KR101227690B1 - Preparation and application of graphene oxide/mwcnt film-based substrate for ldi-ms - Google Patents

Abstract

PURPOSE: Preparation and application of graphene oxide/MWCNT film-based substrate for LDI-MS are provided to efficiently detect small molecules. CONSTITUTION: A graphene oxide-fixed substrate is manufactured by dipping an aminized substrate in graphene oxide aqueous suspension. The graphene oxide-fixed substrate is washed and dried. The dried substrate is heated. The graphene oxide-fixed substrate is dipped in aminized MWCNT(MWCNT-NH2) suspension. The graphene oxide/MWCNT-NH2-thin film substrate is manufactured.

Description

Fabrication and Application of Laser Desorption / Ionization Mass Spectroscopy Based on Graphene and Carbon Nanotube Composite Films {Preparation and Application of Graphene Oxide / MWCNT Film-Based Substrate for LDI-MS}

The present invention relates to a method for preparing graphene oxide / multi-walled carbon nanotube (GO / MWCNT) bilayer thin film and using it as a substrate for laser desorption / ionization mass spectrometry. A method for detecting small molecules using -MS substrate and a method for detecting hydrophobic molecules using reduced GO / MWCNT (RGO / MWCNT) bilayer thin film substrate as LDI-MS substrate.

The matrix-assisted laser desorption / ionization mass spectrometer (MALDI-MS) has been used as a powerful tool for biochemical analysis and proteomics research since it was introduced by Karas and Hillenkamp in the 1980s. Soft ionization, which minimizes fragmentation of analytes, is present as an intact molecular ion peak of the biomolecule, providing an easy-to-interpret mass spectrum. Although MALDI-MS has been successful in analyzing molecules having various molecular weights from 1 to 100 kDa, the use of organic matrix molecules has been shown to interfere with interference in the low mass region ( m / z <500), heterogeneous cocrystallization with analytes. Caused some problems such as hot spot formation and trial and error to find an appropriate matrix composition for successful analysis. Therefore, nanomaterial-assisted laser desorption / ionization (NALDI) and desorption on porous silicon, using inorganic nanoparticles and porous silicon substrates, which can transfer and absorb the energy absorbed from the laser to the analyte for mass spectrometry analysis It is working on the development of matrix-glass laser desorption / ionization mass spectrometers, such as / ionization (DIOS).

Among various nanomaterials, carbon-based nanomaterials such as fullerene, carbon nanotubes, and graphite have been actively studied for use in LDI-MS. In recent years, graphene, monolayer graphite composed of rough honeycomb crystal lattice, has shown considerable potential for application in LDI-MS, as well as its application in nanoelectronics, transparent electrodes and biosensors. In addition to having a high LDI efficiency, the carbon nanomaterial surface can be easily oxidized to provide oxygen functionalities for solubilization in aqueous solvents and proton provision and can be utilized for the enrichment of small amounts of hydrophobic molecules and biomolecules. .

However, NALDI-MS is not widely used due to high cost, cumbersome sample preparation, difficulty in maintaining the quality and stability of nanomaterials, and saturation of the detector due to flight of the nanomaterials themselves. Therefore, development of a matrix-glass LDI platform suitable for the chip format should be preceded for efficient mass spectrometer detection of small molecules. In addition, the chip format for the mass spectrometer is suitable for high throughput analysis strategies without labels or organic matrices. In this respect, DIOS is one of the most attractive platforms for biochips with proper surface chemistry, but it also requires relatively high cost and complicated manipulation procedures for porous silicon production.

MALDI Imaging Mass Spectroscopy (IMS) is another emerging tool in various research fields, such as proteomics, geology, and pharmaceuticals, and can be used to study the spatial distribution of various biomolecules within tissue sections. Compared with current technology, IMS has the following features. Accurate determination of multiple biomolecules based entirely on molecular weight and post-transcriptional modification and location of specific analytes in tissue sections are possible. An important challenge for MALDI-IMS is to distribute the matrix evenly on the tissue surface and to prevent the sublimation of the matrix under high vacuum during ongoing IMS experiments. Therefore, the development of LDI platform suitable for chip type will be a breakthrough to solve these problems in small molecule analysis and tissue imaging using mass spectroscopy.

An object of the present invention is to provide a method for manufacturing a substrate on which graphene oxide / multi-walled carbon nanotubes (GO / MWCNT) bilayer thin films are deposited.

Another object of the present invention is to provide a GO / MWCNT bilayer thin film substrate prepared as the LDI-MS detection substrate for detecting small molecules.

It is still another object of the present invention to provide a method of manufacturing a substrate on which a reduced GO / MWCNT (RGO / MWCNT) bilayer thin film is deposited.

Still another object of the present invention is to provide an RGO / MWCNT bilayer thin film substrate prepared as an LDI-MS detection substrate for detecting hydrophobic molecules.

In order to solve the above problems, the present invention comprises the steps of 1) preparing a GO-immobilized substrate by immersing the aminated substrate in an aqueous suspension of graphene oxide (GO); 2) washing and drying the GO-immobilized substrate; 3) heating the dried substrate; 4) preparing a substrate on which the GO / MWCNT bilayer thin film is deposited by immersing the prepared GO-immobilized substrate in an aminated MWCNT (MWCNT-NH ₂ ) suspension; 5) washing and drying the GO / MWCNT-NH ₂ -thin substrate; And 6) heating the washed GO / MWCNT-NH ₂ -thin substrate to provide a method of manufacturing a substrate on which the GO / MWCNT bilayer thin film is deposited.

Step 1 is a step of immersing the aminated substrate in a GO aqueous suspension to prepare a GO-immobilized substrate, which uses an aminated substrate so that GO can be immobilized to the substrate surface via covalent bonds. The prepared substrate is then used as a support for forming a GO / MWCNT bilayer thin film.

The term "graphene oxide (GO)" of the present invention is a compound in the water-soluble form of graphene, a monoatomic-thick 2D carbon nanomaterial having excellent electronic, thermal and mechanical properties, and can be produced by oxidizing graphite. It is phosphorus material. Recently, it is used in biological applications such as drug delivery, biosensitizer and enzyme activity analysis.

The aminated substrate may be a glass cover slip aminated the surface using APTES ((3-aminopropyl) triethoxysilane), but is not limited thereto. In addition, the immersion time is preferably 1 hour to 48 hours.

Step 2 is a step of washing and drying to remove excess GO not covalently bonded to the substrate, the washing liquid is not limited as long as it is a polar solvent and a volatile solvent, preferably water, ethanol, acetone, methanol , any one or more of n-propanol, I-propanol and ether can be used. More preferably, water and ethanol can be used.

Step 3 is a step of heating the dried substrate in order to chemically bond by heating the GO adsorbed on the aminated substrate to a high temperature, the heating temperature is preferably 100 ℃ to 200 ℃. If it is less than 100 ℃ bond is not made sufficiently, if it exceeds 200 ℃ may be reduced oxygen functional group of GO. In addition, the heating time is preferably 10 minutes to 1 hour, preferably carried out under an inert gas stream such as nitrogen or argon.

In step 4, the GO-immobilized substrate prepared as the support is immersed in the aminated MWCNT (MWCNT-NH ₂ ) suspension so that GO and MWCNT can form a double layer, and the immersion time is 1 hour to 48 hours. It is preferable that it is time.

Step 5 is a step of washing and drying to remove excess carbon nanotubes from the manufactured GO / MWCNT-NH ₂ thin film substrate, the washing liquid is not limited as long as it is a polar solvent and a volatile solvent, preferably May be any one or more of water, ethanol, acetone, methanol, n-propanol, I-propanol and ether. More preferably, water and ethanol can be used.

In step 6, MWCNT-NH ₂ adsorbed on the GO-coated substrate is heated to a high temperature to chemically bond the dried substrate, and the heating temperature is preferably 100 ° C to 200 ° C. If the temperature is less than 100 ° C., the binding may not be sufficiently performed. If the temperature is higher than 200 ° C., a reduction reaction other than the binding may be induced. In addition, the heating time is preferably 10 minutes to 1 hour, preferably carried out under an inert gas stream such as nitrogen or argon.

The GO / MWCNT-NH ₂ bilayer thin film substrate prepared according to the above method can be used for LDI-MS to efficiently analyze small molecules.

1 shows the principle of LDI-MS using a GO / MWCNT bilayer prepared according to the above method. The GO and MWCNT-NH ₂ surfaces are each charged with opposite charges, which in turn can be immobilized on a solid substrate in a stable bilayer structure. The addition of MWCNT-NH ₂ to the GO-film surface is expected to increase the surface roughness and the surface area over which the analyte can be adsorbed, thus increasing the LDI efficiency. As expected from the properties of GO and MWCNT-NH ₂ in solution, the GO / MWCNT-NH ₂ bilayer has high wettability to water due to the charged surface, absorbs ultraviolet rays, and effectively converts the absorbed energy into thermal energy. You can. This feature allows the GO / MWCNT-NH ₂ bilayer to be used as a chip-type LDI platform.

As used herein, the term “small molecule” refers to a low molecular weight organic compound that is not a polymer and is used in pharmacology and biochemistry. Particularly in pharmacology, it is usually used in a limited way to molecules that bind with high affinity to biopolymers such as proteins, nucleic acids, polysaccharides and the like and change their activity or function. The upper molecular weight limit for small molecules is about 800 Daltons, which can rapidly diffuse through the cell membrane to reach intracellular sites of action. These small molecules have a variety of biological functions. For example, it can act as a cell signaling material, a molecular biological tool, or a medicine in medicine. These compounds may be natural or artificial and may have a beneficial effect on the disease or may be harmful. Therefore, in understanding the physiology, it is important to isolate / analyze these small molecules with physiological activity.

In addition, the present invention comprises the steps of: a) reducing the GO / MWCNT-NH ₂ -thin substrate prepared according to the above method by immersing in a hydrazine monohydrate solution; And b) washing and drying the reduced GO (RGO) / MWCNT-NH ₂ -thin substrate.

The step a), GO / MWCNT-NH prepared above in order to reduce the ₂ GO / MWCNT-NH ₂ - a step of immersing the thin film substrate in a solution of hydrazine monohydrate. The reaction temperature is preferably 80 ° C to 150 ° C. If the temperature is lower than 80 ° C., the reduction is not sufficiently performed. If the temperature is higher than 150 ° C., there is a risk that the hydrazine may explode. Moreover, it is preferable that reaction time is 10 minutes-72 hours. In addition, the solvent of the hydrazine monohydrate solution may be a volatile solvent capable of dissolving hydrazine, for example, DMF, water, ethanol, toluene, methanol, ether, ester, chloroform, acetone, DMSO (dimethyl sulfoxide), Propanol or N-propanol can be used.

Step b) is a step of washing to remove the reagents used in the reduction reaction, the washing liquid may be used as a solvent of the hydrarin monohydrate solution.

The RGO / MWCNT-NH ₂ bilayer thin film substrate prepared according to the above method can efficiently analyze hydrophobic small molecules using LDI-MS (Laser Desorption / Ionization Mass Spectrometry). Tissue sections can also be analyzed using Imaging Mass Spectrometry (IMS).

As used herein, the term "hydrophobic" means a property that is not easily combined with water molecules. Generally it is hydrophobic if it is not polar. Hydrophobic materials are lipophilic, with the exception of silicon and fluorine carbide, so they are usually used interchangeably with the term lipophilic.

As described above, the present invention can provide a method for efficiently detecting small molecules by using a GO / MWCNT-NH ₂ bilayer thin film substrate for LDI-ToF MS analysis. The thin film serves as a substrate that absorbs laser energy and efficiently transfers the energy to the deposited analyte for desorption and ionization of the analyte. Therefore, the synergy effect generated at the boundary between GO and MWCNT enables efficient analysis.

According to an embodiment of the present invention, GO / MWCNT-NH ₂ showed excellent efficiency in LDI-MS analysis of small molecules among various GO and MWCNT based substrates, and the reduced form RGO / MWCNT-NH ₂ was hydrophobic. It is expected to show high efficiency in the detection of molecules and to be used for the analysis of tissue sections using IMS. In addition, the GO / MWCNT-NH ₂ bilayer shows excellent detection capability even in repeated use, exhibits high salt resistance, overcomes the disadvantages of the conventional mass spectrometry using organic matrix, and is manufactured by oxidizing graphite. It is expected that it can be used to replace the organic matrix material that is commercially available.

The present invention provides a method for preparing a GO / MWCNT-based substrate and provides a method for efficiently detecting small molecules by performing LDI-ToF MS with the substrate. In addition, the substrates presented above can be fabricated at low cost and used repeatedly, so that they can be widely used in mass spectrometry in place of existing organic matrices.

1 is a simplified diagram of the principle of LDI-MS based on a GO / MWCNT bilayer film-coated substrate.
Figure 2 analyzes the properties of GO and the aminated MWCNT prepared by the oxidation and exfoliation of graphite. Where (a) is an atomic force image of GO, (b) is an FT-IR spectrum, (c) is an atomic force image of MWCHT-NH ₂ , and (d) is a Raman spectrum will be.
3 shows various carbon nanomaterial-based substrates fabricated for LDI efficiency comparison. Here, (a) is a surface fixation film, three kinds of substrate consisting of GO derivative film, MWCNT-NH ₂ film, and GO and MWCNT derivative film, (b) is six different graphene and / or MWCNT-based substrate (C) is an SEM image of the prepared carbon nanomaterial substrate, and the scale bar corresponds to 1 μm.
4 shows the results of LDI-MS analysis of various small molecules obtained by using a GO / MWCNT-NH ₂ bilayer as a substrate. Where (a) is the cation mode and (b) is the mass spectrum obtained in the anion mode, and (c) is a table showing the mass accuracy, resolution, resolution limit and surface density of the analyte (1 nmol / ml) dissolved in distilled water. The aqueous solution of the analyte was coated on a GO / MWCNT bilayer coated on a glass substrate and subjected to LDI-MS.
Figure 5 shows the results of the LDI-MS experiments to determine the detection limit of small molecules when using the GO / MWCNT-NH ₂ bilayer as a substrate. To this end, each sample was diluted to a series of concentrations and analyzed by LDI-MS.
6 shows celloboise ( m / z 365 [M + Na] ⁺ ), phenylalanine ( m / z 188 [M + Na] ⁺ and m / z obtained using GO (a) and RGO (b) dispersed in water. Mass spectra of 210 [M + 2Na] ⁺ ) and Leu-enkephalin ( m / z 577 [M + Na] ⁺ and m / z 593 [M + K] ⁺ ).
7 shows the mass spectra of various small molecules. (a) is Fmoc-Lys (Boc) -OH ( m / z 491 [M + Na] ⁺ and m / z 507 [M + K] ⁺ ), (b) is glucose ( m / z 203 [M + Na ] ⁺ And m / z 219 [M + K] ⁺ ), (c) is lysine ( m / z 169 [M + Na] ⁺ ), (d) is D-mannitol ( m / z 205 [M + Na] ⁺ And m / z 221 [M + K] ⁺ ), (e) is GGDEVDSG ( m / z 799 [M + Na] ⁺ and m / z 815 [M + K] ⁺ ), (f) is phenylalanine ( m / z 188 [M + Na] ⁺ and m / z 204 [M + K] ⁺ ).
8 is a result of LDI-MS analysis of small molecules using various substrates prepared in the present invention. Where (a) is the mass spectra of cellobiose and enkephalin (Leu-enkephalin) obtained from six different carbon nanomaterial-based films as LDI substrates, and (b) is the mass peak of various small molecules obtained from different substrates represented by bar graphs. Intensity comparison, (c) is a schematic showing the synergistic effect between GO and MWCNT as LDI substrate.
9 is an experimental result of the surface properties of the various substrates prepared in the present invention. (A) shows AFM images of six different carbon nanomaterial-based films, (b) shows surface roughness and water contact angle of the carbon nanomaterial chip, and (c) shows the survival rate of BP in the carbon nanomaterial chip. And desorption efficiency.
In Figure 10 (a) is a photograph of the mannitol spot formed on the carbon nanomaterial chip, was obtained by spotting 1 μl of 1 mM mannitol aqueous solution. (b) is a sample spot of each small molecule formed on the GO / MWCNT-NH ₂ bilayer, the concentration and amount of each sample is the same as the mannitol used in (a) (scale bar = 2 mm).
In Figure 11 (a) is a photograph of the water droplets on the carbon nanomaterial chip (b) is a table showing the water contact angle.
In Figure 12 (a) is the mass spectrum of the benzyl pyridinium salt on the various carbon nanomaterial chip, (b) is the chemical structure of the benzyl pyridinium salt ( m / z 170) and its fragment pattern ( m / z 91), And (c) shows the survival rate of the benzyl pyridinium salt calculated on various carbon nanomaterial chips.
Figure 13 (a) shows the results of the LDI-MS analysis at a series of dilution concentrations to determine the detection limit of small molecules dissolved in PBS on the GO / MWCNT-NH ₂ bilayer platform, (b) these The detection limits of small molecules are summarized in a table.
Figure 14 shows the results of mass spectrometry using various concentrations of PBS to confirm salt tolerance, (a) is the mass spectrum of enkephalin ( m / z 577 [M + Na] ⁺ ) (b) is phenylalanine mass spectra of ( m / z 188 [M + Na] ⁺ , m / z 210 [M + 2Na] ⁺ ). Wherein 1 XPBS comprises 0.21 g / L KH ₂ PO ₄ , 9 g / L NaCl and 0.72 g / L NaHPO ₄ .
FIG. 15 shows SEM images of GO / MWCNT-NH ₂ bilayers before and after half-speed use of mass signal intensity changes with repeated use. (a) shows the mass signal intensity changes of cellobiose and phenylalanine on the GO / MWCNT-NH ₂ substrate as a function of the number of repeated uses, and (b) and (c) show GO before and after 15 small molecule analyzes, respectively. / MWCNT-NH ₂ shows an SEM image of a double-layer .GO / MWCNT-NH ₂ is a double-layer was not damaged noticeable even repeated irradiation of the ultraviolet laser.
16 is an experimental result of the distribution of sample spots formed on the GO / MWCNT-NH ₂ substrate. (A) and (b) show optical and mass images (scale bar = 1 mm) of cellobiose ( m / z 365 [M + Na] ⁺ ) uniformly distributed on GO / MWCNT-NH ₂ substrate, respectively. (C) is D-mannitol ( m / z 205 [M + Na] ⁺ ), cellobiose at concentrations of 1, 0.5 and 0.2 nmol, respectively, along the first, second and third columns formed on the GO / MWCNT-NH ₂ substrate. Mass image (scale bar = 2 mm) of small molecule array of ( m / z 375 [M + Na] ⁺ ), and Leu-enkephalin ( m / z 577 [M + Na] ⁺ ), (d ) Is the mass spectrum of small molecules from the spots of the array formed with 0.2 nmol of each analyte.
Figure 17 shows the shape of the sample spot when using the existing MALDI-ToF MS matrix. (a) and (b) show optical and mass images of crystals composed of cellobiose and DHB, which are often used as matrices in conventional MALDI-ToF MS, respectively. Raster width and scale bars are 100 μm and 1 mm, respectively.
18 shows the results of observing chemical reactions and enzyme synthesis with LDI-MS. Here, (a) shows the synthetic scheme, molecular structure and mass spectrum of each molecule of the betulin derivatives (M1, M2, M3), M1 ([M1 + K] ⁺ = m from the GO / MWCNT-NH ₂ bilayer / z 482), M2 ([M2 + Na] ⁺ = m / z 477, [M2 + K] ⁺ = m / z 493) and M3 ([M3 + Na] ⁺ = m / z 479, [M3 + K ] ⁺ = m / z 495) mass peaks were obtained easily. (b) shows the molecular structure of the substrate and product for enzyme-catalyst (alpha-chymotrypsin) esterification. Mass spectra were obtained after 3 and 180 minutes of reaction time from the enzyme reaction mixture. (c) shows the change in the relative mass peak intensities of M4 and M5 in the reaction of (b) as a function of reaction time.
FIG. 19 shows (a) molecular structure of synthesized molecules (M7, M8, and M9) and (b) their mass spectra on GO / MWCNT-NH ₂ chip.
Figure 20 shows the mass spectra obtained from (a) the reaction time from the enzyme reaction mixture for 180 minutes and (b) the mass spectrum of the sample incubated on the substrate for 180 minutes without the enzyme as a control.
Figure 21 shows the results of analyzing the lipid distribution of mouse brain tissue sections by IMS. Where (a) is an optical and mass image of a mouse brain tissue section on an RGO / MWCNT-NH ₂ bilayer matrix, (b) is a mass spectrum from a region designated in the brain tissue section of (a), and (c) is a mass spectrum Summarizes the species identified in, masses corresponding to [GC d18: 0/24: 0 h + K] ⁺ and [GC d18: 1/22: 0 h + Na] ⁺ to reconstruct the composite ion map Peaks were selected.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example 1 Amination of Glass Substrate

The glass substrate was aminated so that graphene oxide (GO) could be fixed to the substrate surface by electrostatic attraction. The glass coverslips were washed with Piranha solution (sulfuric acid: 30% hydrogen peroxide = 3: 1) at 125 ° C. for 10 minutes, washed with water and ethanol, and dried under nitrogen flow. The washed substrate was soaked in 10 mM anhydrous toluene solution of APTES for 30 minutes, sonicated in anhydrous toluene for 2 minutes, washed with water and ethanol and dried under nitrogen flow. This process can be applied to any type of silicon dioxide substrate.

Example 2: Graphene Oxide Suspension Preparation

1.5 g of graphite powder and 0.5 g of sodium nitrate were added to 23 ml of sulfuric acid and stirred in an ice bath. Thereafter, 3 g of potassium permanganate was slowly added to the mixture while maintaining the temperature at 20 ° C. The mixture was diluted with 40 ml of water and stirred for 30 minutes. An additional 100 ml of water was then added in an ice bath to prevent the reaction mixture from boiling rapidly when the temperature of the mixture was raised rapidly to 95 ° C. Finally, 3 ml of hydrogen peroxide (30%) was added slowly to the light yellow mixture. The product solution was filtered and washed with plenty of water until the filtrate was neutralized. The filter cake was dried for 48 hours under reduced pressure. The prepared graphite oxide powder was analyzed by FT-IR spectroscopy by KBr pellet method.

Graphite oxide was sonicated in aqueous solution for one hour to exfoliate to prepare a GO suspension, which was centrifuged at 8000 RPM for 30 minutes to remove large aggregates of GO sheet. To confirm the exfoliation of the graphite oxide, the APTES-functionalized silicon substrate was immersed in GO suspension (0.1 mg / ml) for 1 hour, washed with water and ethanol and dried under nitrogen flow. As a result of the above procedure, electrostatic adsorption of GO on the silicon substrate occurred and the adsorbed GO was observed under an atomic force microscope, and the results are shown in FIGS. 2A and 2C.

2a and 2c show atomic force microscope images and FT-IR spectra of oxidized and exfoliated graphite, respectively. According to this, the thickness of the prepared monolayer GO is about 0.83 nm and the FT-IR spectrum is OH oscillation at 3415 cm ⁻¹ , C═O elongation at 1716 cm ⁻¹ , and C═C of oxidized graphite domain at 1627 cm ⁻¹ . in skeletal vibration, OH strain, and 1079 cm ^-1 at 1400cm ^-1 shows the characteristic peak of the oxygen containing functional group by CO height.

Example  3: MWCNT - NH ₂  Preparation of suspension

40 mg of MWCNTs were sonicated at 40 ° C. in a mixture of nitric acid and sulfuric acid (1: 3) at 60 ° C. for 10 hours to introduce carboxylic acid functional groups onto the sidewalls of the MWCNTs, and filtered through a polymeric carbonate membrane (slit size 400 nm). And washed with a sufficient amount of water until the filtrate was neutralized. The filter cake was washed with ethanol, dried under reduced pressure and rewashed with 40 ml anhydrous DMF. By sonication it was dispersed in 20 ml thionyl chloride containing 1 ml anhydrous DMF and refluxed at 70 ° C. for 24 hours. After the reaction, acylated MWCNTs were obtained from the mixture by rotary evaporation, suspended in 40 ml of ethylenediamine by sonication and refluxed at 125 to 5 days. Finally, the reaction mixture was filtered through an anodic alumina membrane (spacing size 200 nm), washed with ethanol and water and then dispersed in aqueous solution at a concentration of 120 g / ml by sonication.

Atomic force microscopy images of aminated MWCNTs (MWCNT-NH ₂ ) prepared by the above method are shown in FIG. 2B. As shown in FIG. 2B, MWCNT-NH ₂ has a diameter of about 17 nm.

Example 4: GO and GO / MWCNT-NH ₂  Fabrication of Bilayer Thin Films

To induce immobilization of negatively charged GO on positively charged substrate yarns by electrostatic reaction, APTES in an aqueous suspension of GO (1.5 mg / ml), prepared according to the modified Hummer's method The GO-immobilized substrate was prepared by immersing the amarized glass coverslip for 1 hour. It was then washed with water and ethanol, dried under nitrogen flow and then heated to 125 ° C. for 10 minutes under nitrogen flow. Atomic force microscopy images showed that the GO sheet covered the entire surface and overlapped with adjacent GO sheets along the edges. The GO thin film-coated substrate thus obtained was used as a support for preparing a GO / MWCNT-NH ₂ bilayer. The GO-covered substrate was then immersed in an aminoated MWCNT solution (120 μg / ml) for 1 hour to form a GO / MWCNT-NH ₂ bilayer thin film, washed with water and ethanol and dried under nitrogen flow. Finally the GO / MWCNT-NH ₂ bilayer-coated substrate was heated to 125 ° C. for 10 minutes under nitrogen flow. The uniform distribution of MWCNTs immobilized on the GO surface was observed by atomic force microscopy and scanning electron microscopy (SEM) (FIG. 3C).

LDI-MS analysis of small molecules was attempted using the prepared GO / MWCNT-NH ₂ bilayer thin-film glass substrate. Aqueous solutions (1 nmol / μl) of various small molecules including Cellobiose, Leu-enkephalin, glucose, lysine, D-mannitol and phenylalanine were prepared, and 1 nmol of each sample was spotted onto a GO / MWCNT-NH ₂ bilayer film. . After drying the sample spots, the substrate was used for LDI-MS.

The result is a very clean mass spectrum with a low bottom signal showing a mass peak corresponding to the protonated ions, sodium and / or potassium adducts of the analyte small molecules in the cationic mode (cellobiose, m / z 365 [M + Na] ⁺ ; Leu-enkephalin, m / z 576 [M + Na] ⁺ and m / z 592 [M + K] ⁺ ; glucose, m / z 203 [M + Na] ⁺ and m / z 219 [M + K] ⁺ ; Lysine, m / z 147 [M + H] ⁺ , m / z 169 [M + Na] ⁺ and m / z 185 [M + K] ⁺ ; D-mannitol, m / z 205 [M + Na] ⁺ and m / z 221 [M + K] ⁺ ; phenylalanine, m / z 166 [M + H] ⁺ , m / z 188 [M + Na] ⁺ and m / z 204 [M + K] ⁺ ) (FIG. 4A). Metal cation adducts may be due to the extra Na ⁺ and K ⁺ contained in the analyte sample solution with the reagents (ie, potassium permanganate, sulfuric acid and hydrogen peroxide) and / or impurities used to oxidize the graphite to produce GO. will be. These metal cations are strongly adsorbed on the GO surface due to the negative charge of the GO surface and are therefore difficult to remove during the purification process. Protonated species ([M + H] ⁺ ) are sometimes preferred over metal cation adducts, especially for tandem mass spectrometry.

In anionic mode, clear mass spectra were obtained showing peaks corresponding to the respective deprotonated forms for glucose, mannitol, lysine and phenylalanine (glucose, m / z 179 [MH] ⁻ ; mannitol, m / z 181 [ MH] ^-; lysine, m / z 145 [MH] -; phenylalanine, m / z 165 [MH] -) ( Fig. 4b). However, no mass signals of celloboise and Leu-enkephalin were observed. The high LDI efficiency of lysine and phenylalanine in the anion mode may be due to the presence of carboxylic acid groups that can be easily deprotonated.

Mass accuracy, resolution and detection limits obtained in cation mode and the surface density of each small molecule accumulated are summarized in FIG. 4C and the respective mass spectra for determination of detection limits are shown in FIG. 5. LDI was performed using the GO suspension prepared in Example 2 as a control. Mass spectra were obtained after spotting and drying distilled water, small molecules and GO mixed solution on a stainless steel plate. This is shown in Figure 6a. As shown in FIG. 6A, only cellobiose, phenylalanine, and enkephalin in the test analytes showed a mass peak with a relatively high bottom signal in the low mass region ( m / z <200), which was due to destruction of the GO sheet due to strong laser irradiation. And flight. Therefore, surface immobilization of the GO sheet is important to prevent such a high bottom signal.

Therefore, in order to further study the LDI efficiency of surface-fixed carbon materials and to identify which nanomaterials, ie GO or MWCNT-NH ₂ , play a more important role, the methods presented in the examples and comparative examples below, GO, RGO, MWCNT and MWCNT-NH ₂ was used to prepare different substrates in various forms. Schematics, preparation methods, and internuclear microscope images of the prepared GO and GO / MWCNT-NH ₂ bilayer thin film substrates and various other substrates are shown in FIGS. 3A, 3B, and 3C, respectively. As shown in FIG. 3A, these substrates were classified as substrates coated with only three category-GO derivatives, only MWCNT-NH ₂ , and a combination of GO and MWCNT derivatives. MWCNT-NH ₂ -coated substrates were prepared by immobilizing MWCNT-NH ₂ on epoxides present on the glass surface, and three different chips based on GO and MWCNT derivative combinations immobilized MWCNTs on RGO substrates via pp interaction ( RGO / MWCNT), by adsorbing MWCNT-NH ₂ to GO substrate through electrostatic interaction (GO / MWCNT-NH ₂ ), and by chemical reduction of the prepared GO / (RGO / MWCNT-NH ₂ ) (FIG. 3B). Their SEM images, shown in FIG. 3C, show that surface-fixed carbon nanomaterials were successfully prepared with high surface diffusion.

Example 5 Reduced GO (RGO) and RGO / MWCNT-NH ₂  Fabrication of Bilayer Thin Films

GO and GO / MWCNT-NH ₂ bilayer thin-film substrates were immersed in a 20% hydrazine monohydrate solution of DMF at 80 ° C. for one day, washed with DMF, water and ethanol and dried under nitrogen flow. RGO and RGO / MWCNT-NH ₂ bilayer thin film substrates were used as counter pairs to study the effect of the chemical structure of carbon nanomaterials on the LDI efficiency of small molecules.

Comparative Example 1: Preparation of RGO / MWCNT Bilayer Using MWCNT Suspension

MWCNT (10 mg) was dispersed by sonication in 10 ml dichlorobenzene for 1 hour and centrifuged to remove large aggregates of MWCNTs (3099 rcf, 10 minutes). The RGO thin film-coated substrate was immersed in the MWCNT suspension for 1 hour, washed with water and ethanol and dried under nitrogen flow.

Comparative Example 2: MWCNT-NH ₂ -Preparation of film glass substrate

Piranha-treated glass substrates were immersed in 10 mM anhydrous toluene solution of 3-glycidylpropyltriethoxysilane for 30 minutes, washed with ethanol and water and dried under nitrogen flow. The substrate was immersed in a 120 μg / ml MWCNT-NH ₂ suspension for 1 hour, washed with water and ethanol and dried under nitrogen flow. The MWCNT-NH ₂ -coated substrate was heated to 125 ° C. under nitrogen flow.

Experimental Example 1: Mass spectrometry of small molecules and characterization of the prepared substrate

Various analytes, including sugars, peptides, amino acids and protective amino acids, were dissolved in water at a concentration of 1 nmol / μl. 1 μl of the prepared solution was spotted on a carbon nanomaterial chip, dried under atmospheric conditions, inserted into MALDI-ToF MS, and analyzed under the same experimental conditions. This sample preparation protocol was applied to all small organic molecular analyzes on the RGO / MWCNT bilayer platform, including Imaging Mass Spectroscopy (IMS).

Under the same instrument conditions, the small molecule analyte prepared by the above method was analyzed by LDI-MS using six different carbon nanomaterial-based substrates prepared by the method described in the above Examples and Comparative Examples and shown in FIG. 7. . 8A and 8B show bar graphs comparing relative mass peak intensities. As expected, the intensity of the mass peak was highest in the substrates (GO / MWCNT-NH ₂ and RGO / MWCNT-NH ₂ ) using the GO and MWCNT-NH ₂ combinations. Surface wetting due to the presence of surface functional groups such as carboxylic acids and amines also appears to play an important role in improving sample diffusion. It is clear that the binding of MWCNT-NH ₂ to the GO layer markedly improved the LDI efficiency of various small molecules. In contrast, LDI efficiencies of small molecules on RGO / MWCNT-NH ₂ bilayer films vary somewhat compared to their unreduced partners. As a result, for the general purpose of small molecule analysis, the GO / MWCNT-NH ₂ -thin film surface was the best LDI substrate tested. RGO / MWCNT-NH _{2, on the} other hand, is considered the best substrate for hydrophobic analytes such as enkephalin and Fmoc-protected amino acids. Therefore, the interaction between the analyte and the carbon nanomaterial chip plays an important role in the complex LDI process, and it seems that synergistic effect on the efficient LDI exists when the GO / MWCNT bilayer is applied to the LDI-MS. This synergistic effect will occur at the junction of the MWCNT and GO where the analyte can receive energy from both the MWCNT and GO derivatives as shown in FIG. 8C.

To study the relationship between the structure and surface properties of substrates with LDI efficiency, center-line average (CLA) surface roughness and water contact angle were measured. The promising LDI substrates GO / MWCNT-NH ₂ and RGO / MWCNT-NH ₂ have a relatively high CLA surface roughness among the substrates at 6.646 and 5.018 nm, respectively. In contrast, the GO and RGO-coated substrates had a smooth shape with CLA surface roughnesses of 1.117 and 0.904 nm. These results indicate that chemical reduction induces a change in the morphology along with the chemical structure. The CLA surface roughness of the RGO film also increased slightly from 1.117 nm to 1.914 nm after pp interaction-induced adsorption of MWCNTs (RGO / MWCNT), but the surface roughness was increased by TGO / MWCNT- due to the difference in the amount of surface-adsorbed MWCNTs. Lower than NH ₂ . The MWCNT film showed the highest CLA surface roughness (9.079 nm) of the carbon nanomaterial chips produced (FIGS. 9A and 9B). Based on atomic force microscopy characteristics, the efficient LDI substrates GO / MWCNT-NH ₂ and RGO / MWCNT-NH ₂ bilayer films generally exhibited higher CLA surface roughness than those of GO and RGO due to MWCNT adsorption. Although MWCNT films exhibited the highest CLA surface roughness induced at higher adsorption densities of MWCNT-NH ₂ than bilayer films, their LDI efficiency was lower than these bilayer films. These results suggest that GO sheets under MWCNT play an important role in the efficient LDI of analytes. Due to its close relationship with homogeneous sample diffusion, the wettability of the substrate is also an important factor for an efficient LDI platform.

Due to the relatively hydrophilic surface, the analyte solution is easier to diffuse in GO, MWCNT and GO / MWCNT-NH ₂ films compared to the reduced forms-RGO, RGO / MWCNT and RGO / MWCNT-NH ₂ films. 9B, 10 and 11). These results are in good agreement with the higher LDI efficiency of the analytes in GO and GO MWCNT-NH ₂ films than in the reduced pair with the water contact angle values obtained. To further investigate the complex internal energy transfer in the carbon nanomaterial film, estimate the survival rate—the intensity of the precursor ions divided by the total intensity of the fragment and precursor ions—and the desorption efficiency—the absolute total intensity of the fragment and precursor ions— Benzylpyridinium salt (BP) was selected as the model compound for the reaction. These values are known to depend on the internal energy transfer process. The low survival rate of BP in the GO film, 19%, was significantly increased to 60% by incorporating MWCNT-NH ₂ in the GO film (GO / MWCNT-NH ₂ film) and 27% in RGO and RGO / MWCNT-NH ₂ films, respectively. And 76% more increased (Figs. 9C and 12). Increased survival is due to efficient laser energy absorption, restoration of sp2 carbon domains and increased thermal conductivity of GO films through MWCNT bonding. However, despite slightly lower survival rates, the desorption efficiency was significantly higher in the GO / MWCNT-NH ₂ bilayer than in the RGO / MWCNT-NH ₂ bilayer. This low desorption efficiency in RGO / MWCNT-NH ₂ may be due to the loss of absorbed laser energy to the carbon material-based substrate, which is unnecessary but preferred, not the analyte. This energy dissipation appears to be more efficient in RGO / MWCNT-NH ₂ than in GO / MWCNT-NH ₂ due to the restored sp2 carbon domain. These results indicate that the analyte fragmentation and LDI efficiency in the prepared carbon nanomaterial chip are highly dependent on the chemical properties of the carbon nanomaterial chip and the interaction between the analyte and the carbon nanomaterial chip.

Experimental Example  2: of the prepared substrates Salt tolerance  Experiment

From a series of experiments, GO / MWCNT-NH ₂ and RGO / MWCNT-NH ₂ It was concluded that the bilayer was suitable for LDI of small molecules and hydrophobic small molecules, respectively. Therefore, we have further characterized these advantages and performance of the new LDI platform. Salt tolerance is an important component of the LDI platform for the analysis of biological samples containing high salt concentrations. To test the tolerability of the GO / MWCNT-NH ₂ bilayer platform, 1 μmol of the small molecule tested in FIG. 4 was prepared with a ionic strength similar to that of human fluid (0.21 g / L KH ₂ PO ₄ , 9 g / L NaCl and 0.72 g / L NaHPO ₄ ) was dissolved in phosphate buffer (PBS) and spotted on a GO / MWCNT-NH ₂ bilayer and analyzed by LDI-ToF MS (FIGS. 13A and 13B). Although the detection limit of these small molecules has been reduced by one order of magnitude, the GO / MWCNT-NH ₂ bilayer can still be applied for the direct analysis of small molecules dissolved in PB without any desalting or concentration. . In addition, Leu-enkephalin and Phenylalanine dissolved in concentrated PBS with ionic concentrations of 1.68 g / L KH ₂ PO ₄ , 7.2 g / L NaCl and 5.80 g / L NaHPO ₄ were added to GO / MWCNT-NH _2. Successful analysis in the bilayer. This is why GO / MWCNT-NH ₂ High salt resistance of the bilayer was confirmed (FIG. 14).

Experimental Example  3: GO Of MWCNT - NH ₂ Double layer  Reusability Test of Thin Films

Instrumental durability is also an important component of a good LDI platform for stable, continuous, and repeatable applications for nanostructured laser irradiation-induced destruction-free LDI-ToF MS analysis. It has already been reported that GO / MWCNT-NH ₂ bilayers have excellent mechanical durability due to the formation of covalent bonds between the substrate and MWCNT-NH ₂ . Instrumental durability and performance as reusable LDI substrates have been successfully verified by repeated application to LDI-ToF MS crossover analysis of phenylalanine and cellobiose (1 nmol each), including washing and remounting steps.

1 mM (1 nmol) of 1 mM cellobiose and phenylalanine solution were spotted on a GO / MWCNT-NH ₂ bilayer, dried under atmospheric conditions, and inserted into MALDI-ToF MS and analyzed under the same experimental conditions. After analysis, the GO / MWCNT-NH ₂ bilayer was washed thoroughly with water and ethanol and dried under nitrogen flow. The washed GO / MWCNT-NH ₂ bilayer was used repeatedly for cellobiose and phenylalanine analysis. The procedure was repeated up to 15 times.

As shown in FIG. 15, although the mass signal strength of both small molecules gradually decreased in the repeated application of LDI-MS (FIG. 15A), the absolute strength of the small molecules was reduced on the GO / MWCNT-NH ₂ bilayer. It is enough to analyze its existence. Even after 15 repeats of the LDI analysis, no signs of surface destruction (FIGS. 15B and 15C) and contamination of the mass spectrometer detector due to ionization sources and GO / MWCNT-NH ₂ were observed. In the present invention, a single LDI assay cycle is defined as a complete set of a process of spotting an analyte on a substrate, drying the analyte, analyzing the analyte's LDI-ToF MS, washing with water and ethanol, and drying the substrate under a nitrogen flow. do. The results indicate that GO / MWCNT-NH ₂ bilayer film can be repeatedly applied in LDI-MS analysis of small molecules.

Experimental Example  4: uniform distribution of sample

Another important issue for successful LDI-ToF MS analysis is obtaining a uniform mass signal from the entire surface of the LDI-ToF MS platform without sweet-spots. To study mass signal strength from spot to spot on the GO / MWCNT-NH ₂ platform, 1 μl of cellobiose solution (0.1 mM) was spotted onto the GO / MWCNT-NH ₂ bilayer and dried and the entire sample spot was 50 μm. The raster width of was analyzed by IMS.

As shown in FIG. 16, the optical image of the GO / MWCNT-NH ₂ bilayer showed that the analytes formed a circular shape with uniform distribution after solvent evaporation and the mass image showed a single mass signal strength at the mass peak corresponding to cellobiose. The distribution was confirmed to be uniform ( m / z 365 [M + Na] ⁺ ). As a control, 1 μl of a mixed solution of 2,5-dihydroxybenzoic acid (DHB) (10 mg / ml) and celloboise (0.1 mM), which is a typical organic substrate for MALDI-ToF MS, was released. Spotted onto the substrate and analyzed by IMS.

As shown in FIG. 17, the mass image showed undesired formation of sweet-spots due to heterogeneous crystal formation of celloboise-DHB. The formation of uniform sample spots with relatively single mass peak intensity makes the GO / MWCNT-NH ₂ bilayer platform more quantitative and advantageous for monitoring chemical reactions and developing biosensors based on mass spectrometry. In addition, the fabrication of the GO / MCNT-NH ₂ bilayer is suitable for a wide range of processing and array analysis of multiple analytes. In particular, a GO / MWCNT-NH ₂ bilayer was fabricated on a 2 cm ² glass substrate and a 150 μm raster was spotted on top of 1 μl of each solution (1, 0.5, 0.2 mM) of D-mannitol, celloboise, and Leu-enkephalin. The width was analyzed by IMS. All organic molecules were successfully detected as sodium adducts from the respective array spots (3 × 3) formed in the large bilayer chip (FIGS. 16C and 16D).

Experimental Example  5: chemical reaction

Thanks to the surprising performance of the GO / MWCNT-NH ₂ bilayer as a new LDI-ToF MS platform, the platform of the present invention has been extended to various mass spectrometry. GO / MWCNT-NH ₂ bilayer was used to observe the organic reaction based on the change of molecular weight from the starting molecule to the product. Conversion of Betulin (M1) from the initial molecule (432 g / mol) to betulinic acid (M3, 456 g / mol) by betulonic acid (M2, 454 g / mol) was performed by GO / MWCNT-NH ₂ Successful analysis using a bilayer LDI platform (FIG. 18A).

Betulin and betulinic acid have been reported to have anti-cancer, antimalarial, anti-inflammatory and anti-HIV activity and have been widely studied as anticancer agents. Peaks corresponding to the molecules (M1, M2, M3) were not observed in stainless steel or glass substrates without the GO / MWCNT-NH ₂ bilayer. In another example, bound cyclic organic molecules (FIG. 19A) were synthesized and analyzed by LDI-ToF MS on a GO / MWCNT-NH ₂ bilayer platform. This gave a mass peak corresponding to each reaction product (FIG. 19B).

Experimental Example  6: Enzyme-catalyst Synthesis

Next, small molecule enzyme synthesis was performed and the reaction was monitored by LDI-ToF MS on GO / MWCNT-NH ₂ bilayer film. As a model reaction, alpha-chymotrypsin-catalyzed synthesis of N-acetyl-L-tryptophan ethyl ester (M5, 274 g / mol) via esterification of N-acetyl-L-tryptophan (M4, 246 g / mol) Analysis was successful by kinetic measurements using the GO / MWCNT-NH ₂ LDI platform. The results are shown in FIGS. 18B and 18C and 20. Thus, the GO / MWCNT-NH ₂ bilayer platform can be used to monitor the chemical and biological synthesis of small organic molecules and can be considered as a mass spectrometry-based analysis platform of enzyme activity in solution. The specific experimental method is as follows.

29 μl of N-acetyl-L-tryptophan (1.2 mg) and ethanol were dissolved in 500 μl of chloroform and mixed with 100 μl alpha-chymotrypsin solution (10 mg / ml). The mixture was stirred for 3 h at 600 rpm in a transverse shaker. Aliquots of 1 μl on chloroform at 3, 5, 15, 30, 60, 120 and 180 minutes were analyzed by spotting on a GO / MWCNT-NH ₂ bilayer.

Experimental Example  7: Analysis of Mouse Brain Tissue Sections

RGO / MWCNT-NH ₂ was applied to IMS of lipids distributed in mouse brain tissue. The following procedure was performed to obtain brain tissue sections. The mice (C57 / BL6) were beheaded after anesthesia by intraperitoneal injection of a ketamine and xylazine mixture (4: 1, 0.005 mg / g body weight). After the skull is removed, the brain is separated and placed in liquid nitrogen for about 5 minutes. After brain was frozen, brain sections were prepared by cryome (CM 1850, Leica, Nusslach, Germany), cut to 15 μm thickness in the coronal direction at −25 ° C., and immediately mounted on an RGO / MWCNT-NH ₂ bilayer platform. .

For lipid analysis, the RGO / MWCNT-NH ₂ platform, rather than GO / MWCNT-NH _2, was used, which showed higher LDI efficiency and stronger hydrophobic molecules for the RGO / MWCNT-NH ₂ bilayer than the GO / MWCNT-NH ₂ bilayer. This is because affinity can be an advantage for lipids of LDI-MS. Lipids play an important role in biological processes such as signal transduction, neurotransmission, myelination, apoptosis, vesicle tracking and transcription and post-transcriptional regulation, so studies of lipid distribution and identification are important.

To confirm photon energy absorption and energy transfer from the RGO / MWCNT-NH ₂ bilayer to the energy sections, 15 μm thick mouse brain tissue sections were prepared by sagittal cutting and placed side by side on the top surface of the RGO / MWCNT-NH ₂ bilayer. Without using any organic substrates were analyzed by IMS of a laser beam of 50 μm in diameter on a 100 μm raster width and a target plate (FIGS. 21A and 21B). All major ionic species detected from tissues were assigned to glycerophosphocholine (GC) and phosphatidylcholine (PC) adducts, including sodium and potassium, distributed in tissue sections compared to previous reports (FIG. 21C). Total ion maps were constructed using the mass peaks corresponding to [GC d18: 0/24: 0h + K] ⁺ and [GC d18: 1/22: 0h + Na] ⁺ (FIG. 21A).

Tissue slice samples require higher laser power than small molecule samples to obtain acceptable mass spectra, which can cause high background signals ( m / z 100-300) in low mass regions due to destruction of carbon nanomaterials. have. Brain tissue sections 20 μm thick in the substrate of the present invention showed much lower mass peak intensities than those in 15 μm thick brain tissue sections. Thus, for tissue analysis, the possible working mass range of the RGO / MWCNT-NH ₂ bilayer should be at least 300 m / z with thin tissue sections of 15 μm or smaller in thickness. The results showed that the RGO / MWCNT-NH ₂ bilayer platform of the present invention enables direct IMS analysis of tissue sections without the aid of existing organic substrates. Thus, difficulties with homogeneous application to the tissue surface and disadvantages associated with organic substrates such as sublimation during prolonged mass imaging processes have been avoided.

Claims (9)

A method of manufacturing a substrate on which reduced graphene oxide (RGO) / Multi-wall carbon nanotube (MWCNT) bilayer thin film is deposited, comprising the following steps:
1) immersing the aminated substrate in a graphene oxide (GO) aqueous suspension to prepare a GO-immobilized substrate;
2) washing and drying the GO-immobilized substrate;
3) heating the dried substrate;
4) immersing the prepared GO-immobilized substrate in an aminated MWCNT (MWCNT-NH ₂ ) suspension to prepare a GO / MWCNT-NH ₂ -thin substrate on which a GO / MWCNT bilayer thin film is deposited;
5) washing and drying the GO / MWCNT-NH ₂ -thin substrate prepared in step 4); And
6) heating the washed GO / MWCNT-NH ₂ -thin substrate.
7) reducing the GO / MWCNT-NH ₂ -thin substrate heated in step 6) by immersing in a hydrazine monohydrate solution; And
8) washing and drying the reduced GO (RGO) / MWCNT-NH ₂ -thin substrate.
The method of claim 1, wherein the aminated substrate is a glass coverslip.
The process according to claim 1, wherein the washing of step 2) uses at least one selected from the group consisting of water, ethanol, acetone, methanol, n-propanol, I-propanol and ether.
The process according to claim 1, wherein the washing of step 5) uses at least one selected from the group consisting of water, ethanol, acetone, methanol, n-propanol, I-propanol and ether.
delete delete The method of claim 1, wherein the solvent of the hydrazine monohydrate solution of step 7) is DMF, water, ethanol, toluene, methanol, ether, easter, chloroform, acetone, DMSO (dimethyl sulfoxide), propanol, or N-propanol Manufacturing method.
The process of claim 1, wherein the washing of step 8) uses DMF, water, ethanol, toluene, methanol, ether, easter, chloroform, acetone, DMSO (dimethyl sulfoxide), propanol, or N-propanol.
LDI-MS detection of hydrophobic molecules prepared according to the preparation method of claim 1 and RGO / MWCNT-NH ₂ -thin substrate for IMS.

Images