JP3122331U - Sample plate and mass spectrometer equipped with the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sample plate capable of simultaneously measuring MALDI and SALDI.
A The sample plate 2 is perforated by MALDI wells 4 depth D _1, SALDI wells 3 are openings in the form of the bottom opening. Opening depth D ₂ of the wells, so that the D _{1 =} D _2, is adjusted by the recess 7 from the back side. A functional plate 11 having a porous surface and ionization ability is fixed to the recess 7 so as to block the bottom surface of the well 3 to form a functional well 3 for SALDI. The sample dispersion S1 is dropped into the functional well 3, and the matrix and the sample dispersion S2 are dropped into the MALDI well 4 and are ionized by a laser. Since the bottom surfaces of the MALDI well 4 and the SALDI functional well 3 are on the same plane, the focal lengths of the lasers coincide with each other, both measurements can be performed with the same RUN, and MALDI and SALDI can be measured simultaneously. Thus, simultaneous analysis of low-molecular substances and high-molecular substances becomes possible.
[Selection] Figure 5

Description

The present invention relates to a sample plate for holding a sample (sample to be analyzed) and a mass spectrometer equipped with the sample plate, and particularly has a feature in the configuration of the sample plate. This sample plate is used to ionize and release the held sample into the atmosphere by applying energy, and is used for mass spectrometry, particularly desorption / ionization on silicon (DIOS). (Silicon) and surface assisted laser desorption / ionization (SALDI).

In the fields of clinical and biochemistry, mass spectrometers are frequently used to perform qualitative or quantitative analysis on samples such as proteins. This is an analysis from a mass spectrum obtained by performing physical filtering using the mass (m) of ions and the number of charges (z) generated by applying energy to a sample. A time-of-flight mass spectrometer (TOFMS) that performs qualitative / quantitative analysis using the time from the ion generator to the detector is known.

FIG. 1 shows a general configuration example of a time-of-flight mass spectrometer.
The apparatus is roughly composed of an ion generation unit 5, a drift unit 6, and a detection unit (detector) 10, and these rooms are kept at a vacuum degree of about 10 ⁻⁴ to 10 ⁻⁵ Pa. The sample to be analyzed is held on the sample plate 2. When energy is applied to the sample plate 2 by an appropriate method such as irradiating an energy beam such as a laser beam from the light source 8, the sample is ionized and gasified due to the ablation phenomenon. Released into the phase. When an appropriate voltage is applied between the sample plate 2 and the repeller electrode 9, the generated ions having various mass numbers are drawn into the drift portion 6 after being given the same energy. At this time, if the potential difference between the sample plate 2 and the repeller electrode 9 is E, the energy given to the charge q is qE. Therefore, when all of this is converted into kinetic energy, the following relationship is established.

qE = 1/2 · m · v ² Formula (1)

Therefore, as shown in FIG. 2, the ion having a smaller molecular weight m has a higher velocity v, so that it reaches the detector 10 in a shorter time than an ion having a larger m. The time-of-flight mass spectrometer uses this arrival time difference to perform qualitative and quantitative analysis of the sample.

In such an analysis method, particularly when analyzing a biological material such as a protein or a sample having a large molecular weight such as a polymer, the sample is mixed with an organic compound called a matrix and then dropped onto a sample plate and dried. A MALDI (Matrix Assisted Laser Desorption / Ionization) method is generally known in which a sample is desorbed and ionized by irradiating it with an energy beam to perform mass spectrometry. As shown in FIG. 3, the sample plate 2 is provided with a plurality of wells 4 for dropping a sample, and positioning of the sample dropping is performed, and the well 4 is automatically positioned as a target area to perform laser irradiation. By doing this, it is helped to automate the analysis.

On the other hand, a method for ionizing a sample without adding a matrix has been devised and put into practical use. A typical example is the DIOS method (Desorption / Ionization on (porous) Silicon: Patent Document 1). In this ionization method, the ionization ability of a sample is added to the sample plate itself, and as shown in FIG. 4, anodizing treatment (electrochemical anodic oxidation) is performed on the surface of the Si substrate 21 in a spot shape. By performing the method (1), a sample in which a functional well 22 composed of a porous portion of submicron order is formed is used as a sample plate. The left figure of FIG. 4 is an enlarged sectional view of one functional well. Using such a plate, a sample dispersed in an appropriate dispersant is dropped onto the functional well 22 and dried, and then the functional well 22 is positioned as a target area to perform laser irradiation. Ionized (Non-Patent Document 1). The name of DIOS is derived from Si as a base material, but it has been reported that the same effect can be obtained by other matrix free ionization methods such as SALDI method using a material other than Si as a base material. (Non-Patent Documents 2, 3, and 4).

US Pat. No. 6,288,390

http://www.waters.co.jp/product/column/massprep/dios_target.html

Bull.Koream Chem.Soc.2002, Vol.23, No.2 315-319 (Reference: http://66.102.7.104/search?q=cache:mu-nxZU1HioJ:newjournal.kcsnet.or.kr/main/ j_search / j_download.htm% 3Fcode% 3DB020226 +% EF% BC% B3% EF% BC% A1% EF% BC% AC% EF% BC% A4% EF% BC% A9% E3% 80% 80mass & hl = en)

http://www.mssj.jp/Japanese/Conference/2005/program_2005_C1_SY.html (1A-S1-02)

Mei Han, Jan Sunner, J. Am. Soc. Mass Spectrom. 2000, 11, 644-649

By the way, although the MALDI method is highly sensitive as an analytical method, the matrix itself is also decomposed and ionized, so that these become low-molecular-weight background noises and are not suitable for analyzing samples of low-molecular compounds. On the other hand, a matrix-free method such as SALDI that does not use a matrix is inferior in sensitivity, but can be used for analysis of low-molecular compounds.

For this reason, in order to analyze a sample in which a low molecular weight substance and a high molecular weight substance are mixed, it is necessary to perform analysis by both the MALDI method and the matrix free method. Then, since the sample plate to be used is different, there is a problem that it is necessary to replace the sample plate each time.

In addition to the hassle of exchanging, laser irradiation to the sample is configured so that automatic analysis is possible by positioning the laser irradiation position by automatic control by positioning the sample plate before analysis, but when the sample plate is replaced In this case, there is a problem that relative positioning with the automatic control device is required each time.

Although it is conceivable to install both sample plates side by side, in this case, the plate installation area doubles, which causes a new problem that the plate installation table and its drive mechanism are also enlarged.

The present invention has been made in view of these technical issues and required specifications, and it is possible to analyze samples from low molecular weight to high molecular weight at one time without the need to replace the sample plate or reposition the sample. And a mass spectrometer using the sample plate.

In order to achieve the above object, the present invention adopts the following configuration.

(Claim 1)
A sample plate with a plurality of wells for holding samples,
A part of the plurality of wells is a functional well composed of a functional plate whose inner bottom surface has a function of ionizing by applying energy to the sample.

(effect)
The MALDI method and the matrix-free method can be performed on one sample plate, and a wide range of analysis from low molecular weight to high molecular weight can be performed at once without exchanging the sample plate. In addition, since there is no need for replacement, there is no need to re-position for laser irradiation.

(Claim 2)
2. The sample plate according to claim 1, wherein a part of the plurality of wells has an opening at a bottom surface, and the functional well is formed by fixing the functional plate to the opening.

(effect)
In order to produce only some of the multiple wells as functional wells in one sample plate, work steps such as masking of parts other than the non-functional wells are required. Although this is a problem in terms of cost, the manufacturing process and the manufacturing time can be greatly reduced by fixing the separately prepared functional plate to a part of the well with the well bottom surface as an opening.

(Claim 3)
3. The sample plate according to claim 2, wherein the functional well is formed by fixing a functional plate having a larger area than the bottom opening from the back side.

(effect)
It is easier and more efficient to fix a functional plate with a larger area than the bottom opening from the back (opposite the laser irradiation side) rather than fitting the functional plate into the bottom of the well with the bottom open. .

(Claim 4)
The sample plate according to any one of claims 1 to 3, wherein the functional well and the other wells are configured such that their bottom surfaces are on the same plane.

(effect)
When the sample is ionized, an energy beam such as a laser beam is irradiated, and at this time, adjustment is made so that the focus is on the bottom surface of the well. Since the functional well and the other wells are configured so that the bottom surfaces are on the same plane, readjustment of the irradiation light focus becomes unnecessary.

(Claim 5)
The sample plate according to any one of claims 1 to 4, wherein the functional well has a function of DIOS or SALDI.

(effect)
It can be suitably used for matrix-free methods such as SALDI and DIOS.

(Claim 6)
A mass spectrometer comprising at least an ionization unit for ionizing a sample on a sample plate, a mass discrimination unit, and an ion detection unit, wherein the sample plate includes the sample plate according to any one of claims 1 to 5. A mass spectrometer.

(effect)
When used as a sample plate for a mass spectrometer, a mass spectrometer capable of analyzing a wide range of mass numbers at once can be suitably used.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
The mass spectrometer according to the present invention is not different from the conventional one in the overall configuration. For example, the time-of-flight mass spectrometer 1 shown in FIG. 1 is roughly divided into an ion generation unit (ionization unit) 5 and a drift unit (mass discrimination unit) 6, and these rooms have 10 ⁻⁴ to 10 ^−5. The degree of vacuum is about Pa. A sample such as protein, which is an analyte, is held on the sample plate 2, and when energy is applied to the sample plate 2 by an appropriate method such as irradiation with an energy beam such as infrared laser light, the sample is ionized. At the same time, it is released into the gas phase. When an appropriate voltage is applied between the sample plate 2 and the repeller electrode 9, the generated ions having various mass numbers are drawn into the drift portion 6 after being given the same energy. At this time, if the potential difference between the sample plate 2 and the repeller electrode 9 is E, the energy given to the electric charge q is qE. Therefore, when all of this is converted into kinetic energy, the relationship of the above-described equation (1) is established. . Therefore, since the ion with a smaller molecular weight m has a higher velocity v, an ion with a smaller m reaches the detector (ion detector) 10 in a shorter time. In time-of-flight mass spectrometry (TOF-MS), this difference in arrival time is used to perform qualitative and quantitative analysis of the sample.

Here, a mass spectrometer in which the mass discriminating unit is a time-of-flight type is described as an example, but a time-separated mass spectrometer such as an ion trap type may of course be used. Further, it may be a multi-function machine whose main purpose is a function such as microscopic observation other than mass spectrometry, such as a microscopic analyzer. Application to a known technique using a sample plate is possible.

In this type of analyzer, which is frequently used in clinical fields, there are many cases in which a large number of samples of a few μ to ml are generally analyzed. Therefore, it is desired to hold a large number of samples on one sample plate. There is a request. Therefore, it is necessary to provide spots (wells) for holding as many samples as possible on the sample plate and to easily grasp which sample is located at which position. Alternatively, automatic analysis having a function of moving a spot at an arbitrary coordinate on the sample plate to the laser irradiation position by an automatic device is desired. For this reason, the target spot on the sample plate always has regularity. It is desirable that it be formed.

As a configuration of the sample plate according to the present invention used in such a mass spectrometer, a sample plate capable of performing MALDI and DIOS simultaneously is illustrated. Needless to say, the present invention can be applied to other types of matrix free ionization such as SALDI in addition to DIOS.

As shown in FIG. 6, the sample plate main body 2 is a metal flat plate such as an aluminum thin plate, for example, and is capable of electrical contact for TOF-MS analysis. The thickness and material of the thin plate are not particularly limited, but a metal with less outgas is suitable when used in a vacuum. The plate surface is perforated by a plurality of MALDI wells 4 depth D _1. In addition, a part of the wells are opened as DIOS wells 3 in a state of bottom opening in the plate. At this time, opening the depth D ₂ of DIOS wells 3, so that the D _{1 =} D _2, and is adjusted by the recess 7 of the plate 2 back surface.

Well-known processing means such as punching and grinding can be used to form the wells 3 and 4. In addition, it is desirable that the well array conforms to the automatic positioning coordinates of the sample of the mass spectrometer to be applied, but the shape and dimensions can be arbitrarily selected.

A functional plate 11 having a porous surface and having ionization ability is fixed to the recess 7 so as to block the bottom surface of the well 3 to form a functional well (FIGS. 5 and 10). When the functional plate 11 is fixed, both plates may be completely joined or may be simply overlapped with good adhesion. At the time of superposition, it is required to be liquid-tight so that the sample dropped into the well 3 does not leak. The fixing method is not particularly limited as long as the fixing surface is liquid-tight. For example, it may be affixed with a conductive carbon double-sided tape or the like, or may be fixed with a metal paste or an adhesive, but considering the ease of replacement of the functional plate 11, a metal or resin as shown in FIG. It is desirable that the functional plate 11 is suppressed with a clip 13 such as the like, or an itaspring 14 or the like that is welded or bolted as shown in FIG.

As the functional plate 11, as shown in FIG. 7, for example, an anodizing process is performed on one surface of a 0.5 mm Si substrate, and a porous layer 12 is formed on the entire surface of one side of the substrate to form a functional plate. 11 can be used. Even a conventional functional plate in which a porous portion as shown in FIG. 4 is spot-formed can be applied to the present invention, but for spot formation, resist coating → exposure → opening etching → Since a process such as anodizing treatment → resist removal is necessary, the one shown in FIG. 7 that requires only one anodizing process on the entire surface is advantageous in terms of manufacturing process, working time, and manufacturing cost. .

The sample plate 2 according to the present invention shown in FIG. 5 thus produced is a functional well in which the surface of the functional plate is exposed on the bottom surface of the well 3 having a bottom opening, and is suitably used as a well for DIOS. Is done. At the same time, the remaining bottomed well 4 is used as a MALDI well. The sample plate 2 is positioned and placed on the XY stage of the automatic analyzer, and for example, the XY coordinates of the wells 3 and 4 are recognized. An automatic analyzer such as MALDI is a commercial product and is publicly known and publicly used, so the description thereof is omitted here. The sample dispersion S1 is dropped into the functional well 3 for DIOS, and the matrix and the sample dispersion S2 are dropped and dried into the well 4 for MALDI. In this way, the sample is fixed to the bottom surface of each well. The XY stage is driven by the control of the automation device, and the well of arbitrary coordinates sequentially moves to the laser irradiation position, and the laser is irradiated to ionize the sample. In the sample plate 2 according to the present invention, since the bottom surface of the MALDI well 4 and the bottom surface of the DIOS functional well 3 are on the same plane, the focal length of the laser is the same between MALDI and DIOS. Both measurements can be performed with the same RUN, and the measurement throughput can be improved.

In this way, simultaneous measurement of MALDI and DIOS becomes possible, and simultaneous analysis of low molecular weight substances and high molecular weight substances becomes possible. The configuration is not limited to the above-described embodiment, and the configuration can be modified within a range where the same effect can be achieved.

The sample plate according to the present invention is preferably used as a sample plate for a mass spectrometer, but can also be applied to an apparatus that requires ionization of a sample.

It is explanatory drawing which shows the general structural example of a time-of-flight mass spectrometer. It is explanatory drawing which shows the analysis principle of a time-of-flight mass spectrometer. It is explanatory drawing of the sample plate for conventional MALDI. It is explanatory drawing of the sample plate for conventional SALDI. It is sectional drawing which shows the preparation example of the sample plate which concerns on this invention. It is sectional drawing which shows the example of preparation of the sample plate main body which concerns on this invention. It is a figure which shows the example of preparation of the functional plate of the sample plate which concerns on this invention. It is sectional drawing which shows the preparation example of the sample plate which concerns on this invention. It is a figure which shows the example of preparation of the sample plate which concerns on this invention. It is sectional drawing which shows the preparation example of the sample plate which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Time-of-flight mass spectrometer 2 ... Sample plate 3 ... Functional well 4 ... MALDI well 5 ... Ion generating part 6 ... Drift part 8 ... Laser light source 9 ... Repeller electrode 10 ... Detector 11 ... Functional plate 12 ... Porous layer 13 ... Clip 14 ... Ita spring

Claims (6)

A sample plate with a plurality of wells for holding samples,
A part of the plurality of wells is a functional well composed of a functional plate whose inner bottom surface has a function of ionizing by applying energy to the sample.
2. The sample plate according to claim 1, wherein a part of the plurality of wells has an opening on a bottom surface, and the functional well is formed by fixing the functional plate in the opening.
3. The sample plate according to claim 2, wherein the functional well is formed by fixing a functional plate having a larger area than the bottom opening from the back side.
The sample plate according to any one of claims 1 to 3, wherein the functional well and the other wells are configured so that bottom surfaces thereof are on the same plane.
The sample plate according to claim 1, wherein the functional well has a function of DIOS or SALDI.
A mass spectrometer comprising at least an ionization unit for ionizing a sample on a sample plate, a mass discrimination unit, and an ion detection unit, wherein the sample plate includes the sample plate according to any one of claims 1 to 5. A mass spectrometer.

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