阅读说明：本技术 电离法及质量分析方法 (Ionization method and mass analysis method ) 是由 小谷政弘 大村孝幸 三枝大辅 于 2020-01-23 设计创作，主要内容包括：一种电离法,其具备：准备试样支承体的第一工序,该试样支承体具备：具有第一表面、及与第一表面相反侧的第二表面以及开口于第一表面及第二表面的各个的多个贯通孔的电绝缘性的基板、以及安装于基板的电绝缘性的框架；在载置部的载置面上载置试样,以第二表面与试样接触的方式在载置面上载置试样支承体的第二工序；和通过对于第一表面照射带电的微小液滴,将经由多个贯通孔移动至第一表面侧的试样的成分进行电离,吸引被电离的成分的第三工序。(An ionization method, comprising: a first step of preparing a sample support, the sample support comprising: an electrically insulating substrate having a first surface, a second surface opposite to the first surface, and a plurality of through holes opened in the first surface and the second surface, and an electrically insulating frame attached to the substrate; a second step of placing the sample on the placing surface of the placing section, and placing the sample support on the placing surface so that the second surface is in contact with the sample; and a third step of irradiating the first surface with the charged fine droplets to ionize a component of the sample moving to the first surface side through the plurality of through holes, thereby sucking the ionized component.)

1. An ionization method, wherein,

the disclosed device is provided with:

a first step of preparing a sample support, the sample support including: an electrically insulating substrate having a first surface, a second surface opposite to the first surface, and a plurality of through holes opened in the first surface and the second surface, respectively; and an electrically insulating frame attached to the substrate;

a second step of placing a sample on a placement surface of a placement unit, and placing the sample support on the placement surface such that the second surface is in contact with the sample; and

a third step of irradiating the first surface with charged fine droplets to ionize a component of the sample moved to the first surface side through the plurality of through holes, thereby sucking the ionized component.

2. The ionization method according to claim 1,

the third step is performed under a condition of an atmospheric pressure atmosphere to a medium vacuum atmosphere.

3. The ionization method according to claim 1 or 2,

in the third step, the irradiation region of the charged fine droplets is moved relative to the first surface.

4. A method of mass spectrometry, wherein,

the disclosed device is provided with:

the first step, the second step and the third step of the ionization method according to any one of claims 1 to 3; and

a fourth step of detecting the component ionized in the third step.

Technical Field

The present disclosure relates to an ionization method and a mass analysis method.

Background

As a method for ionizing a sample such as a biological sample for mass analysis or the like, Desorption Electrospray Ionization (DESI) is known (for example, see patent document 1). The desorption electrospray ionization method is a method of desorbing and ionizing a sample by irradiating the sample with charged fine droplets (charged-droplets).

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2007-165116

Disclosure of Invention

Technical problem to be solved by the invention

In the desorption electrospray ionization method, for example, in mass analysis, it is required to reliably ionize a component of a sample in order to improve signal intensity (sensitivity).

An object of the present disclosure is to provide an ionization method capable of reliably ionizing components of a sample by irradiation of charged fine droplets, and a mass spectrometry method capable of improving signal intensity.

Technical solution for solving technical problem

An ionization method according to an aspect of the present disclosure includes: a first step of preparing a sample support, the sample support including: an electrically insulating substrate having a first surface, a second surface opposite to the first surface, and a plurality of through holes opened in the first surface and the second surface, respectively; and an electrically insulating frame mounted on the substrate; a second step of placing the sample on the placing surface of the placing section, and placing the sample support on the placing surface so that the second surface is in contact with the sample; and a third step of irradiating the first surface with the charged fine droplets to ionize a component of the sample moving to the first surface side through the plurality of through holes, thereby sucking the ionized component.

In this ionization method, a component of a sample moves from the second surface side to the first surface side through a plurality of through holes on a substrate of a sample support, and stays on the first surface side. Further, since the substrate and the frame of the sample support are electrically insulating members, even when the droplet irradiation portion to which a high voltage is applied is brought close to the first surface, for example, the generation of electric discharge between the droplet irradiation portion and the sample support is suppressed. Therefore, according to this ionization method, the component of the sample that has moved to the first surface side through the plurality of through holes can be reliably ionized by bringing the fine droplet irradiation unit close to the first surface and irradiating the first surface with the charged fine droplets.

In the ionization method of one aspect of the present disclosure, the third step may be performed in an atmospheric pressure atmosphere to a medium vacuum atmosphere (vacuum degree 10)^-3An atmosphere of not less than Torr). This makes it possible to easily replace the sample and to easily observe and analyze the sample.

In the ionization method according to one aspect of the present disclosure, in the third step, the irradiation region of the charged fine droplets may be moved relative to the first surface. Among the components of the sample staying on the first surface side of the substrate, positional information of the sample (information on the two-dimensional distribution of molecules constituting the sample) is maintained. Therefore, by moving the irradiation region of the charged fine droplets relative to the first surface, the components of the sample can be ionized while maintaining the positional information of the sample. This makes it possible to visualize the two-dimensional distribution of molecules constituting the sample in the subsequent step of detecting the ionized component. Further, since the fine droplet irradiation unit can be brought close to the first surface as described above, the irradiation area of the charged fine droplets can be suppressed from being enlarged. Thus, in the subsequent step of detecting the ionized component, the two-dimensional distribution of the molecules constituting the sample can be imaged at high resolution.

A mass spectrometry method according to one aspect of the present disclosure includes: the first step, the second step and the third step of the ionization method; and a fourth step of detecting the ionized component in the third step.

In this mass spectrometry, as described above, the components of the sample are reliably ionized by irradiation of the charged minute droplets, and therefore, the signal intensity at the time of detecting the ionized components can be improved.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present disclosure, it is possible to provide an ionization method capable of surely ionizing components of a sample by irradiation of charged fine droplets, and a mass analysis method capable of improving signal intensity.

Drawings

Fig. 1 is a plan view of a sample support used in a mass spectrometry method according to an embodiment.

Fig. 2 is a sectional view of the sample support taken along line II-II shown in fig. 1.

Fig. 3 is an enlarged image of the substrate of the sample support shown in fig. 1.

Fig. 4 is a diagram showing steps of a mass spectrometry method according to an embodiment.

Fig. 5 is a diagram showing steps of a mass spectrometry method according to an embodiment.

Fig. 6 is a block diagram of a mass spectrometer for implementing a mass spectrometry method according to an embodiment.

FIG. 7 is a mass spectrum obtained by the mass spectrometry of the comparative example.

FIG. 8 is a mass spectrum obtained by the mass spectrometry method of the example.

Fig. 9 is a diagram showing a two-dimensional distribution image of specific ions obtained by the mass spectrometry method of the comparative example.

Fig. 10 is a diagram showing a two-dimensional distribution image of specific ions obtained by the mass spectrometry method of the example.

Detailed Description

Hereinafter, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the drawings, the same or corresponding portions are denoted by the same reference numerals, and redundant description thereof is omitted.

[ sample support ]

As shown in fig. 1 and 2, the sample support 1 includes a substrate 2, a frame 3, and an adhesive layer 4. The substrate 2 has a first surface 2a, a second surface 2b, and a plurality of through holes 2 c. The second surface 2b is a surface opposite to the first surface 2 a. The through holes 2c are opened in the first surface 2a and the second surface 2b, respectively. In the present embodiment, a plurality of through holes 2c are formed in the entire substrate 2 in the same manner (with uniform distribution), and each through hole 2c extends in the thickness direction of the substrate 2 (the direction in which the first surface 2a and the second surface 2b face each other).

The substrate 2 is an electrically insulating member. In the present embodiment, the thickness of the substrate 2 is 1 to 50 μm, and the width of each through-hole 2c is about 1 to 700 nm. The shape of the substrate 2 when viewed from the thickness direction of the substrate 2 is, for example, a substantially circular shape having a diameter of about several mm to several cm. The through-holes 2c are, for example, substantially circular when viewed in the thickness direction of the substrate 2 (see fig. 3). The width of the through-hole 2c refers to the diameter of the through-hole 2c when the shape of the through-hole 2c is circular when viewed from the thickness direction of the substrate 2, and refers to the diameter (effective diameter) of a virtual maximum cylinder accommodated in the through-hole 2c when the shape is other than circular.

The frame 3 has a third surface 3a and a fourth surface 3b, and an opening 3 c. The fourth surface 3b is a surface on the opposite side of the third surface 3a, and is a surface on the substrate 2 side. The opening 3c is opened in each of the third surface 3a and the fourth surface 3 b. The frame 3 is an electrically insulating member, and the frame 3 has a thermal conductivity of 1.0W/m.K or less. In the present embodiment, the material of the frame 3 is PET (polyethylene terephthalate), PEN (polyethylene naphthalate), or PI (polyimide), and the thickness of the frame 3 is 10 to 500 μm (more preferably less than 100 μm). In the present embodiment, the frame 3 has transmissivity with respect to visible light, and the frame 3 has flexibility. The frame 3 has a rectangular shape with one side of about several cm, for example, when viewed from the thickness direction of the substrate 2. The shape of the opening 3c when viewed from the thickness direction of the substrate 2 is, for example, a circle having a diameter of several mm to several cm. The lower limit of the thermal conductivity of the frame 3 is, for example, 0.1W/m.K.

The frame 3 is attached to the substrate 2. In the present embodiment, a region along the outer edge of the substrate 2 in the first surface 2a of the substrate 2 and a region along the outer edge of the opening 3c in the fourth surface 3b of the frame 3 are fixed to each other by the adhesive layer 4. The material of the adhesive layer 4 is, for example, an adhesive material (low melting point glass, vacuum adhesive, or the like) which releases little gas. In the sample support 1, a portion of the substrate 2 corresponding to the opening 3c of the frame 3 functions as an effective region R for moving a component of the sample from the second surface 2b side to the first surface 2a side through the plurality of through holes 2 c.

Fig. 3 is an enlarged image of the substrate 2 when viewed from the thickness direction of the substrate 2. In fig. 3, black portions are through holes 2c, and white portions are partition portions between the through holes 2 c. As shown in fig. 3, a plurality of through holes 2c having a substantially constant width are formed in the substrate 2 in a uniform manner. The aperture ratio of the through holes 2c in the effective region R (the ratio of all the through holes 2c to the effective region R when viewed in the thickness direction of the substrate 2) is practically 10 to 80%, and particularly preferably 60 to 80%. The sizes of the plurality of through holes 2c may be different from each other, or the plurality of through holes 2c may be partially connected to each other.

The substrate 2 shown in fig. 3 is an alumina porous film formed by anodizing Al (aluminum). Specifically, the Al substrate is anodized, and the oxidized surface portion is peeled off from the Al substrate, whereby the substrate 2 can be obtained. The substrate 2 may be formed by anodizing a valve metal other than Al, such as Ta (tantalum), Nb (niobium), Ti (titanium), Hf (hafnium), Zr (zirconium), Zn (zinc), W (tungsten), Bi (bismuth), and Sb (antimony), or may be formed by anodizing Si (silicon).

[ ionization method and Mass analysis method ]

An ionization method and a mass analysis method using the sample support 1 will be described. In fig. 4 and 5, the through-hole 2c and the adhesive layer 4 are not shown in the sample support 1. For convenience of illustration, the sample support 1 shown in fig. 1 and 2 and the sample support 1 shown in fig. 4 and 5 are different in size ratio and the like.

First, the sample support 1 is prepared as a sample support for sample ionization (first step). The sample support 1 may be prepared by manufacturing by a manufacturer of the ionization method and the mass spectrometry method, or may be prepared by transferring from a manufacturer, a seller, or the like of the sample support 1.

Next, as shown in fig. 4 (a), the sample S is placed on the placement surface 6a of the slide glass (placement section) 6 (second step). The sample S is a thin film-like biological sample (aqueous sample) such as a tissue slice, and is in a frozen state. Next, as shown in fig. 4 (b), the sample support 1 is placed on the placement surface 6a so that the second surface 2b of the substrate 2 is in contact with the sample S (second step). At this time, the sample support 1 is disposed so that the sample S is located within the effective region R when viewed from the thickness direction of the substrate 2. Next, as shown in fig. 5 (a), the frame 3 is fixed to the slide glass 6 using an electrically insulating tape 7. When the sample S is thawed in this state, as shown in fig. 5 (b), the component S1 of the sample S moves from the second surface 2b side to the first surface 2a side through the plurality of through holes 2c (see fig. 2) by, for example, capillary action in the substrate 2, and the component S1 of the sample S stays on the first surface 2a side by, for example, surface tension.

Next, if the sample S is dried, as shown in fig. 6, the slide glass 6, the sample S, and the sample support 1 are placed on the stage 21 in the ionization chamber 20 of the mass spectrometer 10. The inside of the ionization chamber 20 is in an atmosphere from atmospheric pressure to a medium vacuum (degree of vacuum 10)^-3An atmosphere of more than or equal to Torr). Next, the region corresponding to the effective region R on the first surface 2a of the substrate 2 is irradiated with the charged fine droplets I, whereby the component S1 of the sample S moving to the first surface 2a side is ionized and attracted as being chargedThe separated sample ion S2 (third step). In the present embodiment, for example, by moving the stage 21 in the X-axis direction and the Y-axis direction, the irradiation region I1 of the charged fine droplet I is moved relative to the region corresponding to the effective region R on the first surface 2a of the substrate 2 (that is, the charged fine droplet I is scanned relative to the region). The first step, the second step, and the third step described above correspond to an ionization method using the sample support 1 (desorption electrospray ionization method in the present embodiment).

In the ionization chamber 20, the charged fine droplets I are ejected from the nozzle 22, and the sample ions S2 are attracted from the suction port of the ion transport tube 23. The nozzle 22 has a double-layered cartridge structure. The solvent is guided in the inner cylinder of the nozzle 22 in a state where a high voltage is applied. This gives a biased charge to the solvent reaching the tip of the nozzle 22. Atomizing gas is directed in the outer barrel of the nozzle 22. Thereby, the solvent is atomized into fine droplets, and solvent ions generated in the process of vaporizing the solvent are emitted as charged fine droplets I.

The sample ions S2 sucked through the suction port of the ion transport tube 23 are transported into the mass analysis chamber 30 through the ion transport tube 23. The inside of the mass analysis chamber 30 is in a high vacuum atmosphere (degree of vacuum 10)^-4An atmosphere of below Torr). In the mass analysis chamber 30, the sample ions S2 are converged by the ion optical system 31 and introduced into the quadrupole mass filter 32 to which a high-frequency voltage is applied. When the sample ions S2 are introduced into the quadrupole mass filter 32 to which the high-frequency voltage is applied, ions having a mass number determined by the frequency of the high-frequency voltage are selectively passed, and the passed ions are detected by the detector 33 (fourth step). The mass number of ions reaching the detector 33 is sequentially changed by sweeping the frequency of the high-frequency voltage applied to the quadrupole mass filter 32, and a mass spectrum in a predetermined mass range is obtained. In the present embodiment, the detector 33 detects ions so as to correspond to the position of the irradiation region I1 of the charged fine droplet I, and images the two-dimensional distribution of the molecules constituting the sample S. The first, second, third and fourth steps described above correspond to those using the sample support 1A method of mass analysis.

[ Effect and Effect ]

In the ionization method using the sample support 1, the component S1 of the sample S moves from the second surface 2b side to the first surface 2a side through the plurality of through holes 2c in the substrate 2 of the sample support 1, and stays on the first surface 2a side. Further, since the substrate 2 and the frame 3 of the sample support 1 are electrically insulating members, even when the nozzle 22, which is a fine droplet irradiation portion to which a high voltage is applied, is brought close to the first surface 2a, for example, the generation of electric discharge between the nozzle 22 and the sample support 1 is suppressed. Therefore, according to the ionization method using the sample support 1, the component S1 of the sample S moving to the first surface 2a side via the plurality of through holes 2c can be reliably ionized by bringing the nozzle 22 close to the first surface 2a and irradiating the first surface 2a with the charged fine droplet I.

In the ionization method using the sample support 1, the atmosphere is from atmospheric pressure to medium vacuum (degree of vacuum 10)^{- 3}An atmosphere of more than or equal to Torr). This makes it possible to easily replace the sample S and to easily observe and analyze the sample S. In particular, under such conditions, the charged fine droplets I ejected from the nozzle 22 easily diffuse in contact with atmospheric gas molecules and the like. Therefore, as described above, it is extremely effective in reliably ionizing the component S1 of the sample S to make the nozzle 22 close to the first surface 2 a.

In the ionization method using the sample support 1, the irradiation region I1 of the charged fine droplet I is moved relative to the region corresponding to the effective region R on the first surface 2a of the substrate 2. In the component S1 of the sample S staying on the first surface 2a side of the substrate 2, positional information of the sample S (information on the two-dimensional distribution of molecules constituting the sample S) is maintained. Therefore, by relatively moving the irradiation region I1 of the charged fine droplet I with respect to the region corresponding to the effective region R on the first surface 2a of the substrate 2, the component S1 of the sample S can be ionized while maintaining the positional information of the sample S. Thus, in the subsequent step of detecting the sample ion S2, the two-dimensional distribution of the molecules constituting the sample S can be imaged. Further, since the nozzle 22 can be brought close to the first surface 2a as described above, the enlargement of the irradiation region I1 of the charged fine droplets I can be suppressed. Thus, in the subsequent step of detecting the sample ion S2, the two-dimensional distribution of the molecules constituting the sample S can be imaged at high resolution.

In the mass spectrometry using the sample support 1, as described above, the component S1 of the sample S is reliably ionized by irradiation of the charged fine droplets I, and therefore, the signal intensity at the time of detecting the sample ions S2 can be improved.

Here, the analysis results of the mass spectrometry methods of the comparative example and the mass spectrometry methods of the examples are described. In the mass spectrometry method of the comparative example, a biological sample is placed on the mounting surface of a slide glass, and the biological sample is irradiated with charged minute droplets, whereby the components of the biological sample are ionized, and a mass spectrum and a two-dimensional distribution image of specific ions are obtained for molecules (ions) constituting the biological sample. In the mass spectrometry method of the embodiment, similarly to the above-described embodiment, the biological sample and the sample support are placed on the placement surface of the slide glass, and the first surface of the substrate of the sample support is irradiated with the charged fine droplets, whereby the components of the biological sample are ionized, and a mass spectrum and a two-dimensional distribution image of specific ions are obtained with respect to molecules (ions) constituting the biological sample.

In the mass spectrometry methods of the comparative examples and the mass spectrometry methods of the examples, frozen sections (40 μm thick) of mouse brain were used as biological samples. In the mass spectrometry method of the comparative example and the mass spectrometry method of the example, respectively, the components of the biological sample are ionized by irradiating the charged fine droplets under the same conditions. In the mass spectrometry method of the comparative example and the mass spectrometry method of the example, mass spectrograms are obtained for molecules (ions) constituting a biological sample under the same conditions. In the mass spectrometry method of the example, the following sample support 1 was used.

Material of the substrate 2: alumina oxide

Thickness of substrate 2: 10 μm

Width of through hole 2 c: 190nm

Aperture ratio of through-hole 2 c: 43 percent

Material of the frame 3: glass

Thickness of frame 3: 130 to 170 μm

Fig. 7 is a mass spectrum obtained by the mass spectrometry of the comparative example, and fig. 8 is a mass spectrum obtained by the mass spectrometry of the example. Fig. 9 is a diagram showing a two-dimensional distribution image of specific ions obtained by the mass spectrometry method of the comparative example, and fig. 10 is a diagram showing a two-dimensional distribution image of specific ions obtained by the mass spectrometry method of the example. In fig. 9 and 10, respectively, (a) is a two-dimensional distribution image with respect to a mass-to-charge ratio (m/z) of 506.32, (b) is a two-dimensional distribution image with respect to a mass-to-charge ratio (m/z) of 528.30, and (c) is a two-dimensional distribution image with respect to a mass-to-charge ratio (m/z) of 534.34. From these results, according to the mass spectrometry of examples, compared with the mass spectrometry of comparative examples, particularly in the mass to charge ratio (m/z) ≦ 600 high signal intensity (for lysophospholipid, for example), also clearly represents the two-dimensional distribution of specific ions. Substances that could not be detected by MALDI, but were only low-sensitive in DESI (comparative), were first unexpectedly detected with high sensitivity by DESI-DIUTHAME (example).

[ modified examples ]

The present disclosure is not limited to the above-described embodiments. For example, the material of the frame 3 may be resin other than PET, PEN, or PI, or may be ceramic or glass. In this case, the electrically insulating frame 3 can be easily obtained. The material of the frame 3 is not particularly limited as long as the frame 3 can achieve electrical insulation. The frame 3 may be colored by, for example, a pigment. This makes it possible to classify the sample support 1 according to the application.

In the above-described embodiment, one effective region R is provided on the substrate 2, but a plurality of effective regions R may be provided on the substrate 2. In the above-described embodiment, the plurality of through holes 2c are formed in the entire substrate 2, but the plurality of through holes 2c may be formed in at least the portion of the substrate 2 corresponding to the effective region R. In the above-described embodiment, the sample S is arranged so that one sample S corresponds to one effective region R, but a plurality of samples S may be arranged so that one effective region R corresponds to the sample S.

Alternatively, an opening different from the opening 3c may be provided in the frame 3, and the sample support 1 may be fixed to the slide glass 6 with the tape 7 through the opening. The sample support 1 may be fixed to the slide glass 6 by a method other than the tape 7 (for example, a method using an adhesive, a fixing member, or the like). When the material of the frame 3 is resin, the sample support 1 may be fixed to the slide glass 6 by static electricity.

The sample S is not limited to a water-containing sample, and may be a dry sample. When the sample S is a dry sample, a solution (e.g., acetonitrile mixture solution) for reducing the viscosity of the sample S is added to the sample S. Thus, for example, the component S1 of the sample S can be moved toward the first surface 2a of the substrate 2 through the plurality of through holes 2c by capillary action.

The components of the above-described embodiments are not limited to the materials and shapes described above, and various materials and shapes can be applied. The configurations of the above-described one embodiment or modification can be arbitrarily applied to the configurations of the other embodiments or modifications.

Description of symbols:

1 … sample support, 2 … substrate, 2a … first surface, 2b … second surface, 2c … through hole, 3 … frame, 6 … slide (mount), 6a … mount surface, I … charged micro droplet, I1 … irradiation area, S … sample, S1 … component, S2 … sample ion (ionized component).