JP2018185200A - Laser desorption ionization mass spectrometry, and organic silica porous membrane substrate for laser desorption ionization mass analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser desorption / ionization mass spectrometry method capable of efficiently performing mass spectrometry with higher detection sensitivity without using a matrix. SOLUTION: The organic silica porous film has an organic group capable of absorbing irradiation laser light in a skeleton, has pores having an average pore diameter of 5 to 50 nm, and has a surface opening ratio of 33 to 70%. On the other hand, after carrying a sample containing a molecule to be measured, the sample-carrying portion of the membrane is irradiated with a laser beam to ionize the molecule to be measured and perform mass spectrometry. Ionized mass spectrometry. [Selection diagram] None

Description

  The present invention relates to laser desorption ionization mass spectrometry and an organic silica porous membrane substrate for laser desorption ionization mass spectrometry.

  Mass spectrometry (MS) is a method in which a sample containing a molecule to be measured is ionized and ions derived from the molecule to be measured are separated and detected by a mass-to-charge ratio (mass / charge (m / z)). This is an analysis method for obtaining information on the chemical structure of the molecule to be measured. In such mass spectrometry (MS), sample ionization is an important process that determines whether analysis is possible and the quality of the spectrum obtained. Various ionization methods have been developed so far to efficiently ionize the sample. It has been.

  As an ionization method employed in such mass spectrometry, for example, laser desorption / ionization (LDI) is known. Particularly in the bio field, so-called matrix-assisted laser desorption ionization is known. The method (matrix-assisted laser destruction / ionization: MALDI) has been widely used. However, mass spectrometry using such matrix-assisted laser desorption / ionization (MALDI) is a substance that has a light absorption property called a matrix, such as molecules (for example, proteins, peptides, saccharides, etc.) to be measured. Is dispersed and the laser is irradiated to ionize the molecules to be measured together with the matrix, so the selection of the matrix to be used and the quality of the mixture of the matrix and the molecules to be measured will determine the success of the analysis. There is a problem that it has a great influence, and there is also a problem that a complicated operation such as pre-scanning is required for measurement. Therefore, mass spectrometry using matrix-assisted laser desorption / ionization (MALDI) cannot always perform mass spectrometry efficiently.

  Under such circumstances, in recent years, development of a method for performing mass spectrometry by ionizing a molecule to be measured without using a matrix while employing a laser desorption ionization method has been advanced. For example, in Japanese Patent Application Laid-Open No. 2014-115187 (Patent Document 1), a sample containing a molecule to be measured is supported on a porous organic silica body having an organic group capable of absorbing irradiation laser light as a skeleton, and then laser light is emitted. A method of performing mass spectrometry by irradiating and ionizing the molecule to be measured is disclosed.

JP 2014-115187 A

  The method described in Patent Document 1 is a method capable of performing mass spectrometry sufficiently efficiently without using a matrix while employing a so-called laser desorption ionization method (LDI). However, in the field of mass spectrometry that uses such LDI, it is desired that an analysis method with higher detection sensitivity will emerge.

  The present invention has been made in view of the above-described problems of the prior art, a laser desorption ionization mass spectrometry method capable of efficiently performing mass spectrometry with higher detection sensitivity without using a matrix, and An object of the present invention is to provide an organic silica porous membrane substrate for laser desorption / ionization mass spectrometry used in the method.

  As a result of intensive studies to achieve the above object, the present inventors have an organic group capable of absorbing irradiation laser light in the skeleton, have pores with an average pore diameter of 5 to 50 nm, and A sample containing the molecule to be measured is supported on the organic silica porous film having a surface aperture ratio of 33 to 70%, and then the sample supporting part of the film is irradiated with laser light to ionize the molecule to be measured. By performing mass spectrometry, it has been found that mass spectrometry can be efficiently performed with higher detection sensitivity without using a matrix, and the present invention has been completed.

  That is, the laser desorption ionization mass spectrometry method of the present invention has an organic group capable of absorbing irradiated laser light in its skeleton, has pores with an average pore diameter of 5 to 50 nm, and a surface aperture ratio of 33. A sample containing a molecule to be measured is supported on an organic silica porous membrane of ˜70%, and then the sample carrying portion of the membrane is irradiated with laser light to ionize the molecule to be measured and perform mass spectrometry. It is the method characterized by performing.

  In the laser desorption ionization mass spectrometry of the present invention, the organic group preferably has an absorption maximum wavelength in the range of 200 to 600 nm. In the laser desorption / ionization mass spectrometry of the present invention, the organic group is preferably an aromatic organic group containing 4 or more carbons. Furthermore, in the laser desorption ionization mass spectrometry of the present invention, it is preferable that triphenylamine is included as the organic group.

  Moreover, the organic silica porous membrane substrate for laser desorption ionization mass spectrometry of the present invention has an organic group capable of absorbing laser light in its skeleton, has pores with an average pore diameter of 5 to 50 nm, and An organic silica porous film having a surface opening ratio of 33 to 70% is included.

  According to the present invention, laser desorption / ionization mass spectrometry capable of performing mass spectrometry efficiently with higher detection sensitivity without using a matrix, and laser desorption / ionization mass spectrometry used for the method It is possible to provide an organic silica porous membrane substrate.

(A) is an AFM image of the porous membrane, (b) is a graph of a frequency distribution curve of height obtained from the AFM image, and (c) is a graph of the AFM image using a threshold value obtained from the graph. Is a binarized image. 2 is a scanning electron micrograph of a cross section of the substrate for mass spectrometry obtained in Example 1. FIG. 4 is a scanning electron micrograph of the cross section of the substrate for mass spectrometry obtained in Example 2. FIG. 2 is a scanning electron micrograph of a cross section of a substrate for mass spectrometry obtained in Comparative Example 1. FIG. 2 is an atomic force microscope image of the surface of an organic silica porous film of a substrate for mass spectrometry obtained in Example 1. FIG. 2 is an atomic force microscope image of the surface of an organic silica porous film of a substrate for mass spectrometry obtained in Example 2. FIG. 2 is an atomic force microscope image of the surface of an organic silica porous film of a substrate for mass spectrometry obtained in Comparative Example 1. FIG. It is a graph which shows the absorption spectrum of the organic silica porous film in the measurement sample (what produced the organic silica porous film (TPA-PMO thin film) on the quartz substrate) utilized for the absorption spectrum measurement experiment. 4 is a graph of mass spectrum measured in Example 3 and a graph of mass spectrum measured in Comparative Example 2. FIG. 4 is a graph of mass spectrum measured in Example 4, a graph of mass spectrum measured in Example 5, and a graph of mass spectrum measured in Comparative Example 3. FIG. 10 is a graph of m-spectrum measured in Example 6. 10 is a graph of m-spectrum measured in Example 7. 10 is a graph of maspectrum measured in Comparative Example 5. 10 is a graph of maspectrum measured in Example 8.

  Hereinafter, the present invention will be described in detail with reference to preferred embodiments thereof.

[Laser desorption ionization mass spectrometry]
The laser desorption ionization mass spectrometry of the present invention has an organic group capable of absorbing irradiation laser light in its skeleton, has pores with an average pore diameter of 5 to 50 nm, and a surface aperture ratio of 33 to 70. % Of the organic silica porous membrane is loaded with a sample containing the molecule to be measured, and then the sample carrying portion of the membrane is irradiated with laser light to ionize the molecule to be measured for mass spectrometry. It is the method characterized by this.

<Organic silica porous membrane>
The organic silica porous membrane used for mass spectrometry in the present invention has an organic group capable of absorbing irradiation laser light in its skeleton, has pores with an average pore diameter of 5 to 50 nm, and a surface aperture ratio of 33. It is an organic silica porous membrane which is ˜70%.

  Such an organic silica porous film has an organic group capable of absorbing irradiated laser light in its skeleton. Here, in the present invention, the “organic group capable of absorbing irradiation laser light” is not particularly limited as long as it is an organic group having a structural part capable of absorbing laser light used in mass spectrometry. Depending on the wavelength of the laser beam used, for example, an organic group having an aromatic ring as a structural part capable of absorbing the laser beam (for example, triphenylamine, fluorene, acridone, methylacridone, quaterphenyl, Among them, an organic group described as X in the general formula (1) described later can be preferably used.

  Moreover, as an organic group which can absorb such irradiation laser light, it is preferable that it is a group which has an absorption maximum wavelength in the range of 200-600 nm (more preferably 250-450 nm, still more preferably 300-400 nm). When the absorption maximum wavelength of the organic group is less than the lower limit, the organic group in the measurement object and the organic silica porous film is decomposed when irradiated with laser light having such a wavelength, and as a result, mass analysis is performed efficiently. On the other hand, if it exceeds the upper limit, it tends to be difficult to obtain light energy necessary for ionization. Thus, when the organic group has an absorption maximum wavelength in the above wavelength range, it becomes possible to more efficiently absorb laser light in a wavelength region used for mass spectrometry. Examples of the organic group having an absorption maximum wavelength in the range of 200 to 600 nm include, for example, triphenylamine, styrylbenzene, fluorene, acridone, methylacridone, quater, which may each have a substituent. Examples include terphenyl and anthracene.

  Furthermore, the organic group capable of absorbing such irradiation laser light is preferably a group having one or more aromatic rings (an aromatic organic group). Such an organic group is more preferably an aromatic organic group containing 4 or more carbon atoms. According to such an aromatic organic group, it becomes possible to absorb laser light more efficiently. Examples of such aromatic organic groups include, for example, triphenylamine, styrylbenzene, fluorene, acridone, methylacridone, quaterphenyl, anthracene, pyrene, styrylbenzene, and acridine, each of which may have a substituent. , Divinylbenzene, divinylpyridine, phenylpyridine, dithienylbenzothiadiazole and the like. Moreover, the said organic silica porous film may have one type of organic group independently as an organic group, or may have a combination of a plurality of types of organic groups. In addition, as such an organic group, from the viewpoint that it exhibits oxidation / reduction activity by light irradiation and is stable, it contains triphenylamine (at least one of the organic groups is triphenylamine). preferable.

In the present invention, “having an organic group in the skeleton” means that the organic group bonded directly or indirectly (via another element) to silicon (Si) forming the silica skeleton of the silica porous membrane. Means that it exists. In addition, as such an organic silica porous film, by having a structure (crosslinked structure) in which silicon atoms forming a siloxane structure (formula:-(Si-O) _n -structure) are crosslinked by an organic group, It is preferable that an organic group is introduced into the skeleton.

  Moreover, as such an organic silica porous film, for example, an organic silica porous film that is a polymer (condensate) of an organic silicon compound having an organic group capable of absorbing irradiation laser light; an organic that can absorb irradiation laser light Organic silica porous film which is a polymer of an organosilicon compound having a group and another organosilicon compound; selected from the group consisting of an organosilicon compound having an organic group capable of absorbing irradiation laser light and silica, alumina, and titania Organic silica porous film which is a polymer with at least one metal oxide; silica porous film (which may or may not have an organic group capable of absorbing irradiated laser light; A silane coupling agent having an organic group capable of absorbing the irradiation laser beam (for example, an irradiation layer) -An organic silica porous film which is a surface modification product of a silica porous film in which the surface of the porous silica film is modified by treatment with an organosilicon compound having an organic group capable of absorbing light); Can do.

  The organosilicon compound having an organic group capable of absorbing such irradiation laser light is not particularly limited, and is a known organosilicon compound (for example, an organosilicon compound described in JP 2008-084836 A, Organosilicon compounds described in Japanese Unexamined Patent Application Publication No. 2009-049346, organosilica materials described in Japanese Unexamined Patent Application Publication No. 2010-196019, organosilane compounds described in Japanese Unexamined Patent Application Publication No. 2010-116512, Among organic silicon compounds described in Japanese Patent Application Laid-Open No. 2008-21496, organic silica-based materials described in Japanese Patent Application Laid-Open No. 2011-241330, and organic silica-based materials described in Japanese Patent Application Laid-Open No. 2011-241331. Therefore, a suitable material may be appropriately selected and used depending on the wavelength of the laser beam to be used.

  Moreover, as an organic silicon compound which has an organic group which can absorb such irradiation laser light, the following general formula (1-i)-(1-iii):

[In the formulas (1-i) to (1-iii), X represents an organic group capable of absorbing irradiation laser light and represents an m-valent organic group, and R ¹ represents an alkoxy group (preferably having a carbon number of 1 to 5 alkoxy group), hydroxyl group (—OH), allyl group (CH ₂ ═CH—CH ₂ —), ester group (preferably an ester group having 1 to 5 carbon atoms) and halogen atom (chlorine atom, fluorine atom, At least one selected from the group consisting of a bromine atom and an iodine atom), R ² represents at least one selected from the group consisting of an alkyl group and a hydrogen atom, and n and (3-n) are each indicates the number of ^{R 1} and ^{R 2} bonded to the silicon atom (Si), n represents an integer of 1~3, m is an integer of 1-4. ]
An organosilicon compound represented by the formula (an organosilicon compound having an organic group capable of absorbing irradiation laser light) is preferred.

  Furthermore, as such an organic silica porous membrane, an organic silicon compound represented by the above general formulas (1-i) to (1-iii) (an organic silicon compound having an organic group capable of absorbing irradiation laser light) is used. At least one polymer selected from the above; or at least one selected from the organosilicon compounds represented by the general formulas (1-i) to (1-iii) and other organosilicon compounds An organic silica porous membrane comprising a polymer (copolymer); and an organic silicon compound represented by the above general formulas (1-i) to (1-iii) (an organic group capable of absorbing irradiation laser light) More preferred is an organic silica porous membrane made of at least one polymer selected from among organic silicon compounds having a In the organic silica porous film made of at least one polymer selected from the organic silicon compounds represented by the general formulas (1-i) to (1-iii), a so-called light collecting antenna function is used. Tends to be able to be expressed more efficiently, thereby tending to ionize the molecule to be measured more efficiently. The “light collecting antenna function” here refers to a function that absorbs light energy and collects the excited energy in the pores when irradiated with light. It tends to be possible to efficiently transfer the light energy of the laser beam by the molecules to be measured supported inside the pores. The definition of such “light collecting antenna function” is the same as the definition described in Japanese Patent Application Laid-Open No. 2008-084836.

In addition, at least one polymer selected from the organosilicon compounds represented by the general formulas (1-i) to (1-iii) has a siloxane structure (formula: — (Si—O)). ₍ structure represented by _y −) having a structure in which silicon atoms are crosslinked by an organic group (crosslinked structure), thereby having a structure having the organic group in the skeleton (so-called “crosslinked type”). Organic silica porous membrane ”. Here, such a crosslinked structure will be described by taking, as an example, a polymerization reaction of an organosilicon compound represented by the above general formula (1-i) and in which R ¹ is an ethoxy group, n is 3, and m is 2. The following general formula (2):

[Wherein, X represents the organic group (an organic group capable of absorbing irradiated laser light and is an m-valent organic group), and p represents an integer corresponding to the number of repeating units. ]
By virtue of the reaction represented by the formula (1), the organic silica porous film obtained after polymerization has a silicon atom that forms a siloxane structure (structure represented by the formula:-(Si-O) _y- ) by the organic group (X). It has a repeating unit having a crosslinked structure (note that the number of p is not particularly limited, but is generally preferably in the range of about 10 to 1000). In addition, when such a crosslinked structure is formed (when the organic silica porous film becomes the crosslinked organic silica porous film), the irradiation laser light is absorbed more efficiently, and the pores of the organic silica porous film are absorbed. The excitation energy tends to move more efficiently with respect to the molecule to be measured supported on the substrate.

As the ^{R 1} in the general formula (1-i) ~ (1 -iii), an alkoxy group and / or hydroxyl group is preferred from the viewpoint of easy control of the condensation reaction (polymerization reaction). In addition, when several R < ¹ > exists in the same molecule, R < ¹ > may be same or different. The alkyl group that can be selected as R ² in the general formula (1) is preferably an alkyl group having 1 to 5 carbon atoms. In addition, when ^two or more R < ² > exists in the same molecule, R < ² > may be the same or different.

In the above general formulas (1-i) to (1-iii), n and (3-n) in the formula represent the numbers of R ¹ and R ² bonded to the silicon atom (Si), respectively. Here, n represents an integer of 1 to 3, and n is particularly preferably 3 from the viewpoint that the structure after condensation can be made more stable.

  Further, m in the general formulas (1-i) to (1-iii) represents the number of silicon atoms (Si) bonded directly or indirectly to the organic group (X). Such m shows the integer of 1-4.

  X in the general formulas (1-i) to (1-iii) is an organic group capable of absorbing the irradiation laser beam, and represents an m-valent organic group. Moreover, as such an organic group (X in the said General formula (1)), as mentioned above, it is more preferable that it is an aromatic organic group containing 4 or more carbon, and especially the following general formula ( 101) to (132):

[In the general formulas (101) to (132), the symbol * indicates that the bond with the symbol is a bond that is bonded to X in the above formulas (1-i) to (1-iii). R ^a represents at least one selected from the group consisting of ^a hydrogen atom and an alkyl group having 1 to 18 carbon atoms, and R ^b each independently represents an ether group, a thioether group, a carbonyl group, a carbonate group, an amino group. , A divalent aliphatic organic group containing at least one group selected from the group consisting of an amide group and a urethane group; an alkylene group; or a single bond. ]
The organic group represented by is particularly preferable. In the organic groups represented by the general formulas (101) to (132), it is more preferable that the bond represented by the symbol * is directly bonded to silicon. Therefore, the organosilicon compound is more preferably a compound represented by the general formula (1-i). In addition, the alkyl group that can be selected as ^Ra in the above general formula has 1 to 18 (more preferably 1 to 12) carbon atoms. When such a carbon number exceeds the upper limit, it tends to be difficult to form a structure as a porous film. As such Ra, ^a methyl group is particularly preferable.

In addition, R ^b in the above general formula each independently contains at least one group selected from the group consisting of an ether group, a thioether group, a carbonyl group, a carbonate group, an amino group, an amide group, and a urethane group. An aliphatic organic group; an alkylene group; or a single bond. The alkylene group preferably has 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. When such a carbon number exceeds the upper limit, it tends to be difficult to form a structure as a porous film. Further, among these alkylene groups, a methylene group, an ethylene group, a propylene group, a butylene group, a pentamethylene group, and a hexamethylene group are particularly preferable, and an ethylene group, a propylene group, and a butylene group are most preferable. The divalent aliphatic organic group preferably further contains a divalent hydrocarbon group having 1 to 12 carbon atoms (more preferably 1 to 6 carbon atoms). As the divalent hydrocarbon group, An alkylene group is preferred. When such a carbon number exceeds the upper limit, it tends to be difficult to form a structure as a porous film.

  Among such organic groups (X in formula (1)), the above general formulas (103), (105), (108), (111), (112), (113), (114), The organic groups represented by (115), (116), (117), (118), (120), (126), (127), (128), (130), (132) are more preferable, The organic group represented by the general formula (116), (117), (118), (126), (130), (132) is more preferred, and the organic group represented by the above general formula (126) (triphenyl) Amine) is particularly preferred. In addition, the organic silica porous membrane having such an organic group in the skeleton may contain one kind of organic group alone, or may contain two or more kinds of organic groups in combination. There may be. In addition, as an organic silica porous membrane which contains 2 or more types of organic groups in combination, it is represented by any one of the general formulas (1-i) to (1-iii) and the types of X are different. Examples thereof include polymers of various organosilicon compounds.

  In addition, when the organic silica porous film is composed of a polymer of an organic silicon compound having an organic group capable of absorbing irradiation laser light and another organic silicon compound, the other organic silicon compound is particularly limited. In addition, a known organosilicon compound other than an organosilicon compound having an organic group capable of absorbing irradiation laser light can be used as appropriate. For example, the following general formulas (A1) and (A2):

[In the general formula (A1), R ²¹ is a hydrocarbon group which may be different from each other, and R ²² may be different from each other, a monovalent organic which has at least one carbon atom and is bonded to a silicon atom. M is an integer of 0-4, n is an integer of 0-4, m and n satisfy the condition of m + n = 4,
In general formula (A2), X is a halogen group that may be different from each other, and R ²³ is a monovalent organic group that has at least one carbon atom and is bonded to a silicon atom, which may be different from each other. , M is an integer from 1 to 3, n is an integer from 1 to 3, and m and n satisfy the condition that m + n = 4. ]
The compound shown by these is mentioned. As such other organosilicon compounds, known compounds can be used as appropriate. Among these, Si (OR ²¹ ) ₄ [R ²¹ is a hydrocarbon from the viewpoints of availability, hydrolysis reaction, etc. Group (more preferably an alkyl group having 1 to 4 carbon atoms, still more preferably a methyl group or an ethyl group). ] The silicon compound represented by this is more preferable.

  Moreover, the organic silica porous membrane according to the present invention has an average pore diameter of 5 to 50 nm. When the average pore diameter is less than the lower limit, it becomes difficult to efficiently disperse the measurement target molecules in the pores and perform mass spectrometry. On the other hand, when the upper limit is exceeded, the measurement target molecules are close to the lower limit. Organic groups in the organic silica porous film are reduced, and it is difficult to perform mass spectrometry efficiently. Further, from the same viewpoint, the average pore diameter is more preferably 7 to 40 nm, and further preferably 10 to 30 nm. Such an average pore diameter (average value of pore diameter) is obtained by scanning the cross section and / or the surface of the porous membrane with a scanning microscope (SEM: for example, a high resolution electrolytic emission scanning electron microscope manufactured by Hitachi High-Technologies Corporation: trade name “SEM” S-5500 ") and can be obtained by measuring and averaging the pore diameters of any 100 or more pores from the obtained SEM image. As a method for measuring such an average pore diameter, for example, by measuring a cross section of any four or more points of the porous membrane with a scanning microscope, any four or more cross-sectional SEM images obtained can be used. By measuring the pore diameter of 25 or more pores, the pore diameter of 100 or more arbitrary pores in total is measured and obtained, and the average value (pore diameter of 100 or more pores) The average pore diameter may be determined by calculating the average pore diameter, and any method may be used that can measure and average the pore diameters of any 100 or more pores. It can be adopted as appropriate. When measuring the diameter of the pore, if the pore shape (cross-sectional shape) in the SEM image is not a perfect circle, the average value of the diameters of the largest circumscribed circle and the smallest circumscribed circle is calculated. Measured as pore diameter (pore diameter). When measuring the pore diameter from the SEM image, measure the diameter of each pore with a ruler, etc., and determine the pore diameter value from the SEM measurement magnification. It may be calculated.

  Furthermore, the organic silica porous membrane according to the present invention has a surface opening ratio of 33 to 70%. If the surface area ratio is less than the lower limit, it is difficult to sufficiently introduce the measurement target molecule into the pores of the organic silica porous membrane, and when the measurement target molecule is supported, it tends to remain in the vicinity of the surface. Yes, it becomes difficult to efficiently transfer the light energy absorbed by the porous organic silica membrane to the molecule to be measured even if it is irradiated with laser, and the molecule to be measured carried on the membrane is ionized at a higher level. It becomes difficult to desorb, and it becomes difficult to further improve the detection sensitivity during mass spectrometry. On the other hand, in the organic silica porous membrane according to the present invention in which the region of 33% (about one third) or more of the surface becomes the opening of the pore, the molecule to be measured is more efficiently contained inside the pore. It is possible to introduce to. Moreover, when the said surface opening ratio exceeds the said upper limit, the quantity of the organic group (wall) of the organic silica porous film which contact | connects a measuring object molecule | numerator becomes inadequate, and it exists in the tendency for efficient mass spectrometry to become difficult. From the same viewpoint, the surface aperture ratio is more preferably 35 to 67%, still more preferably 37 to 65%, and particularly preferably 39 to 60%. When such a surface aperture ratio is in the above range, the ionized desorption performance (performance for desorbing the molecule to be measured by ionization) of the organic silica porous film tends to be further improved. In addition, “the surface opening ratio is 33 to 70%” of the film means that the surface having such an average value of opening ratio (average opening ratio) is provided on at least a part of the surface, It is not intended that the average aperture ratio of all surfaces be in the above range. That is, “the surface aperture ratio is 33 to 70%” of the film has the same meaning as having a surface having an average aperture ratio of 33 to 70%. Here, the “average value of aperture ratio (average aperture ratio)” refers to an average value of aperture ratios in each measurement region described later.

  As such a “surface aperture ratio”, a value measured as follows is adopted. That is, first, a scanning probe microscope (SPM / AFM: trade name “NanoNavi E-sweep” manufactured by Hitachi High-Tech Science Co., Ltd.) is used as a measuring device, and an arbitrary size of 1 μm × 1 μm on the surface of the porous organic silica membrane is used. In a measurement area of 4 points or more, using a micro cantilever made of silicon as a cantilever (silicon cantilever manufactured by Hitachi High-Tech Science Co., Ltd .: trade name “SI-DF20”), the surface state is vertical (X) in dynamic force mode: Analysis is performed under a scanning condition of 256 pixels, horizontal (Y): 256 pixels, and an atomic force microscope (AFM) image is obtained respectively (an AFM image of a measurement area of 1 μm square of four or more arbitrary points is obtained). The obtained AFM image is composed of a total of 65536 (256 × 256) pixels, as is apparent from the scanning conditions, and each pixel has a vertical (X), horizontal (Y), and height ( Information of Z), that is, position data of (X, Y, Z) will be recorded. Further, the height (Z) here is position information in a direction perpendicular to the measurement surface (the surface of the porous membrane) (direction perpendicular to the horizontal plane: height direction). As the reference plane of the value, the position data of the height (Z) is the farthest position from the measurement plane (the position where the distance in the vertical direction from the measurement plane is the longest: the deepest when considered from the depth from the measurement plane A horizontal plane including a pixel (pixel: point) at a position (horizontal plane including a pixel having the lowest height from the ground) is adopted, and the distance (difference) in the vertical direction from the reference plane is set to the height (Z ) Value (unit: nm). Thus, the height (Z) value of the reference surface is set to 0 nm, and the distance in the height direction from the reference surface is used as the height (Z) value (unit: nm) of each pixel. Next, from the AFM image obtained as described above, the vertical axis is the height (Z) value at each pixel, and the ratio of the number of pixels having a specific height to the total number of pixels (specific height). A graph (a graph of height frequency distribution curve) whose horizontal axis is the abundance ratio (unit:%) of pixels having a height value (the same height value), hereinafter simply referred to as “frequency”) is obtained. . The frequency (unit:%) mentioned here is 0.76% because the total number of pixels is 65536 when the number of pixels having a height (Z) of 10 nm is 500, for example, 0.76% [calculation Formula: (500/65536) × 100]. Then, from the height distribution thus obtained (the graph of the height frequency distribution curve), the height value having the maximum frequency (maximum frequency) (the height value is referred to as “H1” for convenience). : The height (Z) value is lower than the number of pixels having the highest value (H1), and half the maximum frequency (0.5 A height value having a frequency of (times) (the height value is referred to as “H2” for convenience). Then, the AFM image is binarized using a value (H2) that is a half of the maximum frequency as a threshold value. By such binarization, the color of the pixel (pixel) where the height (Z) is greater than or equal to H2 and the portion of the pixel (pixel) where the height is less than H2 are divided into two colors. An image is obtained. From such a binarized image, in the present invention, the pixel portion (region) having a height value of H2 or more is defined as the skeleton portion of the porous film (the organic silica portion forming the surface of the film). The pixel portion (region) where the height value is less than H2 is determined as the opening portion of the pore (a void portion based on the pore). In this way, images binarized from the AFM images of any four or more measurement regions (1 μm square) (the organic silica part (membrane skeleton part) forming the surface of the film and the opening part of the pores) 2), and based on such an image, the aperture ratio of each measurement region (the ratio of the pore opening portion to the surface image of each measurement region: the high ratio to the surface image of each measurement region) Percentage of pixel parts (regions) whose height value is less than H2 (calculation formula: ([area of pore opening part (number of pixels whose height value is less than H2)] / [total measurement area] Area (total number of pixels)] × 100 [unit:%]), and by averaging the obtained aperture ratio of each measurement area, the “surface aperture ratio (of any four or more measurement areas) Average value of aperture ratio) ".

  Hereinafter, a method for measuring the surface aperture ratio will be described with reference to FIG. In FIG. 1, (a) is an AFM image of an arbitrary 1 μm square region on the surface of the porous membrane, and (b) shows a frequency distribution curve of height obtained from the data of the AFM image, (C) is an image obtained by binarizing the AFM image. Such an AFM image (FIG. 1A) is recorded with data of (X, Y, Z) as described above, and each pixel (total number of pixels: 65536) of the AFM image is recorded. From the data, a frequency distribution curve of height (a graph showing the distribution of height: FIG. 1B) can be obtained. A height having a value lower than the height having the maximum frequency (the height at the position of H1 in FIG. 1B) and having a frequency half that of the maximum frequency ( The value of the height H2 in FIG. 1B is obtained, and the AFM image is binarized using the height value (H2) as a threshold value. An image binarized from the AFM image in this manner (an image obtained by color-coding the pixel portion whose height value is H2 or more and the pixel portion whose height value is less than H2: FIG. 1 (c) ) In FIG. 1C, the blacked out portion is the opening portion of the pore (void portion of the pore: the portion of the pixel whose height value is less than H2). Then, the area of the opening portion of the pore is obtained from the image, and the ratio of the area of the opening portion of the pore to the total area of the measurement region ([area of the opening portion of the pore] / [total area of the measurement region]) X 100 [unit:%]) is calculated. And in this invention, the ratio with respect to the total area of the measurement area | region of the opening part area of such a pore is each with respect to each of arbitrary 4 points | pieces or more of 1 micrometer squares of the surface of a porous organic silica body. By calculating and averaging this, the surface aperture ratio (the average value of the aperture ratios of any four or more measurement regions) can be obtained. The method for measuring the surface aperture ratio has been described above, but a method for preparing an organic silica porous film that satisfies the conditions for the surface aperture ratio and the average pore diameter as described above will be described later.

  Further, the thickness of such a porous organic silica film is not particularly limited, but is preferably 20 to 500 nm, and more preferably 40 to 300 nm. If the thickness of such a film is less than the lower limit, the absorption efficiency of the laser beam of the organic silica porous film is reduced, and the film absorbs the laser beam sufficiently to efficiently transfer energy from the film to the molecule to be measured. On the other hand, if the upper limit is exceeded, the molecules to be measured introduced into the pores tend to be difficult to desorb.

  In addition, a method that can be suitably used for preparing such an organic silica porous membrane according to the present invention is not particularly limited. For example, the polymer (for example, the general formulas (1-i) to (1- After preparing a thin film (organosilica porous membrane precursor) comprising at least one polymer selected from among the organosilicon compounds represented by iii) and having pores having an average pore diameter of 5 to 50 nm The method for obtaining the porous organic silica film according to the present invention by etching the surface of the thin film so that the surface aperture ratio is 33 to 70% (hereinafter simply referred to as “method (A)” for convenience) Can be adopted.

  In such a method (A), first, a thin film comprising the polymer and having pores having an average pore diameter of 5 to 50 nm is prepared. The method for preparing such a thin film is not particularly limited, and a known method (for example, a method described in JP-A-2008-084836) can be appropriately used. For example, in the presence of a surfactant as a template, The organosilicon compound (for example, at least one compound selected from the organosilicon compounds represented by the above general formulas (1-i) to (1-iii)) is polymerized and then used as a template. By removing the surfactant, a method of obtaining a thin film made of the polymer of the organosilicon compound and having an average pore diameter of 5 to 50 nm can be suitably employed.

  Such a surfactant is not particularly limited, and may be any of cationic, anionic, and nonionic, specifically, alkyltrimethylammonium, alkyltriethylammonium. , Dialkyldimethylammonium, benzylammonium chlorides, bromides, iodides or hydroxides; fatty acid salts; alkyl sulfonates; alkyl phosphates; polyethylene oxide nonionic surfactants (eg, polystyrene-polyethylene oxide) Diblock polymers and the like); primary alkylamines and the like. These surfactants are used alone or in admixture of two or more.

  In addition, from the viewpoint of forming the polymer into a thin film shape, in the method (A), for example, it was obtained by mixing an organic solvent (for example, alcohol), the surfactant, and the organosilicon compound. It is preferable to employ a method in which the mixture is applied on a substrate (for example, by spin coating) to obtain a coating film and then polymerized. In addition, as said base material, when manufacturing porous silica films, such as a silicon base material (Si substrate), an ITO base material, an FTO base material, a quartz base material, a glass base material, various metal base materials, etc., for example. A known base material that can be used can be used as appropriate.

  The method for removing the surfactant after the polymerization of the organosilicon compound is not particularly limited. For example, (i) the thin film in an organic solvent (for example, ethanol, toluene, etc.) having high solubility in the surfactant. (Ii) A method of removing the surfactant by baking the thin film at 200 to 400 ° C., (iii) A method of immersing the thin film in an acidic solution and heating, Examples thereof include an ion exchange method in which an activator is exchanged for hydrogen ions. By removing the surfactant in this manner, a thin film (organosilica porous membrane precursor) made of a polymer of the organosilicon compound having an average pore diameter of 5 to 50 nm efficiently depending on the type of the surfactant. ) Can be formed.

  Moreover, in the said method (A), after preparing the said thin film (organosilica porous membrane precursor), the surface of the said thin film is etched so that surface aperture ratio may be 33 to 70%. In general, when preparing a thin film (organic silica porous membrane precursor) using an organic silica compound, the organic silica portion (silica skeleton portion having an organic group) tends to gather near the gas phase interface during film formation. As a result, a film having a low aperture ratio tends to be formed. Therefore, in order to obtain an organic silica porous film in which a region occupying a wide range of 33% (about one third) or more of the surface becomes the opening of the pores, that is, the surface opening ratio is 33 to 70%. In order to obtain the organic silica porous film according to the present invention, the outermost surface of the thin film is removed by etching or the like so that the internal pores are exposed (the surface opening portion). It is preferable to increase the ratio).

The etching method is not particularly limited, but reactive ion etching (RIE) can be performed because etching can be performed more efficiently so that the outermost surface of the thin film is removed to expose internal pores. ) Treatment is preferred. Such reactive ion etching (RIE) processing is a dry etching method in which etching is performed by supplying plasma and then generating plasma. Such an etching gas is not particularly limited, and a known etching gas can be appropriately used. However, carbon tetrafluoride (CF ₄ ) is used from the viewpoint that it is widely used for etching silica and is effective. It is preferable to use a fluorine-based gas such as. As such an etching gas, a mixture of a fluorine-based gas and another gas (for example, oxygen) may be used. In addition, the conditions employed in the etching are not particularly limited, and optimal conditions may be appropriately set so that the surface aperture ratio is 33 to 70% according to the type of organic silica forming the thin film. . In such etching, for example, the atmospheric pressure is preferably 5 to 30 Pa (more preferably 10 to 20 Pa), and the output of the RF power source (RF output) is 5 to 200 W (more preferably 10 to 50 W). It is preferable that the flow rate of the etching gas is 50 to 200 sccm. The apparatus that can be used for such reactive ion etching is not particularly limited, and a commercially available product (for example, “RIE-10NR” manufactured by Samco Corporation) may be used as appropriate.

  Thus, by removing the outermost surface of the thin film by etching so that the internal pores are exposed, the number of openings on the surface can be increased, and thereby the surface opening ratio is 33 to 70%. Thus, an organic silica porous membrane can be efficiently obtained. In the above method (A), an etching method is employed as a method for setting the surface aperture ratio to 33 to 70%, but the method for setting the surface aperture ratio to 33 to 70% is not particularly limited, and the thin film is formed. After obtaining, a method capable of exposing the pores can be used as appropriate.

  In the present invention, the organic silica porous membrane is used as a substrate for so-called mass spectrometry in mass spectrometry. There is no particular limitation on the form (form of the organic silica porous film used in the mass spectrometry) when used as such a substrate for mass spectrometry, for example, using the one composed only of the above organic silica porous film Alternatively, the organic silica porous membrane may be laminated on a support (for example, the base material (base used during production) so that the surface having a surface opening ratio of 33 to 70% is exposed. The material may be used as a laminate in which the organic silica porous film is laminated on the material). Moreover, as a usage form of the organic silica porous membrane according to the present invention, from the viewpoint of easiness of the preparation, etc., the above-mentioned group is formed so that the surface having a surface opening ratio of 33 to 70% is exposed. It is preferably in the form of a laminate in which the organic silica porous film is laminated on a material (for example, forming an organic silica porous film on the substrate and using it as it is).

<sample>
The sample according to the present invention contains a molecule to be measured. Although it does not restrict | limit especially as such a measuring object molecule | numerator, Since it becomes possible to measure with a higher detection sensitivity by this invention, it is preferable that it is a molecule | numerator derived from a biological body or a molecule | numerator in a biological sample. Such a molecule derived from a living body or a molecule in a biological sample is more preferably a sugar, protein, peptide, glycoprotein, glycopeptide, nucleic acid, glycolipid, etc. It tends to be possible to make it more sophisticated. Such molecules to be measured are prepared from natural products, prepared by partially modifying natural products chemically or enzymatically, and prepared chemically or enzymatically. It may be a thing. Moreover, what has the partial structure of the molecule | numerator contained in a biological body, or the thing imitated by the molecule | numerator contained in a biological body may be used.

  Further, the sample according to the present invention (sample containing the molecule to be measured) may be the molecule to be measured itself, or one containing the molecule to be measured (for example, biological tissue, cell, body fluid or secretion) (For example, blood, serum, urine, semen, saliva, tears, sweat, feces, etc.)). Thus, a biological sample may be used directly as a sample according to the present invention (a sample containing a molecule to be measured). Alternatively, the measurement target molecule may be prepared by carrying out an enzyme treatment or the like after a sample precursor (a precursor of a measurement target molecule or the like) is supported on the porous organic silica membrane. In this case, the treatment is performed after the sample precursor is supported on the organic silica porous film, and as a result, the sample is supported on the organic silica porous film.

  In the present invention, the “measurement target molecule” may be a molecule contained in the sample and the molecule itself whose chemical structure is desired to be determined, or a molecule contained in the sample. In addition, a molecule obtained by derivatizing a molecule whose chemical structure is to be determined (for example, a molecule subjected to mass spectrometry obtained by binding a so-called labeled molecule to a molecule whose chemical structure is to be determined) may be used. Thus, the “molecule to be measured” may be a molecule that has not been derivatized, or may be a molecule that has been derivatized with a labeled molecule. The presence or absence of derivatization is not particularly limited, and may be appropriately determined according to the type of organic group of the organic silica porous film to be used, the type of molecule for which the chemical structure is desired to be determined, and the like. Thus, derivatization is not always necessary depending on the molecule whose chemical structure is to be determined. The molecular weight of such a molecule to be measured is not particularly limited, but it is preferably 160 or more because accurate measurement with other measurement methods is difficult and the characteristics of the present invention are more easily exhibited. , More preferably 500 or more, and particularly preferably 1000 or more.

  Further, when a molecule obtained by derivatizing a molecule whose chemical structure is to be determined is used as the measurement target molecule, the derivatization uses light energy absorbed by the organic group (light energy absorbed by the organic silica porous film). It is preferably carried out by covalently bonding to a label molecule to be acceptable, preferably a label molecule having an absorption band having an overlap of the spectrum with the emission spectrum of the organic silica porous film.

  Such a labeled molecule is not particularly limited as long as it has an effect as an acceptor of energy donated from the organic silica porous membrane, but a molecule commercially available as a fluorescent labeling reagent may be used. Examples of such a labeled molecule include pyrene derivatives, fluorescein derivatives, rhodamine derivatives, cyanine dyes, Alexa Fluor (registered trademark), 2-aminoacridone, 6-aminoquinoline and the like.

  In addition, the combination of the organic silica porous membrane as the energy donor and the labeled molecule as the energy acceptor has the energy transfer efficiency, the overlap between the emission spectrum of the organic silica porous membrane and the absorption spectrum of the molecule to be measured, and the strength of the interaction. It is determined appropriately from such points. For example, when a crosslinked organic silica porous film having a triphenylamine group is used as the organic silica porous film, 2-aminoacridone or the like can be suitably used as the labeling molecule, and methylacrylic acid can be used as the organic silica porous film. When a crosslinked organic silica porous membrane having a don group is used, 4-Fluoro-7-nitrobenzofurazane, 4-Fluoro-7-sulfobenzofurazan, 3-Chlorocarbon-6,7-dimethyl-1-methyl- is used as a labeling molecule. 2 (1H) -quinoxaline etc. can be used suitably. Such a labeled molecule preferably has a functional group that is easily chemically bonded to the target molecule, and derivatization may be performed after being performed in a separate container, or may be performed on an organic silica porous membrane.

  In the present invention, the reason why the organic silica porous membrane can ionize the molecule to be measured more efficiently is not necessarily clear, but the present inventors infer as follows. That is, first, when the organic silica porous film according to the present invention is irradiated with laser light, the organic light in the film absorbs the laser light. By absorbing the laser light in this way, the light energy absorbed by the organic silica porous film can be transferred to the molecule to be measured (energy acceptor). Thus, the organic silica porous membrane according to the present invention acts as an energy donor that moves light energy to a molecule to be measured (energy acceptor) when irradiated with laser light. The energy transfer from the organic silica porous film (energy donor) to the molecule to be measured (energy acceptor) includes energy transfer not via light emission (for example, excitation energy transfer or electron transfer between molecules), and Energy transfer via light emission (for example, energy transfer (energy transfer by light emission reabsorption) in which the molecule to be measured absorbs light emitted from the organic group of the organic silica porous film that has absorbed the laser light) can be considered. The present inventors infer that the energy transfer molecule can ionize the molecule to be measured more efficiently by using the laser beam.In the present invention, the sample containing the molecule to be measured is used. When the organic silica porous membrane is brought into contact with the surface of the organic silica porous membrane, the organic silica porous membrane has a high surface opening ratio. The sample can be efficiently introduced into and supported in the pores. As a result, the molecule to be measured has a larger contact area with respect to the organic silica porous membrane (particularly, the pore inner wall of the organic silica porous membrane). In this way, the present inventors speculate that the molecules to be measured are in contact with the porous organic silica membrane with a larger contact area in order to use the porous membrane having the surface opening ratio. Therefore, the present inventors speculate that the energy transfer between molecules is more easily caused and the above-described effect is exhibited, regardless of whether the light emission is transmitted or not. .

  In the present invention, such energy transfer is considered to enable more efficient ionization of the molecule to be measured using laser light. It is preferable to select so as to satisfy the following relationship. That is, even if the energy transfer (energy transfer from the organic silica porous membrane (energy donor) to the molecule to be measured (energy acceptor)) is any, the energy can be transferred more efficiently. In view of the above, after absorbing the irradiation laser light by the organic group in the organic silica porous film, the spectrum of light emitted from the organic group of the organic silica porous film (emission spectrum from the organic group) and the measurement It is more preferable to select the organic group and the molecule to be measured so that the absorption spectrum of the target molecule overlaps at least at one wavelength. Thus, when the emission spectrum from the organic group and the absorption spectrum of the molecule to be measured overlap at least at one wavelength, the light energy absorbed by the organic silica porous film or the organic silica porous film The excitation energy tends to move more efficiently with the molecule to be measured. In particular, when energy is transferred via light emission, the organic silica porous film emits light by absorbing the irradiation laser beam, and the emission spectrum of the organic silica porous film (emission spectrum from the organic group) and More preferably, the absorption spectrum of the molecule to be measured overlaps at least at one wavelength. This is because the light energy emitted from the porous organic silica film by such light emission tends to move efficiently to the molecule to be measured.

  In addition, regardless of the type of energy transfer (whether it is via light emission or not), the short wavelength end of the emission spectrum of the organic silica porous film is not limited. On the other hand, the emission spectrum of the porous organic silica film and the absorption spectrum of the molecule to be measured overlap at least at one wavelength by being on the shorter wavelength side than the long wavelength end of the absorption spectrum of the molecule to be measured. More preferably. In such a case, the light energy absorbed by the organic silica porous film tends to move more efficiently as light energy or excitation energy with respect to the molecule to be measured.

<Sample loading method>
In the laser desorption ionization mass spectrometry of the present invention, in mass spectrometry, first, a sample containing a molecule to be measured is supported on the organic silica porous film. Although it does not restrict | limit especially as a support method of such a sample, For example, by mounting a sample with respect to the surface which has the said surface opening ratio (33-70% surface opening ratio) of the said organic silica porous film, It is preferable to employ a method of supporting a sample on the membrane. In this way, by placing the sample on the surface of the organic silica porous membrane having the above-described surface opening ratio, the sample easily penetrates into the pores, thereby efficiently in the organic silica porous membrane. It becomes possible to carry a sample. For example, a sample precursor (aqueous solution) is placed on the organic silica porous film by preparing an aqueous solution containing the molecule to be measured and dropping the aqueous solution onto the surface of the organic silica porous film having the surface opening ratio. After that, the sample can be supported on the membrane by drying and removing water as a solvent (in this example, the sample in which the molecule to be measured itself remaining after the removal of water is supported on the organic silica porous membrane is supported. Become). In addition, as described above, by carrying the enzyme treatment after supporting the sample precursor (molecule before enzyme treatment) on the organic silica porous membrane, and preparing the molecule to be measured (enzyme treated product) on the membrane, As a result, a sample containing a molecule to be measured (enzyme-treated product) may be supported on the organic silica porous membrane. Thus, it is possible to carry a sample (measurement molecule itself, a derivative of the measurement molecule, a mixture of the measurement molecule and a standard substance, etc.) finally containing the measurement molecule on the organic silica porous membrane. If so, the method for supporting the sample is not particularly limited.

<Method of mass spectrometry>
In the present invention, the sample containing the molecule to be measured is supported on the organosilica porous membrane as described above, and then the sample-supporting portion of the membrane is irradiated with laser light to ionize the molecule to be measured. To perform mass spectrometry.

  The laser light source used for such mass spectrometry is not particularly limited, and examples thereof include a nitrogen laser (337 nm), a YAG laser triple wave (355 nm), an NdYAG laser (256 nm), a carbon dioxide laser (9400 nm, 10600 nm), and the like. Although a laser light source is mentioned, a nitrogen laser or a YAG laser triple wave laser light source is preferable from the viewpoint that the organic silica porous film is a laser light source having a wavelength capable of efficiently absorbing light.

  In the present invention, the laser light source (for example, a nitrogen laser light source) is used to irradiate the sample carrying portion of the porous organic silica film with laser light. By irradiating the sample carrying part with the laser beam in this way, the molecule to be measured can be ionized. As described above, the ionization mechanism is caused by the irradiation laser being absorbed by the organic group present at the laser irradiation site, and the absorbed light energy being efficiently transferred to the measurement target molecule. The present inventors speculate. In the present invention, the organic silica porous film having a surface opening ratio that satisfies the above conditions is used, and the sample is supported on the surface of the film having the surface opening ratio. In such a case, the dropped molecules to be measured are efficiently introduced into the thin film, and the organic energy of the organic silica porous film is supported on the film in a state where it is easier to receive the light energy. The present inventors speculate that ionization and desorption are further promoted, and mass spectrometry can be performed with higher detection sensitivity than before.

  The laser light irradiation conditions (irradiation intensity, irradiation time, etc.) are not particularly limited, and may be set by appropriately selecting optimum conditions from known mass spectrometry conditions according to the molecule to be measured. .

  In addition, the method for separating and detecting ions for mass spectrometry is not particularly limited. Double focusing method, quadrupole focusing method (quadrupole (Q) filter method), tandem quadrupole (QQ) method, ion A trap method, a time-of-flight (TOF) method, or the like can be employed as appropriate, whereby ionized molecules can be separated and detected according to the mass / charge ratio (m / z). A commercially available apparatus can be used as appropriate for such ion separation and detection, for example, a mass spectrometer manufactured by Bruker Daltonics (trade name “autoflex” or the like), an ion trap time-of-flight mass manufactured by Shimadzu. An analyzer (trade name “AXIMA-QIT etc.”) or the like may be used as appropriate. In this way, mass analysis of the ionized molecule to be measured can be performed.

  The laser desorption / ionization mass spectrometry method of the present invention has been described above. Hereinafter, the organic silica porous membrane substrate for laser desorption / ionization mass spectrometry of the present invention will be described.

[Organic silica porous membrane substrate for laser desorption ionization mass spectrometry]
The organic silica porous membrane substrate for laser desorption ionization mass spectrometry of the present invention has an organic group capable of absorbing laser light in its skeleton, has pores with an average pore diameter of 5 to 50 nm, and has a surface opening. An organic silica porous film having a rate of 33 to 70% is included.

  Such an organic silica porous membrane is the same as that described in the laser desorption / ionization mass spectrometry method of the present invention (suitable conditions for such a membrane and a method for preparing the membrane are also the same). In the organic silica porous membrane substrate for laser desorption / ionization mass spectrometry of the present invention, the laser beam refers to a laser beam used when performing laser desorption / ionization mass spectrometry using the substrate. The absorbable organic group may be appropriately selected according to the laser beam used for the measurement. Such an organic silica porous membrane substrate for laser desorption / ionization mass spectrometry is not particularly limited as long as it includes the organic silica porous membrane, and for example, it is laminated on a specific support. It is good also as a structure. Moreover, as such an organic silica porous membrane substrate for laser desorption ionization mass spectrometry, from the viewpoint of easiness of preparation and the like, the substrate described in the above method (A) (for example, a silicon substrate (Si substrate)) Organic silica porous film on a known porous substrate such as ITO, FTO, quartz, glass, various metal substrates, etc. It is preferable to make a laminated body in which Moreover, it is preferable to employ | adopt the laser desorption ionization mass spectrometry method of the said this invention as a mass spectrometry method using such an organic silica porous membrane board | substrate.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example.

(Example 1)
<Preparation process of organosilica porous membrane precursor>
30 mg of polystyrene-polyethylene oxide dibook polymer (“20 kPS-b-14 kPEO” manufactured by polymer source: surfactant) and triethoxysilyltriphenylamine (TPA organosilane: represented by the above general formula (1-i)) An organic silane compound [wherein m is 3, n is 3, R ¹ is an ethoxy group, and X is an organic group represented by the general formula (126)]. 337 mg of an ethanol solution (TPA organosilane / EtOH solution, content ratio: 0.089 g / g) contained at a rate of 8.17% by mass, 1 mL of tetrahydrofuran (THF), and 0.611 mL of ethanol (EtOH) were mixed. Then, 6 mL of ion-exchange water and 2 mL of 2 mol / L (2M) HCl were added dropwise to the solution, and the solution was added at room temperature ( It was obtained 24 hours with stirring to the sol at 5 about ° C.). Next, the obtained sol was spin coated (rotation speed: 2000 rpm, rotation time: 30 seconds) on a silicon substrate (Si substrate: manufactured by SUMCO, P type, plane orientation [100], resistivity: 0 to 0.02 Ωcm or less). Then, a coating film was formed on the Si substrate to obtain a laminate. Next, the obtained laminate was dried by allowing it to stand overnight (about 15 hours) at room temperature (about 25 ° C.). After the dried laminate was exposed to ammonia vapor at 60 ° C. for 12 hours, it was heated on a hot plate at 100 ° C. for 1 hour to allow the TPA organosilane condensation reaction (polymerization reaction) to proceed in the coating film. A thin film made of a polymer of TPA organosilane was obtained on a Si substrate. Next, the surfactant introduced into the thin film is removed by immersing the Si substrate on which the thin film is formed in toluene and repeatedly performing the process of heating in toluene at 110 ° C. for 24 hours three times. Then, an organic silica porous film precursor (TPA-PMO thin film) was prepared on a Si substrate.

<Reactive ion etching (RIE) treatment>
With respect to the surface of the organic silica porous membrane precursor (TPA-PMO thin film) obtained as described above, the product name “RIE-10NR” manufactured by Samco Co., Ltd. is used as an etching apparatus, and CF ₄ is used as an etching gas. A gas containing O ₂ (CF ₄ / O ₂ mixed gas) is used, and the etching gas is 59 sccm / 10 sccm (CF ₄ / O ₂ ) so that the composition of the mixed gas becomes 59/10 (CF ₄ / O ₂ ). ₂ ), a reactive ion etching (RIE) process was performed under conditions where the atmospheric pressure was 10 Pa, the output of the RF power source (RF output) was 30 W, and the processing time was 10 seconds. Thus, the Si substrate provided with the film after the RIE treatment is immersed in toluene, heat-treated at 110 ° C. for 24 hours, and then vacuum-dried to obtain an organic silica porous film having an RIE-treated surface. It was. In this way, a substrate for mass spectrometry was obtained in which an organic silica porous film having a RIE-treated surface was laminated on a Si substrate.

(Example 2)
During the reactive ion etching (RIE) process, the etching gas is changed from the CF ₄ / O ₂ mixed gas to a gas consisting of only CF ₄ , and the flow rate of the etching gas is 60 sccm (the etching gas is only CF ₄ , O ₂ was 0 sccm), the RF output was changed from 30 W to 25 W, and the treatment time was changed from 10 seconds to 15 seconds, in the same manner as in Example 1 to obtain an organic silica porous film having a RIE-treated surface. It was. In this way, a substrate for mass spectrometry was obtained in which an organic silica porous film having a RIE-treated surface was laminated on a Si substrate. Table 1 shows the reactive ion etching (RIE) processing conditions employed in Examples 1 and 2 so that the processing conditions become clearer.

(Comparative Example 1)
A TPA-PMO thin film was prepared on a Si substrate in the same manner as in Example 1 except that the reactive ion etching (RIE) treatment was not performed, and used as a substrate for mass spectrometry for comparison. Thus, as a substrate for mass spectrometry for comparison, a substrate in which an RIE-untreated organic silica porous film (TPA-PMO thin film) was laminated on a Si substrate was prepared. The RIE-untreated organic silica porous film included in the substrate for mass spectrometry is the same as the “organic silica porous film precursor (TPA-PMO thin film)” described in Example 1.

[Evaluation of characteristics of substrates for mass spectrometry obtained in Examples 1 and 2 and Comparative Example 1]
<SEM measurement (measurement of average pore diameter, thickness, etc. of organic silica porous membrane)>
Using the scanning electron microscope (manufactured by Hitachi High-Technologies Corporation: high resolution electrolytic emission scanning electron microscope SEM S-5500), the cross sections of the substrates for mass spectrometry obtained in Examples 1 and 2 and Comparative Example 1 were respectively used. By measuring under the condition of an acceleration voltage of 10 kV, the average pore diameter and thickness of the organic silica porous membrane were measured, and the surface state and pore state of the membrane were further confirmed.

  In measuring the average pore diameter of such an organic silica porous membrane, first, for each substrate for mass spectrometry, cross sections at arbitrary four points of the substrate were measured by the scanning electron microscope. Next, using each measured cross-sectional SEM image, the pore diameter of any 100 or more (total number) pores in the organic silica porous film is measured for each substrate for mass spectrometry, and the average value is calculated. By calculating | requiring, the average pore diameter (average value of the pore diameters of arbitrary 100 or more pores) of the organic silica porous film formed on each substrate was determined. In measuring the average pore diameter of such an organic silica porous membrane, for each substrate for mass spectrometry, from each cross-sectional SEM image, the pore diameter of any 25 or more pores is measured, The total number of measured arbitrary pores was 100 or more (Example 1: 151, Example 2: 137, Comparative Example 1: 124). Furthermore, the size of the pore diameter was measured with a ruler from each cross-sectional SEM image.

  Further, the thickness of the porous organic silica film was measured from a SEM image of an arbitrary cross section of the substrate with a ruler.

  As a result of the measurement by the scanning electron microscope, cross-sectional SEM images of the organic silica porous films of the substrates for mass spectrometry obtained in Examples 1-2 and Comparative Example 1 are shown in FIG. 2 (Example 1) and FIG. 3 (Example 2) and FIG. 4 (Comparative Example 1). As a result of such measurement, the organic silica porous membrane having the RIE-treated surface obtained in Examples 1 and 2 and the organic silica porous membrane obtained in Comparative Example 1 (RIE untreated) have an average pore diameter. They were found to be 25 nm (Example 1), 27 nm (Example 2), and 24 nm (Comparative Example 1), respectively. 2 to 4, the organic silica porous membrane having the RIE-treated surface obtained in Example 1 has a thickness of 100 nm, and the organic silica having the RIE-treated surface obtained in Example 2 is used. It was confirmed that the porous film had a thickness of 90 nm, and the organic silica porous film (RIE untreated) obtained in Comparative Example 1 had a thickness of 120 nm. From these results, it was confirmed that about 20 to 30 nm of the surface was shaved by the RIE treatment. Moreover, it has confirmed from the result which compared FIGS. 2-4 that the opening part of the surface of the organic silica porous film has increased more by RIE process. In addition, from the cross-sectional SEM images shown in FIGS. 2 to 4, spherical pores are formed in each of the porous films in the substrates for mass spectrometry obtained in Examples 1 and 2 and Comparative Example 1. Was also confirmed.

<Measurement of surface area ratio of organic silica porous membrane>
The aperture ratio (surface aperture ratio) of the surface of the organic silica porous film of the substrate for mass spectrometry obtained in Examples 1-2 and Comparative Example 1 was measured as follows. That is, first, a scanning probe microscope (SPM / AFM: NanoNavi E-sweep manufactured by Hitachi High-Tech Science Co., Ltd.) is used as a measuring device, and a 1 μm square (1 μm × 1) on the surface of the porous organic silica film of the substrate for mass spectrometry. Using a silicon micro-cantilever (trade name “SI-DF20” manufactured by Hitachi High-Tech Science Co., Ltd.) as a cantilever for any four measurement areas with a size of 1 μm), in the dynamic force mode, The surface state was analyzed under scanning conditions of (X): 256 pixels and lateral (Y): 256 pixels to obtain an atomic force microscope (AFM) image. From the position information of the vertical (X), horizontal (Y), and height (Z) of each pixel (pixel) recorded in the AFM image (256 pixels × 256 pixels) obtained in this way, the height ( Z) The value of the vertical distance from the reference plane [unit: nm] is the vertical axis, and the frequency (ratio of the number of pixels having a specific height to the total number of pixels: a specific height value) The graph (the graph of the frequency distribution curve of the height) having the horizontal axis of the abundance ratio (unit:%) of the pixels having was obtained. Then, from the graph of the obtained frequency distribution curve of the height, the frequency having a height value lower than the height value (H1) having the maximum frequency (maximum frequency) and half the maximum frequency. The height value (H2) having Next, the AFM image was binarized using the height value (H2) having a frequency that is ½ of the maximum frequency as a threshold value. By such binarization, for each of four arbitrary measurement areas, the image is color-coded in the pixel portion where the height value is H2 or more and the pixel portion where the height value is less than H2. (Images obtained by binarizing AFM images) were obtained. Based on such a binarized image, the pixel portion (region) where the height value in the image is H2 or more is the skeleton portion of the organic silica porous membrane (the organic silica forming the surface of the porous membrane). And the portion (region) where the height value is less than H2 is determined to be the opening portion of the pores on the surface of the organic silica porous membrane (void portion based on the pores). , Aperture ratio for each measurement region (ratio of pore opening portion in the surface image of each measurement region, calculation formula: ([area of pore opening portion] / [total area of measurement region]) × 100 [unit :%])) Was calculated, and the average value thereof was determined to obtain the surface aperture ratio (average value of aperture ratios of arbitrary four measurement areas).

  As a result of such a measurement, the organic silica porous membrane having the RIE-treated surface obtained in Examples 1 and 2 and the organic silica porous membrane obtained in Comparative Example 1 (RIE untreated) each have a surface opening ratio. It was confirmed that they were 41% (Example 1), 40% (Example 2), and 7% (Comparative Example 1). From these results, it was found that the surface opening ratio of the organic silica porous film can be made higher by performing the RIE treatment.

  In addition, the AFM image of the surface of the organic silica porous film of the substrate for mass spectrometry obtained in Examples 1 and 2 and Comparative Example 1 is shown in FIG. 5 (Example 1), FIG. 6 (Example 2), and FIG. (Comparative example 1) (Note that the AFM image indicated by (b) in each figure is a partial enlarged image of the AFM image indicated by (a)). By directly comparing the results shown in FIGS. 5 to 7, in the organic silica porous membrane (Examples 1 and 2) having the RIE-treated surface, the pores (black contrast portion) exposed on the surface ) Increased significantly compared to the organic silica porous film (RIE untreated) obtained in Comparative Example 1. Thus, it was found that the surface opening ratio of the organic silica porous film can be made higher by performing the RIE treatment.

<Measurement of absorption spectrum>
In order to obtain the absorption spectrum of the organic group in the organic silica porous film of the substrate for mass spectrometry obtained in Examples 1-2 and Comparative Example 1, the following measurements were performed. That is, first, an organic silica porous membrane precursor employed in Example 1 except that a quartz substrate (trade name “synthetic quartz substrate” manufactured by Asahi Techniglass Co., Ltd.) was used instead of the silicon substrate (Si substrate). By adopting the same process as the above preparation process, a TPA-PMO thin film was formed on a quartz substrate to obtain a measurement sample. Then, using this measurement sample (laminated TPA-PMO thin film on a quartz substrate) and using an ultraviolet-visible spectrophotometer JASCO-V670 manufactured by JASCO Corporation as a measuring device, the absorption spectrum of the TPA-PMO thin film Was measured. The absorption spectrum obtained in this way is clearly an absorption spectrum of the organic group in the skeleton when the skeleton of the TPA-PMO thin film is taken into consideration. The absorption spectrum obtained by such measurement is shown in FIG.

  As is clear from the graph of the absorption spectrum shown in FIG. 8, it was confirmed that the TPA-PMO thin film made of a polymer of TPA organosilane has an absorption maximum wavelength at 310 nm. That is, in the TPA-PMO thin film made of a polymer of TPA organosilane, the absorption maximum wavelength of the organic group (triphenylamine) is found to be 310 nm, and further, from the absorption spectrum, a laser having a wavelength of 300 to 400 nm. It was found that light can be absorbed efficiently.

  In addition, all the organic silica porous membranes of the substrates for mass spectrometry obtained in Examples 1 and 2 and Comparative Example 1 are made of a polymer of TPA organosilane, and the type of substrate, presence or absence of surface treatment, and Since it was manufactured by adopting the same method as the above measurement sample except that the conditions of the surface treatment were different, the thin film in the measurement sample and obtained in Examples 1 and 2 and Comparative Example 1 It is apparent that the organic silica porous film of the obtained mass spectrometric substrate basically has the same organic group introduced into the skeleton. Therefore, the organic silica porous films of the substrates for mass spectrometry obtained in Examples 1 and 2 and Comparative Example 1 similarly have the absorption maximum wavelength of the organic group (triphenylamine) of 310 nm and the graph of the absorption spectrum. It was also found that the laser beam having a wavelength of 300 to 400 nm can be efficiently absorbed.

<Characteristics of each organic silica porous membrane>
From the above measurement results, the substrates for mass spectrometry obtained in Examples 1 and 2 have an organic group (triphenylamine) capable of absorbing laser light of 300 to 400 nm in the skeleton, and an average pore diameter of 5 to 5. It was found to have an organic silica porous film having 50 nm pores and a surface aperture ratio of 33 to 70%. On the other hand, the substrate for mass spectrometry obtained in Comparative Example 1 provided with the organic silica porous film (RIE non-treated film) not subjected to RIE treatment has a surface opening ratio (average surface opening ratio) of 7 for the organic silica porous film. It turned out that it was%. From these results, when the TPA organosilane is used, simply preparing it to prepare an organic silica porous membrane has fine pores with an average pore diameter of 5 to 50 nm and a surface aperture ratio of Whereas a porous film of 33% or more could not be formed (Comparative Example 1), the TPA organosilane was polymerized to form a thin film and then subjected to etching treatment such as RIE ( Examples 1-2) It was found that it is possible to prepare an organic silica porous membrane with a sufficiently increased aperture ratio such that the surface aperture ratio is 33% or more.

(Example 3: Mass spectrometry)
Using the substrate for mass spectrometry obtained in Example 1 and using the trade name “autoflex” manufactured by Bruker Daltonics, Inc. as the analyzer, mass spectrometry was performed as follows. That is, first, 1 μL of an aqueous solution containing 4 pmol of a standard peptide, which is a molecule to be measured, is dropped on the surface of the organic silica porous film in the substrate for mass spectrometry (surface with a surface opening ratio of 41%), By natural drying, a standard peptide (measuring molecule) was supported on the organic silica porous membrane. Next, after mounting a substrate having a membrane carrying the standard peptide (measuring molecule) on the cartridge plate of the analyzer, the laser intensity (Laser Power: LP) setting in the analyzer is set to 35%. Then, by irradiating a laser beam having a wavelength of 337 nm to a portion (sample loaded portion) where the sample (standard peptide) of the membrane is loaded (dropped), the standard peptide (measurement target molecule) is ionized to have a mass. Analysis was carried out. As standard peptides, product numbers 8206195 (Bruker # 8206195) [Angiotensin II (M1: 1046.5), Angiotensin I (M2: 1296.7), Substance P (M3: 1347.7), Momnesin (M4: 1619.8), ACTH clip 1-17 (M5: 2093.1), ACTH clip 18-39 (M6: 2465.2), Somatostatin 28 (M7: 3147.5), and the values described in parentheses () are the molecular weight of the H + body] Was used.

(Comparative example 2: mass spectrometry)
Mass spectrometry was performed in the same manner as in Example 3 except that the substrate for mass spectrometry obtained in Comparative Example 1 was used instead of using the substrate for mass spectrometry obtained in Example 1. In addition, the measurement object molecule | numerator was carry | supported on the surface (surface where the surface opening rate is 7%) of the organic silica porous film.

(Example 4: Mass spectrometry)
Mass spectrometry was performed in the same manner as in Example 3 except that the laser intensity (Laser Power) setting in the analyzer was changed from 35% to 30%.

(Example 5: Mass spectrometry)
Mass spectrometry was performed in the same manner as in Example 4 except that the substrate for mass spectrometry obtained in Example 2 was used instead of the substrate for mass spectrometry obtained in Example 1. In addition, the measurement object molecule | numerator was carry | supported on the surface (surface in which the surface opening rate is 40%) of the organic silica porous film.

(Comparative Example 3: Mass spectrometry)
Instead of using the substrate for mass spectrometry obtained in Example 1, the substrate for mass spectrometry obtained in Comparative Example 1 was used, and the laser intensity (Laser Power) setting in the analyzer was set from 30% to 40 Mass spectrometry was performed in the same manner as in Example 4 except that the percentage was changed to%. In addition, the measurement object molecule | numerator was carry | supported on the surface (surface where the surface opening rate is 7%) of the organic silica porous film.

(Comparative Example 4: Mass spectrometry)
Mass spectrometry was performed in the same manner as in Comparative Example 3 except that the laser power (Laser Power) setting was changed from 40% to 30% (the same laser intensity as in Examples 4 and 5 was obtained in Comparative Example 1). When mass spectrometry was performed using the obtained substrate for mass spectrometry), a mass spectrum signal could not be confirmed (peptide could not be detected).

[About the results of mass spectrometry of Examples 3 to 5 and Comparative Examples 2 to 4]
As a result of mass spectrometry of Example 3 and Comparative Example 2, a graph of the mass spectrum obtained in Example 3 and a graph of the mass spectrum obtained in Comparative Example 2 are shown in FIG. Note that Example 3 and Comparative Example 2 were subjected to mass spectrometry by irradiating laser light with the same intensity, and the numerical value in parentheses () next to the peak in the graph of the measured mass spectrum indicates the signal intensity. .

  Moreover, as a result of the mass spectrometry of Examples 4-5 and Comparative Example 3, the graph of the mass spectrum obtained in Example 4, the graph of the mass spectrum obtained in Example 5, and the comparative example 3 are obtained. A graph of the mass spectrum is shown in FIG. In Examples 4 to 5 and Comparative Example 3, mass analysis was performed by irradiating a laser beam at an optimum intensity so that the detection sensitivity was higher, and the side of the peak in the graph of the measured mass spectrum. Numerical values in parentheses () indicate signal intensity.

As is clear from the results shown in FIGS. 9 and 10, in Examples 3 to 5 and Comparative Examples 2 to 3, detection of Substance P (M3) and Mombesin (M4) is performed without using a matrix. Was found to be possible. Further, from the results of mass spectrometry with the same laser intensity (LP = 35%) (results shown in FIG. 9), the performance of each of the substrates for mass spectrometry used in Example 3 and Comparative Example 2 is compared. The signal of the mass spectrum (the mass spectrum measured in Example 3) when the substrate using the prepared organic silica porous film (the substrate for mass spectrometry obtained in Example 1) is used is RIE-untreated organic Compared with the signal of the mass spectrum (mass spectrum measured in Comparative Example 2) when the substrate using the porous silica film (the substrate for mass spectrometry obtained in Comparative Example 1) is used, the intensity of the signal is It became stronger and the detection sensitivity was found to be improved by an order of magnitude (note that the signal intensity of [M3 + Na ⁺ ] and [M4 + Na ⁺ ] was more than 10 times).

  Furthermore, as is clear from the results shown in FIG. 10, in Examples 4 to 5, the peptide was ionized and sufficiently detected even when the laser intensity was lowered to 30% during mass spectrometry. In addition, when mass spectrometry was performed using the substrate for mass spectrometry obtained in Comparative Example 1 at a laser intensity of 30% as in Examples 4 to 5 (Comparative Example 4), a peptide signal could be confirmed. It was also found that by using the mass spectrometric substrates obtained in Examples 1 and 2, the detection sensitivity was further increased, and mass analysis could be performed more efficiently with a lower laser intensity. Further, from the results shown in FIG. 10, when the mass spectrum measured in Examples 4 to 5 and the mass spectrum measured in Comparative Example 3 were compared, the substrate for mass spectrometry obtained in Comparative Example 1 was used. Angiotensin II (M1) and Angiotensin I (M2), which could not be detected when mass spectrometry was performed (Comparative Example 3), were performed using the mass spectrometry substrate obtained in Examples 1-2. (Examples 4 to 5), it was confirmed that detection was possible. From these results, the detection sensitivity was further improved by using the mass spectrometry substrate obtained in Examples 1 and 2. Was found to be high.

(Example 6: Mass spectrometry)
Using the substrate for mass spectrometry obtained in Example 1 and using AXIMA-QIT manufactured by Shimadzu as an analyzer, mass spectrometry was performed as follows. That is, first, on the surface of the organic silica porous film in the mass spectrometric substrate (surface with a surface opening ratio of 41%), the following formula:

[The component present in the portion indicated by A in the chemical formula is a sugar chain, and the component present in the portion indicated by B is a component derived from a labeled molecule (2-aminoacridone) (fixed (coupled) to the sugar chain). Labeled molecule). ]
1 μL of an aqueous solution containing 10 pmol of a saccharide (AMAC-NA2) to which a labeled molecule (labeled dye) represented by the formula (1) is immobilized and dried naturally, thereby supporting AMAC-NA2 (measuring molecule) on the organic silica porous membrane. did. Next, a substrate having a film carrying AMAC-NA2 (measurement target molecule) is mounted on the cartridge plate of the analyzer, and then the film sample (AMAC-NA2) is carried (dropped) on the portion. By irradiating a laser beam having a wavelength of 337 nm, AMAC-NA2 was ionized to perform mass spectrometry. In addition, the measurement was performed in the positive ion mode of the analyzer, and the measurement was performed three times by changing the laser irradiation position for each measurement (mass analysis was performed on three locations). A mass spectrum graph is shown in FIG. 11 as the measurement results of such three mass spectrometry (in addition, the results are described separately as (a), (b), and (c) for each measurement time). In each of FIGS. 11A, 11B, and 11C (in each graph of the mass spectrum measured each time), the signal intensity at the maximum peak was 20 mV, 24 mV, and 28 mV, respectively.

(Example 7: Mass spectrometry)
Mass spectrometry was performed in the same manner as in Example 6, except that the substrate for mass spectrometry obtained in Example 2 was used instead of the substrate for mass spectrometry obtained in Example 1. In addition, the measurement object molecule | numerator was carry | supported on the surface (surface in which the surface opening rate is 40%) of the organic silica porous film. As a measurement result of such mass analysis (for three times), a graph of a mass spectrum is shown in FIG. 12 (note that the result is described separately for (a), (b), and (c) for each measurement time). In each of FIGS. 12A, 12B, and 12C (in each graph of the mass spectrum measured each time), the signal intensity at the maximum peak was 46 mV, 44 mV, and 45 mV, respectively.

(Comparative Example 5: mass spectrometry)
Mass spectrometry was performed in the same manner as in Example 6 except that instead of using the substrate for mass spectrometry obtained in Example 1, the substrate for mass spectrometry obtained in Comparative Example 1 was used. In addition, the measurement object molecule | numerator was carry | supported on the surface (surface where the surface opening rate is 7%) of the organic silica porous film. As a measurement result of such mass analysis (for three times), a graph of a mass spectrum is shown in FIG. 13 (note that the result is described separately as (a), (b), and (c) for each measurement time). In each of FIGS. 13A, 13B, and 13C (in each graph of the mass spectrum measured each time), the signal intensity at the maximum peak is 1.5 mV, 0.6 mV, It was 1 mV.

[About the results of mass spectrometry of Examples 6 to 7 and Comparative Example 5]
As is clear from the results shown in FIGS. 11 to 13, when mass spectrometry was performed by irradiating the sugar (AMAC-NA2, 10 pmol) on which the labeling dye was immobilized with the laser under the same conditions (Examples 6 to 7). In Comparative Example 5), the maximum peak of the mass spectrum measured when the substrate for mass spectrometry (substrate for mass spectrometry obtained in Comparative Example 1) provided with the RIE-untreated organic silica porous film was used. Signal intensity of 0.6 to 1.5 mV (FIG. 13: Comparative Example 5), a substrate for mass spectrometry (obtained in Example 1) provided with an organic silica porous film subjected to RIE treatment When the mass spectrometry substrate and the mass spectrometry substrate obtained in Example 2 are respectively used, the peak signal intensity of the mass spectrum is 20 to 28 mV (FIG. 11: Example 6) and 44, respectively. ~ 46mV FIG. 12: Example 7), and by using the substrate for mass spectrometry obtained in Examples 1 and 2 having a higher surface aperture ratio, the signal intensity of the measured mass spectrum is greatly improved. It was found that the detection sensitivity can be improved by an order of magnitude. From such a result, by using the substrate for mass spectrometry obtained in Examples 1 and 2, it is possible to perform mass analysis without using a matrix and to further increase its detection sensitivity. It was confirmed that it was possible.

(Example 8: Mass spectrometry)
Instead of using an aqueous solution containing 10 pmol of sugar (AMAC-NA2) with a labeled molecule (labeled dye) immobilized, an aqueous solution containing 1 pmol of AMAC-NA2 was used, and the number of measurements was changed from 3 to 2 times. In the same manner as in Example 6, mass spectrometry (mass spectrometry using the substrate for mass spectrometry obtained in Example 1) was performed. A graph of mass spectrum is shown in FIG. 14 as the measurement results of such two mass spectrometry (in addition, the results are described separately for (a) and (b) for each measurement). In each of FIGS. 14 (a) and 14 (b) (in each graph of the mass spectrum measured each time), the signal intensity at the maximum peak was 16 mV and 15 mV, respectively.

(Comparative Example 6)
Mass spectrometry was performed in the same manner as in Example 8 except that the substrate for mass spectrometry obtained in Comparative Example 1 was used instead of the substrate for mass spectrometry obtained in Example 1. A signal could not be confirmed (AMAC-NA2 could not be detected).

[About the results of mass spectrometry of Example 8 and Comparative Examples 5 to 6]
As is clear from the results shown in FIG. 14, even when the amount of AMAC-NA2 supported is 1 pmol, a substrate for mass spectrometry comprising the RIE-treated organic silica porous membrane (the mass obtained in Example 1) In the case of using the (analysis substrate) (in the case of Example 8), the signal intensity (16 mV, 15 mV) of the peak of the mass spectrum was confirmed for each measurement. On the other hand, when a substrate for mass spectrometry (a substrate for mass spectrometry obtained in Comparative Example 1) provided with a RIE-untreated organic silica porous film is used (in the case of Comparative Example 6), AMAC- Under the condition that the amount of NA2 supported was 1 pmol, a mass spectrum signal could not be confirmed. In addition, the signal intensity of the mass spectrum measured by the mass spectrometry of Comparative Example 5 in which the amount of AMAC-NA2 supported is 10 pmol, and the mass spectrum measured by the mass analysis of Example 8 in which the amount of AMAC-NA2 supported is 1 pmol. Even when the signal intensities were compared, it was confirmed by mass spectrometry in Example 8 that the signal intensity was greatly improved. From such comparison, Comparative Example 5 and Example 8 differ in the loading amount of the measurement target molecule (AMAC-NA2), and in the mass spectrometry of Example 8, the loading amount of the measurement target molecule (AMAC-NA2) is different. Despite the small amount, it was found that the detection sensitivity was improved as compared with the mass spectrometry of Comparative Example 5. From these results, when the substrate for mass spectrometry of the present invention (Example 1) is used, it is possible to make the detection sensitivity higher, even when the amount of molecules to be measured is small. It was found that mass analysis can be performed sufficiently efficiently.

  As described above, according to the present invention, laser desorption / ionization mass spectrometry capable of efficiently performing mass spectrometry with higher detection sensitivity without using a matrix, and a laser used in the method It becomes possible to provide an organic silica porous membrane substrate for desorption ionization mass spectrometry. Therefore, the laser desorption ionization mass spectrometry method of the present invention can be widely used in all analysis fields using MS spectra (for example, the field of bioanalysis where only a trace amount of sample is available). is there.

Claims (5)

  Measured with respect to an organic silica porous film having an organic group capable of absorbing irradiation laser light in the skeleton, pores having an average pore diameter of 5 to 50 nm, and a surface opening ratio of 33 to 70%. Laser desorption ionization mass spectrometry characterized in that after carrying a sample containing a molecule of interest, the sample carrying portion of the film is irradiated with laser light to ionize the molecule to be measured and perform mass spectrometry .   The laser desorption ionization mass spectrometry method according to claim 1, wherein the organic group has an absorption maximum wavelength in a range of 200 to 600 nm.   The laser desorption / ionization mass spectrometry method according to claim 1, wherein the organic group is an aromatic organic group containing 4 or more carbon atoms.   The laser desorption / ionization mass spectrometry method according to any one of claims 1 to 3, wherein the organic group includes triphenylamine.   It includes an organic silica porous film having an organic group capable of absorbing laser light in its skeleton, pores having an average pore diameter of 5 to 50 nm, and a surface opening ratio of 33 to 70%. Organic silica porous membrane substrate for laser desorption ionization mass spectrometry.