JP7421170B2 - Organic silica substrate and laser desorption/ionization mass spectrometry method using it - Google Patents

Description

The present invention relates to an organic silica substrate and a laser desorption/ionization mass spectrometry method using the same, and more specifically, an organic silica substrate equipped with an organic silica film capable of absorbing laser light, and a laser desorption method using the same. /Relating to ionization mass spectrometry method.

In conventional general laser desorption/ionization mass spectrometry, a low-molecular-weight organic substance called a matrix is mixed with the target substance, and when laser light is irradiated, the matrix supports the desorption/ionization of the target substance. , which made mass spectrometry possible. However, since the matrix is desorbed/ionized together with the substance to be measured by laser light irradiation, signals derived from the matrix are observed as interference peaks in the low molecular weight region of the obtained mass spectrum (LDI-MS spectrum). There was a problem in that mass spectrometry was difficult.

For this reason, laser desorption/ionization mass spectrometry methods that do not use a matrix are being considered. For example, Japanese Patent Application Publication No. 2018-185200 (Patent Document 1) describes a method that 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 aperture ratio. An organosilica substrate for laser desorption/ionization mass spectrometry is disclosed that includes an organosilica porous membrane having an organic silica porous film having a ionization ratio of 33 to 70%. By using such an organic silica substrate, the above problems have been solved, and laser desorption/ionization mass spectrometry has become possible even for substances to be measured in the low molecular weight range.

However, in laser desorption/ionization mass spectrometry using conventional organic silica substrates, the detection sensitivity of target substances is not necessarily high enough, and a more sensitive laser desorption/ionization mass spectrometry method is required. ing. Therefore, Japanese Patent Application Laid-open No. 2020-165707 (Patent Document 2) discloses an organic silica thin film made of organic silica having an organic group capable of absorbing laser light in its skeleton, and a hydrophobic layer laminated on the surface of the organic silica thin film. the hydrophobic layer is a layer made of a hydrophobic material having a hydrophobic group having an aliphatic carbon skeleton as a main skeleton and a functional group capable of covalently bonding to the silica skeleton of the organic silica, and , an organic silica substrate for laser desorption/ionization mass spectrometry in which the hydrophobic layer has a thickness of 0.2 to 5.5 nm has been proposed, and by using this organic silica substrate, laser desorption with higher sensitivity can be achieved. / It is described that it becomes possible to perform ionization mass spectrometry.

However, when performing laser desorption/ionization mass spectrometry on highly hydrophilic compounds such as bio-related molecules, even if the organic silica substrate described in Patent Document 2 is used, the highly hydrophilic compounds that are the molecules to be measured are It was difficult to detect highly hydrophilic compounds with high sensitivity because they were not uniformly adsorbed onto the surface of the organic silica substrate.

Also, Sunia A. Trauger et al., Anal. Chem. , 2004, Vol. 76, No. 15, pp. 4484-4489 (Non-Patent Document 1), in desorption/ionization mass spectrometry using porous silicon, the surface of porous silicon is treated with a silane compound. It has been reported that molecules to be measured can be detected with high sensitivity by introducing hydrophilic or hydrophobic groups into the surface of porous silicon. There is no mention of such consideration.

Japanese Patent Application Publication No. 2018-185200 Japanese Patent Application Publication No. 2020-165707

Sunia A. Trauger et al., Anal. Chem. , 2004, Vol. 76, No. 15, pp. 4484-4489.

The present invention has been made in view of the above-mentioned problems of the prior art, and includes an organic silica substrate that enables laser desorption/ionization mass spectrometry with high sensitivity even for highly hydrophilic compounds such as biologically related molecules. The object of the present invention is to provide a laser desorption/ionization mass spectrometry method using the same.

As a result of extensive research to achieve the above object, the present inventors have discovered that an organic silica substrate equipped with an organic silica film containing an organic group capable of absorbing laser light in the skeleton has an amide structure in the skeleton. By introducing a group and further introducing a hydrophobic group so that the amide group and the hydrophobic group are arranged uniformly and in a well-balanced manner on the surface of the organic silica membrane, highly hydrophilic compounds such as bio-related molecules can be prepared. The present inventors have discovered that it is also possible to perform laser desorption/ionization mass spectrometry with high sensitivity, and have completed the present invention.

That is, the organic silica substrate of the present invention contains an organic group having a maximum absorption wavelength within a wavelength range of 200 to 600 nm and an amide group bonded to the organic group in its skeleton, and further has a hydrophobic group. It is characterized by comprising a silica membrane.

In the organic silica substrate of the present invention , it is preferable that the hydrophobic group is bonded to a silicon atom of a siloxane bond constituting the organic silica film, and further, it is preferable that the organic group is a naphthalimide ring, and It is preferable that the organic silica film is at least one selected from the group consisting of those having an uneven structure on the surface, those consisting of nanofibers, and those consisting of fine particles.

Further, the laser desorption/ionization mass spectrometry method of the present invention includes preparing a measurement sample by supporting the molecule to be measured on the organic silica substrate of the present invention, and irradiating the measurement sample with laser light. This method is characterized in that the molecules to be measured are desorbed from the organic silica substrate, ionized, and subjected to mass spectrometry.

Although it is not necessarily clear why the use of the organic silica substrate of the present invention makes it possible to perform laser desorption/ionization mass spectrometry with high sensitivity on highly hydrophilic compounds such as bio-related molecules, the present invention They speculate as follows. That is, the organic silica substrate of the present invention includes an organic silica film containing an organic group capable of absorbing laser light and an amide group in its skeleton, and further containing a hydrophobic group. In this organic silica film, as shown in FIG. 1, an amide group is bonded to an organic group capable of absorbing laser light in the skeleton, and a hydrophobic group is bonded to a silicon atom of a siloxane bond. Therefore, amide groups and hydrophobic groups are arranged uniformly and in a well-balanced manner on the surface of the organic silica film. When a measurement sample is prepared by dropping a sample solution containing a highly hydrophilic compound such as a bio-related molecule onto the surface of such an organic silica membrane, as shown in Figure 2, the sample solution is absorbed by the action of the hydrophobic groups. Since the highly hydrophilic compound is concentrated and adsorbed to the amide group, the highly hydrophilic compound is supported on the surface of the organic silica membrane in a uniform and highly dispersed manner. When laser desorption/ionization mass spectrometry is performed using such a measurement sample, the hydrophobic groups reduce the interaction between the surface of the organosilica membrane and the highly hydrophilic compound, thereby allowing the desorption of the highly hydrophilic compound. It is inferred that it is possible to detect highly hydrophilic compounds uniformly with high sensitivity because separation is promoted and signals corresponding to highly hydrophilic compounds can be detected over the entire surface of the organosilica membrane. .

On the other hand, a sample solution containing a highly hydrophilic compound such as a biologically relevant molecule is dropped onto the surface of an organic silica film that has an organic group capable of absorbing laser light and is free of amide groups and hydrophobic groups. When prepared, there are no sites on the surface of the organic silica membrane that strongly adsorb highly hydrophilic compounds, so as shown in Figure 3, the highly hydrophilic compounds aggregate and are uniformly supported on the surface of the organic silica membrane. I can't. Therefore, when performing laser desorption/ionization mass spectrometry using such a measurement sample, the amount of highly hydrophilic compounds supported varies depending on the measurement point on the surface of the organic silica membrane, so The intensity of the signal detected by the highly hydrophilic compound also varies, and the detection intensity of the highly hydrophilic compound becomes uneven on the surface of the organosilica membrane.Furthermore, since the hydrophobic group cannot promote the desorption of the highly hydrophilic compound, It is inferred that the detection sensitivity for sexual compounds will be lower.

In addition, a sample solution containing a highly hydrophilic compound such as a biologically relevant molecule is dropped onto the surface of an organic silica film that has an organic group and an amide group that can absorb laser light and no hydrophobic groups. As shown in Figure 4, when a highly hydrophilic compound is adsorbed to the amide group, the sample solution spreads over the surface of the organosilica membrane due to the action of the hydrophilic amide group, resulting in a highly hydrophilic compound per unit area. The concentration of the compound is lower. Therefore, when laser desorption/ionization mass spectrometry is performed using such a measurement sample, the intensity of the signal corresponding to a highly hydrophilic compound at the measurement point on the surface of the organosilica membrane is low, indicating that the highly hydrophilic compound due to the hydrophobic group is It is presumed that the detection sensitivity of highly hydrophilic compounds will be low because the compound cannot be affected by the effect of promoting desorption of the compound.

Furthermore, a sample solution containing a highly hydrophilic compound such as a biologically relevant molecule is dropped onto the surface of an organic silica film that has an organic group capable of absorbing laser light and a hydrophobic group, but does not have an amide group, and a sample solution for measurement is prepared. As shown in Figure 5, when the sample solution is prepared, the sample solution is concentrated due to the action of the hydrophobic groups, but since there are no sites on the surface of the organic silica membrane where highly hydrophilic compounds can be adsorbed, the highly hydrophilic compounds do not aggregate. However, it cannot be uniformly supported on the surface of the organic silica film. Therefore, when laser desorption/ionization mass spectrometry is performed using such a measurement sample, the hydrophobic groups promote the desorption of highly hydrophilic compounds, so although highly hydrophilic compounds are detected with high sensitivity, Since the amount of highly hydrophilic compounds supported varies depending on the measurement point on the surface of the organic silica membrane, the intensity of the signal corresponding to the highly hydrophilic compound also varies, and the detection sensitivity of highly hydrophilic compounds is lower than that of the organic silica membrane. It is assumed that the surface becomes non-uniform.

According to the present invention, it is possible to perform laser desorption/ionization mass spectrometry with high sensitivity even on highly hydrophilic compounds such as biologically relevant molecules.

FIG. 1 is a schematic diagram showing the skeletal structure of an organic silica film constituting the organic silica substrate of the present invention. FIG. 2 is a schematic diagram showing an embodiment in which a highly hydrophilic compound is supported on the surface of an organic silica substrate of the present invention. FIG. 2 is a schematic diagram showing a mode in which a highly hydrophilic compound is supported on the surface of a conventional organic silica substrate. FIG. 2 is a schematic diagram showing a mode in which a highly hydrophilic compound is supported on the surface of a conventional organic silica substrate. FIG. 2 is a schematic diagram showing a mode in which a highly hydrophilic compound is supported on the surface of a conventional organic silica substrate. 3 is an atomic force microscope photograph showing the surface shape of organic silica thin film b produced in Production Example 2. 3 is an optical micrograph showing the state of nanofibers in the nanofiber dispersion prepared in Production Example 3. 3 is a scanning electron micrograph showing the surface shape of the dried coating film produced in Production Example 3. 3 is a scanning electron micrograph showing the surface shape of organic silica thin film C produced in Production Example 3. 3 is an atomic force micrograph showing the surface shape of organic silica thin film g produced in Comparative Production Example 4. 1 is a graph showing a mass spectrum of a measurement sample (organic silica thin film A, angiotensin I) prepared in Example 1. 2 is a graph showing a mass spectrum of a measurement sample (organic silica thin film B, amyloid β) prepared in Example 2. FIG. 3 is a diagram showing a mapping image of amyloid β in the measurement sample (organic silica thin film B, amyloid β) prepared in Example 2. 3 is a graph showing a mass spectrum of a measurement sample (organic silica thin film B, amyloid β) prepared in Example 3. 3 is a graph showing the mass spectrum of the measurement sample (organic silica thin film B, phosphatidylcholine) prepared in Example 4. 3 is a graph showing a mass spectrum of a measurement sample (organic silica thin film B, insulin) prepared in Example 5. 3 is a graph showing the mass spectrum of the measurement sample (organic silica thin film B, amyloid β in plasma solution) prepared in Example 6. 3 is a graph showing a mass spectrum of a measurement sample (organic silica thin film B) consisting only of plasma solution analyzed in Example 6. 7 is a graph showing a mass spectrum of a measurement sample (organic silica thin film C, amyloid β) prepared in Example 7. 7 is a graph showing a mass spectrum of a measurement sample (organic silica thin film C, insulin) prepared in Example 8. 2 is a graph showing a mass spectrum of a measurement sample (organic silica thin film C, a mixture of verapamil, phosphatidylcholine, and angiotensin II) prepared in Example 9. 2 is a graph showing a mass spectrum of a measurement sample (organic silica thin film C, a mixture of verapamil, phosphatidylcholine, and angiotensin II) prepared in Example 9. 2 is a graph showing a mass spectrum of a measurement sample (organic silica thin film C, a mixture of verapamil, phosphatidylcholine, and angiotensin II) prepared in Example 9. 1 is a graph showing a mass spectrum of a measurement sample (organic silica thin film a, amyloid β) prepared in Comparative Example 1. 2 is a graph showing a mass spectrum of a measurement sample (organic silica thin film e, amyloid β) prepared in Comparative Example 2. FIG. 7 is a diagram showing a mapping image of amyloid β in the measurement sample (organic silica thin film A, amyloid β) prepared in Example 10. FIG. 3 is a diagram showing a mapping image of amyloid β in a measurement sample (organic silica thin film f, amyloid β) prepared in Comparative Example 3. This is a graph showing the average value of the signal intensity corresponding to the proton adduct of amyloid β in the measurement sample prepared in Example 10 (organic silica thin film A) and the measurement sample prepared in Comparative Example 3 (organic silica thin film f). be. FIG. 7 is a diagram showing a mapping image of amyloid β in the measurement sample (organic silica thin film D, amyloid β) prepared in Example 11. FIG. 4 is a diagram showing a mapping image of amyloid β in a measurement sample (organic silica thin film h, amyloid β) prepared in Comparative Example 4. This is a graph showing the average value of the signal intensity corresponding to the proton adduct of amyloid β in the measurement sample prepared in Example 11 (organic silica thin film D) and the measurement sample prepared in Comparative Example 4 (organic silica thin film h). be.

Hereinafter, the present invention will be explained in detail based on its preferred embodiments.

[Organic silica substrate]
First, the organic silica substrate of the present invention will be explained. The organic silica substrate of the present invention includes an organic silica film containing in its skeleton an organic group and an amide group having a maximum absorption wavelength within a wavelength range of 200 to 600 nm, and further having a hydrophobic group.

(Organic silica film)
The organic silica film according to the present invention contains in its skeleton an organic group having a maximum absorption wavelength within the wavelength range of 200 to 600 nm (hereinafter also referred to as "laser light absorbing organic group"). When the maximum absorption wavelength of the laser light absorbing organic group is within the above wavelength range, the laser light irradiated in laser desorption/ionization mass spectrometry can be efficiently absorbed. On the other hand, if the maximum absorption wavelength of the laser light-absorbing organic group is less than the lower limit, when irradiated with laser light with a wavelength less than the lower limit in laser desorption/ionization mass spectrometry, the molecule to be measured or the organic silica On the other hand, if the maximum absorption wavelength of the laser light-absorbing organic group exceeds the upper limit, laser light with a wavelength exceeding the upper limit may be used in laser desorption/ionization mass spectrometry. Even when irradiated with , it may not be possible to ionize the molecules to be measured due to insufficient energy. Further, the wavelength range in which the maximum absorption wavelength of the laser light absorbing organic group exists is preferably 250 to 450 nm, more preferably 300 to 400 nm.

As such a laser light absorbing organic group, an organic group containing an aromatic hydrocarbon group having 10 or more carbon atoms is preferable from the viewpoint of being able to efficiently absorb laser light, and specifically, a naphthalimide ring, a naphthalimide ring, Triphenylamine ring, pyrene ring, diphenylpyrene ring, tetraphenylpyrene ring, perylene ring, perylene bisimide ring, acridone ring, methylacridone ring, styrylbenzene ring, divinylbenzene ring, fluorene ring, quaterphenyl ring, anthracene ring, acridine ring, phenylpyridine ring, divinylpyridine ring, porphine ring, phthalocyanine ring, diketopyrrolopyrrole ring, dithienylbenzothiazole ring, and the like. Among such organic groups containing aromatic hydrocarbon groups having 10 or more carbon atoms, naphthalimide rings, triphenylamine rings, pyrene rings, perylene rings, and acridone rings are preferred from the viewpoint of chemical stability against laser beam irradiation. is more preferred, and a naphthalimide ring is even more preferred. Note that such an organic group containing an aromatic hydrocarbon group having 10 or more carbon atoms can be appropriately selected depending on the wavelength of the laser light to be irradiated. Further, the organic silica film according to the present invention may contain one type of such organic group containing an aromatic hydrocarbon group having 10 or more carbon atoms, or may contain two or more types. .

In the organic silica film according to the present invention, the laser light absorbing organic group is contained in the skeleton of the organic silica film, and is directly or indirectly attached to silicon (Si) in the main skeleton of the organic silica film. (via another element), and more preferably to silicon (Si) constituting a siloxane bond. Further, in the organic silica film according to the present invention, it is preferable that two or more silicon (Si) atoms are bonded to one laser light absorbing organic group. When only one silicon (Si) is bonded to one laser-absorbing organic group, the density of the laser-absorbing organic group decreases, so the absorption intensity of laser beam tends to decrease. On the other hand, the upper limit of the number of silicon (Si) molecules bonded to one laser light absorbing organic group is not particularly limited, but is preferably 6 or less, more preferably 4 or less, and particularly preferably 3 or less. . When the number of silicon (Si) bonded to one laser light absorbing organic group exceeds the above upper limit, the proportion of the laser light absorbing organic group decreases, and the laser light absorption efficiency tends to decrease.

In the organic silica film, the mass ratio of silicon forming the organic silica film to the laser light absorbing organic group ([mass of silicon]/[laser light absorbing organic group]) is 0.05 to 0.50 ( It is more preferably within the range of 0.10 to 0.40, still more preferably 0.10 to 0.35, particularly preferably 0.15 to 0.35). When the mass ratio is less than the lower limit, the crosslinking density of the organic silica film becomes low, and a sufficiently cured organic silica film tends to be difficult to obtain.On the other hand, when it exceeds the upper limit, the relative laser light absorption Since the density of organic groups decreases, the absorption intensity of laser light tends to decrease, and it also tends to become difficult to form an uneven structure on the surface of the organic silica film by nanoimprinting or the like.

In the organic silica membrane according to the present invention, an amide group is contained within the skeleton of the organic silica membrane. The presence of amide groups in the skeleton of organosilica membranes improves the adsorption of molecules to be measured on the organosilica membrane surface, especially highly hydrophilic compounds such as bio-related molecules, in laser desorption/ionization mass spectrometry. However, it becomes possible to uniformly support molecules to be measured on the entire surface of the organic silica film. Moreover, it is preferable that the amide group is bonded directly or indirectly (via another element) to the laser light absorbing organic group. As a result, the amide groups are regularly arranged within the skeleton of the organosilica membrane, so in laser desorption/ionization mass spectrometry, the adsorption sites for the analyte molecules become homogeneous, and the detection intensity of the signal corresponding to the analyte molecules becomes uniform. uniformity is improved. Furthermore, in the organic silica film according to the present invention, it is sufficient that one or more amide groups are bonded to one laser light absorbing organic group. Further, there is no particular upper limit to the number of amide groups bonded to one laser light absorbing organic group, but it is preferably 6 or less, more preferably 4 or less, and particularly preferably 2 or less. If the number of amide groups bonded to one laser light-absorbing organic group exceeds the above upper limit, the target molecule supported on the organic silica film tends to be difficult to desorb in laser desorption/ionization mass spectrometry. be.

The organic silica membrane according to the present invention contains hydrophobic groups. Due to the presence of hydrophobic groups in the organic silica membrane, the sample solution containing the molecules to be measured that is dropped onto the organic silica membrane is concentrated during laser desorption/ionization mass spectrometry. It is supported at high density on the surface, increasing the intensity of the signal corresponding to the molecule to be measured. Furthermore, since the interaction between the surface of the organic silica film and the molecules to be measured is reduced, the desorption of the molecules to be measured is promoted, making it possible to detect signals corresponding to the molecules to be measured with high sensitivity. It is preferable that such a hydrophobic group is bonded to a silicon atom of a siloxane bond constituting the organic silica film. As a result, hydrophobic groups and amide groups are uniformly present in a well-balanced manner on the surface of the organic silica membrane, so that molecules to be measured are uniformly supported on the surface of the organic silica membrane in laser desorption/ionization mass spectrometry. It becomes possible to detect signals corresponding to molecules on the entire surface of the organic silica membrane.

Furthermore, in the organosilica membrane according to the present invention, the molar ratio of hydrophobic groups to amide groups (hydrophobic groups/amide groups) is preferably 4/1 to 0.5/1, and 3/1 to 1/1. More preferably, 2/1 to 1.5/1 is particularly preferred. If the hydrophobic group/amide group is less than the above lower limit, sufficient hydrophobicity will not be imparted to the surface of the organic silica film containing the laser light absorbing organic group and the amide group. The concentration effect of the sample solution containing the target molecule and the desorption promoting effect of the target molecule cannot be obtained sufficiently, and the signal corresponding to the target molecule cannot be detected with high sensitivity.On the other hand, if the above upper limit is exceeded, In laser desorption/ionization mass spectrometry, the adsorption of molecules to be measured on the surface of an organic silica membrane tends to decrease, making it difficult to support molecules to be measured uniformly over the entire surface of the organic silica membrane.

Such hydrophobic groups are not particularly limited as long as they can impart hydrophobicity to the organic silica film containing a laser light-absorbing organic group and an amide group, and include, for example, alkyl groups, alkynyl groups, alkenyl groups, Examples thereof include a fluorine atom-containing group, a halogen atom-containing group other than a fluorine atom, an alkoxy group, and an aromatic ring.

The number of carbon atoms in each of the alkyl group, alkynyl group and alkenyl group is preferably 1 to 40, more preferably 1 to 30, particularly preferably 1 to 20. When the number of carbon atoms in the alkyl group, alkynyl group, and alkenyl group exceeds the above upper limit, it becomes difficult to obtain an organic silane compound having an alkyl group, an alkynyl group, or an alkenyl group, and steric hindrance is large, resulting in laser light absorption. When introducing an alkyl group, an alkynyl group, or an alkenyl group into an organosilica film containing an organic group and an amide group, the reaction of an organosilane compound having an alkyl group, an alkynyl group, or an alkenyl group becomes difficult to proceed. Sufficient hydrophobicity cannot be imparted to an organic silica film containing a light-absorbing organic group and an amide group, and the density of laser light-absorbing organic groups in the organic silica film decreases, resulting in a decrease in laser light absorption performance. There is a tendency to Further, the alkyl group, alkynyl group, and alkenyl group may be linear or branched, but they have little steric hindrance and can be used to form a laser-absorbing organic group and an amide group. When introducing an alkyl group, an alkynyl group, or an alkenyl group into the containing organosilica film, a linear one is preferable from the viewpoint that the reaction of an organosilane compound having an alkyl group, an alkynyl group, or an alkenyl group proceeds easily. .

Examples of such alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and cyclohexyl groups. Examples of the alkynyl group include an ethynyl group, a propynyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, and the like. Examples of the alkenyl group include ethenyl group, propenyl group, butenyl group, pentenyl group, hexenyl group, heptenyl group, octenyl group, and the like. Further, these alkyl groups, alkynyl groups and alkenyl groups may be substituted with a heterocyclic group such as a glycidoxy group.

Examples of the fluorine atom-containing group include a fluoroalkyl group and a fluoroether group. The fluoroalkyl group is an alkyl group in which some or all of the hydrogen atoms are substituted with fluorine atoms, and may be linear or branched, but may be sterically hindered. When introducing a fluoroalkyl group into an organic silica film containing a small, laser-absorbing organic group and an amide group, a straight-chain Preferably.

The number of carbon atoms in the fluoroalkyl group (the number of carbon atoms in the alkyl chain) is preferably 1 to 16, more preferably 2 to 10, particularly preferably 3 to 8. If the number of carbon atoms in the fluoroalkyl group exceeds the above upper limit, steric hindrance will be large, and when introducing a fluoroalkyl group into an organic silica film containing a laser light absorbing organic group and an amide group, Because the reaction of the silane compound is difficult to proceed, sufficient hydrophobicity cannot be imparted to the organic silica film containing laser light absorbing organic groups and amide groups, and the density of the laser light absorbing organic groups in the organic silica film There is a tendency that the absorption performance of laser light decreases.

Examples of such fluoroalkyl groups include trifluoromethyl group (CF ₃ -), pentafluoroethyl group (CF ₃ -CF ₂ -), heptafluoropropyl group (CF ₃ -CF ₂ -CF ₂ -), etc. Perfluoroalkyl group in which all of the hydrogen atoms are substituted with fluorine atoms; 2,2,2-trifluoroethyl group (CF ₃ -CH ₂ -), 3,3,3-trifluoropropyl group (CF ₃ - CH ₂ -CH ₂ -), 2,2,3,3,3-pentafluoropropyl group (CF ₃ -CF ₂ -CH ₂ -), 3,3,4,4,4-pentafluorobutyl group (CF ₃ -CF ₂ -CH ₂ -CH ₂ -), 3,3,4-trifluorobutyl group (F-CH ₂ -CF ₂ -CH ₂ -CH ₂ -), 1H, 1H, 2H, 2H-nonafluoro Hexyl group (CF ₃ -CF ₂ -CF ₂ -CF ₂ -CH 2 _{-CH 2} -), 1H, 1H, 2H, 2H-tridecafluorooctyl group (CF ₃ -CF 2 -CF _{2 -CF 2} -CF ₂ -CF ₂ -CH ₂ -CH ₂ -), 1H,1H,2H,2H-heptadecafluorodecyl group (CF ₃ -CF _{2 -CF 2 -CF} 2 _{-CF 2} -CF ₂ -CF ₂ -CF ₂ Examples include fluoroalkyl groups in which at least some of the hydrogen atoms are substituted with fluorine atoms, such as -CH ₂ -CH ₂ -). Among these fluoroalkyl groups, 3,3,3- Trifluoropropyl group (CF ₃ -CH ₂ -CH ₂ -), 1H, 1H, 2H, 2H-nonafluorohexyl group (CF ₃ -CF ₂ -CF _{2 -CF 2} -CH ₂ -CH ₂ -), 1H , 1H, 2H, 2H-tridecafluorooctyl group (CF ₃ -CF ₂ -CF 2 -CF ₂ -CF _{2 -CF 2 -CH 2} -CH ₂ -), 1H, 1H, 2H, 2H-heptadecafluoro A decyl group (CF ₃ -CF ₂ -CF ₂ -CF ₂ -CF 2 -CF ₂ -CF _{2 -CF 2 -CH 2} -CH ₂ -) is more preferable, since the hydrophobizing effect is sufficiently expressed, and during the reaction From the viewpoint of being less susceptible to steric hindrance, 3,3,3-trifluoropropyl group and 1H,1H,2H,2H-nonafluorohexyl group are particularly preferred.

As the fluoroether group, for example, the following formula (i):
R ¹ -O-(R ² -O) _k - (i)
[In the formula, R ¹ represents an alkyl group or a fluoroalkyl group, R ² represents a fluoroalkylene group, and k is an integer of 1 or more. ]
Examples include a group having a structure represented by:

The alkyl group and fluoroalkyl group that are R ¹ may be linear or branched, but they have little steric hindrance and are suitable for laser light absorbing organic groups and amide groups. When introducing a fluoroether group into an organic silica film containing a fluoroether group, a linear one is preferable from the viewpoint that the reaction of an organic silane compound having a fluoroether group proceeds easily. The number of carbon atoms in such alkyl groups and fluoroalkyl groups (the number of carbon atoms in the alkyl chain) is preferably 1 to 16, more preferably 1 to 10, and particularly preferably 1 to 8. If the number of carbon atoms in the alkyl group and fluoroalkyl group exceeds the above upper limit, steric hindrance will be large, and when a fluoroether group is introduced into an organic silica film containing a laser light-absorbing organic group and an amide group, the fluoroether group Because the reaction of the organosilane compound having a laser-absorbing organic group with The density of the group decreases, and the laser light absorption performance tends to decrease.

Examples of such alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and cyclohexyl groups. Examples of fluoroalkyl groups include all hydrogen atoms such as trifluoromethyl group (CF ₃ -), pentafluoroethyl group (CF ₃ -CF ₂ -), heptafluoropropyl group (CF ₃ -CF ₂ -CF ₂ -), etc. is substituted with a fluorine atom; 2,2,2-trifluoroethyl group (CF ₃ -CH ₂ -), 3,3,3-trifluoropropyl group (CF ₃ -CH ₂ -CH ₂ -), 2,2,3,3,3-pentafluoropropyl group (CF ₃ -CF ₂ -CH ₂ -), 3,3,4,4,4-pentafluorobutyl group (CF ₃ -CF ₂ - CH ₂ -CH ₂ -), 3,3,4-trifluorobutyl group (F-CH ₂ -CF ₂ -CH ₂ -CH ₂ -), 1H,1H,2H,2H-nonafluorohexyl group (CF ₃ -CF ₂ -CF ₂ -CF ₂ -CH ₂ -CH ₂ -), 1H, 1H, 2H, 2H-tridecafluorooctyl group (CF ₃ -CF ₂ -CF 2 -CF _{2 -CF 2} -CF ₂ -) CH ₂ -CH ₂ -), 1H,1H,2H,2H-heptadecafluorodecyl group (CF ₃ -CF _{2 -CF 2} -CF ₂ -CF ₂ -CF _{2 -CF 2} -CF ₂ -CH ₂ -CH Examples include fluoroalkyl groups in which at least some of the hydrogen atoms are substituted with fluorine atoms, such as _2- ). Among these alkyl groups and fluoroalkyl groups, perfluoroalkyl groups are preferred from the viewpoint of imparting sufficient hydrophobicity to the organic silica film containing a laser light absorbing organic group and an amide group, and have low steric hindrance. When introducing a fluoroether group into an organic silica film containing a laser light-absorbing organic group and an amide group, trifluoromethyl groups ( More preferred are CF ₃ -), pentafluoroethyl group (CF ₃ -CF ₂ -), and heptafluoropropyl group (CF ₃ -CF ₂ -CF ₂ -).

The fluoroalkylene group that is R ² may be linear or branched, but it has low steric hindrance and contains a laser light absorbing organic group and an amide group. When introducing a fluoroether group into an organic silica film, a linear one is preferable from the viewpoint that the reaction of an organic silane compound having a fluoroether group proceeds easily. The number of carbon atoms in such a fluoroalkylene group (the number of carbon atoms in the alkylene chain) is preferably 1 to 16, more preferably 1 to 10, and particularly preferably 1 to 8. If the number of carbon atoms in the fluoroalkylene group exceeds the above upper limit, steric hindrance will be large, and when introducing a fluoroether group into an organic silica film containing a laser light absorbing organic group and an amide group, Because the reaction of the silane compound is difficult to proceed, sufficient hydrophobicity cannot be imparted to the organic silica film containing laser light absorbing organic groups and amide groups, and the density of the laser light absorbing organic groups in the organic silica film There is a tendency that the absorption performance of laser light decreases.

Such fluoroalkylene groups include difluoromethylene group (-CF ₂ -), tetrafluoroethylene group (-CF ₂ -CF ₂ -), hexafluoropropylene group (-CF ₂ -CF ₂ -CF ₂ -), Octafluorobutylene group (-CF ₂ -CF ₂ -CF ₂ -CF ₂ -), 1-trifluoromethyl-1-fluoromethylene group (-CF(CF ₃ )-), 2-trifluoromethyl-1,1 , 2-trifluoroethylene group (-CF(CF ₃ )-CF ₂ -), 3-trifluoromethyl-1,1,2,2,3-pentafluoropropyl group (-CF(CF ₃ )-CF ₂ -CF ₂ -), 2-trifluoromethyl-1,1,2,3,3-pentafluoropropyl group (-CF ₂ -CF(CF ₃ )-CF ₂ -), etc., all of the hydrogen atoms are fluorine atoms perfluoroalkylene group substituted with; 2,2-difluoroethylene group (-CF ₂ -CH ₂ -), 3,3-difluoropropylene group (-CF ₂ -CH ₂ -CH ₂ -), 2,2, 3,3-tetrafluoropropylene group (-CF ₂ -CF ₂ -CH ₂ -), 3-trifluoromethyl-3-fluoropropylene group (-CF(CF ₃ )-CH ₂ -CH ₂ -), 3, 3-difluorobutylene group (-CH ₂ -CF ₂ -CH ₂ -CH ₂ -), 3,3,4,4-tetrafluorobutylene group (-CF ₂ -CF 2 -CH _{2 -CH 2} -), 1H , 1H, 2H, 2H-octafluorohexylene group (-CF ₂ -CF ₂ -CF ₂ -CF ₂ -CH ₂ -CH ₂ -), 1H, 1H, 2H, 2H-dodecafluorooctylene group (-CF ₂ -CF ₂ -CF 2 -CF _{2 -CF 2 -CF 2} -CH 2 _{-CH 2} -), 1H, 1H, 2H, 2H-hexadecafluorodecylene group (CF ₃ -CF ₂ -CF ₂ -CF Examples include fluoroalkylene groups in which at least some of the hydrogen atoms are substituted with fluorine atoms, such as ₂ -CF _{2 -CF 2} -CF 2 -CF ₂ -CH ₂ -CH ₂ -). Among these fluoroalkylene groups, it is possible to impart sufficient hydrophobicity to an organic silica film containing a laser light-absorbing organic group and an amide group, and also has low steric hindrance. When introducing a fluoroether group into an organic silica film containing a difluoromethylene group (-CF ₂ -), a tetrafluoroethylene group (- CF ₂ -CF ₂ -), hexafluoropropylene group (-CF ₂ -CF ₂ -CF ₂ -), octafluorobutylene group (-CF ₂ -CF 2 -CF _{2 -CF 2} -), 1-trifluoromethyl -1-fluoromethylene group (-CF(CF ₃ )-), 2,2-difluoroethylene group (-CF ₂ -CH ₂ -), 3-trifluoromethyl-3-fluoropropylene group (-CF(CF ₃ )-CH ₂ -CH ₂ -), 3,3-difluorobutylene group (-CH ₂ -CF ₂ -CH ₂ -CH ₂ -), 3,3,4,4-tetrafluorobutylene group (-CF ₂ - CF ₂ --CH ₂ --CH ₂ --) is preferred.

In the formula (i), k is preferably 1 to 100, more preferably 1 to 40. When k exceeds the above upper limit, reaction control of the organic silane compound containing a fluoroether group tends to become difficult. Furthermore, when k is 2 or more, a plurality of R ² 's in the formula (i) may be the same or different.

Examples of the halogen atom-containing group other than the fluorine atom include those having the same structure as the fluorine atom-containing group except that another halogen atom is included instead of the fluorine atom. Examples of such groups containing halogen atoms other than fluorine atoms include halogenated alkyl groups containing halogen atoms other than fluorine atoms, halogenated ether groups containing halogen atoms other than fluorine atoms, and halogen atoms other than fluorine atoms.

The halogenated alkyl group containing a halogen atom other than a fluorine atom is an alkyl group in which part or all of the hydrogen atoms are substituted with a halogen atom other than a fluorine atom, and even if it is a linear one, it is a branched one. However, when introducing a halogenated alkyl group into an organic silica film that has low steric hindrance and contains a laser light absorbing organic group and an amide group, an organic silane having a halogenated alkyl group is used. From the viewpoint that the reaction of the compound progresses easily, linear ones are preferable.

The halogen atom of such a halogenated alkyl group is preferably a chlorine atom, a bromine atom, or an iodine atom, more preferably a chlorine atom or a bromine atom, from the viewpoint of versatility, stability, and reproducibility. is particularly preferred. Further, the number of carbon atoms in such a halogenated alkyl group is preferably 1 to 5, more preferably 1 to 4, and particularly preferably 1 to 3. If the number of carbon atoms in the halogenated alkyl group exceeds the above upper limit, steric hindrance will be large, and when introducing the halogenated alkyl group into an organic silica film containing a laser light absorbing organic group and an amide group, the halogenated alkyl group Because the reaction of the organosilane compound having a laser-absorbing organic group with The density of the group decreases, and the laser light absorption performance tends to decrease.

Examples of such halogenated alkyl groups include trichloromethyl group (CCl ₃ -), tribromomethyl group (CBr ₃ -), pentachloroethyl group (CCl ₃ -CCl ₂ -), pentabromoethyl group (CBr ₃ -CBr ₂ -), 1,1-dichloroethyl group (CH ₃ -CCl ₂ -), 1,1-dibromoethyl group (CH ₃ -CBr ₂ -), etc., all of the hydrogen atoms are substituted with halogen atoms. perhalogenated alkyl groups; chloromethyl group (Cl-CH ₂ -), bromomethyl group (Br-CH ₂ -), 2-chloroethyl group (Cl-CH ₂ -CH ₂ -), 2-bromoethyl group (Br- CH ₂ -CH ₂ -), 3-chloropropyl group (Cl-CH ₂ -CH ₂ -CH ₂ -), 3-bromopropyl group (Br-CH ₂ -CH ₂ -CH ₂ -), 3,3, 4,4,4-pentachlorobutyl group (CCl ₃ -CCl ₂ -CH ₂ -CH ₂ -), 3,3,4,4,4-pentabromobutyl group (CBr ₃ -CBr ₂ -CH ₂ -CH Examples include halogenated alkyl groups in which at least some of the hydrogen atoms are substituted with halogen atoms, such as _2- ). Furthermore, examples of the halogenated ether group include 2-chloromethyloxyethyl group (Cl-CH ₂ -O-CH ₂ -CH ₂ -).

Examples of the alkoxy group include propoxy group, t-butoxy group, pentyloxy group, and allyloxy group. Further, examples of the aromatic ring include a phenyl group, a 1-naphthyl group, a benzyl group, a styryl group, and the like.

Among these hydrophobic groups, organic silica membranes containing laser light-absorbing organic groups and amide groups can be imparted with higher hydrophobicity, and in laser desorption/ionization mass spectrometry, molecules to be measured are From the viewpoint of being supported at high density on the molecule and promoting the elimination of the molecule to be measured, alkyl groups, fluoroalkyl groups, and phenyl groups are preferable, and alkyl groups and fluoroalkyl groups are more preferable.

The organic silica film according to the present invention is preferably at least one selected from the group consisting of those having an uneven structure on the surface, those consisting of nanofibers, and those consisting of fine particles. Since these organic silica membranes have a large surface area, in laser desorption/ionization mass spectrometry, the amount of molecules to be measured on the surface of the organic silica membrane increases, and the intensity of the signal corresponding to the molecules to be measured increases.

Examples of organic silica films having an uneven structure on the surface include organic silica films having a porous structure on the surface in which pores consisting of columnar voids are formed, and organic silica films having on the surface a pillar nanoarray structure in which columnar bodies are arranged. Examples include membranes. Note that "column shape" here refers to so-called column shapes such as a substantially circular column and a substantially polygonal column, as well as the size of both ends (diameter, length, etc.) such as a substantially conical shape, a substantially polygonal pyramid shape, etc. ) is a concept that also includes those with different shapes.

In the porous structure, the average diameter of the pores is preferably 5 to 1000 nm, more preferably 5 to 500 nm, particularly preferably 5 to 300 nm. If the average diameter of the pores is less than the lower limit, it becomes difficult to adsorb molecules with large molecular weights to the walls of the pores or the membrane surface, and the effect of increasing the surface area due to the porous structure tends to be insufficient. If the above upper limit is exceeded, the surface area of the organic silica film tends not to increase sufficiently. Further, the average depth of the pores is more preferably 20 to 1500 nm, and even more preferably 50 to 500 nm. When the average depth of the pores is less than the above lower limit, the surface area of the organic silica film tends not to increase sufficiently. On the other hand, if the average depth of the pore exceeds the above upper limit, in laser desorption/ionization mass spectrometry, even when irradiated with laser light, target molecules supported deep within the pore will be desorbed and ionized. This tends to be difficult. Further, the average distance between the centers of adjacent pores is preferably 20 to 1000 nm, more preferably 20 to 500 nm, even more preferably 20 to 300 nm. If the average distance between the centers of adjacent pores is less than the lower limit, it tends to be difficult to fabricate a mold used to form a porous structure, while if it exceeds the upper limit, the surface area of the organic silica film will decrease. tends not to increase sufficiently.

Further, in the pillar nanoarray structure, the average value of the distance between adjacent columnar bodies (distance between wall surfaces of adjacent columnar bodies) is preferably 5 to 500 nm, more preferably 5 to 400 nm, and 5 to 250 nm. Particularly preferred. When the average value of the distance between adjacent columnar bodies is less than the above lower limit, it becomes difficult to adsorb molecules with a large molecular weight to the wall surfaces of the columnar bodies, and the effect of increasing the surface area due to the porous structure tends to be insufficient. . On the other hand, if the average value of the distance between adjacent columnar bodies exceeds the above upper limit, the surface area of the organic silica film tends not to increase sufficiently. Further, the average height of the columnar bodies is more preferably 20 to 1500 nm, and even more preferably 50 to 500 nm. When the average height of the columnar bodies is less than the lower limit, the surface area of the organic silica film tends not to increase sufficiently. On the other hand, if the average height of the columnar bodies exceeds the above-mentioned upper limit, even if laser light is irradiated in laser desorption/ionization mass spectrometry, the molecules to be measured supported deep in the voids between the columnar bodies will be desorbed. It tends to be difficult to ionize. Furthermore, the average diameter of the cross section of the columnar bodies is preferably 10 to 1000 nm, more preferably 10 to 500 nm, and particularly preferably 10 to 300 nm. If the average diameter of the cross section of the columnar bodies is less than the lower limit, it tends to be difficult to fabricate a mold used to form a pillar nanoarray structure, whereas if it exceeds the upper limit, the surface area of the organic silica film is insufficient. It tends not to increase. Further, the average distance between the centers of the cross sections of adjacent columnar bodies is preferably 20 to 1000 nm, more preferably 20 to 500 nm, and even more preferably 20 to 300 nm. If the average distance between the centers of the cross-sections of adjacent columnar bodies is less than the lower limit, it tends to be difficult to fabricate a mold used to form a pillar nanoarray structure.On the other hand, if it exceeds the upper limit, organic The surface area of the silica film tends not to increase sufficiently.

In addition, the average diameter and average depth of the pores in the porous structure, the average distance between the centers of adjacent pores, the average value of the distance between adjacent columnar bodies in the pillar nanoarray structure, and the average value of the distance between adjacent columnar bodies, The average height, the average diameter of the cross sections of the columnar bodies, and the average distance between the centers of the cross sections of adjacent columnar bodies can be measured using an atomic force microscope.

Such an uneven structure on the surface of an organic silica film can be efficiently formed by nanoimprinting. For example, by performing nanoimprinting using a mold having a pillar array structure, a porous structure in which pores consisting of columnar voids are formed on the surface of an organic silica film is formed. Further, by performing nanoimprinting using a mold having a porous structure in which pores consisting of columnar voids are formed, a pillar nanoarray structure is formed on the surface of the organic silica film. Such nanoimprinting is not particularly limited, and conventionally known methods can be employed.

The organic silica film made of nanofibers is made of nanofibers of organic silica (that is, organic silica containing a laser light absorbing organic group and an amide group in the skeleton and further having a hydrophobic group) constituting the organic silica film. Examples include aggregates and laminates. The average cross-sectional diameter of such nanofibers is preferably 5 to 800 nm, more preferably 10 to 500 nm, and particularly preferably 20 to 300 nm. If the average cross-sectional diameter of the nanofibers is less than the lower limit, it tends to be unable to effectively adsorb relatively bulky molecules such as proteins; on the other hand, if it exceeds the upper limit, the surface area is not sufficiently increased. Therefore, in laser desorption/ionization mass spectrometry, the adsorption amount of target molecules tends to decrease. Further, the average aspect ratio of the nanofibers is preferably 3 to 1000, more preferably 4 to 500, and particularly preferably 5 to 100. If the average aspect ratio of the nanofibers is less than the above lower limit, it will be difficult to recover the nanofibers during synthesis, and productivity will tend to decrease; on the other hand, if it exceeds the above upper limit, it will be difficult to form a homogeneous organic silica thin film. It tends to be difficult to homogeneously support molecules to be measured in laser desorption/ionization mass spectrometry. Note that the average diameter and average aspect ratio of the cross section of the nanofibers can be measured using an atomic force microscope.

The organic silica film made of fine particles is an aggregate of fine particles of organic silica (i.e., organic silica containing a laser light absorbing organic group and an amide group in its skeleton and further having a hydrophobic group) constituting the organic silica film. can be mentioned. The average diameter of such fine particles is preferably 10 to 2,500 nm, more preferably 20 to 1,000 nm, and particularly preferably 50 to 800 nm. If the average diameter of the fine particles is less than the lower limit, it will be difficult to recover the fine particles from the solution during synthesis, and productivity will tend to decrease.On the other hand, if it exceeds the upper limit, the effect of increasing the surface area will decrease, and The sample solution tends to flow out due to the voids. Note that the average diameter of the fine particles can be measured using an atomic force microscope.

The average thickness of the organic silane film according to the present invention is preferably 20 to 2000 nm, more preferably 50 to 1000 nm, and particularly preferably 100 to 500 nm. If the average thickness of the organosilane film is less than the lower limit, the organosilane film will not be able to absorb enough laser light even when irradiated with laser light in laser desorption/ionization mass spectrometry, and the molecules to be measured will be desorbed/ Ionization efficiency tends to decrease, and on the other hand, when the above upper limit is exceeded, the proportion of the membrane that does not participate in analysis increases in laser desorption/ionization mass spectrometry, which tends to increase costs and increases the risk of interference with the substrate. Adhesion tends to decrease.

(Organic silica substrate)
The organic silica substrate of the present invention includes such an organic silica film, and is usually obtained by forming an organic silica film on a base material, but is not limited thereto. The base material is not particularly limited as long as it can support the organic silica film; for example, silicon base material (Si base material), ITO base material, FTO base material, quartz base material, glass base material. , various metal substrates, various thin film substrates, and other known substrates used in producing silica films can be used as appropriate; however, the organic silica substrate of the present invention can be used for laser desorption/ionization mass spectrometry. In this case, conductive base materials such as stainless steel, silicon base material, ITO base material, ZnO base material, SnO base material, FTO base material, etc. are preferable. Further, the shape of such a base material is not particularly limited, but a flat plate shape is preferable.

Such an organic silica substrate of the present invention can be manufactured, for example, by the following method. That is, first, a sol solution is prepared by partially polymerizing an organic silane compound (preferably an alkoxysilane compound) containing a laser light absorbing organic group and an amide group in its skeleton, and this sol solution is used to After forming a coating film on the surface of a base material, it is cured to form an organic silica film containing a laser light absorbing organic group and an amide group in its skeleton on the base material.

The laser light absorbing organic group in the organic silane compound corresponds to the laser light absorbing organic group in the organic silica film described above. This laser light absorbing organic group is contained in the skeleton of the organic silane compound, and is directly or indirectly attached to silicon (Si) in the main skeleton of the organic silane compound (via other elements). ) is preferably bonded, and more preferably bonded to silicon (Si) constituting a silyl group (more preferably an alkoxysilyl group). Further, in the organic silane compound, it is preferable that two or more silyl groups (more preferably alkoxysilyl groups) are bonded to one laser light absorbing organic group. When the number of silyl groups bonded to one laser light absorbing organic group is one, the density of the laser light absorbing organic group decreases, and therefore the absorption intensity of laser light tends to decrease. On the other hand, the upper limit of the number of silyl groups bonded to one laser light absorbing organic group is not particularly limited, but is preferably 6 or less, more preferably 4 or less, and particularly preferably 3 or less. When the number of silyl groups bonded to one laser light absorbing organic group exceeds the above upper limit, the proportion of the laser light absorbing organic groups decreases, and the laser light absorption efficiency tends to decrease.

In addition, in such an organosilane compound, the mass ratio of silicon forming the organosilane compound to the laser light-absorbing organic group ([mass of silicon]/[laser light-absorbing organic group]) is 0.05 to It is preferably within the range of 0.50 (more preferably 0.10 to 0.40, still more preferably 0.10 to 0.35, particularly preferably 0.15 to 0.35). When the mass ratio is less than the lower limit, the crosslinking density of the organic silica film becomes low, and a sufficiently cured organic silica film tends to be difficult to obtain.On the other hand, when it exceeds the upper limit, the relative laser light absorption Since the density of organic groups decreases, the absorption intensity of laser light tends to decrease, and it also tends to become difficult to form an uneven structure on the surface of the organic silica film by nanoimprinting or the like.

The amide group in the organosilane compound corresponds to the amide group in the organosilica film described above. This amide group is contained within the skeleton of the organosilane compound. The presence of an amide group in the skeleton of the organosilane compound improves the adsorption of molecules to be measured, particularly highly hydrophilic compounds such as bio-related molecules, in laser desorption/ionization mass spectrometry, and the molecules to be measured are improved. It is possible to obtain an organic silica film that can uniformly support the entire surface of the organic silica film. Moreover, it is preferable that the amide group is bonded directly or indirectly (via another element) to the laser light absorbing organic group. By using an organosilane compound in which an amide group is bonded to a laser-absorbing organic group, it is possible to efficiently desorb and ionize target molecules from an organosilica substrate in laser desorption/ionization mass spectrometry. An organic silica film can be obtained. Furthermore, in the organic silane compound, one or more amide groups may be bonded to one laser light absorbing organic group. Further, there is no particular upper limit to the number of amide groups bonded to one laser light absorbing organic group, but it is preferably 6 or less, more preferably 4 or less, and particularly preferably 2 or less. If the number of amide groups bonded to one laser light-absorbing organic group exceeds the above upper limit, the target molecule supported on the organic silica film tends to be difficult to desorb in laser desorption/ionization mass spectrometry. be.

The sol solution (colloidal solution) is obtained by partially polymerizing an organic silane compound containing a laser-absorbing organic group and an amide group in its skeleton. Such a sol solution can be appropriately formed by employing a known method known as the so-called sol-gel method in the field of manufacturing silica structures, except for using the organic silane compound. Note that such a sol solution is preferably a solution containing a partial polymer obtained by partially hydrolyzing and condensing the organic silane compound. The solvent used for such a solution is not particularly limited, and any known solvent used in the so-called sol-gel method can be used as appropriate, such as methanol, ethanol, methoxyethanol, 1-propanol, isopropanol, acetone, and tetrahydrofuran. , N,N-dimethylformamide, 1,4-dioxane, acetonitrile, and other organic solvents. Among such solvents, methoxyethanol, 1-propanol, isopropanol, 1,4-dioxane, and tetrahydrofuran are preferred from the viewpoint of volatility near room temperature and high solubility of organic compounds.

Further, when preparing such a sol solution, the conditions (temperature and reaction time) for partially polymerizing the organosilane compound are not particularly limited, and may vary depending on the type of the organosilane compound used, for example. The reaction temperature may be about 0 to 100°C, and the reaction time may be about 5 minutes to 24 hours. Further, from the viewpoint of efficiently advancing such partial polymerization, it is preferable to use an acid catalyst. Examples of such acid catalysts include mineral acids such as hydrochloric acid, nitric acid, and sulfuric acid.

As a method for preparing such a sol solution, for example, a solution containing the organosilane compound, the solvent, and the acid catalyst is prepared, and the solution is heated at room temperature (20 to 28°C, preferably 25°C). A method may be employed in which the organic silane compound is partially polymerized (partial hydrolysis and partial polycondensation) by stirring for about 0.25 to 12 hours to prepare a sol solution. In such a case where the reaction is carried out by stirring, if the stirring time is less than the lower limit, the hydrolysis reaction of the silyl group becomes insufficient, and the curing reaction of the film after film formation tends to be difficult to proceed.

The sol solution may contain organic silane compounds other than the organic silane compound (for example, tetramethoxysilane, Tetraalkoxysilanes such as tetraethoxysilane and tetrabutoxysilane) may also be included.

Further, in the sol solution, the content of the organic silane compound in the solvent is preferably 0.2 to 20% by mass, more preferably 0.5 to 7% by mass. If the content of such organosilane compounds is less than the above lower limit, it tends to be difficult to manufacture a uniform film while controlling the thickness, whereas if it exceeds the above upper limit, it becomes difficult to control the reaction in the sol solution. It tends to be difficult to prepare a stable sol solution.

Further, in such a sol solution, the content of the organic silane compound in the solvent is preferably 2 to 200 g/L, more preferably 5 to 150 g/L. If the content of such organosilane compounds is less than the above lower limit, it tends to be difficult to manufacture a uniform film while controlling the thickness, whereas if it exceeds the above upper limit, it becomes difficult to control the reaction in the sol solution. It tends to be difficult to prepare a stable sol solution.

In addition, such a sol solution is formed by partially polymerizing the organic silane compound, and then filtered with a membrane filter or the like to form a film from the viewpoint of preventing contamination during production and ensuring higher smoothness. It is preferable to use it for

Moreover, the method of forming a coating film obtained from the above-mentioned sol solution is not particularly limited, and methods of casting the sol solution into a mold or applying it to a substrate by various coating methods are suitably employed. Further, as such a coating method, a known method (for example, a method of coating using a bar coater, a roll coater, a gravure coater, etc., a method of dip coating, a spin coating, a spray coating, etc.) may be adopted as appropriate. can.

Further, the thickness of the film (uncured or semi-cured) obtained from such a sol solution is preferably 0.1 to 100 μm, more preferably 0.1 to 25 μm. If the thickness of such a film is less than the above lower limit, it tends to be difficult to maintain the film thickness uniformly over the entire surface of the substrate, whereas if it exceeds the above upper limit, the film thickness may become uneven due to flow or dripping. It tends to be easy to do.

As a method for curing such a coating film, it is sufficient to appropriately adopt conditions that allow the hydrolysis and condensation reactions to proceed depending on the type of organic silane compound used, and the temperature, heating time, etc. are particularly determined. Although not limited, it is preferable to heat at a temperature of about 25 to 150° C. for a period of about 1 to 48 hours. By heating in this way, it becomes possible to further advance the hydrolysis and condensation reaction of the organosilane compound and/or the partial polymer of the organosilane compound, thereby improving the coating film obtained from the sol solution. It becomes possible to form the organic silica film by curing. In addition, in such a curing step, in order to more efficiently hydrolyze the remaining alkoxy groups and harden the film, the coating film is heated in the above temperature range (25 to 150°C) and heated for 1 to 150°C. Preferably, the exposure to hydrochloric acid vapor is for about 48 hours. Such exposure to hydrochloric acid vapor makes it possible to promote reactions not only on the surface of the coating film but also inside it, making it possible to more efficiently hydrolyze the remaining alkoxy groups and harden the film. It also becomes possible to expose hydroxyl groups on the surface of the resulting membrane.

In addition, when forming an uneven structure on the surface of an organic silica film in such a method, after forming a coating film using the sol solution and before curing, the uneven structure is formed on the coating film by nanoimprinting. Preferably, it is formed and then cured.

Furthermore, when forming an organic silica film made of nanofibers, it is preferable to use the aforementioned organic silane compound having two or more amide groups in one molecule. When such organosilane compounds having two or more amide groups in one molecule are partially polymerized, the molecules stack and one-dimensional crystal growth is promoted, resulting in nanofiber-shaped organosilane compounds. A partially polymerized product is formed. This nanofiber-shaped partial polymer of the organic silane compound maintains its shape even when it is cured to form organic silica, and an organic silica film made of organic silica nanofibers is obtained.

Further, when forming an organic silica film made of fine particles, known particle lamination techniques such as spin coating, spray atomization, and dip coating can be appropriately employed.

Next, a hydrophobic group is introduced by bringing an organic silane compound containing a hydrophobic group into contact with the thus formed organic silica film containing a laser light absorbing organic group and an amide group in the skeleton. Can be done.

The hydrophobic group in the organic silane compound containing a hydrophobic group corresponds to the hydrophobic group in the organic silica film described above. When the organosilane compound containing the hydrophobic group is brought into contact with the organosilica film, the silicon atoms of the siloxane bonds constituting the organosilica film react with the silyl groups of the organosilane compound containing the hydrophobic group. , an organic silica film containing a laser light absorbing organic group and an amide group in its skeleton and further containing the hydrophobic group is obtained. There is no particular limitation on the method of bringing the organic silane compound containing a hydrophobic group into contact with the organic silica film, for example, a method of applying a solution containing the organic silane compound containing a hydrophobic group to the organic silica film. , a method of immersing the organic silica film in a solution containing the organic silane compound containing the hydrophobic group, a method of exposing the organic silica film to the vapor of the organic silane compound containing the hydrophobic group, and other conventionally known methods. can be mentioned.

When introducing a hydrophobic group into an organic silica film containing a laser light-absorbing organic group and an amide group in its skeleton, the molar ratio of the hydrophobic group to the amide group (hydrophobic group/amide group) is 4/1 to 0. .5/1 is preferred, 3/1 to 1/1 is more preferred, and 2/1 to 1.5/1 is particularly preferred. If the hydrophobic group/amide group is less than the above lower limit, sufficient hydrophobicity will not be imparted to the surface of the organic silica film containing the laser light absorbing organic group and the amide group. It tends to be difficult to obtain an organic silica membrane that has the effect of concentrating the sample solution containing the target molecule and the effect of promoting the desorption of the target molecule, making it possible to detect signals corresponding to the target molecule with high sensitivity. On the other hand, if the upper limit is exceeded, an organic silica film with high adsorption properties for molecules to be measured and capable of uniformly supporting molecules to be measured over the entire surface can be obtained in laser desorption/ionization mass spectrometry. tends to be difficult.

Examples of organic silane compounds containing such hydrophobic groups include alkylsilane compounds, alkynylsilane compounds, alkenylsilane compounds, silane compounds containing fluorine atoms, silane compounds containing halogen atoms other than fluorine atoms, alkoxysilane compounds, and aromatic rings. Examples include containing silane compounds.

The alkyl group in the alkylsilane compound corresponds to the alkyl group in the organic silica film described above, and the alkynyl group in the alkynylsilane compound corresponds to the alkynyl group in the organic silica film described above. The alkenyl group corresponds to the alkynyl group in the organic silica film described above. Furthermore, the fluorine atom-containing group in the fluorine atom-containing silane compound corresponds to the fluorine atom-containing group in the organic silica film described above, and the halogen atom-containing group other than the fluorine atom in the silane compound containing a halogen atom other than the fluorine atom corresponds to the fluorine atom-containing group in the organic silica film described above. , corresponds to the halogen atom-containing group other than fluorine atom in the above-mentioned organic silica film, and the alkoxy group in the alkoxysilane compound corresponds to the alkoxy group in the above-mentioned organic silica film, and the aromatic ring-containing silane compound The aromatic ring corresponds to the aromatic ring in the organic silica film described above.

Examples of the alkylsilane compound include alkylalkoxysilanes such as methoxytrimethylsilane, ethoxytrimethylsilane, 8-glycidoxyoctyltrimethoxysilane, and cyclohexyltrimethoxysilane. Examples of the alkenylsilane compound include alkenylalkoxysilanes such as 7-octenyltrimethoxysilane.

Examples of the fluorine atom-containing silane compound include trimethoxy(3,3,3-trifluoropropyl)silane, trimethoxy(1H,1H,2H,2H-nonafluorohexyl)silane, and triethoxy(1H,1H,2H,2H). 3,3,3-trifluoropropyldimethylchlorosilane, (heptadecafluoro-1, Examples include fluoroalkyldialkylchlorosilanes such as 1,2,2-tetrahydrodecyl)dimethylchlorosilane.

Examples of the silane compounds containing halogen atoms other than fluorine atoms include trialkylchlorosilanes such as trimethylchlorosilane, triethylchlorosilane, and tripropylchlorosilane; alkyltrichlorosilanes such as octadecyltrichlorosilane; 2-chloroethyltrimethoxysilane, 3-chlorosilane, and the like; Examples include chloroalkyltrialkoxysilanes such as propyltrimethoxysilane. Examples of the alkoxysilane compound include triethoxy(isobutyl)silane, hexyltriethoxysilane, decyltriethoxysilane, and octadecyltrimethoxysilane. Examples of the aromatic ring-containing silane compound include dimethoxydiphenylsilane, trimethoxyphenylsilane, benzyltriethoxysilane, and trimethoxy(4-vinylphenyl)silane.

[Laser desorption/ionization mass spectrometry method]
Next, the laser desorption/ionization mass spectrometry method of the present invention will be explained. In the laser desorption/ionization mass spectrometry method of the present invention, a measurement sample is prepared by supporting a molecule to be measured on the organic silica substrate of the present invention, and the measurement sample is irradiated with a laser beam. This is a method in which target molecules are desorbed from the organic silica substrate, ionized, and subjected to mass spectrometry. By using the organic silica substrate of the present invention, molecules to be measured can be detected with high sensitivity.

In the laser desorption/ionization mass spectrometry method of the present invention, first, a sample to be measured in laser desorption/ionization mass spectrometry (a sample containing a molecule to be measured) is supported on the organic silica substrate of the present invention. Next, the organic silica substrate supporting the sample containing the molecule to be measured is irradiated with laser light. This allows the molecules to be measured to be desorbed from the organic silica substrate, ionized, and subjected to mass spectrometry.

There is no particular restriction on the sample containing the analyte molecule to which the laser desorption/ionization mass spectrometry method of the present invention can be applied, but by using the organic silica substrate of the present invention, compounds with high affinity can be detected. Bio-related molecules such as amino acids, proteins, sugars, phospholipids, hormones, nucleic acids, and their metabolites are suitable from the viewpoint that they can be detected with high sensitivity.

Furthermore, there are no particular limitations on the laser light used in the laser desorption/ionization mass spectrometry method of the present invention, and examples include nitrogen laser (wavelength: 337 nm), YAG laser triple harmonic (wavelength: 355 nm), NdYAG laser (wavelength: 355 nm), :256 nm), carbon dioxide laser (wavelength: 9400 nm and 10600 nm), and the like. There are no particular restrictions on the laser light irradiation conditions (irradiation intensity, irradiation time, etc.), and the optimum conditions may be appropriately selected from known mass spectrometry conditions depending on the molecule to be measured.

Furthermore, there are no particular limitations on the method for separating and detecting ions for mass spectrometry in the laser desorption/ionization mass spectrometry method of the present invention, such as double focusing method, quadrupole focusing method (quadrupole (Q) filter method). ), tandem quadrupole (QQ) method, ion trap method, time of flight (TOF) method, etc. can be employed.

In the laser desorption/ionization mass spectrometry method of the present invention, since amide groups and hydrophobic groups are uniformly present in a well-balanced manner on the surface of the organic silica substrate, the molecules to be measured are distributed over the entire surface of the organic silica substrate. It is uniformly supported at a high concentration, and aggregation of the molecules to be measured is suppressed. Furthermore, the action of the hydrophobic group promotes the desorption of the analyte molecule after laser irradiation, making it easier to ionize it. As a result, the desorption/ionization of the analyte molecules by laser light irradiation is promoted, the signal corresponding to the analyte molecules becomes stronger, and the signal/noise (S/N) ratio becomes higher, resulting in high detection sensitivity. mass spectrometry becomes possible.

EXAMPLES Hereinafter, the present invention will be described in more detail based on Examples and Comparative Examples, but the present invention is not limited to the following Examples.

(Synthesis example 1)
First, under a nitrogen atmosphere, 1,8-naphthalic anhydride (5.95 g, 30.0 mmol), N-acetylethylenediamine (8.17 g, 80.0 mmol) and dimethylacetamide (DMAc, 120 ml) were mixed to obtain a mixture. The resulting mixture was stirred while heating at 125°C for 17 hours, and the following formula:

The reaction represented by was performed. After cooling the resulting solution to room temperature, water (350 ml) was added and stirred, and the resulting precipitate was collected by suction filtration and vacuum dried to obtain a solid component (yield: 7.18 g, rate: 85%).

The obtained solid component was dissolved in deuterated chloroform (CDCl ₃ ), and ^{the 1} H-NMR spectrum was measured and identified using an NMR measuring device (JNM-ECX400P manufactured by JEOL Ltd.), and the amide group was bonded. It was confirmed that it was a naphthalimide compound [NI (amide)]. The results are shown below.
¹ H-NMR (CDCl ₃ , δ in ppm): 1.90 (s, 3H), 3.65 (m, 2H), 4.40 (m, 2H), 6.23 (bs, 1H), 7 .76 (m, 2H), 8.22 (m, 2H), 8.60 (m, 2H).

Next, under a nitrogen atmosphere, a naphthalimide compound [NI(amide)] (4.00 g, 14.17 mmol) with an amide group bonded to it, carbonyldihydridotris(triphenylphosphine)ruthenium [RuH ₂ (CO) (PPh ₃ ) ₃ ] (322 mg, 0.35 mmol) and mesitylene (100 ml) were mixed, triisopropoxyvinylsilane (13.4 ml, 11.6 g, 50.0 mmol) was added, and the resulting mixture was heated at 160°C for 3 Stir while heating for an hour, then use the following formula:

The reaction represented by was performed. After cooling the obtained solution to room temperature, using neutral silica gel column chromatography (developing solvent: chloroform/hexane = 1/1 (v/v) → chloroform/ethanol = 3/1 (v/v)) A fraction containing an organic silane compound containing a naphthalimide ring to which an amide group was bonded was separated and collected. After removing the solvent from this fraction using a rotary evaporator, the residue was recrystallized using cold hexane. The obtained crystals were collected by suction filtration, washed with cold hexane, and then vacuum dried to obtain a solid component (yield: 9.70 g, yield: 92%).

The obtained solid component was dissolved in deuterated chloroform (CDCl ₃ ), and ^{the 1} H-NMR spectrum was measured and identified using an NMR measuring device (JNM-ECX400P manufactured by JEOL Ltd.), and the amide group was bonded. It was confirmed that the product was a reaction product [NI(amide)-Si(IP)] between the naphthalimide compound and triisopropoxyvinylsilane. The results are shown below.
¹ H-NMR (CDCl ₃ , δ in ppm): 1.04 (m, 4H), 1.24 (d, J = 6.0Hz, 36H), 1.91 (s, 3H), 3.51 ( m, 4H), 3.64 (m, 2H), 4.30 (m, 6H), 4.43 (m, 2H), 6.60 (bs, 1H), 7.55 (d, J=8 .4Hz, 2H), 8.02 (d, J=8.4Hz, 2H).

(Synthesis example 2)
First, 1,8-naphthalic anhydride (5.95 g, 30.0 mmol), pyridine (60 ml) and ethylenediamine (60 ml) were mixed under a nitrogen atmosphere, and the resulting mixture was heated at 110°C for 72 hours. Stir and use the following formula:

The reaction represented by was performed. After removing most of the pyridine and ethylenediamine from the resulting solution using a rotary evaporator, the residue was recrystallized using cold acetonitrile. The obtained crystals were collected by suction filtration and dried under vacuum to obtain a solid component (yield: 5.10 g, yield: 71%).

The obtained solid component was dissolved in deuterated chloroform (CDCl ₃ ), and identified by measuring the ¹ H-NMR spectrum using an NMR measuring device (JNM-ECX400P manufactured by JEOL Ltd.). -aminoethyl)-1,8-naphthalimide. The results are shown below.
¹ H-NMR (CDCl ₃ , δ in ppm): 3.08 (t, J = 6.6 Hz, 2H), 4.29 (t, J = 6.6 Hz, 2H), 7.76 (m, 2H ), 8.22 (m, 2H), 8.61 (m, 2H).

Next, under a nitrogen atmosphere, N-(2-aminoethyl)-1,8-naphthalimide (2.40 g, 10.0 mmol), succinic acid (0.53 g, 4.50 mmol), 4-dimethylaminopyridine ( DMAP, 48.9 mg, 0.40 mmol) and dichloromethane (40 ml) were mixed, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 3.50 g, 18.3 mmol) was added. , the resulting mixture was stirred at room temperature for 3 days and the following formula:

The reaction represented by was performed. After adding ethanol (150 ml) to the resulting suspension and stirring for 1 hour, insoluble solid components were collected by suction filtration. This solid component was redispersed in acetonitrile and stirred while heating at 70° C. for 5 minutes. After the resulting suspension was cooled to room temperature, insoluble solid components were collected by suction filtration and vacuum dried (yield: 1.81 g, yield: 71%).

The obtained solid component was dissolved in deuterated dimethyl sulfoxide (DMSO-d ₆ ), and the ¹ H-NMR spectrum was measured using an NMR measuring device (JNM-ECX400P manufactured by JEOL Ltd.) for identification. It was confirmed that the product was a naphthalimide dimer [NI(amide)-d] to which an amide group was bonded. The results are shown below. Note that one of the methylene peaks is thought to overlap with the DMSO peak (δ ~ 3.3 ppm).
¹ H-NMR (DMSO-d ₆ , δ in ppm): 2.06 (s, 4H), 4.10 (m, 4H), 7.85 (m, 4H), 7.91 (m, 2H) , 8.45 (m, 8H).

Next, under a nitrogen atmosphere, a naphthalimide dimer [NI(amide)-d] (0.62 g, 1.10 mmol) with an amide group bonded to it, carbonyldihydridotris(triphenylphosphine)ruthenium [RuH ₂ ( CO)(PPh3) _{3 ]} (55.1 mg, 0.06 mmol) and dimethylacetamide (DMAc, 20 ml) were mixed, and further triisopropoxyvinylsilane (2.35 ml, 2.05 g, 8.80 mmol) was added. The resulting mixture was stirred while heating at 160°C for 3 hours, and the following formula:

The reaction represented by was performed. After removing most of the DMAc from the resulting solution using a rotary evaporator, the residue was mixed with ethyl acetate (10 ml), further hexane (150 ml) was added, and the resulting mixture was cooled in a refrigerator for 12 hours. . The obtained crystals were collected by suction filtration, washed with cold hexane, and then vacuum dried to obtain a solid component (yield: 1.22 g, yield: 74%).

The obtained solid component was dissolved in deuterated chloroform (CDCl ₃ ), and ^{the 1} H-NMR spectrum was measured and identified using an NMR measuring device (JNM-ECX400P manufactured by JEOL Ltd.), and the amide group was bonded. It was confirmed that the product was a reaction product [NI(amide)-d-Si(IP)] between the naphthalimide dimer and triisopropoxyvinylsilane. The results are shown below.
¹ H-NMR (CDCl ₃ , δ in ppm): 1.03 (m, 8H), 1.23 (d, J=6.0Hz, 72H), 2.40 (s, 4H), 3.50 ( m, 12H), 4.28 (m, 12H), 4.34 (m, 4H), 6.69 (bs, 2H), 7.53 (d, J=8.4Hz, 4H), 7.99 (d, J=8.4Hz, 4H).

(Comparative synthesis example 1)
First, 1,8-naphthalic anhydride (2.97 g, 15.0 mmol), 3-aminopentane (3.49 g, 40.0 mmol) and pyridine (20.0 ml) were mixed under a nitrogen atmosphere, and the mixture was heated at 110°C. The following formula:

The reaction represented by was performed. After the resulting solution was cooled to room temperature, 1M hydrochloric acid (100 ml) was added and stirred, and the resulting precipitate was collected by suction filtration. After washing the obtained precipitate with water, acetonitrile (50 ml) was added and stirred while heating at 60°C for 1 hour, and the obtained solution was left standing at -20°C overnight. Thereafter, the precipitate was collected by suction filtration, washed with acetonitrile at -20°C, and then dried under vacuum to obtain a solid component (yield: 3.82 g, yield: 95%).

The obtained solid component was dissolved in deuterated chloroform (CDCl ₃ ), and identified by measuring the ¹ H-NMR spectrum using an NMR measuring device (JNM-ECX400P manufactured by JEOL Ltd.). It was confirmed that it was a compound (Pe-NI). The results are shown below.
¹ H-NMR (CDCl ₃ , δ in ppm): 0.91 (t, J=7.3Hz, 6H), 2.26 (m, 2H), 5.06 (m, 1H), 7.76 ( dd, J = 7.3, 8.2 Hz, 2H), 8.21 (d, J = 8.2 Hz, 2H), 8.58 (d, J = 7.3 Hz, 2H).

Next, under a nitrogen atmosphere, the above (Pe-NI) (1.00 g, 3.74 mmol), carbonyldihydridotris(triphenylphosphine)ruthenium [RuH ₂ (CO) (PPh ₃ ) ₃ ] (91.8 mg, (0.10 mmol) and mesitylene (20 ml) were mixed and heated to 100°C to obtain a homogeneous solution. Triisopropoxyvinylsilane (4.65 g, 20.0 mmol) was added to this solution, stirred while heating at 160°C for 5 hours, and the following formula:

The reaction represented by was performed. After cooling the obtained solution to room temperature, neutral silica gel column chromatography (developing solvent: chloroform/hexane = 1/1 (v/v) → chloroform only → chloroform/ethanol = 40/3 (v/v)) A fraction containing a naphthalimide ring-containing organosilane compound was obtained. After removing the solvent from this fraction using a rotary evaporator, the residue was dissolved in chloroform (100 ml), and a metal scavenger ("SiliaMetS (R) DMT" manufactured by SiliCycle) (10 g) was added. The remaining RuH ₂ (CO) (PPh ₃ ) ₃ was removed by stirring for 3 days. Thereafter, the metal trapping agent was removed by suction filtration, and the solvent was removed using a rotary evaporator, followed by vacuum drying to obtain a solid component (yield: 2.72 g, yield: 99%).

The obtained solid component was dissolved in deuterated chloroform (CDCl ₃ ), and identified by ¹ H-NMR spectrum measurement using an NMR measuring device (JNM-ECX400P manufactured by JEOL Ltd.). -NI) and triisopropoxyvinylsilane (Pe-NI-Si(IP)). The results are shown below.
¹ H-NMR (CDCl ₃ , δ in ppm): 0.88 (t, J = 7.4Hz, 6H), 1.07 (m, 4H), 1.24 (d, J = 6.2Hz, 36H ), 1.87 (m, 2H), 2.23 (m, 2H), 3.49 (m, 4H), 4.32 (sep, J=6.2Hz, 6H), 5.06 (m, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.96 (d, J = 8.3 Hz, 2H).

(Comparative production example 1)
NI(amide)-Si(IP) (90 mg) obtained in Synthesis Example 1 was dissolved in a mixed solution of ethanol (0.99 ml) and diethylene glycol monomethyl ether (0.01 ml), and 2M hydrochloric acid (12 μl) was added. and stirred at room temperature for 20 minutes to prepare a sol solution. After filtering this sol solution through a membrane filter, it was spin-coated onto a silicon substrate (1500 rpm for 3 seconds) and dried at room temperature. An organic silica substrate provided with a silica thin film a was produced.

(Preparation example 1)
In a sealable Teflon (registered trademark) container, an organic silica substrate comprising an organic silica thin film a having a naphthalimide ring to which an amide group is bonded, prepared in the same manner as in Comparative Preparation Example 1, and trimethoxy (1H, 1H, 2H) , 2H-nonafluorohexyl) silane (25 μl) was placed therein, and the Teflon (registered trademark) container was sealed. This container was heated at 150°C for 1 hour in an argon atmosphere to introduce a nonafluorohexyl group as a hydrophobic group into the organic silica thin film a. An organic silica substrate having a smooth organic silica thin film A was obtained.

(Preparation example 2)
First, a sol solution was prepared in the same manner as in Preparation Example 1. After filtering this sol solution with a membrane filter, it was spin-coated on a silicon substrate (1500 rpm for 3 seconds) and immediately molded into a polyethylene terephthalate nanomold ("FleFimo" manufactured by Soken Kagaku Co., Ltd., nanopillar array, pitch: 250 nm, Pillar diameter: 150 nm, pillar height: 250 nm) were stacked and pressed using a flat plate press at 1 ton at 80°C for 3 hours to form organic silica thin film b having naphthalimide rings with amide groups bonded. did. After removing the nanomold, the surface of the organic silica thin film b was observed using an atomic force microscope (AFM). The results are shown in FIG. As shown in FIG. 6, it was confirmed that the pillar structure of the nanomold was transferred to the surface of the organic silica thin film b, and a vertically aligned nanoporous structure was formed.

Next, a nonafluorohexyl group was added to the organic silica thin film b as a hydrophobic group in the same manner as in Preparation Example 1, except that an organic silica substrate having an organic silica thin film b was used instead of an organic silica substrate having an organic silica thin film a. An organic silica substrate was obtained which included an organic silica thin film B having a naphthalimide ring and a nonafluorohexyl group to which an amide group was introduced and having an uneven structure on its surface.

(Preparation example 3)
First, NI(amide)-d-Si(IP) (30 mg) obtained in Synthesis Example 2 and hexane (3.0 ml) were placed in a glass bottle and heated at about 70°C to dissolve the NI(amide)- d-Si(IP) was dissolved. The glass bottle was submerged in water and rapidly cooled to room temperature to precipitate the nanofibers made of NI (amide)-d-Si (IP), and then subjected to ultrasonication for 15 minutes to separate the nanofiber dispersion. Obtained. FIG. 7 shows an optical micrograph of the nanofiber dispersion.

Next, the nanofiber dispersion was spin-coated on the silicon substrate (500 rpm for 3 seconds → 800 rpm for 3 seconds → 1500 rpm for 20 seconds) and dried at room temperature. The surface of the obtained dried coating film was observed using a scanning electron microscope. The results are shown in FIG. 8A. As shown in FIG. 8A, it was confirmed that the dried coating film was formed of nanofibers made of the NI(amide)-d-Si(IP).

Next, this dried coating film is exposed to 2M hydrochloric acid vapor at 100° C. for 3 hours to polycondense the nanofibers to form an organic silica thin film c having a naphthalimide ring to which an amide group is bonded, and further, This organic silica thin film c was exposed to vapor of 1H, 1H, 2H, 2H-nonafluorohexyldimethylchlorosilane at 120°C for 3 hours to introduce nonafluorohexyl groups as hydrophobic groups into the organic silica thin film c, and the amide groups were bonded. An organic silica substrate comprising an organic silica thin film C having a naphthalimide ring and a nonafluorohexyl group was obtained. The surface of this organic silica thin film C was observed using a scanning electron microscope. The results are shown in FIG. 8B. As shown in FIG. 8B, it was confirmed that the organic silica thin film C was formed of organic silica nanofibers. That is, even when a dry coating film formed from nanofibers made of NI(amide)-d-Si(IP) is subjected to steam treatment with hydrochloric acid and 1H, 1H, 2H, 2H-nonafluorohexyldimethylchlorosilane, It was confirmed that in the obtained organic silica thin film C, the morphology of the nanofibers was maintained.

(Preparation example 4)
A methyl group was introduced as a hydrophobic group into the organic silica thin film b in the same manner as in Preparation Example 2, except that methoxytrimethylsilane (25 μl) was used instead of trimethoxy(1H,1H,2H,2H-nonafluorohexyl)silane. An organic silica substrate having an organic silica thin film D having a naphthalimide ring bound to an amide group and a methyl group and having an uneven structure on the surface was obtained.

(Comparative production example 2)
Pe-NI-Si (IP) (90 mg) obtained in Comparative Synthesis Example 1 was dissolved in 1-propanol (1 ml), 2M hydrochloric acid (12 μl) was added, and the sol solution was stirred at room temperature for 60 minutes. Prepared. After filtering this sol solution through a membrane filter, it was spin-coated onto a silicon substrate (1400 rpm for 4 seconds) and dried at room temperature, resulting in a smooth surface with a naphthalimide ring to which no amide group was bonded. An organic silica substrate comprising an organic silica thin film e was prepared.

(Comparative production example 3)
Instead of the organic silica substrate comprising the organic silica thin film a having a naphthalimide ring to which an amide group is bonded, an organic silica thin film e having a naphthalimide ring to which no amide group is bonded, prepared in the same manner as in Comparative Preparation Example 2. A nonafluorohexyl group was introduced as a hydrophobic group into the organic silica thin film e in the same manner as in Preparation Example 1 except that an organic silica substrate having An organic silica substrate having an organic silica thin film f having a smooth surface was obtained.

(Comparative production example 4)
First, a sol solution prepared in the same manner as in Comparative Preparation Example 2 was used as a sol solution, and amide groups were bonded onto a silicon substrate in the same manner as in Preparation Example 2, except that spin coating was performed at 1400 rpm for 4 seconds. An organic silica thin film g having no naphthalimide rings was formed. The surface of this organic silica thin film g was observed using an atomic force microscope (AFM). The results are shown in FIG. As shown in FIG. 9, it was confirmed that the pillar structure of the nanomold was transferred to the surface of the organic silica thin film g, and a vertically aligned nanoporous structure was formed.

Next, a methyl group was introduced as a hydrophobic group into the organic silica thin film g in the same manner as in Preparation Example 4, except that an organic silica substrate having an organic silica thin film g was used instead of an organic silica substrate having an organic silica thin film b. An organic silica substrate was obtained, which was provided with an organic silica thin film h having a naphthalimide ring to which no amide group was bonded, a methyl group, and an uneven structure on the surface.

Table 1 summarizes the presence or absence of naphthalimide rings, amide groups, and hydrophobes in the organic silica thin films constituting the organic silica substrates obtained in Production Examples 1 to 4 and Comparative Production Examples 1 to 4, as well as the surface shape.

(Example 1)
<Preparation of measurement sample>
A sample solution was prepared by dissolving angiotensin I (molecular weight: 1296.5) as a molecule to be measured at a concentration of 1 pmol/μl in an aqueous solution containing trifluoroacetic acid as an ionizing agent at a concentration of 0.1 vol%. 1 μl of this sample solution was dropped onto the organic silica thin film A obtained in Preparation Example 1, and then air-dried to support angiotensin I on the surface of the organic silica thin film A to prepare a measurement sample.

<Laser desorption/ionization mass spectrometry>
Using a laser desorption/ionization mass spectrometer (“autoflex maX” manufactured by Bruker Daltonics), Nd/YAG was added to the same location in the region carrying angiotensin I of the obtained measurement sample in reflectron mode. Laser (wavelength: 355 nm) was irradiated 100 times with a laser intensity of 50%, and the obtained spectra were integrated to obtain a mass spectrum (LDI-MS spectrum). The results are shown in FIG. As shown in Figure 10, in the obtained LDI-MS spectrum, a signal corresponding to the proton adduct of angiotensin I (m/z = 1296.7) was detected at a signal/noise (S/N) ratio of 80. clearly observed. From this result, angiotensin I can be desorbed by laser with high sensitivity by using an organic silica substrate having a naphthalimide ring bonded with an amide group and a nonafluorohexyl group, and having an organic silica thin film A with a smooth surface. /Ionization mass spectrometry was found to be possible.

(Example 2)
<Preparation of measurement sample>
Amyloid β (molecular weight: 4329.8) was added as a molecule to be measured at a concentration of 0.5 pmol/in a mixed solvent of 30 vol% of a 0.1 vol% trifluoroacetic acid solution containing citric acid as an ionizing agent at a concentration of 4 nmol/μl and 70 vol% of acetonitrile. A sample solution was prepared by dissolving the sample at a concentration of μl. After dropping 1 μl of this sample solution onto the organic silica thin film B obtained in Preparation Example 2, it was air-dried to support amyloid β on the surface of the organic silica thin film B to prepare a measurement sample.

<Laser desorption/ionization mass spectrometry>
Using a laser desorption/ionization mass spectrometer (“autoflex maX” manufactured by Bruker Daltonics), an Nd/YAG laser was applied to the same location within the amyloid β-bearing region of the obtained measurement sample in a linear mode. (wavelength: 355 nm) at a laser intensity of 50% 100 times, and the obtained spectra were integrated to obtain a mass spectrum (LDI-MS spectrum). The results are shown in FIG. As shown in Figure 11, in the obtained LDI-MS spectrum, a signal corresponding to the proton adduct of amyloid β (m/z = 4331) was clearly detected with a signal/noise (S/N) ratio = 209. Observed. Based on these results, amyloid β can be removed by laser desorption with high sensitivity by using an organic silica substrate with an organic silica thin film B that has a naphthalimide ring bonded to an amide group and a nonafluorohexyl group and has an uneven structure on its surface. It was found that separation/ionization mass spectrometry can be performed.

In addition, the above laser desorption/ionization mass spectrometry was performed at 100 μm intervals within the dropping region of the sample solution of the measurement sample, and the proton adduct of amyloid β (m/ A mapping image of amyloid β was created based on the signal corresponding to z=4331). The results are shown in FIG. As shown in Figure 12, a signal corresponding to the proton adduct of amyloid β was detected in the entire area where the sample solution was dropped, confirming that amyloid β was uniformly supported on the surface of organic silica thin film B. It was done. From this result, amyloid β can be absorbed into the organic silica thin film by using an organic silica substrate comprising an organic silica thin film B that has a naphthalimide ring bound to an amide group and a nonafluorohexyl group and has an uneven structure on its surface. It was found that it was supported uniformly and ionized with high desorption efficiency.

(Example 3)
A measurement sample was prepared in the same manner as in Example 2, except that the concentration of amyloid β in the sample solution was changed to 0.5 fmol/μl (1/1000 times that of Example 2), and the laser desorption/ionization mass Analysis was carried out. The results are shown in FIG. As shown in Figure 13, in the obtained LDI-MS spectrum, a signal corresponding to the amyloid β proton adduct (m/z = 4331) was clearly detected at a signal/noise (S/N) ratio of 13. Observed. From this result, it was found that by using an organic silica substrate comprising an organic silica thin film B having a naphthalimide ring bonded with an amide group and a nonafluorohexyl group, and having an uneven structure on the surface, amyloid β at a low concentration can be improved. It was found that laser desorption/ionization mass spectrometry can be performed with high sensitivity.

(Example 4)
<Preparation of measurement sample>
An ethanol solution in which the phospholipid phosphatidylcholine (molecular weight: 758.1) as a molecule to be measured is dissolved at a concentration of 0.1 mM is diluted with an aqueous trifluoroacetic acid solution at a concentration of 0.1 vol% so that the phosphatidylcholine concentration is 100 fmol/μl. A sample solution was prepared. After dropping 1 μl of this sample solution onto the organic silica thin film B obtained in Preparation Example 2, it was air-dried to support phosphatidylcholine on the surface of the organic silica thin film B to prepare a measurement sample.

<Laser desorption/ionization mass spectrometry>
Using a laser desorption/ionization mass spectrometer (“autoflex maX” manufactured by Bruker Daltonics), an Nd/YAG laser was applied to the same location in the region supporting phosphatidylcholine of the obtained measurement sample in reflectron mode. (wavelength: 355 nm) at a laser intensity of 40% 100 times, and the obtained spectra were integrated to obtain a mass spectrum (LDI-MS spectrum). The results are shown in FIG. As shown in Figure 14, in the obtained LDI-MS spectrum, a signal corresponding to the proton adduct of phosphatidylcholine (m/z = 735.7) was clearly detected at a signal/noise (S/N) ratio of 5. was observed. Furthermore, a fragment ion (m/z=551.5), which is often observed in the ionization of phosphatidylcholine, was not detected. From these results, phosphatidylcholine can be softly ionized by using an organic silica substrate comprising an organic silica thin film B having an amide group-bonded naphthalimide ring and a nonafluorohexyl group and having an uneven structure on the surface. It was found that laser desorption/ionization mass spectrometry can be performed with high sensitivity.

(Example 5)
<Preparation of measurement sample>
A sample solution was prepared by dissolving insulin (molecular weight: 5807), a type of peptide hormone, as a molecule to be measured at a concentration of 10 fmol/μl in an aqueous solution containing trifluoroacetic acid as an ionizing agent at a concentration of 0.1 vol%. After dropping 1 μl of this sample solution onto the organic silica thin film B obtained in Preparation Example 2, it was air-dried to support insulin on the surface of the organic silica thin film B to prepare a measurement sample.

<Laser desorption/ionization mass spectrometry>
An Nd/YAG laser ( Wavelength: 355 nm) was irradiated 100 times at a laser intensity of 50%, and the obtained spectra were integrated to obtain a mass spectrum (LDI-MS spectrum). The results are shown in FIG. As shown in Figure 15, in the obtained LDI-MS spectrum, a signal corresponding to the proton adduct of insulin (m/z = 5808) was observed at a signal/noise (S/N) ratio = 3. . From these results, it was found that by using an organic silica substrate comprising an organic silica thin film B having a naphthalimide ring with an amide group bonded to it and a nonafluorohexyl group and having an uneven structure on the surface, it is possible to obtain low concentrations of insulin with good sensitivity. It was found that laser desorption/ionization mass spectrometry can be performed using this method.

(Example 6)
<Preparation of measurement sample>
The same volume of acetonitrile was added to commercially available plasma and held at 4°C for 15 minutes. This solution was centrifuged, and the resulting supernatant was diluted 10 times with a mixed solvent of 30 vol% of a 0.1 vol% trifluoroacetic acid solution containing citric acid at a concentration of 4 nmol/μl as an ionization aid and 70 vol% of acetonitrile. A plasma solution was prepared by diluting it to A sample solution was prepared by dissolving amyloid β (molecular weight: 4329.8) as a molecule to be measured in this plasma solution at a concentration of 5.0 pmol/μl. After dropping 1 μl of this sample solution onto the organic silica thin film B obtained in Preparation Example 2, it was air-dried to support amyloid β on the surface of the organic silica thin film B to prepare a measurement sample.

<Laser desorption/ionization mass spectrometry>
Using a laser desorption/ionization mass spectrometer (“autoflex maX” manufactured by Bruker Daltonics), an Nd/YAG laser was applied to the same location within the amyloid β-bearing region of the obtained measurement sample in a linear mode. (wavelength: 355 nm) at a laser intensity of 50% 100 times, and the obtained spectra were integrated to obtain a mass spectrum (LDI-MS spectrum). The results are shown in FIG. 16A. As shown in Figure 16A, in the obtained LDI-MS spectrum, a signal corresponding to the amyloid β proton adduct (m/z = 4329) was clearly detected at a signal/noise (S/N) ratio of 5. Observed. On the other hand, when the plasma solution containing no amyloid β was subjected to laser desorption/ionization mass spectrometry in the same manner as above, no characteristic signal was detected as shown in FIG. 16B. From these results, it was found that by using an organic silica substrate comprising an organic silica thin film B having a naphthalimide ring with an amide group bonded to it and a nonafluorohexyl group, and having an uneven structure on the surface, it was found that a mixed solution with plasma components could be prepared. Only amyloid β signals can be selectively detected in laser desorption/ionization mass spectrometry, and amyloid β can be detected with high sensitivity in laser desorption/ionization mass spectrometry even in the presence of body fluids or in the presence of multiple components. was suggested. Therefore, an organic silica substrate having an organic silica thin film B having an amide group-bonded naphthalimide ring and a nonafluorohexyl group and having an uneven structure on the surface can be used for laser desorption/ionization of biomolecules existing in the body. It was confirmed that it is useful for analysis.

(Example 7)
<Preparation of measurement sample>
Amyloid β (molecular weight: 4329.8) was added as a molecule to be measured at a concentration of 1.0 pmol/in a mixed solvent of 30 vol% of a 0.1 vol% trifluoroacetic acid solution containing citric acid as an ionizing agent at a concentration of 4 nmol/μl and 70 vol% of acetonitrile. A sample solution was prepared by dissolving the sample at a concentration of μl. After dropping 1 μl of this sample solution onto the organic silica thin film C obtained in Preparation Example 3, it was air-dried to support amyloid β on the surface of the organic silica thin film C to prepare a measurement sample.

<Laser desorption/ionization mass spectrometry>
Using a laser desorption/ionization mass spectrometer (“autoflex maX” manufactured by Bruker Daltonics), an Nd/YAG laser was applied to the same location within the amyloid β-bearing region of the obtained measurement sample in a linear mode. (wavelength: 355 nm) at a laser intensity of 50% 100 times, and the obtained spectra were integrated to obtain a mass spectrum (LDI-MS spectrum). The results are shown in FIG. As shown in Figure 17, in the obtained LDI-MS spectrum, a signal corresponding to the amyloid β proton adduct (m/z = 4330) was clearly detected at a signal/noise (S/N) ratio of 140. Observed. From this result, amyloid β can be laser desorbed/ionized with high sensitivity by using an organic silica substrate equipped with an organic silica thin film C made of organic silica nanofibers having a naphthalimide ring bonded with an amide group and a nonafluorohexyl group. It turned out that mass spectrometry can be performed.

(Example 8)
<Preparation of measurement sample>
A sample solution was prepared by dissolving insulin (molecular weight: 5807) as a molecule to be measured at a concentration of 1 pmol/μl in an aqueous solution containing trifluoroacetic acid as an ionizing agent at a concentration of 0.1 vol%. After dropping 1 μl of this sample solution onto the organic silica thin film C obtained in Preparation Example 3, it was air-dried to support insulin on the surface of the organic silica thin film C to prepare a measurement sample.

<Laser desorption/ionization mass spectrometry>
An Nd/YAG laser ( Wavelength: 355 nm) was irradiated 100 times at a laser intensity of 50%, and the obtained spectra were integrated to obtain a mass spectrum (LDI-MS spectrum). The results are shown in FIG. As shown in Figure 18, in the obtained LDI-MS spectrum, a signal corresponding to the proton adduct of insulin (m/z = 5808) was clearly observed at a signal/noise (S/N) ratio = 12. It was done. From these results, it was found that by using an organic silica substrate equipped with an organic silica thin film C made of organic silica nanofibers having a naphthalimide ring to which an amide group is bonded and a nonafluorohexyl group, insulin can be desorbed by laser with high sensitivity and mass ionized. It turns out that it can be analyzed.

(Example 9)
<Preparation of measurement sample>
Verapamil (molecular weight: 454.6), phosphatidylcholine (molecular weight: 734.0), and angiotensin II (molecular weight: 1046.2) were added as measurement target molecules to an aqueous solution containing trifluoroacetic acid as an ionizing agent at a concentration of 0.1 vol%. A sample solution was prepared by dissolving it at a concentration of 1 pmol/μl. After dropping 1 μl of this sample solution onto the organic silica thin film C obtained in Preparation Example 3, it was air-dried to support verapamil, phosphatidylcholine, and angiotensin II on the surface of the organic silica thin film C, and a measurement sample was prepared.

<Laser desorption/ionization mass spectrometry>
Using a laser desorption/ionization mass spectrometer (“autoflex maX” manufactured by Bruker Daltonics), Nd/ YAG laser (wavelength: 355 nm) was irradiated 100 times at a laser intensity of 50%, and the obtained spectra were integrated to obtain a mass spectrum (LDI-MS spectrum). The results are shown in FIGS. 19A to 19C. As shown in FIGS. 19A to 19C, in the obtained LDI-MS spectra, proton adducts of each component (verapamil: m/z = 455.6, phosphatidylcholine: m/z = 735, angiotensin II: m/ A signal corresponding to z=1047.5) was clearly observed. From this result, by using an organic silica substrate equipped with an organic silica thin film C made of organic silica nanofibers having a naphthalimide ring to which an amide group is bonded and a nonafluorohexyl group, each component of the mixture of verapamil, phosphatidylcholine, and angiotensin II can be extracted. It was found that laser desorption/ionization mass spectrometry can be performed with high sensitivity.

(Comparative example 1)
Instead of the organic silica substrate provided with the organic silica thin film C obtained in Production Example 3, the organic silica substrate provided with the organic silica thin film a obtained in Comparative Production Example 1 was used to deposit amyloid β on the surface of the organic silica thin film a. A measurement sample was prepared in the same manner as in Example 7 except that it was supported, and laser desorption/ionization mass spectrometry was performed. The results are shown in FIG. As shown in FIG. 20, in the obtained LDI-MS spectrum, no clear signal corresponding to the proton adduct of amyloid β (m/z=4331) was observed. From this result, even if the organic silica thin film has a naphthalimide ring with an amide group bonded to it and has a smooth surface, it is clear that the organic silica thin film a has not been subjected to hydrophobization treatment (no hydrophobic group). The substrate was found to be unsuitable as a substrate for laser desorption/ionization mass spectrometry of amyloid β.

(Comparative example 2)
Instead of the organic silica substrate provided with the organic silica thin film C obtained in Production Example 3, the organic silica substrate provided with the organic silica thin film e obtained in Comparative Production Example 2 was used to coat amyloid β on the surface of the organic silica thin film e. A measurement sample was prepared in the same manner as in Example 7 except that it was supported, and laser desorption/ionization mass spectrometry was performed. The results are shown in FIG. As shown in FIG. 21, in the obtained LDI-MS spectrum, no clear signal corresponding to the proton adduct of amyloid β (m/z=4331) was observed. From this result, even if the organic silica thin film has a naphthalimide ring and has a smooth surface, it is clear that the organic silica thin film e has no amide groups and has not been subjected to hydrophobic treatment (no hydrophobic groups). The substrate was also found to be unsuitable as a substrate for laser desorption/ionization mass spectrometry of amyloid β.

(Example 10)
Instead of the organic silica substrate provided with the organic silica thin film B obtained in Production Example 2, an organic silica substrate provided with the organic silica thin film A obtained in Production Example 1 was used to support amyloid β on the surface of the organic silica thin film A. A measurement sample was prepared in the same manner as in Example 2, except that a mapping image of amyloid β was created. The results are shown in FIG.

(Comparative example 3)
Instead of the organic silica substrate provided with the organic silica thin film B obtained in Production Example 2, the organic silica substrate provided with the organic silica thin film f obtained in Comparative Production Example 3 was used to deposit amyloid β on the surface of the organic silica thin film f. A measurement sample was prepared in the same manner as in Example 2 except that it was supported, and a mapping image of amyloid β was created. The results are shown in FIG.

As shown in FIG. 22, in the organic silica thin film A obtained in Preparation Example 1, a signal corresponding to the proton adduct of amyloid β was detected in the entire region where the sample solution was dropped. On the other hand, as shown in FIG. 23, in the organic silica thin film f obtained in Comparative Preparation Example 3, the range in which the signal corresponding to the proton adduct of amyloid β was detected was limited.

Furthermore, in each of the mapping images shown in FIGS. 22 and 23, the average value of signal intensities at 15 measurement points where strong signals were detected was calculated. The results are shown in FIG. As shown in FIG. 24, the average signal intensity of the organic silica thin film A obtained in Production Example 1 was higher than that of the organic silica thin film F obtained in Comparative Production Example 3.

From these results, even if the organic silica thin film has a naphthalimide ring and nonafluorohexyl group and has a smooth surface, amyloid β can be uniformly distributed on the surface of the organic silica thin film if there are no amide groups. It was found that the range in which the signal corresponding to the proton adduct of amyloid β could not be supported was limited, and the average value of the signal intensity was low. On the other hand, when an amide group is bonded to the naphthalimide ring, amyloid β is uniformly supported on the surface of the organic silica thin film, so the signal corresponding to the proton adduct of amyloid β is transmitted throughout the dropping area of the sample solution. It was found that the average signal intensity was also high. That is, when an organic silica substrate having an organic silica thin film A having a naphthalimide ring to which an amide group is bonded and a nonafluorohexyl group and a smooth surface is used, naphthalimide to which an amide group is not bonded is used. It was found that laser desorption/ionization mass spectrometry of amyloid β can be performed with higher sensitivity than when using an organic silica substrate having a ring and a nonafluorohexyl group and having a smooth surface. Ta.

(Example 11)
Instead of the organic silica substrate provided with the organic silica thin film C obtained in Production Example 3, an organic silica substrate provided with the organic silica thin film D obtained in Production Example 4 was used to support amyloid β on the surface of the organic silica thin film D. A measurement sample was prepared in the same manner as in Example 7, and a mapping image of amyloid β was created in the same manner as in Example 2, except that this measurement sample was used. The results are shown in FIG.

(Comparative example 4)
Instead of the organic silica substrate provided with the organic silica thin film C obtained in Production Example 3, the organic silica substrate provided with the organic silica thin film h obtained in Comparative Production Example 4 was used to deposit amyloid β on the surface of the organic silica thin film h. A measurement sample was prepared in the same manner as in Example 7, except that it was supported, and a mapping image of amyloid β was created in the same manner as in Example 2, except that this measurement sample was used. The results are shown in FIG.

As shown in FIG. 25, in the organic silica thin film D obtained in Preparation Example 4, a signal corresponding to the proton adduct of amyloid β was detected in the entire region where the sample solution was dropped. On the other hand, as shown in FIG. 26, in the organic silica thin film h obtained in Comparative Preparation Example 4, the range in which the signal corresponding to the proton adduct of amyloid β was detected was limited.

Furthermore, in each of the mapping images shown in FIGS. 25 and 26, the average value of signal intensities at 15 measurement points where strong signals were detected was calculated. The results are shown in FIG. As shown in FIG. 27, the organic silica thin film D obtained in Production Example 4 had a higher average signal intensity than the organic silica thin film h obtained in Comparative Production Example 4.

From these results, even if the organic silica thin film has a naphthalimide ring and methyl group and has an uneven structure on the surface, amyloid β can be uniformly distributed on the surface of the organic silica thin film if there are no amide groups. It was found that the range in which the signal corresponding to the proton adduct of amyloid β could not be supported was limited, and the average value of the signal intensity was low. On the other hand, when an amide group is bonded to the naphthalimide ring, amyloid β is uniformly supported on the surface of the organic silica thin film, so the signal corresponding to the proton adduct of amyloid β is transmitted throughout the dropping area of the sample solution. It was found that the average signal intensity was also high. That is, when using an organic silica substrate having an organic silica thin film D having a naphthalimide ring to which an amide group is bonded and a methyl group and having an uneven structure on the surface, a naphthalimide ring to which an amide group is not bonded is used. It has been found that amyloid β can be subjected to laser desorption/ionization mass spectrometry with higher sensitivity than when using an organic silica substrate comprising an organic silica thin film h having a methyl group and an uneven structure on its surface.

From the above results, by using an organic silica substrate equipped with an organic silica film containing a naphthalimide ring to which an amide group is bonded in its skeleton and further having a hydrophobic group, bio-related molecules can be uniformly distributed on the surface of the organic silica film. It was also found that it can be supported in a highly dispersed manner, making it possible to perform laser desorption/ionization mass spectrometry of biologically relevant molecules with high sensitivity.

As explained above, according to the present invention, it is possible to obtain an organic silica substrate capable of uniformly supporting a highly hydrophilic compound such as a biologically relevant molecule over the entire surface at a high concentration. Further, by using this organic silica substrate, it becomes possible to perform laser desorption/ionization mass spectrometry with high sensitivity even for highly hydrophilic compounds such as biologically related molecules.

Claims (5)

An organic silica film containing in its skeleton an organic group having a maximum absorption wavelength within a wavelength range of 200 to 600 nm and an amide group bonded to the organic group, and further having a hydrophobic group. Silica substrate. The organic silica substrate according to claim 1, wherein the hydrophobic group is bonded to a silicon atom of a siloxane bond constituting the organic silica film. The organic silica substrate according to claim 1 or 2, wherein the organic group is a naphthalimide ring. 4. The organic silica film according to claim 1, wherein the organic silica film is at least one selected from the group consisting of a film having an uneven surface, a film made of nanofibers, and a film made of fine particles. The organic silica substrate according to any one of the items. A measurement sample is prepared by supporting a molecule to be measured on the organic silica substrate according to any one of claims 1 to 4 ,
A laser desorption/ionization mass spectrometry method, characterized in that the measurement sample is irradiated with laser light to desorb the molecules to be measured from the organic silica substrate, ionized, and subjected to mass spectrometry.