paper, and fully washing by using 1M sodium bisulfite until no residual treatment solution exists on the surface of the graphene film. Next, the graphene surface was modified and protected by a mixed solution (pH 5.0) of 5.0mM 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), 5.0mM N-hydroxy-sulfosuccinimide (sulfo-NHS) and 0.10M 2- (N-morpholino) ethanesulfonic acid (MES)For about 50 minutes. Thereafter, the graphene mesh was activated in 50mM TBE buffer (pH 8.5) containing 11.3mM nitrilotriacetic acid for a certain period of time (about 2 hours), and then 11.3mM nickel sulfate (NiSO) was added dropwise₄) The solution was reacted for a period of time (about 1 hour). Finally, the modified graphene films were washed with deionized water and used for subsequent characterization and protein capture.

2. Analysis of results

The prepared suspended graphene film is functionalized through a series of chemical reactions to realize the specific function of the bioactive ligand Ni-NTA, and the quality of the bioactive ligand functionalized graphene film is represented by a series of microscopes and spectral analysis. The suspended graphene film can withstand multiple operations during the functionalization process, so that no additional damage is introduced after the chemical modification. Compared with unmodified suspended graphene films, the surface of the bioactive ligand functionalized graphene film is rougher and has additional structural features (fig. 1(d) and fig. 1 (e)). The surface height fluctuation of the bioactive ligand functionalized graphene film is on average-1.5 nm (fig. 1(f) and fig. 4), similar to the vertical height of the attached Ni-NTA molecules, indicating that the bioactive ligand is successfully prepared on the graphene surface. It is noteworthy that the average height of the attached ligands is much smaller than the thickness of amorphous carbon thin film (-5 nm) used in conventional cryoelectron microscopy, and thus it is possible to significantly reduce the background noise of the transmission electron microscopy support film. Scanning transmission electron microscope imaging and corresponding energy dispersive X-ray spectroscopy elemental distribution of bioactive ligand functionalized graphene films demonstrate the uniform distribution of Ni atoms on the graphene surface, demonstrating the successful introduction of Ni-NTA on graphene films (fig. 1 (g)). High resolution scanning transmission electron microscopy of bioactive ligand functionalized graphene thin films showed dispersed high contrast features, possibly corresponding to individual nickel atoms on the surface of the modified graphene (fig. 1 (h)).

The invention analyzes the element composition and the electronic structure of the bioactive ligand functionalized graphene film by using X-ray photoelectron spectroscopy (XPS). Calibrate all XPS spectra to sp²284.0eV binding energy of carbon. Functionalized graphene for bioactive ligandsThe XPS spectra of the films showed signals for C1 s, O1 s, N1 s, Ni 2p and Au 2p (1s, 2p correspond to the electronic configuration of the electrons in the atom) at atomic ratios of 83.2%, 13.6%, 1.0%, 0.8% and 1.3%, respectively (FIG. 5). The spectral signal of Au comes from the gold-supported mesh supporting the porous carbon. The high resolution Ni 2p spectra show two prominent bands at 856.4eV and 874.1eV (FIG. 1(i)), corresponding to Ni 2p3/2 and Ni 2p1/2, respectively, and Ni²⁺Literature values for the electron binding energy of cations are consistent. The characteristic peak of N1 s centered at 400.3eV in the high-resolution N1 s spectrum can be assigned to C-NH₂A bond derived from NTA covalently attached to graphene thin film (fig. 1 (i)).

Further, the invention uses water contact angle measurement to evaluate the hydrophilicity of the bioactive ligand functionalized graphene thin film. The effect of the functionalization treatment on the hydrophilicity of the graphene film grown on the surface of the copper foil (fig. 6(a)) and the transfer to the Au carrier net (fig. 6(b)) was investigated. Fig. 6(a) shows that the water contact angle of graphene on copper foil decreased from 88 ° to 53 ° after functionalization treatment. On the transferred Au-loaded mesh, the water contact angle dropped from 56 ° to 29 °, indicating a higher hydrophilicity after treatment (fig. 6 (b)).

The integrity of the carbon-carbon bonds in graphene is critical to its particular physical properties. The invention represents the Raman spectra of the suspended graphene film and the bioactive ligand functionalized graphene film. The results showed 2D (2690 cm)^-1Binaural from second order Raman Scattering) and G (1580 cm)^-1First order raman scattering) peaks with intensity ratio close to 8 and no defect peaks, demonstrating high quality of suspended graphene films (fig. 6 (c)). After the functionalization treatment, the shapes of G and 2D peaks of graphene are well maintained, and weak D peaks (1350 cm) appear^-1Defect-induced second order raman scattering, fig. 6(c)), indicating that the graphene lattice change is small. This ensures high thermal and electrical conductivity of the bioactive ligand functionalized graphene thin film and its potential ability to reduce damage to the sample by electron radiation. The effect of electron radiation on the stability of the bioactive ligand functionalized graphene thin films was studied by characterizing the diffraction pattern changes of the graphene thin films in transmission electron microscopy (fig. 6(d) and 6 (e)). Biologically active ligand workThe peak in the diffraction pattern of the graphene film can be converted to the peak at 200kV

The suspended graphene films after 1s of exposure to the accelerated electron dose rate were very similar (fig. 6(d) and 6(e)), indicating that the bioactive ligand functionalized graphene films retained a good lattice structure. The relative intensity of the Bragg reflection of the graphene film and the biologically active ligand functionalized graphene film, defined as the ratio of the third-order integrated intensity to the first-order integrated intensity of the Bragg peak, even at up to 200,000 e-

Also shows small decay at high electron emission (fig. 6 (f)). Negligible radiation damage on the grid demonstrates the high stability of bioactive ligand functionalized graphene thin films under electron beams due to the high electron conductivity of continuous large area single crystal graphene thin films.

Example 2 study of biological application Properties of bioactive ligand functionalized graphene film

1. Test method

Under refrigeration, a certain concentration of fluorescent protein with a specific binding tag with small ligand molecules is dripped into the bioactive ligand functionalized graphene film prepared in the embodiment 1, the film is kept still for a certain time and then repeatedly washed by using a buffer solution, and then a fluorescence microscope is used for detecting a fluorescence signal on the surface of graphene. A plurality of control groups are simultaneously arranged in the experiment, and the specific binding capacity of the bioactive ligand functionalized graphene film to the protein with the label is tested.

Negative staining EM analysis of His-tagged PNPase: one drop of about 5 μ L of a 100nM PNPase sample was added drop wise to the freshly modified graphene mesh and incubated for 15 minutes in a high humidity chamber at 4 ℃. Then with sample buffer (20mM HEPES (pH 7.5), 50mM NaCl, 1mM MgCl₂) And lightly washing the carrier net for 3-5 times. Next, 3 μ Ι _ of 2% uranyl acetate was pipetted onto the bioactive ligand functionalized graphene thin film and allowed to stain for about 1 minute. After staining, the grid was blotted dry and air dried. For miaowAzole wash experiments, the sample incubated mesh was washed 5 times with 300mM imidazole solution and then negatively stained with 2% uranyl acetate. Negative staining micrographs were obtained under a FEI Tecnia Spirit 120 electron microscope equipped with a Gatan US4000 CCD camera.

Frozen samples were made using Vitro bot: dripping 4 microliters of biomacromolecules (such as PNPase) with a certain concentration and a certain concentration to the bioactive ligand functionalized graphene film, and preparing a frozen sample under the conditions that the humidity is 100%, the temperature is 12 ℃, the blotting pressure is 2.5 units, and the blotting time is 2 s. In addition, the ratio of 1: 1 molar ratio-5 μ L of droplets of the mixed protein solution of His-tagged 20S proteasome and 60S ribose precursor were pipetted onto the graphene-modified carrier network (bioactive ligand functionalized graphene thin film) and the control carrier network. After incubation for 15min, the mesh was gently washed 3-5 times and then transferred to a FEI Vitrobot. In sample preparation, the suction pressure and the suction time are adjusted according to the conditions, so that the proper thickness of the disordered ice layer of the sample is ensured. After the sample is prepared, adding the frozen sample into a Cassette according to a Titan krois sample loading process, automatically collecting data by Auto-EMation, researching the influence of the novel graphene net-carrying material on the ice layer thickness, the sample distribution uniformity, the orientation, the integrity, the anti-irradiation capability of the sample, the irradiation drift condition and the like of the prepared sample in detail, feeding back the problems occurring in the middle to the preparation process, and correcting, perfecting and optimizing the problems. And selecting protein particles according to the collected electron microscope photos, constructing a three-dimensional model, and calculating to obtain the resolution of biomacromolecule structure analysis.

Frozen electron tomography: the cryo-electron tomography micrographs were obtained on FEI Titan Krios, accelerated at 300 kv, Gatan K2 camera,

dose rate and total cumulative dose

The magnification for data acquisition is 64,000 ×, the calibrated pixel size is

For each series of tilts, the tilt angle is from +51 ° to-51 °, with a step size of 3 °. All tilt series were obtained at an under-focus of about-5.0 μm. The location of the 20S proteasome particle in the cryo-electron tomographic density map is determined primarily by template matching methods.

2. Analysis of results

(1) Biological binding activity of bioactive ligand functionalized graphene film

The binding ability of the bioactive ligand functionalized graphene thin film was first verified by fluorescence microscopy using His-tagged Red Fluorescent Protein (RFP).

And 2mL of His-tagged red fluorescent protein was applied to the bioactive ligand functionalized graphene thin film surface at a concentration of 2 mg/mL. After incubation with protein solution for 15min at room temperature, the bioactive ligand functionalized graphene film surface was washed with protein buffer and characterized under a fluorescence microscope. A high density of red fluorescent spots was clearly identified on the surface (FIG. 7(b)), indicating the presence of His-tagged red fluorescent protein. To verify whether the accumulation of His-tagged red fluorescent protein on the surface of the bioactive ligand functionalized graphene thin film was specific, a control experiment was performed by washing the red fluorescent protein-bound bioactive ligand functionalized graphene thin film with 300mM imidazole, and it was found that almost all of the red signal was depleted (fig. 7 (c)). In parallel experiments, Ni ions were intentionally omitted in the chemical modification step. The lack of Ni ions completely eliminated the binding capacity of the bioactive ligand functionalized graphene thin film, and thus no red fluorescent protein signal was detected on the surface of the bioactive ligand functionalized graphene thin film (fig. 7 (a)). To test the affinity of Ni-NTA bioactive ligand functionalized graphene films, their binding ability to His-tagged PNPase proteins was characterized using negative staining electron microscopy (fig. 8). Consistent with the fluorescence microscopy characterization results, almost no protein particles were identified in the absence of Ni ions (fig. 7(d) and volume 7 (g)). When Ni ions were present during the chemical modification, the bioactive ligand functionalized graphene thin film could adsorb monodisperse protein particles with high signal-to-noise ratio (fig. 7(e) and 7(h)), these attached PNPase particles disappeared after washing with 300mM imidazole (fig. 7(f) and 7(i)), confirming that His-tagged protein specifically binds to the bioactive ligand functionalized graphene thin film by Ni-NTA chelation.

(2) Application of bioactive ligand functionalized graphene film in three-dimensional reconstruction of electron microscope

Further, a mixed solution of His-tagged 20S proteasome and 60S ribosome precursor was used at a ratio of-1: the molar ratio of 1 further characterizes the bioactive ligand functionalized graphene thin films used for frozen electron microscope sample preparation.

As a control, a frozen electron microscope sample was also made using an amorphous carbon support mesh (fig. 9(a)) and an NTA functionalized graphene thin film (without Ni ions) support mesh (fig. 9 (b)). It was found that bioactive ligand functionalized graphene thin film loading mesh (fig. 9(c)) effectively reduced background noise and retained more structural detail than continuous amorphous carbon loading mesh (fig. 9 (a)). The protein particles (20S proteasome or 60S ribosomal precursor) on the bioactive ligand functionalized graphene thin film carrier web showed higher contrast and lower background noise signal in the original micrograph than on the amorphous carbon coated carrier web. On the premise of approximately the same number of particles, the two-dimensional classification of 20S proteasome and 60S ribosomal precursor on bioactive ligand functionalized graphene thin film carrier on average has more detail than the two-dimensional classification of amorphous carbon carrier on average (fig. 9(c) and fig. 9(a) inset). To further confirm that the selectivity of the bioactive ligand functionalized graphene thin film loaded network to His-tagged protein was higher than that of the unlabeled protein, images of mixed His-tagged 20S proteasome and 60S ribosome precursor samples on the bioactive ligand functionalized graphene thin film loaded network lacking Ni ions (fig. 9(b)) and the Ni-NTA modified bioactive ligand functionalized graphene thin film loaded network (fig. 9(c)) were classified and calculated by single particle cryoelectron microscopy analysis. Indeed, on the Ni-NTA modified bioactive ligand functionalized graphene thin film mesh, the particle ratio of His-tagged 20S proteasome to 60S ribosomal precursor increased 9-fold over the NTA modified graphene film mesh (fig. 9 (d)). Clearly, there is still some amount of non-specific adsorption of 60s ribosomal precursors on the carrier web. This is probably due to the large number of negatively charged RNAs in the 60s ribosomal precursor.

Cryo-electron tomography (Cryo-ET) of His-tagged 20S proteasome was next performed on the bioactive ligand functionalized graphene thin film mesh to further characterize the frozen specimens (fig. 10(c), 10(d), and 12). Electron tomography clearly shows that almost all 20S particles are adsorbed to the graphene film in the same layer on the bioactive ligand functionalized graphene thin film carrier web. In contrast, in the control sample in which 20S proteasome was applied to a porous carbon mesh (Quantifoil), most of the proteasome particles were distributed to the upper or bottom air-water interface of the glass ice (fig. 10(a), fig. 10(b) and fig. 11). Notably, when graphene thin film network-supported proteins were functionalized by bioactive ligands, almost all orientations of the 20S proteasome particles were considered as a side (rectangular) view (fig. 12), consistent with the affinity binding strategy for proteasomes containing beta subunits labeled with histidine. For particles supported by porous carbon mesh, top (circular) and side (rectangular) views of 20S proteasome can be identified at the air-water interface as well as in vitreous ice (fig. 12), suggesting that His-tagged proteasomes specifically bind to bioactive ligand functionalized graphene thin films by interacting with Ni-NTA ligands on their surfaces. In summary, these experimental results show that the bioactive ligand functionalized graphene film supported network has the advantage of preventing the target macromolecule from being adsorbed to the air-water interface.

In practice, it was found that when using bioactive ligand functionalized graphene thin film grids, making frozen samples with an optimal ice thickness of 20-0 nm was more controllable (fig. 10(d) and fig. 12), and this ice thickness was considered to be an ideal choice for embedding most proteins and did not introduce additional background noise. Glass ice on the bioactive ligand functionalized graphene thin film was more uniform than on the porous carbon supported mesh (fig. 10 (a)). Furthermore, since most protein particles are in the same plane, graphene is functionalized during data processing, such as with bioactive ligandsThe estimation of the contrast transfer function of the particles on the film carrier web, such as the under-focus value of the particles, becomes more reliable. In contrast, particles supported by a porous carbon carrier mesh are adsorbed into the upper or bottom air-water interface, resulting in large variations in height and making data processing, especially CTF estimation, more complex and less accurate. Thus, from the exact same number (6,095) of particle images, the final resolution of 3D reconstruction of 20S proteasome on bioactive ligand functionalized graphene thin films is reported as

Much higher than that of the continuous carbon film and porous carbon coating

And

the final resolution of (c). (FIG. 13 (a)). The euler angle distribution of the 20S proteasome particles also showed some differences. In addition to the side view, a part of the particles on the continuous carbon or porous carbon has other views (fig. 13 (b)). However, these other views are rarely obtained on bioactive ligand functionalized graphene thin films, providing another evidence that His-tagged 20S proteasomes are captured onto bioactive ligand functionalized graphene thin films through specific interaction of Ni-NTA and His-tag. On a bioactive ligand functionalized graphene film carrier net

Resolution 3D cryoelectron microscopy reconstruction of His-tagged 20S proteasome was sufficient to unambiguously track the backbone of asymmetric alpha-and beta-subunits and identify some bulky side chains (fig. 10(e) -fig. 10 (g)). The interaction interfaces between subunits in the 20S protease body can also be solved with high precision to determine the molecular mechanism of the complex biological function (fig. 14). In conclusion, the bioactive ligand functionalized graphene film carrying net has the specificity of capturing protein with His labels, and is suitable for serving as a support material for three-dimensional reconstruction of a freezing electron microscope with atomic resolution.

Claims

1. The preparation method of the bioactive ligand functionalized graphene film comprises the following steps:

1) transferring the graphene film to an electron microscope carrier net;

3) activating the graphene film by using nitrilotriacetic acid, and then reacting with nickel salt to obtain the bioactive ligand functionalized graphene film;

the structure of the bioactive ligand functionalized graphene film is as follows:

the bioactive ligand is connected to the graphene film and is a Ni-nitrilotriacetic acid ligand;

before step 3), the method further comprises the following modification steps:

treating the graphene film with a mixed aqueous solution of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride, N-hydroxy-sulfosuccinimide and 2- (N-morpholino) ethanesulfonic acid;

in the mixed aqueous solution, the concentration of the 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride is 1-6 mM, the concentration of the N-hydroxy-sulfosuccinimide is 1-6 mM, and the concentration of the 2- (N-morpholino) ethanesulfonic acid is 10-200 mM;

the pH value of the mixed aqueous solution is 4.0-6.0;

the treatment time is 1-60 minutes.

2. The method of claim 1, wherein: in the step 2), the alkaline compound is sodium hydroxide or potassium hydroxide;

in the mixed aqueous solution, the concentration of the potassium permanganate is 0-0.4M but not zero, and the concentration of the alkaline compound is 0-0.2M but not zero;

the treatment time is 10-60 minutes.

3. The production method according to claim 1 or 2, characterized in that: in the step 3), the graphene film is placed in a TBE buffer solution containing the nitrilotriacetic acid to carry out the activation step;

the activation time is 0.1-4 hours;

the concentration of the nitrilotriacetic acid is 1.0-15 mM;

the pH value of the TBE buffer solution is 7-9.

4. The production method according to claim 3, characterized in that: in the step 3), the nickel salt is nickel sulfate, nickel chloride or nickel nitrate;

dropwise adding the aqueous solution of the nickel salt to the graphene film;

the reaction time is 0.1-1.5 hours;

the concentration of the nickel salt aqueous solution is 1.0-15 mM.