CN113960078B - Application of multifunctional graphene grid in three-dimensional reconstruction of cryoelectron microscope - Google Patents
Application of multifunctional graphene grid in three-dimensional reconstruction of cryoelectron microscope Download PDFInfo
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Abstract
The invention discloses an application of a multifunctional graphene grid in three-dimensional reconstruction of a cryoelectron microscope. The preparation method of the multifunctional graphene-supported net comprises the following steps: etching the copper foil covered by the graphene film grown by CVD to obtain the graphene net; carrying out oxygen plasma treatment on the graphene net; then processing the graphene net with MES buffer solution containing EDC and NHS; then, adopting nitrilotriacetic acid and biotin to activate the graphene net; finally reacting with nickel salt to obtain the product. The multifunctional graphene grid can specifically anchor target biomolecules on the surface of the multifunctional graphene grid, so that the problem of the interface between air and water is avoided, particle orientation distribution can be controlled, related protein purification labels are constructed on different directions of each biomacromolecule, abundant Euler angles are provided, and the structure analysis of the biomacromolecules is more reliable and more efficient, therefore, the multifunctional graphene grid can promote the universality and repeatability of applying a cryoelectron microscope in structural biology analysis.
Description
Technical Field
The invention relates to an application of a multifunctional graphene grid in three-dimensional reconstruction of a cryoelectron microscope, belonging to the technical field of biological cryoelectron microscopes.
Background
Due to the continuous development of hardware and software associated with cryoelectron microscopy, cryoelectron microscopy (cryo-EM) has now become a routine approach to addressing the atomic resolution structure of biological macromolecules, greatly expanding the understanding of biological mechanisms at the molecular level. However, this method still faces some key technical challenges, especially for frozen sample preparation. In practice, a small amount of a solution containing biomolecules is added dropwise to a fresh glow-discharge grid covered by a supporting membrane (e.g. a porous carbon membrane) and then quenched in liquid ethane. Ideally, it is expected that biomolecules will be directionally enriched embedded in a thin layer of vitrified ice supported by a noiseless membrane, but in practice this is not the case and the direction of embedding of the biomolecules is not controllable. In most cases, the biomolecule particles are absorbed into the air-water interface, which can lead to denaturation and dominant orientation problems of the biomolecules, making subsequent three-dimensional reconstruction impractical or unreliable.
Faced with these problems, thin amorphous carbon layers were explored as support films in frozen sample preparation. However, carbon films have many disadvantages, such as poor electron conductivity and strong background noise, which cause problems with sample charging and target signal immersion during cryo-electron microscopy data collection. Graphene and its derivatives are also used as a support film in a cryoelectron microscope due to their superior properties of ultra-high thermal/electrical conductivity, strong mechanical strength and nearly no background noise. In fact, these films only partially solve the air-water interface problem and hardly control the particle distribution in frozen samples. Some affinity networks (e.g., functionalized graphene, lipid monolayers or carbon films and streptavidin two-dimensional crystal films can anchor target molecules to the surface of the film, thereby keeping the target particles away from the air-water interface and in a healthier state.
Disclosure of Invention
The invention aims to provide an application of a multifunctional graphene supported mesh (MFG supported mesh) in three-dimensional reconstruction of a cryoelectron microscope, wherein the MFG supported mesh can specifically anchor target biomolecules on the surface of the MFG supported mesh, so that the problem of an air-water interface is avoided, the particle orientation distribution can be controlled, rich Euler angles are provided by constructing related protein purification labels to different directions of each biomacromolecule, and the biomacromolecule structure analysis is more reliable and efficient, so that the MFG supported mesh can promote the universality and repeatability of the cryoelectron microscope in structural biological analysis.
The multifunctional graphene net can be prepared according to the following method:
1) Etching the copper foil covered by the CVD-grown graphene film to obtain the graphene net (namely the electron microscope net and the covered graphene film) as an electron microscope net;
the electron microscope carrier mesh is provided with a hole array;
2) Carrying out oxygen plasma treatment on the graphene carrying net;
3) Treating the graphene net with 2- (N-morpholino) ethanesulfonic acid (MES) buffer solution;
the 2- (N-morpholino) ethanesulfonic acid buffer solution contains 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride and N-hydroxy-sulfosuccinimide;
4) Then, activating the graphene net treated in the step 3) by adopting nitrilotriacetic acid and biotin;
5) And 4) reacting the graphene net treated in the step 4) with nickel salt to obtain the multifunctional graphene net.
In the preparation method, in the step 1), the CVD method and the etching can be carried out according to the existing method;
the diameter of the holes on the electron microscope carrier net is 30-50 microns, and preferably 30 microns.
When the carrier net paved with 1-4 layers of graphene films is used as a starting material, the suspended graphene is ultra-clean, and carbon atoms on etched copper holes and a Moire pattern of double-layer graphene can be well recognized when the carrier net is imaged by a high-resolution STEM (figure 1 (C)); an atomic-force microscope (AFM) was also used to characterize the suspended graphene, whose surface was very flat and free of contamination. Therefore, the preparation method of the graphene net without the polymer and the transfer greatly maintains the integrity and the cleanness of the graphene film.
In the preparation method, in the step 2), the oxygen plasma treatment time is 6 to 12 seconds, namely, the graphene net-carrying is subjected to glow discharge treatment in an oxygen environment of a reactive ion etching machine, and the graphene film is activated by a low-damage oxygen plasma charging method; in contrast, the graphene film without oxygen plasma treatment is substantially hydrophobic and Ni-NTA (nickel-nitrilotriacetic acid ligand) and biotin cannot be modified on its surface (fig. 1 (D)), when graphene is treated with oxygen plasma for 6S or 12S, S2 p3/2 (168.7 eV), ni 2p3/2 (856.4 eV), ni 2p1/2 (874.1 eV) signals can be clearly identified by XPS, and the relative abundance of these elements is roughly positively correlated with charge time; the S signal and Ni are derived from biotin and Ni-NTA, respectively.
After the graphene net is subjected to oxygen plasma treatment, the graphene net is subjected to multiple functionalization by adopting Ni-NTA (nitrilotriacetic acid) and biotin.
In the step 3), in the 2- (N-morpholino) ethanesulfonic acid buffer solution, the concentration of the 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride is 5 to 10mM, preferably 5mM, and the concentration of the N-hydroxy-sulfosuccinimide salt is 5 to 10mM, preferably 5mM;
in the step 3), the treatment time is 30-60 min, such as 50min.
In the preparation method, before the step 4), the method further comprises a step of washing the graphene supported net with a TBE buffer solution.
In the preparation method, in the step 4), the graphene net-carrying is activated by using a TBE buffer solution containing the nitrilotriacetic acid and the biotin;
in the TBE buffer solution, the concentration of the nitrilotriacetic acid is 10-15 mM, and the concentration of the biotin is 30-40 mM;
the biotin may be O- (2-aminoethyl) -O' - [2- (biotinylamino) ethyl]Eight polyethylene glycol (biotion-PEG-NH) 2 ) O- (2-aminoethyl) etherRadical) -O' - [2- (biotinylamino) ethyl]Dimer polyethylene glycol, O- (2-aminoethyl) -O' - [2- (biotinylamino) ethyl]Decaethylene glycol, and the like;
the activation time is 1 to 3 hours, preferably 1.5 hours.
In the above preparation method, in step 5), the nickel salt may be nickel sulfate, nickel chloride or nickel nitrate;
dropwise adding the aqueous solution of the nickel salt to the graphene net;
the reaction time can be 0.1-1.5 h;
the concentration of the aqueous solution of the nickel salt may be 1.0 to 15mM.
The graphene film (MFG-supported web) after the multiple functionalization through steps 4) and 5) becomes very rough and has structural features (fig. 1 (E)) compared to the non-functionalized graphene film, and the height of these raised features is mostly in the range of 1 to 2 nm. The EDX elemental map of the MFG-supported mesh showed that both S and Ni were uniformly and dispersedly distributed on the graphene surface, demonstrating successful modification of biotin and Ni-NTA (fig. 1 (F)). High Angle Annular Dark Field (HAADF) imaging by STEM determines a monodisperse high contrast spot (upper graph in fig. 1 (G)) assigned to Ni and verified by the corresponding EELS spectrum (lower graph in fig. 1G). Peak value of energy loss of
And Ni L 2,3 The theoretical values of. No biotin signal is present in HAADF imaging, probably because it consists of light elements (e.g., carbon, hydrogen, and sulfur) and is susceptible to the ultra-high electron dose used in STEM. To evaluate the modification efficiency, the nickel atom density on the graphene film was calculated (fig. 1 (G)) based on the HAADF image (fig. 1 (G)), and it was found that there was more than one Ni-NTA modification site per two square nanometers on the graphene surface. Such a modification density is not only suitable for the binding of target macromolecules, which typically have diameters between a few nanometers and tens of nanometers, but also has little damage to graphene. The invention further uses TEM to image the multifunctional graphene film, and finds that the film is pollution-free and the graphene crystal lattice is well preserved, therebyAs indicated by the electron diffraction (SAED) pattern of the selected regions (inset in fig. 1 (H)).
The MFG carrier web of the present invention is hydrophilic and resistant to electron beam radiation. The invention examines hydrophilicity by a Water Contact Angle (WCA) test. According to the invention, WCA of the graphene supported net and the multifunctional graphene supported net is measured, and the angle is reduced from 97.4 degrees to 39.1 degrees after functionalization (fig. 2 (A)), so that the method is suitable for subsequent preparation of biological electron microscope specimens. The superior properties of graphene, such as ultra-high thermal and electrical conductivity and strong mechanical strength, are mainly attributed to the perfect two-dimensional hexagonal lattice formed by sp2 hybridized carbon atoms. Raman spectrum shows that no D peak (1350 cm) exists in the graphene film before modification -1 ) Indicating that graphene has few defects (fig. 2 (B), upper blue curve). After multiple functionalization, a weak D peak appears, and the intensity ratio of the D peak to the G peak (1580 cm) -1 ) Is about 0.2 (fig. 2 (B), lower red curve), much lower than graphene oxide. Further, 2D (2690 cm) of graphene -1 ) And the G peak, which indicates that the intrinsic lattice of graphene is well preserved after the multifunctionalization treatment of the present invention. In order to measure the mechanical properties of the MFG, the present invention performed AFM nanoindentation experiments on the suspended MFG. As shown in fig. 3 (C), the elastic coefficient of the MFG is only slightly lower than that of the original graphene (left side), indicating that the sigma bond is maintained in the graphene lattice and corresponds to the raman result of the MFG with weak D peak (fig. 2 (B)). Further, the present invention characterizes radiation damage on graphene and multifunctional graphene films using a TEM with a transmittance of about 100e-/A2/s (FIG. 2 (D)), records the integrated intensities of the third-order (I3 rd) and first-order (Imax) Bragg diffraction peaks in series and plots the relative intensities (I3 rd/Imax) against the cumulative dose (FIG. 2 (D)), and found that up to about 100e-/A2/s
The relative intensity of the multifunctional graphene is only slightly attenuated under the irradiation of (2), which is very suitable for imaging under TEM.
The MFG-supported mesh has binding specificity to a target biomolecule. The invention relates to aThe binding specificity of the multifunctional graphene membrane is verified by a fluorescence microscope. In biochemical assays, ni-NTA can specifically bind to histidine-tagged (His-tag) proteins, while biotin can capture streptavidin with high affinity. Specifically, the present invention first performs multiple functionalization of graphene films grown on copper foil with Ni-NTA and biotin ligands, and then incubates about 500 μ l of a protein solution containing His-tagged Red Fluorescent Protein (RFP) and streptavidin-FITC with MFG. FITC will produce green fluorescence if excited. Thereafter, the mfg. mesh-carrying surface was thoroughly washed with various buffers (fig. 3). When imaged with dual wavelength excitation under a fluorescence microscope, high density of red and green spots were visible on the functionalized graphene regions after washing with normal sample buffer (fig. 3 (a)), indicating that His-tagged RFP and streptavidin-FITC bound to the graphene surface. In contrast, the unfunctionalized regions had little red or green signal. Imidazole can competitively chelate nickel ions. In the presence of imidazole, the red signal was almost completely washed away, while the green signal was retained (fig. 3 (B)). At the same time, high concentration
The biotin solution washed away most of the green signal but had little effect on the red signal (FIG. 3 (C)). When the MFG surface was washed with imidazole and high concentration biotin solution simultaneously, both red and green signals disappeared (fig. 3 (D)). In addition, the present invention also performed a binding experiment on single ligand functionalized graphene by fluorescence microscopy using Ni-NTA or biotin, and obtained the same result. The above results indicate that the multifunctional graphene realized by Ni-NTA and biotin ligand can bind to the target protein with high specificity.
The MFG grid provided by the invention can well solve the problems of air-water interface adsorption and dominant orientation. To demonstrate the ability of mfg. networks to avoid the adsorption of biomolecules at the air-water interface, the present invention uses the frozen Electron Tomography (ET) method to reconstruct frozen specimens and locate individual 20S proteasome particles, whose beta subunit is tagged with a histidine tag and alpha subunit is tagged with an SBP tag. These two different tags are constructed on different sides of the 20S proteasome, the His-tag on the side and the SBP on the top, and they specifically bind to various ligands modified on the MFG cargo net. As a control, conventional porous carbon and graphene films were characterized. In porous carbon webs, protein particles are distributed predominantly on the air-water interface of the specimen and ice thickness of about 80nm is estimated by measuring the distance between the two particle distribution layers (fig. 4 (a) and, furthermore, there is a serious dominant orientation problem for 20S proteasomes, the main orientation of which is a side view, i.e. a rectangular view (fig. 4 (a), right).
To further test whether the MFG mesh can allow direction control, the present invention compares the euler angle distribution of mono-ligand functionalized graphene films to MFG. In 3D reconstruction based on 20S proteasome particles collected on mono-ligand functionalized graphene membranes, the preferred directions were found to be the side view of Ni-NTA functionalized graphene (fig. 5 (a)) and the top view of biotin functionalized graphene (fig. 5 (B)), respectively. These data are consistent with previous statements that Ni-NTA binds to His tag constructed laterally to the 20S proteasome, whereas biotin-streptavidin binds to SBP tag on the upper surface of the 20S proteasome. As expected, the euler angle distribution becomes more balanced when the 20S proteasome is applied to a multifunctional graphene mesh (fig. 5 (C)), and finally the cryo-electron microscopy structure of the 20S proteasome was determined, in which the MFG mesh supported 12,451 particles (fig. 5 (D)). The changed Euler angle distribution caused by the modification of different affinity ligands proves that the MFG grid has great potential for designing the orientation distribution of target biomolecules and ensures that the structural reconstruction of the cryoelectron microscope is more reliable.
The air-water interface is always a big problem in the preparation of biological cryoelectron microscope samples, and the dispersibility of biomolecules and the directional distribution of ice are difficult to control, so that the structural solving efficiency of the cryoelectron microscope is greatly limited. Based on the multifunctional graphene supported net, experiments confirm that the multifunctional graphene supported net can specifically anchor target biomolecules on the surface of the multifunctional graphene supported net, so that the problem of an interface between air and water is avoided. Furthermore, multifunctional graphene enables the artificial design of particle orientation distributions compared to single ligand modification. It is therefore expected that a mfg. mesh modified with multiple affinity ligands will facilitate the versatility and reproducibility of the application of cryoelectron microscopy in structural biological analysis.
Drawings
FIG. 1 is a representation of Ni-NTA and biotin functionalized graphene loaded mesh (MFG loaded mesh); fig. 1 (a) is an optical microscope image of a graphene net made by directly etching a copper foil. Fig. 1 (B) is a low magnification TEM image of graphene supported mesh showing aligned pores of about 30 μm diameter, fig. 1 (C) is a high resolution STEM image showing the moire pattern of double layer suspended graphene, fig. 1 (D) is an XPS examination as a function of modification efficiency and oxygen plasma treatment time, fig. 1 (E) is a typical AFM image of MFG with significant protrusions on it, fig. 1 (F) is EDX element mapping of S and Ni on MFG, fig. 1 (G) is a HAADF STEM image of MFG and corresponding EELS data, fig. 1 (H) is a TEM micrograph of MFG, and the inset is SAED mode.
FIG. 2 is an energy absorption test of graphene and MFG webs; wherein fig. 2 (a) is hydrophilicity of graphene (above) and mfg. mesh (below) as measured by WCA; FIG. 2 (B) is a comparison of Raman spectra of graphene (top) and MFG mesh (bottom) showing 2D (2690 cm) -1 )、D(1350cm -1 ) And G (1580 cm) -1 ) A peak; FIG. 2 (C) is the elastic coefficients of graphene (right-hand curve) and MFG grid (left-hand curve) characterized by AFM nanoindentation experiments; fig. 2 (D) is the ratio of the third-order diffraction point intensity (I3 rd) to the first-order (Imax) point intensity versus dose accumulation (upper curve for graphene, lower curve for MFG mesh).
FIG. 3 is the result of characterization of the binding specificity of MFG-loaded mesh by fluorescence microscopy; FIG. 3 (A) shows that red and green signals were detectable on MFG after incubation with His-tagged RFP and streptavidin-FITC; FIG. 3 (B) shows only green spots on MFG when washed with imidazole solution; FIG. 3 (C) shows that most of the green spots were washed away and the red spots were retained after washing with the high concentration biotin solution; FIG. 3 (D) shows that the red and green spots disappeared when washed with imidazole and high concentration biotin solution.
FIG. 4 is a graph of particle location in a cryogenic sample prepared by using different support membranes; wherein, FIG. 4 (A) is a distribution of protein particles on a porous carbon film; FIG. 4 (B) shows the distribution of protein particles on a graphene membrane; FIG. 4 (C) is the protein particle distribution of protein particles on the MFG; the left graph shows the uptake of protein particles at the air-water interface, the middle graph shows the particle position, each spot represents an individual particle, and the right graph shows the distribution cross section of the 20S proteasome in the ice layer reconstructed using cryo-electron tomography.
FIG. 5 is an Euler angle distribution of 20S proteasomes on a mono-functional or multifunctional graphene membrane; wherein fig. 5 (a) is listed for euler angle distribution on Ni-NTA functionalized graphene film and corresponding dominant orientation, fig. 5 (B) is euler angle distribution on biotin functionalized graphene, fig. 5 (C) is euler angle distribution on multiple affinity ligand functionalized graphene film (MFG), fig. 5 (D) is 3D reconstruction based on 20S proteasome particles supported by MFG mesh.
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.
Characterization of graphene loading nets in the following examples:
the structure of the graphene carrier web was characterized by Optical Microscopy (OM) (Nikon, DS-Ri 2), SEM (acceleration voltage 1-2kV, hitachi S-4800), AFM (scansyst mode, bruker size icon, hint: SNL-A), raman spectrometer (532nm, labRAM HR-800, horiba), XPS (Kratos Analytical Ltd.), and high resolution aberration-corrected STEM (60kV, nion U-HERMS 200).
AFM nanoindentation testing of graphene loaded screens in the following examples:
nanoindentation testing was performed on pristine and multifunctional suspended graphene films using a commercial AFM system (Icon) with commercial cues. The suspended graphene film was scanned with tapping mode prior to each indentation to find and ensure complete suspended graphene on the pores. The AFM tip was then centered on the center of the circular hole. In performing mechanical testing, a force-displacement mode was used. The elastic coefficient can be quantified using a force-displacement curve and previously reported theoretical models.
Protein sample preparation in the following examples:
RFP protein with His-tag at the N-terminus was purified from E.coli, while streptavidin-FITC protein was purchased from Thermo Fisher (catalog SA 1001). Construction and purification of the 6 × His and SBP 9-tagged 20S proteasome were as follows: 1) The N-terminus of the β -subunit was introduced with 6 histidines and the C-terminus of the α -subunit was added with SBP by PCR, respectively. 2) Co-transforming these engineered β and α subunit plasmids into escherichia coli competent cells; 3) Expression and assembly of the β and α subunits in vivo into the full 20S proteasome; 4) Coli cells were disrupted by sonication, and the cell lysate containing the 20S proteasome was centrifuged and applied to a nickel column. After incubation, the nickel column bound 20S proteasome was competitively eluted with 300mM imidazole buffer and further purified by molecular sieve system using imidazole-free buffer (50 mM Tris-HCl,100mM NaCl).
Cryo-electron microscopy experiments and structural analysis in the following examples:
first 0.02mg/ml streptavidin solution was incubated with the multifunctional graphene carrier for about 20 minutes, and then the carrier was washed 3-5 times with buffer (50 mM Tris-HCl,100mM NaCl). About 5 μ L of 20S proteasome solution was pipetted onto freshly processed MFG mesh and incubated in a high humidity chamber for about 20min. Then, the net is used
Protein buffer (50 mM Tris-HCl,150mM NaCl) was gently washed 3 times and then transferred to Vitrobot (Thermo Fisher Scientific) at a temperature of 12 ℃ and a humidity of 100%. The time for the filter paper clip to absorb water is set as 3s, and the suction force of the filter paper clip is set as-2. After blotting, the mesh was quickly immersed in liquid ethane and then stored in liquid nitrogen for further cryoelectron microscopy.
Frozen samples were transferred to a Titan Krios microscope (Thermo Fisher Scientific) at an acceleration voltage of 300keV in liquid nitrogen. Then using a K2 direct electron detector camera (Gatan Corp.) at an under-focus range of-1.5 μm to-2.5 μm, at a magnification of 22500X and
collects data. Total cumulative electron exposure dose for a single photograph is->
Each photograph was divided into 32 frames and applied to subsequent movement corrections by the MotionCor2 program. During reconstruction, the CTF and under-focus values of all these motion corrected micrographs were first estimated by the CTFFIND4 software package. The particles were then automatically selected and subjected to several rounds of two-dimensional classification by relion 3.1. And removing the particles with wrong classification shape categories, and using the rest particles for further three-dimensional classification and refined reconstruction. In the final refinement step, the orientation distribution analysis was performed using the D7 symmetry and plotting the euler angle for each particle.
Cryoelectron microscopy tomography analysis in the following examples:
the cryo-ET data set was collected using SerialEM software on a Titan Krios microscope (Thermo Fisher Scientific,300 keV) equipped with a K2 direct electron detector (Gatan Corp.). For each tilt series, the acquisition was from +51 ° to-51 ° in steps of +3 °, with an under-focus value of-5.0 μm. The electron dose is about
The total dosage is about
The enlarged physical pixel size of all photos is ≥>
After the oblique series of photographs were centered, they were reconstructed using the IMOD program, and all photographs were bin4 compressed. Coordinates of 20S proteasome particles during Cryo-ET reconstruction were located by manual picking.
Example 1 preparation and structural characterization of MFG Carrier webs
1) Preparation of graphene carrier net
Firstly, growing a graphene film on a copper foil by adopting a CVD (chemical vapor deposition) method, and then directly and selectively etching the copper foil into an electron microscope grid micro-grid to obtain an ultra-clean and firm graphene grid, wherein the grid comprises holes arranged in an array (figure 1 (A) and figure 1 (B)).
During the preparation process of the low-temperature sample, the coverage degree and the integrity on the carrying net of the graphene depend on the pore diameter and the number of graphene layers, and when the pore diameter of the double-layer or multi-layer graphene is about 30 mu m, the coverage degree and the integrity on the carrying net of the graphene can be well maintained. This example uses a mesh coated with 1 to 4 layers of graphene as the starting material. The suspended graphene is ultra clean and when imaged by high resolution STEM, the carbon atoms on the etched copper pores and the moire pattern of the bi-layer graphene are well recognized (fig. 1 (C), the STEM image is gaussian blurred to reduce noise). Atomic-force microscopy (AFM) was also used to characterize suspended graphene, whose surface was very flat and free of contamination. Therefore, the manufacturing method of the graphene net without polymer and transfer greatly maintains the integrity and cleanness of the graphene film.
2) Functionalization of graphene-supported networks
The graphene grid array was first glow-discharged for 12 seconds in a 5sccm oxygen atmosphere in a reactive ion etcher (Pico SLS, diener), and then 5. Mu.l of 100mM MES buffer (pH 5.0) containing 5.0mM 1-ethyl-3- (3-bis-MES) was immediately aspiratedMethyl-aminopropyl) carbodiimide hydrochloride (EDC, sigma-Aldrich Company, CAS 25952-53-8) and 5.0mM N-hydroxy-sulfosuccinimide (sulfo-NHS, sigma-Aldrich Company, CAS 106627-54-7)) were placed on a freshly glow-discharged graphene mesh. After 50min incubation at room temperature, the graphene loaded mesh was washed with 50mM TBE buffer (pH 8.5) and 11.3mM NTA-NH dissolved 2 And 30mM biotion-PEG-NH 2 (O- (2-aminoethyl) -O' - [2- (biotinylamino) ethyl ] ethyl]Octa polyethylene glycol) of the buffer solution, activating the graphene net for 1.5h, and then reacting with 11.3mM NiSO 4 The solution was incubated for 1h. Finally, the carrier web is washed with distilled water or buffer.
In order to avoid the copper foil from being oxidized under harsh chemical conditions, the invention uses a low-damage oxygen plasma charging method to activate the graphene film and optimizes the processing time of oxygen plasma to obtain a proper functionalization result characterized by X-ray photoelectron spectroscopy (XPS). In contrast, graphene films that were not oxygen plasma treated were essentially hydrophobic, and Ni-NTA and biotin could not be modified at their surface (fig. 1 (D)). When graphene is treated with oxygen plasma for 6S or 12S, S2 p3/2 (168.7 eV), ni 2p3/2 (856.4 eV), ni 2p1/2 (874.1 eV) signals can be clearly identified by XPS, and the relative abundance of these elements is roughly in positive correlation with the charge time. The S signal is from the Ni of biotin and Ni-NTA, respectively. However, the S and Ni contents were not increased at the 18S and 24S treatment times, indicating that the 12S treatment of oxygen plasma may be sufficient to saturate the modification of the graphene surface (fig. 1 (D)).
AFM characterization was first performed on the surface of multiple affinity ligand functionalized graphene (MFG), which became very rough and structurally characterized compared to unfunctionalized graphene films (fig. 1 (E)). The height of these raised features is mostly in the range of 1-2 nm. The EDX elemental map of the MFG-supported mesh showed that both S and Ni were distributed uniformly and dispersedly on the graphene surface, demonstrating successful modification of biotin and Ni-NTA (fig. 1 (F)). High Angle Annular Dark Field (HAADF) imaging by STEM determines a monodisperse high contrast spot (FIG. 1 (G) top view) assigned to Ni and processed by the corresponding EELS spectrumVerification (fig. 1 (G) lower panel). Peak value of energy loss of
With Ni L 2,3 The theoretical values of (a) and (b) are identical. No biotin signal is present in HAADF imaging, probably because it consists of light elements (e.g., carbon, hydrogen, and sulfur) and is susceptible to the ultra-high electron dose used in STEM. To evaluate the modification efficiency, the nickel atom density on the graphene film was calculated (fig. 1 (G)) based on the HAADF image (fig. 1 (G)), and it was found that there was more than one Ni-NTA modification site per two square nanometers on the graphene surface. Such a modification density is not only suitable for the binding of target macromolecules, which typically have diameters between a few nanometers and tens of nanometers, but also has little damage to graphene. Further imaging of the multifunctional graphene film using TEM found that the film was non-contaminating and the graphene lattice was well preserved, as indicated by the electron diffraction (SAED) mode of its selected regions (inset in fig. 1 (H)).
Example 2 bioapplication Performance Studies of MFG-Supported nets
1. The MFG carrier is hydrophilic and resistant to electron beam radiation
The invention examines hydrophilicity by a Water Contact Angle (WCA) test. According to the invention, WCA of the graphene supported mesh and the multifunctional graphene supported mesh is measured, and the angle is reduced from 97.4 degrees to 39.1 degrees after functionalization (shown in figure 2 (A)), so that the method is suitable for subsequent preparation of biological electron microscope specimens. The superior properties of graphene, such as ultra-high thermal and electrical conductivity and strong mechanical strength, are mainly attributed to the perfect two-dimensional hexagonal lattice formed by sp2 hybridized carbon atoms. Raman spectrum shows that no D peak (1350 cm) exists in the graphene film before modification -1 ) Indicating that graphene has few defects (fig. 2 (B), upper blue curve). After multiple functionalization, a weak D peak appears, and the intensity ratio of the D peak to the G peak (1580 cm) -1 ) Is about 0.2 (fig. 2 (B), lower red curve), much lower than graphene oxide. Further, 2D (2690 cm) of graphene -1 ) And the G peak are well preserved, which shows that the graphene is subjected to the multi-functionalization treatment of the inventionThe intrinsic lattice is well preserved. In order to measure the mechanical properties of the MFG, the present invention performed AFM nanoindentation experiments on the suspended MFG. As shown in fig. 2 (C), the elastic coefficient of the MFG is only slightly lower than that of the original graphene (left curve), indicating that the sigma bonds are retained in the graphene lattice and correspond to the raman results for the MFG with weak D peaks (fig. 2 (B)). Next, radiation damage on graphene and multifunctional graphene films was characterized using a TEM with a transmittance of about 100e-/A2/s (FIG. 2 (D)). The integrated intensities of the third (I3 rd) and first (Imax) order bragg diffraction peaks were recorded in series and the relative intensities (I3 rd/Imax) were plotted against the cumulative dose (fig. 2D). Found in up to
The relative intensity of the multifunctional graphene is only slightly attenuated under the radiation of (2), which is very suitable for imaging under TEM.
2. MFG carrier networks having binding specificity for target biomolecules
The binding specificity of the multifunctional graphene membrane is verified through a fluorescence microscope. In biochemical assays, ni-NTA can specifically bind to histidine-tagged (His-tag) proteins, while biotin can capture streptavidin with high affinity. Graphene films grown on copper foil were first multi-functionalized with Ni-NTA and biotin ligands and then incubated with MFG for about 500 μ l of a protein solution containing His-tagged Red Fluorescent Protein (RFP) and streptavidin-FITC. FITC will produce green fluorescence if excited. Thereafter, the MFG surface was thoroughly washed with various buffers (fig. 3). When imaged with dual wavelength excitation under a fluorescence microscope, high density of red and green spots were visible on the functionalized graphene regions after washing with normal sample buffer (fig. 3 (a)), indicating that His-tagged RFP and streptavidin-FITC bound to the graphene surface. In contrast, the unfunctionalized regions had little red or green signal. Imidazole can competitively chelate nickel ions. In the presence of imidazole, the red signal was almost completely washed away, while the green signal was retained (fig. 3 (B)). At the same time, high concentration
The biotin solution of (a) washed away most of the green signal but had little effect on the red signal (fig. 3 (C)). When the MFG surface was washed with imidazole and high concentration biotin solution simultaneously, both red and green signals disappeared (fig. 3 (D)). In addition, the present invention also performed a binding experiment on single ligand functionalized graphene by fluorescence microscopy using Ni-NTA or biotin, and obtained the same result. The above results indicate that the multifunctional graphene realized by Ni-NTA and biotin ligand can bind to the target protein with high specificity.
3. The MFG well solves the problem of air-water interface adsorption and the problem of dominant orientation
To demonstrate the ability of MFG to avoid adsorption of biomolecules at the air-water interface, the present invention uses the frozen Electron Tomography (ET) method to reconstruct frozen specimens and locate individual 20S proteasome particles, whose beta subunit is tagged with a histidine tag and alpha subunit is tagged with an SBP tag. These two different tags are constructed on different sides of the 20S proteasome, the His-tag on the side and the SBP on the top, and they specifically bind to various ligands modified on the MFG cargo net. As a control, conventional porous carbon and graphene films were characterized. In porous carbon grids, protein particles are mainly distributed at the air-water interface of the specimen, and ice thickness of about 80nm is estimated by measuring the distance between two particle distribution layers (fig. 4 (a)), and there is a serious problem of dominant orientation of the 20S proteasome, whose main orientation is a side view, i.e., a rectangular view (fig. 4 (a), right panel). In the unfunctionalized graphene carrier web, many particles were found to be absorbed onto the air-water interface, although many particles lay down on the graphene surface (fig. 4 (B), left and middle panels). The side view is again the preferred orientation of the graphene surface and air-water interface (fig. 4 (B), right panel). In contrast, MFG supports that almost all protein particles in the frozen sample are located on the same layer of the MFG surface, and their orientation is relatively rich in both top (circular) and side (rectangular) views (fig. 4 (C), right). The results show that the MFG membrane can anchor target protein particles on its surface, thereby solving the problem of air-water interfacial adsorption.
To further test whether the MFG mesh can allow direction control, the present invention compares the euler angle distribution of mono-ligand functionalized graphene films to MFG. In 3D reconstruction based on 20S proteasome particles collected on mono-ligand functionalized graphene membranes, the preferred directions were found to be the side view of Ni-NTA functionalized graphene (fig. 5 (a)) and the top view of biotin functionalized graphene (fig. 5 (B)), respectively. These data are in agreement with the above, i.e. Ni-NTA binding to His tag constructed laterally to the 20S proteasome, whereas biotin-streptavidin binds to SBP tag on the upper surface of the 20S proteasome. As expected, the euler angle distribution becomes more balanced when the 20S proteasome is applied to a multifunctional graphene mesh (fig. 5 (C)), and finally the cryo-electron microscopy structure of the 20S proteasome was determined, in which the MFG mesh supported 12,451 particles (fig. 5 (D)). The changed Euler angle distribution caused by the modification of different affinity ligands proves that the MFG grid has great potential for designing the orientation distribution of target biomolecules and ensures that the structural reconstruction of the cryoelectron microscope is more reliable.
Although the number of high resolution structures of macromolecules resolved using single particle electron microscopy techniques has continued to increase in recent years, the generality and reliability of this technique are severely challenged by the problems of gas-liquid interfaces in the preparation of samples and the dominant orientation of particles, which require a great deal of effort in actual sample preparation. The present invention provides multifunctional graphene (MFG) as a support membrane for frozen samples, which exhibits accuracy of binding to biomacromolecule purification tags by covalently modifying the graphene membrane with multi-affinity ligands, and prevents the occurrence of gas-liquid interface problems of these biomacromolecules during frozen sample preparation. Then, according to the characteristic that the multi-affinity ligand graphene (MFG) has different affinity ligands, the distribution of the particle direction can be designed, and related protein purification labels are constructed on different directions of each biomacromolecule, so that abundant Euler angles are provided, and the structure analysis of the biomacromolecules is more reliable and more efficient.
Claims (1)
1. The application of the multifunctional graphene net in the three-dimensional reconstruction of a freezing electron microscope;
the multifunctional graphene net-carrying specifically binds to a target biomolecule or protein and is anchored on the surface of the target biomolecule or protein;
the multifunctional graphene net carrier controls the particle orientation distribution of the target biomolecule or the protein;
the multifunctional graphene grid is prepared according to a method comprising the following steps:
1) Etching a copper foil covered by a CVD (chemical vapor deposition) grown graphene film to be used as an electron microscope grid, so as to obtain the graphene grid;
the electron microscope carrier mesh is provided with a hole array and is obtained by the etching step;
the diameter of the mesh on the electron microscope carrier is 30-50 microns;
2) Carrying out oxygen plasma treatment on the graphene net;
the time of the oxygen plasma treatment is 6 to 12 seconds;
3) Treating the graphene net with a 2- (N-morpholino) ethanesulfonic acid buffer solution;
the 2- (N-morpholino) ethanesulfonic acid buffer solution contains 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride and N-hydroxy-sulfosuccinimide;
in the 2- (N-morpholino) ethanesulfonic acid buffer solution, the concentration of the 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride is 5-10 mM, and the concentration of the N-hydroxy-sulfosuccinimide salt is 5-10 mM;
the treatment time is 30-60 min;
4) Then, activating the graphene net treated in the step 3) by adopting nitrilotriacetic acid and biotin;
activating the graphene net with a TBE buffer solution containing the nitrilotriacetic acid and the biotin;
in the TBE buffer solution, the concentration of the nitrilotriacetic acid is 10-15 mM, and the concentration of the biotin is 30-40 mM;
the biotin is O- (2-aminoethyl) -O ' - [2- (biotinylamino) ethyl ] octapolyethylene glycol, O- (2-aminoethyl) -O ' - [2- (biotinylamino) ethyl ] dipolypolyethylene glycol or O- (2-aminoethyl) -O ' - [2- (biotinylamino) ethyl ] decapolyethylene glycol;
the activation time is 1-3 h;
before the step 4), the method further comprises the step of washing the graphene net with a TBE buffer solution;
5) Reacting the graphene net treated in the step 4) with nickel salt to obtain the multifunctional graphene net;
the nickel salt is nickel sulfate, nickel chloride or nickel nitrate;
dropwise adding the aqueous solution of the nickel salt to the graphene net;
the reaction time is 0.1 to 1.5 hours;
the concentration of the nickel salt aqueous solution is 1.0-15 mM.
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