WO2024122903A1

WO2024122903A1 - Matrix for sample ionization, sample preparation method for maldi, and maldi-tof mass spectrometry method

Info

Publication number: WO2024122903A1
Application number: PCT/KR2023/018030
Authority: WO
Inventors: 변재철; 박형호
Original assignee: 연세대학교 산학협력단
Priority date: 2022-12-07
Filing date: 2023-11-10
Publication date: 2024-06-13
Also published as: KR20240084867A

Abstract

Disclosed are: a matrix for sample ionization; a sample preparation method for MALDI; and a MALDI/TOF mass spectrometry method. The matrix for sample ionization may comprise: a silica aerogel including silicon dioxide (SiO2); and an organic matrix filling pores of the silica aerogel.

Description

Matrix for sample ionization, sample preparation method for Maldi and Maldi mass spectrometry method

The present invention relates to a matrix for sample ionization, a sample preparation method for Maldi, and a Maldi mass spectrometry method.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Maldytop mass spectrometry) has been widely used in the analysis of large molecular weight biomolecules such as proteins and nucleic acids. In this method, most of the sample is ionized into a protonated form using organic molecules called a matrix to deliver laser energy to the sample. During the ionization process, the matrix molecules are fragmented into a form that cannot be reproduced by laser irradiation. Fragmented matrix molecules produce mass peaks in the low m/z range. For this reason, Malditop mass analysis of small molecules with molecular weights below a certain level has been limited.

Because it is performed after drying the sample on a Malditop mass spectrometry target plate, the sample crystals generally have a non-uniform shape, and the intensity of the mass peak from the sample crystal varies depending on the laser irradiation position even for a sample spot of the same concentration. , quantitative measurements for conventional malditope mass spectrometry were limited.

Porous silicon has been reported as an inorganic matrix for malditop mass spectrometry, and many research groups have attempted to develop matrices based on inorganic materials to replace organic matrices. This method is called LDI-MS (Laser Desorption/Ionization Mass Spectrometry). Inorganic matrices have been effectively used in the quantitative analysis of small molecules such as metabolites, drugs, and neurotransmitters because inorganic materials produce mass peaks without being fragmented by laser irradiation during measurement. Inorganic matrices have previously been fabricated using carbon-based nanomaterials such as carbon nanotubes and graphene, semiconductor-based nanomaterials such as Si, SiO ₂ , ZnO, SnO 2 , TiO 2 , and metal nanoparticles such as gold, silver, and platinum. .

One object of the present invention is to provide a matrix for sample ionization that does not generate mass peaks during Maldi analysis.

Another object of the present invention is to provide a method for preparing a sample for Maldi using the sample ionization matrix.

Another object of the present invention is to provide a Maldi mass spectrometry method using the sample preparation method for Maldi.

A matrix for sample ionization according to an embodiment of the present invention includes silica airgel containing silicon dioxide (SiO ₂ ); and an organic matrix filling the pores of the silica airgel.

In one embodiment, the organic matrix may include alpha-4-hydroxycinnamic acid or 2,5-Dihydroxybenzoic acid.

In one embodiment, the weight ratio of the organic matrix to the weight of the silica airgel may be about 5 to 390,000 times.

In one embodiment, the weight ratio of the organic matrix to the weight of the silica airgel may be about 25 to 79,000 times.

In one embodiment, the weight ratio of the organic matrix to the weight of the silica airgel may be about 120 to 3,100 times.

In one embodiment, the sample ionization matrix may have a porosity of about 74 to 91%.

The sample preparation method for maldi according to an embodiment of the present invention includes a first step of producing silica airgel nanoparticles by pulverizing silica airgel; A second step of preparing a suspension containing the silica airgel nanoparticles and an organic matrix material; and a third step of applying the suspension on a substrate to form a matrix and applying a sample on the matrix.

In one embodiment, the silica airgel nanoparticles may be pulverized to have a radius of about 100 to 200 nm.

In one embodiment, the organic matrix material may include alpha-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. .

In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 5 to 390,000 times.

In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 25 to 79,000 times.

In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 120 to 3,100 times.

The Maldi mass spectrometry method according to an embodiment of the present invention includes a first step of forming a matrix by applying a suspension containing silica airgel nanoparticles and an organic matrix material onto a substrate; A second step of applying a sample on the matrix; A third step of ionizing the sample by irradiating the matrix and the sample with a laser; and a fourth step of performing mass analysis of the ionized sample.

In one embodiment, the sample may contain dodecanoly-L-carnitine.

The matrix for sample ionization according to an embodiment of the present invention has a low detection amount of the matrix when ionizing the sample through laser irradiation, enabling analysis of small molecule samples.

Through the sample preparation method for Maldi according to an embodiment of the present invention, a sample capable of Maldi analysis can be prepared even for low-molecular-weight samples.

The Maldi mass spectrometry method according to an embodiment of the present invention can be used to analyze small molecule samples, and in particular, can be used for diagnosis by analyzing cancer biomarkers.

Figure 1 is a diagram schematically showing the structure of a matrix for sample ionization according to an embodiment of the present invention.

Figure 2 is a flow chart showing a sample preparation method for maldi according to an embodiment of the present invention.

Figure 3 is a flow chart showing the Maldi mass spectrometry method according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. Since the present invention can be subject to various changes and can have various forms, specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, and should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention. While describing each drawing, similar reference numerals are used for similar components. In the attached drawings, the dimensions of the structures are enlarged from the actual size for clarity of the present invention.

The terms used in this application are only used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, or a combination thereof described in the specification, but are not intended to indicate the presence of one or more other features or numbers. It should be understood that this does not exclude in advance the possibility of the existence or addition of steps, operations, components, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

Referring to Figure 1, the matrix 100 for sample ionization according to an embodiment of the present invention includes a silica airgel 101 containing silicon dioxide (SiO ₂ ); and an organic matrix 102 filling the pores of the silica airgel 101. Since Figure 1 schematically shows the silica airgel 101 and the organic matrix 102, the structure of the silica airgel 101, the size of the pores, the position and particle shape of the organic matrix 102, etc. are exemplary, and the present invention The scope is not limited to this.

The silica airgel 101 is a material containing silicon dioxide and may have porosity. In the context of this specification, porosity includes a significant number of pores, wherein the pores are formed to have a size that has a specific physical property and a property that emerges from the physical property, and refers to a material or member having such properties. can do. The silica airgel 101 may have appropriate porosity and corresponding porosity, and may have physical properties derived thereby. In addition, as will be described below, since the organic matrix 102 may always fill at least a portion of the pores of the silica airgel 101, the porosity of the silica airgel 101 is determined by the relative amount of the organic matrix 102 and the sample. When the ionization matrix 100 is used for actual analysis, the behavior of the organic matrix 102 may be affected when irradiated with a laser. In one embodiment, the sample ionization matrix 100 may have a porosity of about 74 to 91%.

The organic matrix 102 is not different from a conventional matrix used for sample ionization through a laser, and the type of organic matrix 102 is not limited as long as it can be used for sample ionization through a laser. In one embodiment, the organic matrix 102 may include alpha-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. You can. In one embodiment, at least a portion of the organic matrix 102 may fill the pores of the silica airgel 101.

The ratio of the silica airgel 101 and the organic matrix 102 can be appropriately adjusted depending on the properties of the desired sample ionization matrix 100. In one embodiment, the weight ratio of the organic matrix 102 to the weight of the silica airgel 101 may be about 5 to 390,000 times. At the above weight ratio, the peak of the matrix relative to the sample may be derived to be significantly low. In one embodiment, the weight ratio of the organic matrix 102 to the weight of the silica airgel 101 may be about 25 to 79,000 times. At the above weight ratio, the peak of the matrix relative to the sample can be derived to be significantly lower. In one embodiment, the weight ratio of the organic matrix 102 to the weight of the silica airgel 101 may be about 120 to 3,100 times. At the above weight ratio, a significant improvement in signal-to-noise ratio can be seen when analyzing the sample ionization matrix 100.

Referring to Figure 2, the sample preparation method for maldi (200) according to an embodiment of the present invention includes a first step (S210) of producing silica airgel nanoparticles by pulverizing silica airgel; A second step (S220) of preparing a suspension containing the silica airgel nanoparticles and an organic matrix material; and a third step (S230) of applying the suspension on a substrate to form a matrix and applying a sample on the matrix.

The description of the sample preparation method 200 for maldi according to the embodiment of the present invention may be applied in the same or similar manner to the same or similar configuration as the description of the matrix for sample ionization according to the embodiment of the present invention. Therefore, clearly inferred content may be omitted to avoid repetition, and should be construed as not intended to be excluded from the scope of the present invention.

The first step (S210) is a step of manufacturing silica airgel nanoparticles, and the silica airgel nanoparticles can be obtained by grinding pre-manufactured silica airgel. Since the silica airgel nanoparticles are then mixed with the organic matrix material in the solution, the silica airgel nanoparticles can be pulverized to an appropriate size to obtain the desired properties. In one embodiment, in the first step (S210), the silica airgel nanoparticles may be pulverized to have a radius of about 100 to 200 nm.

The second step (S220) is a step of preparing a suspension. As described above, the silica airgel nanoparticles prepared in the first step (S210) are added to the solution together with the organic matrix material and mixed to prepare a suspension. It's a step. Through the second step (S220), rearrangement may occur so that the organic matrix material fills the pores of the silica airgel nanoparticles. In one embodiment, the organic matrix material may include alpha-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. .

In the second step (S220), a suspension is prepared and the ratio of the silica airgel nanoparticles and the organic matrix material can be determined. As described above, the physical properties of the finally prepared sample ionization matrix and other properties resulting therefrom can be determined depending on the ratio of the silica airgel nanoparticles and the organic matrix material. In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 5 to 390,000 times. In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 25 to 79,000 times. In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 120 to 3,100 times.

The third step (S230) is the final step of preparing a sample target that can be ionized using a laser. The sample target may be formed by applying the suspension on a substrate to form a matrix, and then applying a sample on the matrix. The application method of the suspension and sample is not particularly limited, but methods such as spin-coating or drop-coating may be selected. In one embodiment, the sample may contain dodecanoly-L-carnitine.

Referring to FIG. 3, the Maldi mass spectrometry method 300 according to an embodiment of the present invention is a first step (S310) of forming a matrix by applying a suspension containing silica airgel nanoparticles and an organic matrix material on a substrate. ; A second step of applying a sample on the matrix (S320); A third step (S330) of ionizing the sample by irradiating a laser to the matrix and the sample; and a fourth step (S340) of performing mass analysis on the ionized sample.

The description of the Maldi mass spectrometry method 300 according to the embodiment of the present invention includes the description of the matrix for sample ionization according to the embodiment of the present invention or the description of the sample preparation method for Maldi according to the embodiment of the present invention. It may be applied identically or similarly to the same or similar configuration. Therefore, clearly inferred content may be omitted to avoid repetition, and should be construed as not intended to be excluded from the scope of the present invention.

The first step (S310) is a step of forming a matrix, and the matrix can be formed by applying a suspension containing silica airgel nanoparticles and an organic matrix material. In one embodiment, the suspension may be the suspension described in the sample preparation method for maldi according to the embodiment of the present invention. In one embodiment, the matrix formed by applying the suspension may be a matrix for sample ionization according to an embodiment of the present invention. The method of application is not particularly limited, but may be applied by spin-coating or drop-coating. In one embodiment, the organic matrix material may include alpha-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. . In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 5 to 390,000 times. In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 25 to 79,000 times. In one embodiment, the suspension may be prepared so that the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is about 120 to 3,100 times.

The second step (S320) is a step of applying the sample to be analyzed on the matrix. The sample to be analyzed is not particularly limited, but in one embodiment, the sample may contain dodecanoly-L-carnitine. The dodecanoly-L-carnitine is a substance known to be used as a biomarker for colon cancer, etc., and in one embodiment, the Maldi mass spectrometry method 300 according to an embodiment of the present invention can be used to diagnose colon cancer. there is. The method of application is not particularly limited, but may be applied by spin-coating or drop-coating.

The third step (S330) is a step of ionizing the sample by irradiating a laser to the applied analysis target sample and matrix. The laser may be any type of laser that can be used for sample ionization, and is not particularly limited. Fragmentation and ionization of the matrix can be derived through irradiation of the laser, and thereby (assisted) the sample to be analyzed can also be ionized.

The fourth step (S340) is a step of performing mass analysis on the ionized sample. The method of performing mass analysis of an ionized sample is not particularly limited as long as it is a method known at the time of filing the present application. The ionized sample can be accelerated for mass analysis, and electromagnetic methods can be used to accelerate the ionized sample. The behavior of the accelerated ionized sample varies depending on the mass, and the method for observing the behavior is not particularly limited as long as it is a method known at the time of filing the present application. In one embodiment, the behavior can be fractionated by mass and analyzed qualitatively and/or quantitatively by a time of flight (TOF) analyzer.

Hereinafter, embodiments of the present invention will be described in detail. However, the examples described below are only some embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

재료ingredient

Dodecanoyl-L-carnitine (DC), proline, serine, glutamic acid, histidine, alpha-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), human serum, fluorescein, Acetonitrile, 0.1% trifluoroacetic acid (TFA), formic acid, ammonium fluoride (NH4F), sulfuric acid (H ₂ SO ₄ ), trimethylchlorosilane (TMCS), methanol, isopropyl alcohol, and n-hexane. It was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Sodium silicate solution was purchased from Duksan Chemical Co. (Ansan, Korea). Double-distilled deionized water (18 MΩ·cm) was obtained using a Milli-Q® water purification system (Millipore, Billerica, MA, USA).

시료 제작Sample preparation

For the CHCA organic matrix, the matrix solution was prepared in a mixed solution of 50% acetonitrile/0.1% trifluoroacetic acid (1:1, v/v). An organic matrix with SiO ₂ airgel was prepared in EtOH to disperse the airgel in suspension form while maintaining the same concentration of CHCA for further applications. Samples for matrix model analysis were prepared by dissolving DC in DW/MeOH (1:1, v/v) and vortexing for 30 s. Standard DC samples for calibration were prepared by serial dilution of DC samples with DW/MeOH (1:1, v/v). DC-spiked serum samples were prepared by spiking human serum products with known concentrations of DC. DC spiked serum samples were processed using the acetonitrile formic acid extraction method. In this method, serum samples (100 μL) are diluted with acetonitrile/formic acid (99:1, v/v, 400 μL) and incubated in an ice bath for 30 minutes. The mixture was centrifuged at 14,000 rpm and 4°C for 10 min, and the supernatant (300 μL) was transferred to a new EP tube for further analysis. Serum samples from colorectal cancer patients (n = 20) were obtained from the National Cancer Center (Goyang, Korea), and control samples were collected from healthy volunteers (n = 10). The study protocol was approved by the Institutional Review Board (IRB) of Severance Hospital (IRB No. 4-2017-0321). All study procedures were performed in accordance with relevant guidelines and regulations. Patient serum samples were subjected to previously described extraction methods.

SiO2 에어로겔을 포함하는 매트릭스 성질Matrix properties containing SiO2 airgel

The LDS-MS matrix must be able to ionize the target sample without mass peaks of matrix molecules appearing at low m/z values of 1000 or less (Figure 4a). In the case of SiO ₂ airgel mixed with CHCA, the matrix peak of CHCA disappeared, and only the mass peak of the sample was obtained. These results imply that the SiO ₂ airgel/CHCA mixture can be used as a solid matrix for analyzing small molecules. In the present invention, SiO ₂ airgel was synthesized by a conventional sol-gel method using Na ₂ SiO ₂ as a precursor in distilled water. After aging in alcohol, the SiO ₂ airgel was prepared into millimeter-sized particles (FIG. 4a). From Brunauer, Emmett, and Teller (BET) analysis, the specific surface area was measured to be 540 m ² /g, the total pore volume was measured to be 1.3 cm ³ /g, and the average pore size was measured to be 9.3 nm. . To apply the SiO ₂ airgel to LDS-mass spectrometry, the SiO ₂ airgel was ground into powder using a microbead homogenizer. By DLS analysis, the hydrodynamic radius of SiO ₂ airgel particles was measured to be 100 to 200 nm, and the zeta potential was measured to be -2504 ± 247 mV (n=5). These results show that the SiO ₂ airgel has a porous structure with a porosity greater than 82.1%.

The applicability of SiO ₂ airgel as a solid matrix for LDI-MS was investigated step by step by mixing it with a conventional organic matrix (CHCA) (Figure 4b). The model amino acid (histidine, Mw = 155.2 Da) itself did not produce a mass peak without an organic matrix in a conventional Malditop mass spectrometer (case 1 in Figure 4b). It is generally known that matrices fragment when exposed to lasers. When an organic matrix such as CHCA was used as a sample for Maldives mass spectrometry (case 2 in Figure 4b), there were unreproducible mass peaks at low m/z below 500, as in the Dongrae study. Therefore, the mass peaks of amino acids with a molecular weight range of 100 to 200 Da were difficult to separate from the mass peaks of fragmented matrix molecules. Individually, when the SiO ₂ airgel was used as a sample for LDI-Top mass spectrometry (case 3 in Figure 4b), no mass peak was detected because the airgel itself was not ionized and desorbed by the laser. No mass peaks were detected when SiO ₂ airgel and amino acids were mixed as model samples (case 4 in Figure 4b) and in the case of the conventional organic matrix of CHCA (case 5 in Figure 4b). Therefore, it was confirmed that SiO ₂ airgel itself is not sufficient to ionize amino acids and other organic molecules (CHCA). The mass peak of the model amino acid (histidine) was observed only when the SiO ₂ airgel was mixed with the conventional organic matrix (CHCA) (case 6 in Figure 4b). These results showed that SiO ₂ airgel can be used for mass peak removal from organic matrix molecules and ionization of target samples after mixing with conventional organic matrix (CHCA), named as combi matrix (SiO ₂ airgel + CHCA). The ionization mechanism of the sample in the combi matrix can be explained based on the properties of the airgel. No mass peaks were detected for the SiO ₂ airgel (case 3 in Figure 4b) and the combination matrix without sample (case 4 in Figure 4b). These results are identical to the results of the parylene matrix chip, which has been reported as a matrix system suitable for small molecule analysis by using a nanoporous parylene film as a mixing area between the matrix and the sample, preventing excessive energy radiation by the laser to the organic matrix. . When the combi-matrix is mixed with the sample, as in the case of the parylene-matrix chip, mixing of the matrix molecules and the target sample occurs within the nanopores of the airgel, which leads to a mass peak from the target sample (case 6 in Figure 4b). . Therefore, the porous structure of SiO ₂ airgel is considered to provide a mixing area for the target sample and organic matrix molecules.

The properties of the combi matrix (SiO ₂ airgel + CHCA) were compared using different types of amino acids with nonpolar (proline), polar (serine), acidic (glutamic acid), and basic (histidine) side chains, and the same properties of ionizing amino acids were compared. This was observed. To test the feasibility of different types of organic matrices, a combi matrix was created using SiO ₂ airgel and DHB. The same types of amino acids were then analyzed using DHB and combi matrix. Amino acid analysis was judged to be possible as in the case of the CHCA-based combi matrix. First, the optimal ratio of components for the combi matrix was assumed to produce no CHCA mass peak (case 6 in Figure 4b). To optimize the amount of airgel needed as a matrix to remove the mass peak in CHCA, the amount of SiO ₂ airgel in the combi matrix was controlled in the range from 2.4 × 10 ^-5 to 2.0 mg/mL and the organic matrix was 10 mg/mL ( was fixed at 52.8 μmol). The amount of SiO ₂ airgel corresponded to the specific surface area in the previous BET analysis using a conversion factor of 5.4 x 10 ³ cm ² /mg. The ratio (M/A) of the organic matrix (M, CHCA) and SiO ₂ airgel (A) was adjusted to 1:0.2 to 1:2.6 × 10 ^-6 (weight per weight, w/w), which is the surface area of the airgel This corresponds to 4.9×10 ^-3 to 4.1×10 ² μmol/cm ² considering 5.4×10 ³ cm ² /mg (FIG. 5a). For each measurement, the amount of combi matrix was controlled to be approximately 5.0 μg per sample point. The intensities of the combi matrix peaks were calculated by averaging the mass peaks in the m/z range from 100 to 400, and the error bars were estimated to be 3 times the standard deviation (σ). The effective M/A ratio for removing organic matrix noise signals was estimated to be less than 6.3×10 ² (w/w), which is greater than 1.6×10 ⁻² mg of SiO ₂ airgel for 10.0 mg of organic matrix (CHCA). Corresponds to sheep (Figure 5b). These results show that the combi-matrix can be optimized as a solid matrix for LDI-mass spectrometry without interference of the mass peak of CHCA at a ratio (M/A) of 6.3 × 10 ² (w/w). In the next step, DC was used as the target sample to analyze the optimal combination matrix without interference from the mass peak of CHCA. Similar to the previous experiment, the ratio (M/A) of the organic matrix (M, CHCA) and SiO ₂ airgel (A) was adjusted to the range of 1:0.2 to 1:2.6 × 10 ^-6 (w/w), and the target The sample (DC) was set to 10 μg/mL at a fixed concentration. When the ratio (M/A) was less than 1.3 × 10 ² (w/w), the matrix peak completely disappeared (Figure 5c). As in the previous experiment, the intensity of the combi matrix peak was calculated by averaging the mass peaks in the m/z range from 100 to 400 (AU), the signal-to-noise (S/N) ratio was calculated, and compared with the mass intensity in DC ([DC +H] ⁺ =344.5). Referring to Figure 5d, it shows the optimal ratio (M/A) estimated to be 6.3×10 ² (w/w), and the highest signal-to-noise ratio is 154.2 (AU). These results show that the maximum S/N ratio can be achieved for the combi matrix at controlled ratios of organic matrix and SiO ₂ airgel.

SiO2 에어로겔을 매트릭스로 사용하는 정량 분석Quantitative analysis using SiO2 airgel as matrix

LDI-MS was used for quantitative analysis of small molecules. However, conventional Malditop mass spectrometry was performed using dried samples; Therefore, even at the same sample point, non-uniform crystals were formed on the target plate and the amount of sample ions generated was different depending on the laser irradiation location. For this reason, direct quantification of samples using Malditop mass spectrometry was difficult, and for quantification, a method of mixing internal standards at known concentrations was typically used. Typically, LDI-MS uses inorganic nanostructures instead of organic matrices. In the case of an inorganic matrix, even if the sample crystal of each nanostructure has a non-uniform shape, the sample spot has a uniform distribution of the sample on the target plate, and the amount of sample ions in each sample spot is statistically the same depending on the laser irradiation location. (Figure 6a). Therefore, quantification of samples using LDI-MS was influenced by the homogeneous and concentrated distribution of sample crystals fabricated on nanostructures.

Direct quantification was performed using the combi matrix as the matrix for LDI mass spectrometry. First, the distribution of SiO ₂ airgel was observed on the target plate using the crystal of Figure 6b, and the purpose was to form uniform sample crystals using a combi matrix using a fluorescent dye as a sample. The morphology of the sample crystals was compared for different concentrations of SiO ₂ airgel (first row in Figure 6b), and the fluorescence images were compared for a homogeneous distribution of the sample (second row in Figure 6b). For the sample crystals without airgel, the sample crystals in the combi matrix showed uniform fluorescence in the sample crystals. Then, the fluorescence image (second row in Figure 6b) was analyzed from the fluorescence intensity of the sample crystals using commercial software from Teledyne Photometrics (Tuscon, AZ, USA) to show the distribution of the sample (fluorescent dye) within the sample crystals. . The 3D image of the sample crystal (third row in Figure 6b) shows the distribution of the sample using the fluorescence signal. As can be seen in the fluorescence image (second row of Figure 6b), it was observed that the coffee-ring effect was minimized as the concentration of airgel increased for the combi matrix. The distribution of the sample was analyzed along the lines shown in the 3D image and plotted as a 2D drawing image (4th row in Figure 6b). These 2D images also showed that the coffee-ring effect was observed to be minimal as the concentration of airgel increased for the combi matrix. These results show that a homogeneous and dense distribution of the sample in the target plate can be achieved by SiO ₂ airgel for quantitative analysis using LDI-mass spectroscopy.

As mentioned earlier, quantification of samples using conventional Maldi-MS is difficult because non-uniform crystals are formed on the target plate. Therefore, even at the same sample point, the number of ionized samples varies depending on the laser irradiation location. The combi matrix produced a uniform and concentrated distribution of nano-sized crystals in the organic matrix, and direct quantification of samples without the use of internal standards was demonstrated. Quantifying samples using LDI-MS requires achieving high reproducibility in repeated measurements, such as spot-to-spot and shot-to-shot reproducibility. For reproducibility estimation, DC samples at a concentration of 1 pmol/μL were used, and the combi matrix determined to be optimal in previous experiments was prepared at 6.3 × 10 ² (w/w) (Figure 5d). Measurements were performed for 1,000 shots using the Random Work Function. First, the average signal (m) was calculated from five sample spots, and then the standard deviation (σ) between the average signals was calculated (n=5). Relative standard deviation (RSD=m/σ×100 in %) and reproducibility (%) were calculated as 100-RSD. Spot-to-spot reproducibility was estimated to be 61.5% for conventional Maldi-Top mass spectrometry and 96.0% for LDI-Top mass spectrometry across five independent sample spots (Figure 7a). Shot-to-shot reproducibility was estimated at five different locations of the same sample point and measurements were also performed for 1,000 shots using the Random Work Function (Figure 7b). The shot-to-shot reproducibility was estimated to be 61.5% for the conventional Maldi-Top mass spectrometer and 90.4% for the LDI-Top mass spectrometer using a combi matrix using the same DC sample. These results indicate that Combi-Matrix can be effectively used for quantitative analysis of samples with much higher spot-to-spot and shot-to-shot reproducibility compared to conventional Maldi-Top MS.

To investigate the influence of the thermal properties of SiO ₂ airgel in LDI-MS, samples from a conventional Maldi MS were exposed to a laser, ionized and evaporated on a target plate made of stainless steel. Due to the insulating properties of SiO ₂ airgel, the dissipation of thermal energy through the target plate by laser irradiation can be minimized in the combi matrix (Figure 8a). DSC measurements were made at a ratio of 5 to 3.9 × 10 ⁵ (w/w) for the combi-matrix, and the temperature range was set at 200 to 300°C at a temperature rise rate of 15.0°C/s (FIG. 8b). The DSC graph of organic matrix without SiO ₂ aerogel (CHCA) showed that heat flow started at a temperature of 266.1 °C (onset temperature) and reached the peak of heat flow at 270.2 °C. As the relative amount of SiO ₂ airgel increased, the melting transition of CHCA occurred and the thermal parameters such as peak temperature, onset temperature, and melt enthalpy decreased. Additionally, the thermal conductivity of the combi matrix sample was calculated from the DSC thermogram using the modified Fourier law:

k = QL/AΔT

Here, k is the thermal conductivity (W/Km), Q is the heat transfer rate, L is the thickness of the sample in the DSC pan, A is the cross-sectional area of the sample, and ΔT is the temperature change.

The thermal conductivity of the sample was calculated by replacing Q/ΔT in the above equation with the slope of the DSC curve. The slope decreased as the airgel restricted heat flow to the sample. This indicates that the thermal conductivity decreased from 9.0 × 10 ^-2 (W/Km) to 3.0 × 10 ^-2 (W/Km), indicating an increase in insulation (Figure 8c). In the previous experiment (Fig. 5b), the combi matrix was used as a composite of CHCA at a ratio (M/A) of 6.3 × 10 ² (w/w), which allows the combi matrix to have a sufficiently low thermal conductivity to avoid dissipation of laser energy through the target plate. It could be optimally used as a solid matrix for LDI-mass spectrometry without interference of mass peaks. These results showed that by using SiO ₂ airgel in the combi-matrix, heat transfer to the target plate can be minimized and radiant energy can be used more effectively for ionization and desorption of the sample in LDI-MS.

The thermal properties of DSC analysis were also used to illustrate the principles of quantitative analysis using combi matrices. In the case of quantitative analysis using LDI-MS, nanostructured sample crystals are densely and uniformly distributed (Figure 6a). As previously discussed, the organic matrix occupied the pores of the SiO ₂ airgel to form combi-matrix nanocrystals. In DSC analysis of the combi-matrix, the formation of nanocrystals in the pore organic matrix (CHCA) of SiO ₂ airgel was confirmed by estimating the size of the combi-matrix crystals using the Gibbs-Thomson equation. The Gibbs-Thomson equation relates the change in melting temperature from bulk to nanocrystal size:

r _crystal = (2T ₀ σV _m )/(ΔH _fus (T ₀ -T))

where r is the radius of the crystal (m), T ₀ is the melting temperature in the bulk state (K), σ is the interfacial tension of the crystal (N/m), V _m is the molar volume of the crystal, and ΔH _fus is the enthalpy of fusion (J/m). mol), T is the melting temperature (K) of the nanostructure. The DSC graph of the organic matrix without SiO ₂ aerogel (CHCA) shows that the heat flow starts at 266.1 °C (onset temperature) and peaks at 270.2 °C. As the relative amount of SiO ₂ airgel increased from 2.4 × 10 ^-5 to 2.0 mg/mL, the onset temperature decreased from 266.1 to 259.6 °C (Figure 8b). In DSC measurements, when the relative amount of SiO ₂ airgel was changed from 0 to 6.3 × 10 ² (w/w), the change in melting temperature (ΔT) for the optimized combi matrix was estimated to be 1.4 K. From the Gibbs-Thomson equation, the average crystal radius (r) of the organic matrix (CHCA) of the combi matrix was calculated to be 463.2 nm, which is much smaller than the bulk state (r _bulk > 29.1 μm) as shown (Figure 8d). These results show that quantification of samples is possible using the combi-matrix from the homogeneous and concentrated distribution of combi-matrix nanocrystals.

DC 분석에의 적용Application to DC analysis

Combi-Matrix was applied as a matrix for quantitative analysis of dodecanoly-L-carnitine (DC) using LDI-MS. DC has been reported as a biomarker for colon cancer, and an increased concentration of DC is observed in patient serum due to increased lipid metabolism (beta-oxidation) in the mitochondria of cancer cells compared to a concentration of 0.1 pmol/μL DC in normal serum. It is known. DCs were analyzed using a combi-matrix-based LDI mass spectrometer. In the spectrum, the matrix peak was significantly reduced compared to mass spectrometry using a conventional organic matrix (CHCA), and the intensity of the mass peak of DC ([DC+H] ⁺ ) increased to the range of 0.01 to 100 pmol/μL. The standard curve showed a linear correlation in the concentration range from 0.01 to 100 pmol/μL, the linear coefficient (R ² ) was 0.943, and the limit of detection (LOD) was determined to be 0.1 pmol/μL (Figure 9a). Because of the low concentration of DC in serum, the response of the samples was clearly expressed on a logarithmic scale. The dose response curve was fitted linearly as log ₁₀ (intensity) = 0.56 × log ₁₀ ([DC]) + 4.7. For quantitative measurement of DC in serum, samples (DC) were extracted using acetonitrile with formic acid as an extraction solution. Standard samples of DCs in serum were analyzed using LDI-mass spectrometry based on combi matrix as matrix. The spectra showed a significant reduction in matrix peaks compared to conventional mass spectrometry using an organic matrix (CHCA). The intensity of the mass peak of DC ([DC+H] ⁺ ) increased quantitatively in the concentration range from 0.01 to 100 pmol/μL. The standard curve yielded a linear correlation in the concentration range 0.01 to 100 pmol/μL with a coefficient of linearity (R ² ) of 0.967 and the limit of detection (LOD) was estimated to be 0.01 pmol/μL (Figure 9b). The dose-response curve was fitted linearly as log ₁₀ (intensity) = 0.41 × log ₁₀ ([DC]) + 4.2. These results showed that the intensity of the mass peak from losses in the extraction step was significantly reduced compared to that of the standard sample in the same concentration of buffer. However, even after the extraction step, the dynamic range of the measurement was observed to increase significantly.

Sera from healthy volunteers (n = 10) and patients (n = 20) were used for quantitative analysis of DC, and intensity plots of mass peaks for real samples are shown (Figure 10a). Results were compared with the cutoff intensity value (3137.6 (a.u.)) for medical diagnosis of colon cancer. Some patient sera showed relatively higher intensities than normal samples from healthy volunteers. The mass peak intensity of the actual sample was converted to the concentration of DC and plotted on a standard curve to distinguish patient and normal serum (FIG. 10b).

The test results of real samples were compared with those of conventional LC-MS, and the agreement of the two different methods, LDI-mass spectrometry and LC-MS, was statistically analyzed using Blund-Altman plots and Passing-Bablok regression ( Figure 10c). The Bland-Altman plot suggests that the signal difference was distributed within the 95% confidence level (1.96±σ). This test indicates that both methods are strongly correlated with similar results found for DC analysis of serum samples. As a result of MS analysis of the two methods distributed within the 95% confidence level (1.96±σ) in Passing-Bablok regression, the Spearman correlation coefficient (ρ) was found to be 0.947 (P<0.0001). In these results, a Spearman correlation coefficient (ρ) greater than 0.5 indicates good correlation between the two different analysis methods. These two analysis results show that statistically consistent results can be obtained using combi-matrix-based LDI-MS and conventional LC-MS. Therefore, combi-matrix-based LDI-MS can be applied to the medical diagnosis of colorectal cancer using DC as a biomarker.

Although the present invention has been described above with reference to preferred embodiments, those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the present invention as set forth in the following patent claims. You will understand that it is possible.

[Explanation of symbols]

100 Matrix for sample ionization

101 Silica Airgel

102 Organic Matrix (Substance)

Sample Preparation Method for 200 Maldi

300 Maldi mass spectrometry method

Claims

In a matrix that transmits laser energy to a sample to induce desorption and ionization of the sample,

Silica airgel containing silicon dioxide (SiO 2 ); and

Containing; an organic matrix filling the pores of the silica airgel;

Matrix for sample ionization.
According to paragraph 1,

The organic matrix includes alpha-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid,

Matrix for sample ionization.
According to paragraph 2,

The weight ratio of the organic matrix to the weight of the silica airgel is 5 to 390,000 times,

Matrix for sample ionization.
According to paragraph 3,

The weight ratio of the organic matrix to the weight of the silica airgel is 25 to 79,000 times,

Matrix for sample ionization.
According to paragraph 4,

The weight ratio of the organic matrix to the weight of the silica airgel is 120 to 3,100 times,

Matrix for sample ionization.
According to paragraph 1,

The matrix for sample ionization has a porosity of 74 to 91%,

Matrix for sample ionization.
A first step of producing silica airgel nanoparticles by pulverizing silica airgel;

A second step of preparing a suspension containing the silica airgel nanoparticles and an organic matrix material; and

A third step of applying the suspension on a substrate to form a matrix and applying a sample on the matrix.

Sample preparation method for maldi.
In clause 7,

Grinding the silica airgel nanoparticles so that they have a radius of 100 to 200 nm,

Sample preparation method for maldi.
In clause 7,

The organic matrix material includes alpha-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid,

Sample preparation method for maldi.
According to clause 9,

In the suspension, the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is prepared to be 5 to 390,000 times,

Sample preparation method for maldi.
According to clause 10,

In the suspension, the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is prepared to be 25 to 79,000 times,

Sample preparation method for maldi.
According to clause 11,

In the suspension, the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is prepared to be 120 to 3,100 times,

Sample preparation method for maldi.
A first step of forming a matrix by applying a suspension containing silica airgel nanoparticles and an organic matrix material onto a substrate;

A second step of applying a sample on the matrix;

A third step of ionizing the sample by irradiating the matrix and the sample with a laser; and

A fourth step of performing mass analysis of the ionized sample; comprising,

Maldi mass spectrometry method.
According to clause 13,

The sample contains dodecanoly-L-carnitine,

Maldi mass spectrometry method.
According to clause 13,

The organic matrix material includes alpha-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid,

Maldi mass spectrometry method.
According to clause 15,

In the suspension, the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is prepared to be 5 to 390,000 times,

Maldi mass spectrometry method.
According to clause 16,

In the suspension, the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is prepared to be 25 to 79,000 times,

Maldi mass spectrometry method.
According to clause 17,

In the suspension, the weight ratio of the organic matrix to the weight of the silica airgel nanoparticles is prepared to be 120 to 3,100 times,

Maldi mass spectrometry method.