JP7492065B1 - Sample support and method for producing the same - Google Patents

Abstract

[Problem] To provide a sample support capable of effectively improving the detection sensitivity of sample components, and a method for manufacturing said sample support. [Solution] The sample support 1 is a sample support for ionizing a sample Sa. The sample support 1 comprises a substrate 2 having a first surface 2a, a second surface 2b opposite to the first surface 2a, and an irregular porous structure 3 that opens at least to the first surface 2a. The porous structure 3 is formed by a plurality of large particles 31 connected to each other, and a plurality of small particles 32 having a diameter smaller than that of the large particles 31. At least a portion of the plurality of small particles 32 is held by being sandwiched between two or more large particles 31 that constitute the first surface 2a. [Selected Figure] Figure 2

Description

The present disclosure relates to a sample support and a method for manufacturing a sample support.

Desorption electrospray ionization (DESI) is known as a method for ionizing samples such as biological samples. As a sample support suitable for this desorption electrospray ionization, a sample support is known that includes a substrate having a first surface, a second surface opposite to the first surface, and an irregular porous structure that opens at least to the first surface (see, for example, Patent Document 1). In the above sample support, for example, the sample transferred onto the first surface is irradiated with charged droplets, thereby desorbing and ionizing the sample.

JP 2022-43571 A

In the above-mentioned sample support, there is a demand for improved detection sensitivity of sample components, for example, in mass spectrometry using an ionization method such as the above-mentioned desorption electrospray ionization method.

The present disclosure aims to provide a sample support that can effectively improve the detection sensitivity of sample components and a method for manufacturing the sample support.

The present disclosure includes the following sample supports [1] to [9] and a method for manufacturing a sample support [10].

[1] A sample support for ionization of a sample, comprising:
a substrate having a first surface, a second surface opposite the first surface, and an irregular porous structure opening at least to the first surface;
the porous structure is formed by a plurality of first particles connected to each other and a plurality of second particles having a smaller diameter than the first particles,
A sample support, wherein at least a portion of the plurality of second particles are sandwiched and held between two or more of the first particles constituting the first surface.

In the sample support of [1] above, the porous structure is composed of not only a plurality of first particles, but also second particles sandwiched and held between two or more first particles constituting the first surface. This makes it possible to reduce the gaps in the sample support on the first surface (i.e., spaces where no particles constituting the porous structure exist) when the sample support is viewed from a direction facing the first surface. In addition, on the first surface, not only the joints between the first particles but also the joints between the first particles and the second particles and the joints between the second particles are added. This makes it possible to suitably retain the sample to be measured on the first surface (particularly on the joints described above). Therefore, according to the sample support of [1] above, it is possible to efficiently ionize the components of the sample that are retained on the first surface, and therefore it is possible to effectively improve the detection sensitivity of the components of the sample.

[2] A sample support according to [1], in which the second particles are formed from the same material as the first particles.

If the first particles and the second particles are made of different materials, there is a risk that during ionization, a signal caused by a material (the material of the second particles) different from the base material (the material of the first particles) may be generated as noise. According to the configuration [2] above, the occurrence of the above-mentioned problems can be avoided. In addition, since the melting points and thermal expansion coefficients of the first particles and the second particles are equal, the substrate (irregular porous structure) can be manufactured easily and with stable quality.

[3] The sample support of [2], in which the first particles and the second particles are formed of an insulating material.

According to the above configuration [3], a substrate in which a plurality of first particles and a plurality of second particles are integrated can be manufactured by a simple method such as sintering.

[4] The sample support of [3], wherein the insulating material is glass.

According to the above configuration [4], by forming the first particles and the second particles from glass, which has a relatively low melting point among insulating materials, a substrate having an irregular porous structure can be obtained conveniently and inexpensively.

[5] Any of the sample support of [1] to [4], in which, when the first surface is viewed from a position facing the first surface along a direction in which the first surface and the second surface face each other, the following formula (1) is satisfied, where R1 is an average particle size of the first particles included in a unit area of a predetermined size, and R2 is an average particle size of the second particles included in the unit area:
R1×1/100≦R2≦R1×1/2 (1)

According to the configuration of [5] above, it is possible to preferably realize a configuration in which one or more second particles are sandwiched and held between two or more first particles constituting the first surface. As a result, it is possible to preferably obtain the effect of improving the detection sensitivity as described above.

[6] The substrate is
a first layer including the first surface and including a plurality of the first particles and a plurality of the second particles;
and a second layer located closer to the second surface than the first layer, the second layer being made of a plurality of the first particles and not containing the second particles.

In the configuration of [6] above, a first layer containing a mixture of first and second particles is provided to facilitate retention of the sample on the first surface, while a second layer is provided below the first layer (on the second surface side) that does not contain second particles and thus allows liquid to pass through more easily than the first layer. This makes it possible to prevent excess liquid components from overflowing onto the first surface and impeding measurement (ionization of sample components remaining on the first surface) when, for example, a sample to be measured that contains liquid components is transferred or dripped onto the first surface of the sample support.

[7] The sample support of [6], wherein the thickness of the first layer in a first direction in which the first surface and the second surface face each other is 1/5 or less of the thickness of the second layer in the first direction.

According to the configuration of [7] above, the effect of [6] above can be preferably obtained by ensuring a sufficient thickness of the second layer relative to the first layer.

[8] The substrate further includes a third layer including the second surface and in which a plurality of the first particles and a plurality of the second particles are mixed;
the second layer is located between the first layer and the third layer;
A sample support according to [6] or [7], wherein at least a portion of the plurality of second particles included in the third layer are sandwiched and held between two or more of the first particles constituting the second surface.

According to the configuration of [8] above, both the first surface and the second surface can be used as the measurement surface (i.e., the surface that supports the sample to be measured). This improves convenience because the user (measurer) of the sample support does not need to specify which surface of the sample support is the measurement surface when transferring or dripping the sample to be measured onto the sample support.

[9] A sample support according to any one of [1] to [8], further comprising a conductive layer that does not block the openings of the porous structure in the first surface and covers the first surface along the uneven shape of the first surface formed by the first particles and the second particles.

According to the configuration of [9] above, it becomes possible to use the sample support for laser desorption ionization and the like without impairing the effect of [1] above. More specifically, when using laser desorption ionization and the like, that is, when it is necessary to apply a voltage to the first surface to guide the components of the sample ionized on the first surface to the ion detector (ground electrode), it becomes possible to apply a voltage appropriately via the conductive layer.

[10] A method for producing a sample support according to any one of [1] to [9], comprising the steps of:
a first sintering step of sintering the plurality of first particles to obtain a sintered body having substantially the same outer shape as the substrate;
an adding step of adding the plurality of second particles to a surface of the sintered body corresponding to the first surface;
a second sintering step of sintering the sintered body obtained in the adding step and the plurality of second particles to obtain the porous structure.

According to the manufacturing method [10] above, a highly reliable porous structure can be obtained by performing a two-stage sintering process. That is, first, in the first sintering process, a structure having high strength and stability is obtained only from a plurality of first particles, and then, by going through the addition process and the second sintering process, a sample support having the effect of [1] above can be easily and stably obtained.

The present disclosure makes it possible to provide a sample support that can effectively improve the detection sensitivity of sample components and a method for manufacturing the sample support.

FIG. 2 is a perspective view showing an embodiment of a sample support. 2 is a SEM image of region A shown in FIG. 1 . 1A to 1C are diagrams illustrating examples of a form in which small particles are held by large particles that constitute a first surface. FIG. 2 is a diagram showing a schematic layer structure of the sample support of FIG. 1; 2 is a SEM image showing a cross section of the sample support of FIG. 1; FIG. 6 is an enlarged view of a portion including an area A1 in FIG. 5 . FIG. 2 is a diagram showing a second step in a mass spectrometry method using the sample support of FIG. 1. FIG. 2 is a diagram showing an example of the configuration of a mass spectrometer for carrying out the above-described mass spectrometry method. FIG. 2 is a diagram showing an example of an irradiation area of microdroplets in an example and a comparative example. FIG. 13 is a diagram showing measurement results of detection sensitivity per irradiation region in an example and a comparative example. FIG. 13 is a diagram showing a schematic layer structure of a sample support according to a first modified example. FIG. 13 is a diagram showing a schematic layer structure of a sample support according to a second modified example. FIG. 13 is a diagram showing a schematic configuration example of large particles, small particles, and a conductive layer on a first surface of a sample support according to a second modified example.

The following describes in detail an embodiment of the present invention with reference to the drawings. Note that the same or corresponding parts in each drawing are given the same reference numerals, and duplicated explanations will be omitted.

[Sample Support]
As shown in FIG. 1, the sample support 1 includes a substrate 2. As an example, the substrate 2 is formed in a rectangular plate shape. The substrate 2 has a first surface 2a and a second surface 2b opposite to the first surface 2a. The first surface 2a is insulating (electrically insulating). In this embodiment, the substrate 2 is an insulating member. Therefore, not only the first surface 2a but the entire substrate 2 is insulating. The thickness of the substrate 2 (the distance from the first surface 2a to the second surface 2b) is, for example, about 100 μm to 1500 μm.

2, the substrate 2 has an irregular porous structure 3 that opens to the first surface 2a. In this embodiment, the entire substrate 2 is formed by the porous structure 3. Here, the "irregular porous structure" refers to, for example, a structure in which voids (pores) extend in irregular directions and are irregularly distributed in three dimensions. For example, the irregular porous structure includes a structure in which the pores enter the substrate 2 from one inlet (opening) on the first surface 2a side and branch into multiple paths, or a structure in which the pores enter the substrate 2 from multiple inlets (openings) on the first surface 2a side and merge into one path. On the other hand, a structure in which multiple pores extending from the first surface 2a to the second surface 2b along the thickness direction of the substrate 2 (i.e., the direction D1 (first direction) in which the first surface 2a and the second surface 2b face each other) are provided as the main pores (i.e., a regular structure composed of pores extending mainly in one direction) is not included in the irregular porous structure.

The porous structure 3 is formed by an aggregate of a plurality of particles. An aggregate of a plurality of particles is a structure in which a plurality of particles are gathered so as to be in contact with each other. An example of an aggregate of a plurality of particles is a structure in which a plurality of particles are bonded or adhered to each other. That is, in order to form a structure in which a plurality of particles are fixed in a state in which they are in contact with each other, the plurality of particles may be directly connected by fusion or the like, or may be indirectly connected via another member. In this embodiment, the plurality of particles are bonded to each other by fusion. In this embodiment, the porous structure 3 is formed by a plurality of large particles 31 (first particles) connected to each other and a plurality of small particles 32 (second particles) having a diameter smaller than that of the large particles. The small particles 32 are formed of the same material as the large particles 31. In this embodiment, the large particles 31 and the small particles 32 are formed of an insulating material. For example, the large particles 31 and the small particles 32 are formed of glass. In this embodiment, soda glass, which has a relatively low melting point among glasses, is used as the material for the large particles 31 and the small particles 32 from the viewpoint of facilitating the manufacture of the aggregate structure. Additionally, the large particles 31 and the small particles 32 are both spherical beads (glass beads).

As shown in FIG. 2, in the SEM image taken from the direction opposite to the first surface 2a, although there is some variation in shape and size for each particle, each of the multiple particles constituting the porous structure 3 can be visually classified into either large particles 31 or small particles 32. That is, the porous structure 3 is composed of particle groups that can be clearly distinguished into two groups in terms of "size". More specifically, each of the multiple large particles 31 has some variation in shape and size, but has a diameter larger than the small particles 32 at least to the extent that it can be distinguished from the SEM image, and is distinguishable from the small particles 32. Similarly, each of the multiple small particles 32 has some variation in shape and size, but has a diameter smaller than the large particles 31 at least to the extent that it can be distinguished from the SEM image, and is distinguishable from the large particles 31.

When the first surface 2a is viewed from a position facing the first surface 2a along a direction D1, the average particle size of the large particles 31 contained in a unit area of a predetermined size (e.g., an area of several hundred μm to 1 mm square) is R1, and the average particle size of the small particles 32 contained in the unit area is R2, the following formula (1) is satisfied. More preferably, the following formula (2) is satisfied.
R1×1/100≦R2≦R1×1/2 (1)
R1×1/10≦R2≦R1×2/5 (2)

The above average particle sizes R1 and R2 can be calculated, for example, based on an SEM image as shown in FIG. 2. For example, first, all spherical objects are extracted by performing known image processing (edge detection, etc.) on the SEM image of FIG. 2. Note that the above objects may be extracted visually instead of the above image processing. Next, an object having a maximum diameter is extracted from the extracted objects, and an object having a diameter with an error from the maximum diameter of a certain amount or less (for example, 30% or less of the maximum diameter) is classified as a large particle 31. Next, the remaining objects that are not classified as a large particle 31 are classified as a small particle 32. Next, the average diameter of the objects classified as a large particle 31 is calculated as the average particle size R1 of the large particle 31, and the average diameter of the objects classified as a small particle 32 is calculated as the average particle size R2 of the small particle 32. The average particle sizes R1 and R2 can be calculated by the above processing. Note that the above calculation method is an example, and the average particle sizes R1 and R2 may be calculated by other methods. As an example, the average particle size R1 of the large particles 31 is approximately 50 μm, and the average particle size R2 of the small particles 32 is approximately 5 μm to 20 μm.

2, the first surface 2a of the substrate 2 is composed of the surfaces (upper surfaces) of the large particles 31 and the small particles 32 located on the uppermost surface (outermost layer) when the direction from the second surface 2b toward the first surface 2a is taken as the upward direction. In FIG. 2, the black parts are parts where the large particles 31 and the small particles 32 constituting the first surface 2a are not present, and correspond to the gaps (openings) between the particles. When the first surface 2a is viewed along the direction D1 from a position facing the first surface 2a, it is preferable that in a unit area of a predetermined size (for example, an area of several hundred μm to 1 mm square), the area occupied by the large particles 31 is the largest, and the area occupied by the small particles 32 is next larger than the area occupied by the gaps (openings) between the particles. The porous structure 3 opens to the first surface 2a at such openings. The liquid that has permeated into the porous structure 3 from the openings on the first surface 2a can pass through the inside of the porous structure 3 and escape to the outside of the second surface 2b from the openings on the second surface 2b side. That is, in this embodiment, the porous structure 3 is open on both the first surface 2a and the second surface 2b, and the openings on the first surface 2a and the openings on the second surface 2b are connected to each other via the gaps between the particles inside the porous structure 3.

As shown in FIG. 2, at least a portion of the small particles 32 contained in the porous structure 3 is sandwiched and held between two or more large particles 31 (i.e., the large particles 31 located on the uppermost surface) constituting the first surface 2a. Examples of the form in which the small particles 32 are held by the two or more large particles 31 constituting the first surface 2a include a form in which one small particle 32 is held between two large particles 31 as shown in FIG. 3A, and a form in which multiple (two in this example) small particles 32 that are in contact with each other are held between two large particles 31 as shown in FIG. 3B. However, the form in which the small particles 32 are held by the two or more large particles 31 constituting the first surface 2a is not limited to the above example. For example, there may be a form in which one small particle 32A is sandwiched and held between three large particles 31, as in some small particles 32A shown in FIG. 2. As shown in Figures 2 and 3, the porous structure 3 has a configuration in which one or more small particles 32 are held by two or more large particles 31 that make up the first surface 2a, and therefore has joints J1 between the large particles 31, joints J2 between the large particles 31 and the small particles 32, and joints J3 between the small particles 32 on the first surface 2a.

Here, most of the small particles 32 are distributed on the first surface 2a (i.e., between the large particles 31 constituting the first surface 2a) and are not present inside the substrate 2 (porous structure 3) at a distance of a certain distance from the first surface 2a toward the second surface 2b. That is, as shown in FIG. 4, when the layer structure of the substrate 2 constituted by the porous structure 3 is shown, the substrate 2 has a mixed layer 21 (first layer) and a large particle layer 22 (second layer). The mixed layer 21 is a layer including the first surface 2a and containing a mixture of a plurality of large particles 31 and a plurality of small particles 32. The large particle layer 22 is located on the second surface 2b side of the mixed layer 21 and is a layer consisting of a plurality of large particles 31 and does not contain small particles 32.

Figure 5 is an SEM image of a cross section of a portion including the mixed layer 21 and a portion of the large particle layer 22 (a portion adjacent to the mixed layer 21) of the substrate 2. Figure 6 is an enlarged view of a portion including the region A1 shown in Figure 5. In Figure 6, only some of the small particles 32 confirmed in the cross section of the region A1 are labeled. In the example shown in Figures 5 and 6, if the large particles 31 located in the outermost layer (i.e., the large particles 31 constituting the first surface 2a) are considered to be the first layer, the portion from the first surface 2a to the second and third layers constitutes the mixed layer 21. That is, in this example, the mixed layer 21 is constituted by the portion above the line L1 shown in Figure 5 (the first surface 2a side), and the large particle layer 22 is constituted by the portion below the line L1 (the second surface 2b side). In this embodiment, as can be seen from Figures 5 and 6, substantially only the multiple large particles 31 are in contact with the virtual plane constituting the outermost surface of the first surface 2a (i.e., the plane along the line above the region A1). In other words, the small particles 32 are sandwiched between the large particles 31, and are of such a size and amount that they do not come into contact with the imaginary plane.

The thickness of the mixed layer 21 in the direction D1 is 1/5 or less of the thickness of the large particle layer 22 in the direction D1. In this embodiment, as an example, the mixed layer 21 is about 1/10 of the total thickness of the substrate 2 (porous structure 3). In other words, the thickness of the mixed layer 21 is about 1/9 of the thickness of the large particle layer 22.

[Method of manufacturing the sample support]
The sample support 1 (porous structure 3) is manufactured, for example, as follows. First, a sintered body is obtained by sintering a plurality of large particles 31 (first sintering step). Specifically, the plurality of large particles 31 are compressed by a press or the like and heated at a high temperature below the melting point of the large particles 31, so that the surfaces of the plurality of large particles 31 are fused and bonded to each other, and a sintered body consisting of only the plurality of large particles 31 is obtained. The sintered body has approximately the same external shape as the substrate 2 to be finally obtained. Next, a plurality of small particles 32 are added to the surface of the sintered body corresponding to the first surface 2a (i.e., the surface that will eventually become the first surface 2a) (addition step). For example, a plurality of small particles 32 are sprinkled on the surface of the sintered body. Next, the sintered body and the plurality of small particles 32 added to the sintered body are sintered (resintered) in a mixed state, and a porous structure 3 in which the surfaces of the plurality of large particles 31 and the plurality of small particles 32 are fused and bonded to each other is obtained (second sintering step).

However, the method of manufacturing the sample support 1 is not limited to the above method. For example, the sample support 1 (porous structure 3) may be manufactured by mixing a plurality of large particles 31 and a plurality of small particles 32 and then performing a single sintering process. However, if the plurality of large particles 31 are not fixed (sintered), the plurality of small particles 32 may be contained in large quantities inside the sintered body (i.e., inside the substrate 2), and the above-mentioned large particle layer 22 may not be formed properly. Therefore, from the viewpoint of reliably and easily obtaining the above-mentioned layer structure (i.e., the mixed layer 21 and the large particle layer 22), it is preferable to obtain the sample support 1 (porous structure 3) by the two-stage sintering process as described above. The sample support 1 manufactured by the above-mentioned manufacturing method (first sintering process, addition process, and second sintering process) can also be regarded as having a structure in which a plurality of small particles 32 are added as a separate member from the substrate to the first surface of the substrate as a sintered body formed by the plurality of large particles 31 so as to be able to maintain a certain outer shape. That is, the multiple large particles 31 can be considered as elements that make up the substrate 2 of the sample support 1, and the multiple small particles 32 can be considered as elements that are added (filled) to a portion of the substrate 2, including the first surface 2a, in order to increase the surface area and seams (seams J2, J3) of the first surface 2a of such substrate 2 (multiple large particles 31).

[Ionization method and mass spectrometry method]
The ionization method and mass spectrometry method using the sample support 1 will be described. First, the above-mentioned sample support 1 is prepared as a sample support for ionizing a sample (first step). The sample support 1 may be prepared by being manufactured by the practitioner of the ionization method and mass spectrometry method, or may be prepared by being transferred from the manufacturer or seller of the sample support 1.

Next, as shown in FIG. 7, the sample Sa is transferred to the first surface 2a of the substrate 2 (second step). In the example of FIG. 7, the sample Sa is a slice of fruit (lemon). For example, the sample Sa is pressed against the first surface 2a of the substrate 2, so that a portion of the sample Sa is attached onto the first surface 2a.

8, the slide glass 6 and the sample support 1 are placed on the stage 41 in the ionization chamber 40 of the mass spectrometer 10. Then, the component Sa1 on the first surface 2a of the substrate 2 is irradiated with the charged microdroplets I to an area (hereinafter referred to as the "target area") including the area where the transferred sample Sa exists, and the ionized component, sample ion Sa2, is attracted (third step). In this embodiment, for example, the stage 41 is moved in the X-axis and Y-axis directions to move the irradiation area I1 of the charged microdroplets I relative to the target area (i.e., the charged microdroplets I are scanned relative to the target area). The above first, second, and third steps correspond to the ionization method using the sample support 1 (in this embodiment, the desorption electrospray ionization method).

In the ionization chamber 40, charged microdroplets I are sprayed from the nozzle 42, and sample ions Sa2 are sucked in from the suction port of the ion transport tube 43. The nozzle 42 has a double-cylinder structure. The solvent is guided into the inner cylinder of the nozzle 42 while a high voltage is applied. This gives a one-sided charge to the solvent that has reached the tip of the nozzle 42. A nebulizing gas is guided into the outer cylinder of the nozzle 42. This causes the solvent to be sprayed as microdroplets, and the solvent ions generated in the process of evaporating the solvent are ejected as charged microdroplets I.

The sample ions Sa2 sucked in from the suction port of the ion transport tube 43 are transported into the mass spectrometry chamber 50 by the ion transport tube 43. The mass spectrometry chamber 50 is under high vacuum conditions (vacuum level of 10 ⁻⁴ Torr or less). In the mass spectrometry chamber 50, the sample ions Sa2 are focused by the ion optical system 51 and introduced into the quadrupole mass filter 52 to which a radio frequency voltage is applied. When the sample ions Sa2 are introduced into the quadrupole mass filter 52 to which a radio frequency voltage is applied, ions having a mass number determined by the frequency of the radio frequency voltage are selectively passed, and the passed ions are detected by the detector 53 (fourth step). By scanning the frequency of the radio frequency voltage applied to the quadrupole mass filter 52, the mass number of the ions reaching the detector 53 is sequentially changed to obtain a mass spectrum in a predetermined mass range. In this embodiment, the detector 53 detects ions corresponding to the position of the irradiation region I1 of the charged microdroplet I, and an image of the two-dimensional distribution of the molecules constituting the sample Sa is obtained. The above first, second, third and fourth steps correspond to a mass spectrometry method using the sample support 1.

[Action and Effect]
In the above-mentioned sample support 1, the porous structure 3 is configured to include not only a plurality of large particles 31 but also small particles 32 sandwiched and held between two or more large particles 31 constituting the first surface 2a. This makes it possible to reduce the gaps of the sample support 1 at the first surface 2a (i.e., spaces where no particles constituting the porous structure 3 exist) when the sample support 1 is viewed from a direction facing the first surface 2a. That is, as can be seen from the SEM image of FIG. 2, the gaps between the large particles 31 at the outermost layer are filled by the plurality of small particles 32 being held between the plurality of large particles 31 at the outermost layer. Also, as shown in FIG. 2, in the first surface 2a, not only the joints J1 between the large particles 31 but also the joints J2 between the large particles 31 and the small particles 32 and the joints J3 between the small particles 32 are added in addition. The sample Sa transferred or dropped on the first surface 2a is particularly likely to remain at such joints J1, J2, and J3. That is, by increasing the number of such joints J1, J2, and J3, the sample Sa to be measured can be suitably retained on the first surface 2a (particularly on the joints J1, J2, and J3 described above). Therefore, according to the sample support 1, it is possible to efficiently ionize the component Sa1 of the sample Sa retained on the first surface 2a, and therefore the detection sensitivity of the component Sa1 of the sample Sa (i.e., the detection sensitivity of the sample ion Sa2) in mass spectrometry using the sample support 1 (for example, the first to fourth steps in the above embodiment) can be effectively improved. Furthermore, since the sample Sa is more likely to remain on the first surface 2a as described above, the area on the first surface 2a where the sample Sa is stained becomes larger and the stain becomes darker. This improves the visibility of the sample Sa attached to the measurement surface (first surface 2a) of the sample support 1, and also provides the effect of facilitating the work of determining the irradiation range of the microdroplets I for ionization.

9 and 10, the above effects will be supplemented. In the example of FIG. 9 and FIG. 10, by providing a conductive layer 4 described later, laser desorption ionization is used in which ionization is performed by irradiating laser light instead of the microdroplets I, rather than the desorption electrospray ionization method of the above embodiment. However, (A) and (B) of FIG. 9 show the state before the conductive layer 4 is provided in the example and the comparative example. (A) of FIG. 9 shows an example of a single irradiation range R of laser light in the example (i.e., the sample support 1B having the conductive layer 4 described later). (B) of FIG. 9 shows an example of a single irradiation range R of laser light in the comparative example. The sample support according to the comparative example has a substrate (porous structure) composed only of a plurality of large particles 31, and differs from the example in that it does not contain a plurality of small particles 32. FIG. 10 shows mass spectra obtained by performing mass analysis (laser desorption ionization method) of a sample Sa (Angiotensin II, as an example) using each of the above examples and comparative examples. That is, in FIG. 10, the horizontal axis indicates the mass-to-charge ratio (m/z), and the vertical axis indicates the signal intensity (arbitrary unit: arb.unit). FIG. 10 shows a mass spectrum M1 of the embodiment and a mass spectrum M2 of the comparative example. In order to easily compare the mass spectrum M1 of the embodiment and the mass spectrum M2 of the comparative example, the origin of the signal intensity of the mass spectrum M1 of the embodiment (i.e., the value corresponding to the signal intensity "0") is shifted upward (by about +0.8). In addition, the mass spectra M1 and M2 are normalized with the peak intensity of Na citrate in each of the embodiment and the comparative example set to 100% (1.0). As shown in FIG. 10, according to the embodiment, a higher signal intensity was obtained at the position corresponding to the sample Sa (Angiotensin II) than in the comparative example. That is, according to the embodiment, it was confirmed that the component Sa1 of the sample Sa is more likely to remain on the first surface 2a than in the comparative example, and thus the detection sensitivity of the component Sa1 of the sample Sa is significantly improved. As described above, FIG. 10 shows the measurement results when the laser desorption ionization method is performed using a sample support having a conductive layer 4, but it is believed that similar results will be obtained when the mass analysis by the desorption electrospray ionization method described above (the first to fourth steps described above) is performed using a sample support not having a conductive layer 4. That is, the component Sa1 of the sample Sa is more likely to remain on the first surface 2a in the example (sample support 1) than in the comparative example (i.e., a sample support having a substrate (porous structure) composed only of a plurality of large particles 31 and not including a plurality of small particles 32), and therefore it is believed that high detection sensitivity can be obtained in the mass analysis by the desorption electrospray ionization method described above (the first to fourth steps described above).

In the sample support 1, the small particles 32 are formed of the same material as the large particles 31. If the large particles 31 and the small particles 32 were formed of different materials, there is a risk that a signal caused by a material (the material of the small particles 32) different from the base material (the material of the large particles 31) may be generated as noise during ionization (in this embodiment, during ionization of the component Sa1 by irradiation with the microdroplets I). In contrast, by forming the large particles 31 and the small particles 32 from the same material as in this embodiment, the occurrence of the above-mentioned problems can be avoided. In addition, since the melting points and thermal expansion coefficients of the large particles 31 and the small particles 32 are equal, the second sintering step described above can be easily performed (i.e., it can be performed under the same temperature conditions as the first sintering step), and the substrate 2 (porous structure 3) can be manufactured easily and with stable quality.

In the sample support 1, the large particles 31 and the small particles 32 are formed of an insulating material. According to the above configuration, the substrate 2 in which the multiple large particles 31 and the multiple small particles 32 are integrated can be manufactured by a simple method such as sintering. Furthermore, since the substrate 2 can be made insulating, the sample support 1 suitable for the above-mentioned desorption electrospray ionization method can be realized. Furthermore, in this embodiment, the large particles 31 and the small particles 32 are formed of glass. According to the above configuration, by forming the large particles 31 and the small particles 32 from glass having a relatively low melting point, the heating temperature required for the above-mentioned first sintering step and second sintering step can be relatively low, so that the substrate 2 having the porous structure 3 can be obtained suitably and inexpensively.

In the sample support 1, the sizes of the multiple large particles 31 and multiple small particles 32 contained in the porous structure 3 are adjusted so as to satisfy the above formula (1) (i.e., "R1 x 1/100 ≦ R2 ≦ R1 x 1/2"). With the above configuration, it is possible to preferably realize a configuration in which one or more small particles 32 are sandwiched and held between two or more large particles 31 constituting the first surface 2a. As a result, the effect of improving the detection sensitivity as described above can be preferably obtained. Note that, from the viewpoint of more preferably realizing a configuration in which small particles 32 are held between large particles 31 as described above, it is more preferable to satisfy the above formula (2) (i.e., "R1 x 1/10 ≦ R2 ≦ R1 x 2/5").

In the sample support 1, the substrate 2 has a mixed layer 21 including the first surface 2a and a large particle layer 22 located on the second surface 2b side of the mixed layer 21. That is, in the sample support 1, the mixed layer 21 is provided with a mixture of large particles 31 and small particles 32 to make it easier to retain the sample Sa on the first surface 2a, while the large particle layer 22 is provided below the mixed layer 21 (on the second surface 2b side) and does not contain small particles 32, so that the liquid passes through it more easily than the mixed layer 21. This allows the liquid component to escape appropriately from the mixed layer 21 to the large particle layer 22 when the sample Sa to be measured, which contains a liquid component, is transferred or dropped onto the first surface 2a of the sample support 1. As a result, it is possible to prevent excess liquid components from overflowing onto the first surface 2a, and to prevent the measurement (i.e., ionization of the component Sa1 of the sample Sa that remains on the first surface 2a) from being hindered by the generation of such excess liquid components.

In the sample support 1, the thickness of the mixed layer 21 in the direction D1 is 1/5 or less of the thickness of the large particle layer 22 in the direction D1. According to the above configuration, the thickness of the large particle layer 22 relative to the mixed layer 21 is sufficiently ensured, so that the above-mentioned effect (i.e., the effect of allowing liquid components that may interfere with the measurement to escape from the mixed layer 21 to the large particle layer 22) can be preferably obtained.

The manufacturing method of the sample support 1 also includes the above-mentioned first sintering step, the addition step, and the second sintering step. According to this manufacturing method, a highly reliable porous structure 3 can be obtained by performing a two-stage sintering step. That is, first, in the first sintering step, a structure having high strength and stability is obtained only from a plurality of large particles 31 (i.e., the part that constitutes the framework of the porous structure 3), and then, by going through the addition step and the second sintering step, a sample support 1 that exhibits the above-mentioned effects can be easily and stably obtained.

In the above ionization method, in the third step, the irradiation area I1 of the charged microdroplets I is moved relative to the first surface 2a. In the component Sa1 of the sample Sa remaining on the first surface 2a side of the substrate 2, the position information of the sample Sa (two-dimensional distribution information of the molecules (component Sa1) constituting the sample Sa) is maintained. Therefore, by moving the irradiation area I1 of the charged microdroplets I relative to the first surface 2a (target area), the component Sa1 of the sample Sa can be ionized while maintaining the position information of the sample Sa. As a result, in the later step of detecting the sample ions Sa2, the two-dimensional distribution of the molecules constituting the sample Sa can be imaged. Furthermore, since the nozzle 42 can be brought closer to the first surface 2a as described above, the irradiation area I1 of the charged microdroplets I can be suppressed from expanding. As a result, in the later step of detecting the sample ions Sa2, the two-dimensional distribution of the molecules constituting the sample Sa can be imaged with high resolution.

In addition, in the mass spectrometry method using the sample support 1, as described above, the component Sa1 of the sample Sa is suitably ionized by irradiation with the charged microdroplets I, so that the signal strength can be improved when detecting the sample ions Sa2.

[Modification]
The present disclosure is not limited to the above-described embodiment. The material and shape of each component are not limited to the above-described material and shape, and various materials and shapes can be adopted. In addition, some components included in the sample support 1 according to the above embodiment may be omitted or modified as appropriate. For example, in the above embodiment, some characteristic components included in the sample support 1 and some effects exerted by each component have been described, but the sample support according to the present disclosure does not necessarily need to be configured to exert all the effects described in the above embodiment, and may be configured to exert only some of the effects described in the above embodiment. In the latter case, the sample support only needs to have a configuration essential for exerting at least the part of the effect, and the configuration that is not essential for exerting the part of the effect may be omitted or modified as appropriate. Note that, when focusing on one effect, the configuration essential for exerting the one effect should be reasonably understood based on technical common sense and the description of this specification, with the person skilled in the art as the standard. Below, some specific modified examples of the sample support of the present disclosure are illustrated.

(First Modification)
A sample support 1A according to a first modified example will be described with reference to Fig. 11. The sample support 1A differs from the sample support 1 in that it has a symmetrical structure in the direction D1, i.e., the portion on the first surface 2a side and the portion on the second surface 2b side have the same structure, and both the first surface 2a and the second surface 2b are configured to be usable as measurement surfaces (surfaces onto which the sample Sa is transferred or dropped).

That is, the substrate 2A (porous structure 3A) of the sample support 1A includes a second surface 2b and has a mixed layer 23 (third layer) in which a plurality of large particles 31 and a plurality of small particles 32 are mixed, and the large particle layer 22 is located between the mixed layer 21 and the mixed layer 23. The second surface 2b is configured in the same manner as the first surface 2a. That is, at least a portion of the plurality of small particles 32 contained in the mixed layer 23 is sandwiched and held between two or more large particles 31 that constitute the second surface 2b.

The sample support 1A makes it possible to use both the first surface 2a and the second surface 2b as measurement surfaces (i.e., surfaces that support the sample Sa to be measured). This improves convenience because the user of the sample support 1A (i.e., the person who performs measurements using the sample support 1A) does not need to specify which surface of the sample support 1A is the measurement surface when transferring or dripping the sample Sa to be measured onto the sample support 1A.

(Second Modification)
A sample support 1B according to the second modification will be described with reference to Figures 12 and 13. The sample support 1B differs from the sample support 1 in that it includes a conductive layer 4. By including the conductive layer 4, the sample support 1B can be used in an ionization method (such as laser desorption ionization) that requires application of a voltage on the first surface 2a to detect a component Sa1 of an ionized sample Sa.

The conductive layer 4 covers the first surface 2a so as not to block the openings of the porous structure 3 on the first surface 2a. That is, the conductive layer 4 is provided so as not to completely block the openings (gaps between particles) of the porous structure 3 on the first surface 2a. As a result, even in the sample support 1B, the liquid components contained in the sample Sa transferred or dropped onto the first surface 2a (strictly speaking, onto the conductive layer 4 formed on the first surface 2a) can penetrate into the porous structure 3 and escape to the large particle layer 22. That is, in the sample support 1B, compared to the case where the conductive layer 4 is formed on a substrate composed of only large particles 31, for example, the conductive layer 4 is also formed on the surface of the small particles 32, so that a more continuous conductive layer 4 can be formed and the conductivity on the first surface 2a can be suitably secured. In addition, the conductive layer 4 is in a state in which gaps are provided to the extent that at least the liquid components contained in the sample Sa can escape to the large particle layer 22.

The conductive layer 4 covers the first surface 2a along the uneven shape of the first surface 2a formed by the large particles 31 and the small particles 32 (for example, the recessed shapes at the joints J1, J2, J3, etc.). That is, the thickness of the conductive layer 4 is made very thin compared to the size (diameter) of the large particles 31 and the small particles 32. As a result, the shape of the outer surface of the conductive layer 4 formed on the surfaces of the large particles 31 and the small particles 32 follows the original surface shapes of the large particles 31 and the small particles 32. Therefore, as shown in FIG. 13, the uneven shape of the first surface 2a (particularly the recessed shapes at the joints J1, J2, J3) is maintained even after the conductive layer 4 is formed.

The conductive layer 4 is formed of a conductive material. It is preferable that the material of the conductive layer 4 is a metal that has low affinity (reactivity) with the sample Sa and high conductivity. From this perspective, it is preferable that the material of the conductive layer 4 is, for example, Au (gold), Pt (platinum), etc. The conductive layer 4 is formed to a thickness of about 1 nm to 350 nm by, for example, a plating method, an atomic layer deposition method (ALD), a vapor deposition method, a sputtering method, etc. Note that, for example, Cr (chromium), Ni (nickel), Ti (titanium), etc. may be used as the material of the conductive layer 4.

13A and 13B, the conductive layer 4 is formed by performing the above-mentioned deposition method, sputtering method, etc. from the first surface 2a side so as to cover the surfaces of the large particles 31 and small particles 32 constituting the first surface 2a that are exposed to the first surface 2a. On the other hand, when the conductive layer 4 is formed by ALD, the conductive layer 4 can be formed not only on the surfaces exposed to the first surface 2a side but also on the surfaces of the large particles 31 and small particles 32 facing the second surface 2b side by penetrating into the gaps of the porous structure 3. In other words, the conductive layer 4 is formed so as to cover at least the surfaces exposed to the first surface 2a side of the large particles 31 and small particles 32 constituting the first surface 2a, and may be provided so as to cover the entire surfaces of the large particles 31 and small particles 32 constituting the first surface 2a.

The sample support 1B makes it possible to use the sample support 1B for laser desorption ionization and the like while still achieving the same effect as the sample support 1 (i.e., without impeding the effect of the sample support 1 due to the presence of the conductive layer 4). More specifically, when using the laser desorption ionization and the like, that is, when it is necessary to apply a voltage to the first surface 2a in order to guide the component Sa1 of the sample Sa ionized on the first surface 2a to the ion detector (ground electrode side), it becomes possible to apply a voltage appropriately via the conductive layer 4.

(Other Modifications)
The configurations of the above-described embodiment, the first modification, and the second modification may be appropriately combined. For example, the first modification and the second modification may be combined. In this case, the conductive layer 4 is provided on each of the first surface 2a and the second surface 2b of the sample support 1A.

In the above embodiment, the porous structure 3 is configured such that the multiple large particles 31 and the multiple small particles 32 are fused to each other, so that the small particles 32 are sandwiched and held between two or more large particles 31 that make up the first surface 2a, but the small particles 32 do not necessarily have to be fused to adjacent large particles 31.

In the above embodiment, the sample support 1 is configured to include only the substrate 2, but the sample support 1 may include a member other than the substrate 2. For example, a support member (such as a frame) for supporting the substrate 2 may be provided on a part of the substrate 2 (such as a corner).

The sample Sa is not limited to the slice of fruit (lemon) exemplified in the above embodiment. The sample Sa may have a flat surface or an uneven surface. The sample Sa may be something other than fruit, such as a plant leaf. In this case, by transferring the components of the surface of the leaf, which is the sample Sa, to the first surface 2a, it is possible to perform imaging mass spectrometry of the surface (veins) of the leaf.

In the above embodiment, the entire substrate 2 is constituted by the porous structure 3, but the porous structure 3 may be formed in a part of the substrate 2. For example, the porous structure 3 may be formed only in a central region (a part of the first surface 2a) defined as a measurement region for transferring or dropping the sample Sa in the substrate 2. In this case, the porous structure 3 may not be formed in other parts of the substrate 2. In addition, the porous structure 3 may not be formed over the entire area from the first surface 2a to the second surface 2b. That is, the porous structure 3 only needs to be open at least to the first surface 2a, and may not be open to the second surface 2b. For example, the substrate 2 may be constituted by a flat plate including the second surface 2b and a porous structure 3 provided on the surface of the plate opposite to the second surface 2b. As an example, the substrate 2 may be constituted by a glass plate and a sintered body (porous structure 3) of glass beads provided on the glass plate.

In the above embodiment, the first surface 2a is insulating so that the sample support 1 can be used in the desorption electrospray ionization method. More specifically, the substrate 2 (porous structure 3) itself is made of an insulating material, so that the first surface 2a is insulating. However, the sample support 1 can be configured to be used in the desorption electrospray ionization method by a configuration other than the above. For example, the substrate 2 (porous structure 3) may be made of a conductive material. In this case, an insulating coating may be applied to the first surface 2a of the substrate 2, so that the first surface 2a has insulating properties. By applying such an insulating coating, the first surface 2a of the substrate 2 can be made insulating, so that the substrate 2 formed of a conductive material can be used. For example, in this case, the porous structure 3 may be formed of an aggregate of multiple particles (large particles 31 and small particles 32) made of metal. In this way, when an insulating coating is provided, the freedom of selection of the substrate material (i.e., the material of the large particles 31 and the small particles 32) can be improved.

In addition, the material of the large particles 31 and small particles 32 constituting the porous structure 3 may be a metal oxide (e.g., alumina, etc.) or an insulating coated metal, in addition to the insulating material exemplified in the above embodiment (glass (soda glass) is used as an example in the above embodiment). In addition, the shape of the large particles 31 and small particles 32 constituting the porous structure 3 is not limited to a spherical shape, and may have a shape other than a spherical shape. In the latter case, the diameter (average particle diameter R1, R2) of the particles (large particles 31 or small particles 32) described in the above embodiment may be read as the effective diameter of the particle observed when the substrate 2 is viewed along the direction D1 from a position facing the first surface 2a (i.e., the maximum diameter of a virtual cylinder that fits within the area occupied by the particle).

1, 1A, 1B... sample support, 2, 2A... substrate, 2a... first surface, 2b... second surface, 3, 3A... porous structure, 4... conductive layer, 21... mixed layer (first layer), 22... large particle layer (second layer), 23... mixed layer (third layer), 31... large particles (first particles), 32, 32A... small particles (second particles), J1, J2, J3... joints, Sa... sample, Sa1... component, Sa2... sample ions (ionized components).

Claims (9)

A sample support for ionization of a sample, comprising:
a substrate having a first surface, a second surface opposite the first surface, and an irregular porous structure opening at least to the first surface;
the porous structure is formed by a plurality of first particles connected to each other and a plurality of second particles having a smaller diameter than the first particles,
at least a portion of the second particles are sandwiched and held between two or more of the first particles constituting the first surface,
The substrate is
a first layer including the first surface and including a plurality of the first particles and a plurality of the second particles;
a second layer located closer to the second surface than the first layer, the second layer being made up of a plurality of the first particles and not including the second particles. The sample support of claim 1, wherein the second particles are formed from the same material as the first particles. The sample support according to claim 2, wherein the first particles and the second particles are formed of an insulating material. The sample support of claim 3, wherein the insulating material is glass. 2. The sample support according to claim 1, wherein, when the first surface is viewed from a position facing the first surface along a direction in which the first surface and the second surface face each other, the following formula (1) is satisfied, where R1 is an average particle size of the first particles contained in a unit area of a predetermined size, and R2 is an average particle size of the second particles contained in the unit area:
R1×1/100≦R2≦R1×1/2 (1) 2. The sample support of claim 1 , wherein the thickness of the first layer in a first direction in which the first surface and the second surface face each other is 1/5 or less of the thickness of the second layer in the first direction. the substrate further includes a third layer including the second surface and in which a plurality of the first particles and a plurality of the second particles are mixed;
the second layer is located between the first layer and the third layer;
The sample support according to claim 1 or 6 , wherein at least a portion of the plurality of second particles contained in the third layer are sandwiched and held between two or more of the first particles constituting the second surface. The sample support according to claim 1, further comprising a conductive layer that does not block the openings of the porous structure in the first surface and covers the first surface along the uneven shape of the first surface formed by the first particles and the second particles. 1. A method for producing a sample support for ionization of a sample , comprising the steps of:
the sample support comprises a substrate having a first surface, a second surface opposite the first surface, and an irregular porous structure opening at least to the first surface;
the porous structure is formed by a plurality of first particles connected to each other and a plurality of second particles having a smaller diameter than the first particles,
at least a portion of the second particles are sandwiched and held between two or more of the first particles constituting the first surface,
The manufacturing method includes:
a first sintering step of sintering the plurality of first particles to obtain a sintered body having substantially the same outer shape as the substrate;
an adding step of adding the plurality of second particles to a surface of the sintered body corresponding to the first surface;
a second sintering step of sintering the sintered body obtained in the adding step and the plurality of second particles to obtain the porous structure.

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