Functionalized nanomembrane, a method for preparation thereof and their use

Abstract

The present invention discloses functionalized nanomembranes, a method for preparation and their use. The functionalized nanomembrane comprises a) a first layer comprising a nanomaterial, b) a second layer comprising a biorepulsive material, the second layer being attached to at least one side of the first layer, and c) affinity groups, attached to the second layer.

Claims

1. Functionalized nanomembrane, comprising a) a first layer consisting of a nanomaterial, selected from a carbon nanomembrane and a film of amorphous carbon, b) a second layer comprising a biorepulsive material, the second layer being attached to at least one side of the first layer, and c) affinity groups, attached to the second layer, wherein one or more chemical bonds between respective functional groups of the nanomaterial, the biorepulsive material, and the affinity groups are comprised in a functionalized nanomembrane, wherein the biorepulsive material substantially forms an outer surface of the second layer, wherein said outer surface of the second layer forms an interface between the first layer and the second layer and the second layer substantially consists of the biorepulsive material; wherein the carbon nanomembrane has a thickness in a range of 0.5-4 nm; and wherein when the functionalized nanomembrane is a functionalized carbon nanomembrane, the functionalized carbon nanomembrane has a thickness in a range of 3-25 nm.

2. Functionalized nanomembrane according to claim 1, wherein the biorepulsive material consists of polyglycerol (PG), polyethyleneglycol (PEG), oligoethyleneglycol (OEG), peptides, proteins, oligo-carbohydrates, or (zwitter-)ionic polymers.

3. Functionalized nanomembrane according to claim 1, wherein the affinity group is one species selected from a specific recognition pair.

4. Method for preparing a functionalized nanomembrane according to claim 1, comprising the steps a) providing a first layer comprising a nanomaterial, b) functionalization of the first layer with a biorepulsive material for obtaining a second layer comprising the biorepulsive material, and c) functionalization of the second layer with affinity groups.

5. Method according to claim 4, wherein the first layer, consisting of the nanomaterial, is a nanomaterial supported on a TEM grid.

6. A method of structural analysis of biomolecules, comprising using the functionalized nanomembrane, according to claim 1, as support film in transmission electron microscopy (TEM).

7. The method of structural analysis, comprising using the functionalized nanomembrane according to claim 6, wherein the functionalized nanomembrane is supported on a TEM grid.

8. Functionalized nanomembrane according to claim 1, wherein the functionalized carbon nanomembrane has a thickness in a range of 3-10 nm.

9. Functionalized nanomembrane according to claim 1, wherein the biorepulsive material consists of polyglycerol (PG), polyethyleneglycol (PEG), oligoethyleneglycol (OEG), peptides, proteins, oligo-carbohydrates, or (zwitter-)ionic polymers.

10. Functionalized nanomembrane according to claim 1, wherein the affinity group is one species selected from a specific recognition pair.

11. Functionalized nanomembrane according to claim 1, wherein the affinity group is one species selected from a specific recognition pair comprising chelate complexes/oligo-His, biotin/(strept)avidin, or specific DNA/RNA sense/antisense pairs.

12. Functionalized nanomembrane according to claim 10, wherein the specific recognition pair comprises chelate complexes/oligo-His, biotin/(strept)avidin, or specific DNA/RNA sense/antisense pairs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is now further illustrated by the accompanying figures and detailed description from which further features and advantages may be taken. It is to be noted that the following explanations are presented for the purpose of illustration and description only; they are not intended to be exhaustive or to limit the invention to the precise form disclosed.

(2) FIG. 1 schematically illustrates chemical nanolithography of a carbon nanolayer arranged on a support material.

(3) FIG. 2 shows (a) a functionalized carbon nanomembrane having PG as biorepulsive material and an EDTA derivative as affinity group, (b) a X-ray photoelectron spectra (XPS) and (c) a low-resolution helium ion microscopy (HIM) image of the free-standing functionalized carbon nanomembrane.

(4) FIG. 3 shows a schematic view of using a functionalized carbon nanomembrane for in situ separation/isolation of appropriately tagged biomolecules from cell lysate.

(5) FIG. 4 shows TEM images of negatively stained polyhistidine-tagged (His-Tag) biomolecules specifically bound to PG and EDTA functionalized carbon nanomembranes.

(6) FIG. 5 shows (a) a CNM transferred to a graphene sheet and (b) functionalized CNM patterned with graphene stripes.

(7) FIG. 6 shows a TEM image of negatively stained His-tagged thermosome from P. furiosus on functionalized amorphous carbon film mounted on Quantifoil grids.

DETAILED DESCRIPTION

(8) FIG. 1 shows chemical nanolithography of a carbon nanomembrane arranged on a support material via electron irradiation or extreme UV (EUV) light. Electron irradiation of aromatic SAMs results in their lateral cross-linking (A. Turchanin et al., Proc. Surf Sci. 2012, 87, 108; W. Geyer et al., Appl. Phys. Lett. 1999, 75, 2401; A. Turchanin et al., Langmuir 2009, 25, 7342). The cross-linking converts the SAM into a mechanically stable molecular nanolayer with a thickness of one molecule, which can be tuned from 0.5 to 3 nm (A. Turchanin et al., ACS Nano 2013, 7, 6489; U.S. Pat. No. 8,377,243 B2). In case of chemical nanolithography of nitro group-terminated biphenyl SAMs, cross-linked amino-terminated areas in a 4-nitro-1,1-biphenyl-4-thiol (NBPT) SAM on gold may be produced. In particular, nitro groups are reduced into the respective amino groups, which may be modified for the preparation of functionalized nanomembranes (U.S. Pat. No. 6,764,758 B1).

(9) A similar cross-linking of the aromatic SAMs can be attained with extreme UV (EUV) light. In addition, EUV opens new opportunities for the fabrication of nanopatterned nanomembranes by using EUV interference lithography (EUV-IL). EUV-IL combines the advantages of a parallel fabrication process with very high resolution below 10 nm. Its nanopatterning capability is far beyond that of photolithography, electron beam lithography, and scanning probe lithography, in terms of resolution or throughput. It may be used for making free-standing patterned nanomembranes of various shapes.

(10) Free-standing carbon nanomembranes may also be chemically functionalized. In some cases, even a second face on the carbon nanomembrane is available for modifications. These free-standing bifacial carbon nanomembranes are usually known as Janus nanomembranes.

(11) FIG. 2 shows a functionalized carbon nanomembrane having PG as biorepulsive material and EDTA as affinity groups. It has been shown that the amino-terminated, cross-linked surfaces could not only be rendered biorepulsive by a grafting process, but can also be modified by acylation chemistry. Preferably, multidentate ligands, such as EDTA, are used as affinity groups, which are capable of reversibly binding Ni.sup.2+ ions. FIG. 2b shows X-ray photoelectron spectra (XPS) of a pristine functionalized carbon nanomembrane (top), the same membrane after incubation with Ni.sup.2+ (center) and after removal of Ni.sup.2+ with EDTA solution (bottom). XPS analysis shows that the functionalized carbon nanomembranes bind Ni.sup.2+ reversibly, which means that a reversible attachment/detachment of the specimen becomes possible. Furthermore, free-standing PG and EDTA functionalized carbon nanomembranes were transferred from the original gold substrate onto TEM grids. FIG. 2c shows a low-resolution helium ion microscopy (HIM) image of a free-standing PG and EDTA functionalized carbon nanomembrane on a Quantifoil TEM grid.

(12) The inventive functionalized nanomembranes may be used as TEM support films for the specific immobilization of biomolecules on their surface via bio-recognition reactions. FIG. 3 shows the concept of using the inventive carbon nanomembranes. Selective immobilization of biomolecules is achieved just by immersion of the functionalized nanomembrane into a raw mixture, for example, a cell lysate. The hydrogel intermediate layer prevents the unspecific binding of constituents of the cell lysate to the nanomembrane. The final assembly is suitable for vitrification and subsequent structural analysis by cryoTEM.

(13) FIG. 4 shows TEM images of negatively stained His-tagged biomolecules specifically bound to carbon nanomembranes functionalized with PG and EDTA. TEM analysis reveals that His-tagged thermosome molecules bind to functionalized carbon nanomembranes, whereas thermosomes without His-Tag do not bind to functionalized carbon nanomembranes. FIG. 4a shows a TEM image of negatively stained His-tagged thermosome molecules from Pyrococcus furiosus attached to the functionalized carbon nanomembrane. FIG. 4c shows a TEM image of a control experiment. It shows a functionalized carbon nanomembrane after incubation with bare thermosome without His-Tag and subsequent negative staining. Apart from dark deposits no thermosome particles are visible.

(14) FIG. 5 shows how functionalized carbon nanomembranes can be coupled to graphene to enhance the conductivity to reduce sample charging during the TEM measurements. FIG. 5a shows the deposition of the functionalized nanomembrane onto a separately fabricated graphene sheet. Charges collected or formed in the nanomembrane can be easily transferred to the highly conductive graphene sheet due to the proximity (e.g. by tunneling). In FIG. 5b parts of the carbon nanomembrane have been transformed into graphene stripes, e.g. by prolonged local treatment with electrons before the remaining parts of the CNM become functionalized. During TEM analysis of biomolecules, charges are then collected by the conductive graphene stripes, which are electrically grounded. Both strategies keep the samples electrically neutral and reduce scattering effects by charge/charge interaction.

(15) FIG. 6 shows a TEM image of negatively stained His-tagged thermosome from P. furiosus on functionalized amorphous carbon film mounted on Quantifoil grids.

EXAMPLES

(16) 1) Formation of a Carbon Nanomembrane (CNM)

(17) CNMs are prepared by electron irradiation induced crosslinking of 4-nitro-1,1-biphenyl-4-thiol (NBPT) self-assembled monolayers (SAMs) on gold. To form the SAMs, 300 nm films of thermally evaporated Au on mica are used. The substrates are cleaned in a UV/ozone-cleaner, rinsed with ethanol and blown dry in a stream of nitrogen. For the SAM formation, two methods can be used. Either the substrates are immersed in a 10 mmol solution of NBPT in dry, degassed dimethylformamide (DMF) for 72 h in a sealed flask under nitrogen. Afterwards samples are rinsed with DMF and ethanol and blown dry with nitrogen. Alternatively, NBPT is evaporated from a Knudsen cell onto the Au films at vacua better than 10.sup.5 mbar. Crosslinking to the CNM and conversion of the nitro groups to amino groups is achieved in high vacuum (<5*10.sup.7 mbar) with an electron floodgun at an electron energy of 100 eV and a dose of 50 mC/cm.sup.2.

(18) 2) Formation of the Second Layer (Here: Polyglycerol)

(19) The CNM on its gold-on-mica substrate is deposited into dry and clean polytetrafluoroethylene (PTFE) containers filled with a 10% (w/w) solution of glycidol in dry N-methylpyrrolidinone (NMP). After closing the PTFE vessels tightly, it is heated in an oven to 150 C. for 10 h. After cooling, the films are taken out, washed with NMP, water, and acetone, and then dried in the ambient under exclusion of dust.

(20) 3) Functionalization of the Second Layer with Affinity Groups (Here: EDTA)

(21) The disodium salt of ethylenediaminotetraacetic acid (Na.sub.2EDTA2 H.sub.2O, 510 mg) was dispersed in dry dimethylformamide (DMF, 15 mL) and thionylchloride (0.3 mL) was added. After stirring at room temperature for 2 h, the mixture was heated to 80 C. for 45 min. After cooling, the film system was immersed immediately in this reaction mixture and shaken at 50 rpm for 2 h. Then the films were taken out and purged with DMF, ethanol, water, and acetone. Drying took place in the ambient under exclusion of dust.

(22) 4) Lifting the Film Systems Off and Transfer to the TEM Grids

(23) The functionalized CNMs are transferred onto TEM grids using a protecting layer of poly(methyl methacrylate) (PMMA) dissolved in chlorobenzene or ethyl acetate. This layer is used for mechanical stabilization of the CNMs during the transfer process. Two layers of this polymer of overall thickness of 400 nm are spin-coated in sequence onto the CNM. First, a layer of low molecular weight PMMA (50 K), then a layer of high molecular weight PMMA (950 K) are spin-cast each for 30 s at 4000 rpm and cured on a hot plate at 90 C. for 5 min. The underlying mica support is separated from the gold/functionalized CNM/PMMA structure by a slight dipping into water of one of the edges/corners of the multilayered sample that finally (after separation) floats on the air/water interface. Further, the sample is transferred by using a mica piece from the water surface to an I.sub.2/KI/H.sub.2O etching bath (1:4:10) where the gold film is dissolved within 15 min. Then the CNM is transferred to pure water for complete cleaning of the membrane from iodine contamination. Finally, the CNM/PMMA structure is fished out by the target substrate, a TEM grid, and the PMMA layer is dissolved in acetone using a critical point dryer to minimize damage of the freestanding parts.

(24) 5) Electron Microscopy of Biological Samples on Functionalized CNM (Here: Based on EDTA/Ni.sup.2+/His-tag Interaction)

(25) The functionalized CNM on a TEM grid was incubated with 3 l NiCl.sub.2 (1 mg/mL Ni.sup.2+ in PBS buffer) for 30 seconds and rinsed with distilled water. Subsequently, 3 l of protein solution (His-tagged thermosome from Pyrococcus furiosus, 0.2 mg/ml) were applied to the functionalized CNM for 30 seconds, rinsed with distilled water and negatively stained with 1% uranylacetate solution. Samples were analyzed in a FEI Tecnai Spirit transmission electron microscope at an accelerating voltage of 120 kV. Images were acquired with a 4 k4 k CCD camera (Gatan).

(26) 6) Functionalization of Amorphous Carbon Film

(27) Continuous amorphous carbon films on gold-on-mica substrate are treated with oxygen plasma. Films having a defined thickness ranging from 2 to 10 nm were utilized. A thickness of 4 to 5 nm was found to exhibit advantageous balance between mechanical stability and transparency. Analogously to the functionalization of CNMs, the plasma treated amorphous carbon film on its gold-on-mica substrate is deposited into dry and clean PTFE containers filled with a 10% (w/w) solution of glycidol in dry NMP. After closing the PTFE vessels tightly, it is heated in an oven to 140 C. for 6-24 h. After cooling, the films are taken out, immersed for 10 min in water, and then dried in a stream of nitrogen. By way of example, the second layer was functionalized with EDTA groups by heating the substrate in a 0.1% (w/w) solution of EDTA monoanhydride in absolute DMF to 90 C. for 1 h. By varying the EDTA monoanhydride concentration, the functional group loading of the surface can be varied. The functionalized amorphous carbon films are transferred to TEM grids following the protocol (see example 4). By way of example, films were transferred to Quantifoil holey carbon coated TEM grids. Transfer to holey gold or holey/lacey carbon coated or pure TEM grids can be achieved analogously. The functionalized amorphous carbon films can be coupled to graphene (as shown for CNMs in FIG. 5) to enhance the conductivity to reduce sample charging during the TEM measurements.

(28) 7) Electron Microscopy of Biological Samples on Functionalized Amorphous Carbon Film (Here: Based on EDTA/Ni.sup.2+/His-tag interaction)

(29) To activate the functionalized amorphous carbon film, 3 l 1 mM NaOH were added, blotted, and washed once with distilled water. Subsequently, 3 l 0.1% NiSO4 solution were added and left for 30 s. The sample was washed twice with 3 l distilled water. 3 l of sample was added to the grid and left for 30 s. The grid was blotted with filter paper. The grid was washed twice with 3 l distilled water, blotted, and stained with Uranyl acetate. Samples were analyzed in an FEI Tecnai Spirit at an acceleration voltage of 120 kV.

(30) The features disclosed in the foregoing description, claims and the drawings may, both separately or in any combination, be material for realizing the invention in diverse forms thereof.