JP2008249389A - Mass spectrometry and mass spectrometer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mass spectrometry capable of measuring easily a planar distribution of components of a sample. <P>SOLUTION: This mass spectrometry for ionizing the sample by a field desorption ionization method by using a plurality of emitters arranged flatly, and performing mass spectrometry of generated ions has a process for installing the sample so that a measuring surface of the sample corresponds to the plurality of emitters arranged flatly, and that each corresponding part of the sample is arranged on each emitter; a process for driving each emitter independently, generating an electric field successively, and ionizing a part corresponding to each emitter of the sample corresponding thereto; and a process for performing mass spectrometry of ions generated by ionization in correspondence with each emitter driven for generating the ions. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a mass spectrometry method and a mass spectrometer using a field desorption ionization method.

  Techniques for analyzing the planar distribution of organic substances are important in evaluating organic materials. For example, there are various spectroscopic imaging techniques such as IR imaging using the inherent infrared absorption of organic substances. In addition, in a biological sample such as a cell or tissue, it is extremely important to scan and image the spatial distribution of the biological material contained therein to obtain position information.

  Mass spectrometry is widely used to identify organic molecules. The ionization methods include electrospray ionization (ESI), atmospheric pressure ionization (API), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), laser desorption ionization (DPI), and fast atom bombardment (FAB). Is mentioned. Liquid secondary ion mass spectrometry (LSIMS), field desorption (FD), electron impact (EI), chemical ionization (CI), matrix-assisted laser desorption ionization (MALDI), time-of-flight secondary ionization (TOF- SIMS) and the like are known.

  As a method for analyzing the planar distribution of an organic substance using the obtained molecular weight information using a mass spectrometry technique, a method using a matrix-assisted laser desorption / ionization (MALDI) mass spectrometer can be given. MALDI is highly useful in that it can perform mass spectrometry by ionizing molecules that are difficult to ionize, such as proteins. In MALDI, a matrix agent for promoting ionization is added to a sample, and molecules are ionized by laser light. Examples of imaging brain cell sections by MALDI are known (Non-Patent Documents 1 to 5).

  Further, a time-of-flight secondary ionization (TOF-SIMS) mass spectrometer is widely used as a technique for imaging the spatial distribution of molecular weight information of a measurement object. This mass spectrometer irradiates a solid sample surface with a high-speed pulsed ion beam (primary ion) in a high vacuum, and positively or negatively charged ions (secondary ions) of the surface components generated by this irradiation. ) To obtain molecular weight information. By scanning the primary ion beam, an ion image (mapping) of the sample surface can be measured.

  One of the ionization methods in mass spectrometry is a field desorption ionization method. A mass spectrometry method using this ionization method is called field desorption ionization mass spectrometry (FD-MS). In the FD-MS, sample molecules are ionized by applying a strong electric field to an emitter to which a sample is applied, and ions are desorbed by repulsion of positive charges between the ions and the emitter.

  FIG. 1 schematically shows a double-focusing mass spectrometer equipped with a general field desorption ion source. In the figure, an emitter 100 is formed by densely growing countless fine whiskers on a tungsten wire having a diameter of several tens of micrometers. When a high voltage of about 10 kV is applied between the emitter 100 and the counter electrode 101, a strong electric field is generated at the tip of the whisker, and ionization is performed on the emitter 100 through current. The generated ions are mass-separated through the sector magnet 104 and a mass spectrum is obtained by the detector 106. The FD-MS ion source can directly ionize hardly volatile molecules applied to the emitter 100, and has an advantage that a strong molecular ion peak can be observed. Therefore, it is an effective ionization method for determining the molecular weight of organic substances. Further, an external additive such as a matrix agent is not required.

  On the other hand, a field emission display (FED) is one in which emitters are two-dimensionally arranged in a plane and the emitter is used as an electron emission source. The FED has an emitter in a portion corresponding to one pixel, an anode electrode and a phosphor disposed so as to face the emitter, and a display is configured by forming the one pixel in a matrix. ing. Electrons are emitted from the emitter into the vacuum, and collide with the phosphor to emit light. This emitter has a minute protrusion shape, and is arranged in a grid pattern in the same number as the pixels, and emits electrons toward the phosphors arranged facing each other. The two-dimensionally arranged emitters can sequentially emit electrons from the emitters in a position-selective manner.

In this field emission device, the emitter is made of, for example, tungsten, molybdenum or the like, and is formed in a substantially conical shape on the cathode electrode exposed from the pore. The bottom surface is electrically connected to the cathode electrode. In addition, it has been proposed to two-dimensionally arrange carbon nanotubes as electron emission sources in a planar manner (for example, Patent Document 1).
Caprioli, R.M. M.M. et al. , Anal. Chem. 1997, 69, 4751-4760. Stoeckli, M .; et al. , J .; Am. Soc. Mass Spectrom, 1999, 10, 67-71 Chaurand, P.A. et al. , Anal. Chem. 1999, 71, 5263-5270. Stoeckli, M .; et al. , NATURE MEDICINE, 2001, 7, 493 Stonele, R.M. et al. , Anal. Chem. 2001, volume 73, p1999-1402. JP-A-10-149760

  As described above, a technique for analyzing the planar distribution (positional information) of an organic substance in a biological sample such as a cell or tissue is important in organic material evaluation, and a better analysis technique is required.

  Mass spectrometry capable of identifying organic molecules is applied to imaging measurement because highly sensitive and accurate information can be obtained from molecular mass information. Among them, MALDI is highly useful in that mass spectrometry can be performed by ionizing molecules such as proteins that are difficult to ionize. In this measurement by MLDI, a matrix agent for accelerating ionization is added to a sample, and a laser is irradiated to ionize molecules. However, when MALDI is used, there is a problem that mixing of the measurement sample and the matrix agent is essential.

  Moreover, TOF-SIMS can perform imaging measurement of molecular weight information distributed on the sample surface. However, since the sample is sputtered with ions of very strong energy, the molecules are destroyed and it is difficult to obtain high molecular weight information of the sample.

  An object of the present invention is to provide a mass spectrometric method and a mass spectroscopic device capable of easily measuring a planar distribution of constituent components of a sample.

  According to the present invention, there is provided a mass spectrometry method in which a sample is ionized by a field desorption ionization method using a plurality of emitters arranged in a plane and mass analysis is performed on the generated ions. The step of placing the sample so that the corresponding measurement surfaces of the sample correspond to each other and the corresponding portion of the sample is disposed on each emitter, and the emitters are driven independently to sequentially generate an electric field, and in accordance with the sample There is provided a mass spectrometric method comprising the steps of ionizing a portion corresponding to each of the emitters, and mass analyzing the ions generated by the ionization in correspondence with the emitters driven to generate the ions.

  Further, according to the present invention, a plurality of emitters arranged in a plane are provided, each emitter can be driven independently, and an ionization unit that performs field desorption ionization of a sample and ions generated in the ionization unit are mass-separated. There is provided a mass spectrometer having a mass analyzer for detecting mass-separated ions.

  ADVANTAGE OF THE INVENTION According to this invention, the mass spectrometry method and mass spectrometer which can measure the planar distribution of the structural component of a sample easily can be provided.

  The present invention relates to a method of ionizing a sample by using a field desorption ionization method and mass-analyzing the generated ions, and a mass spectrometry method capable of imaging detection of a planar distribution state of a constituent component of a sample, and its A mass spectrometer used in the method can be provided.

  In the present invention, the emitters contributing to field desorption ionization are arranged in a plane (two-dimensional array). For example, these emitters can be arranged in a matrix with equal spacing between adjacent emitters.

  The sample measurement surface is made to correspond to these emitters, and the sample is set two-dimensionally so that the corresponding part of the sample is arranged on each emitter. For example, the sample can be placed by contacting a thin film of the sample on these emitters or by transferring a corresponding portion of the sample onto each emitter.

  The ionization of the sample is performed by driving these emitters independently, sequentially generating an electric field in a position-selective manner, and locally desorbing portions corresponding to the respective emitters of the sample accordingly.

  Ions generated by this ionization are mass-separated and detected in the mass analyzer. By performing ion detection corresponding to the emitter driven to generate the ion, it is possible to obtain position information of the measurement component corresponding to the emitter position along with the identification of the ions. That is, the two-dimensional distribution information of the measurement component on the measurement surface of the sample can be obtained together with the molecular weight information of the ion from the position of each emitter and the mass analysis result of the ion corresponding to each emitter. As a result, the detection (imaging) of the two-dimensional distribution state of the specific component on the measurement surface of the sample can be performed.

  In the present invention, since ionization is performed by the field desorption ionization method, ionization can be performed without destroying the measurement component, and it is easy to obtain high molecular weight information of the sample.

  Such a mass spectrometry method includes a plurality of emitters arranged in a plane, each emitter can be driven independently, and an ionization unit that performs field desorption ionization of a sample and analyzes ions generated in the ionization unit It can implement using the mass spectrometer which has a mass spectrometer part. This mass analysis unit has a mass separation unit for flying and mass-separating ions generated in the ionization unit, and a detection unit for detecting the mass-separated ions.

  In FIG. 2, the structure of the ionization part which performs ionization in this invention is shown typically.

As shown in FIG. 2, an anode electrode 201 is formed on a substrate 200, and an insulating layer 202 is laminated on the anode electrode 201. As the substrate 200, a glass substrate such as quartz glass, Pyrex (registered trademark) glass, and blue plate glass, a silicon wafer, and a laminated plate provided with an insulating layer on the surface can be used. The laminate silicon substrate having an insulating film such as SiO ₂ is provided on the surface, stainless steel plate insulating film such as SiO ₂ is provided on the surface, an aluminum plate anodized film of the barrier type is provided on the surface Can be mentioned.

  The insulating layer 202 is provided with pores 203 and 205 penetrating to the anode electrode 201, and emitters 204 and 206 are formed in these pores, respectively. Each emitter in the pore is formed on a common anode electrode. An emitter made of a general emitter material can be used. For example, a material selected from tungsten, molybdenum, nickel, gold, silver, copper, gallium, arsenic, cobalt, carbon, silicon, and a mixture containing these can be used.

  As the emitter material, gold has heat resistance and is dissolved by aqua regia, so that it can be easily etched and the electrode sharpened. In addition, since the surface of the emitter made of gold has a large chemical adsorption force with respect to sulfur atoms, the sample molecules are easily adhered to the emitter. Furthermore, by chemically modifying the surface, the component molecules contained in the measurement sample can be efficiently adhered to the emitter surface by coordination, bonding or adsorption.

  As an emitter material, diamond can produce a diamond-projected emitter having a very sharp tip, and can be arranged and arranged at a predetermined position of the diamond. Further, by chemically modifying the surface of diamond, component molecules contained in the measurement sample can be efficiently adhered to the emitter surface by coordination, bonding or adsorption.

  As an emitter material, silicon can form an emitter with a very sharp tip by a combination of reactive ion etching (RIE) and thermal oxidation. Further, by chemically modifying the surface, the component molecules contained in the measurement sample can be efficiently adhered to the emitter surface by coordination, bonding or adsorption.

  It is desirable that the emitter has a sharp tip so that the highest possible electric field strength can be obtained. For example, it may have a conical shape as shown in FIG. 2, or may have a very thin wire shape such as carbon nanotube 208 as shown in FIG. 3.

  Each emitter is partitioned by an insulator. The spatial resolution of two-dimensional distribution imaging is determined by the distance between the emitters. The interval between the emitters can be set to 1 mm or less, and is preferably 100 μm or less, more preferably 10 μm or less, and particularly preferably 5 μm or less from the viewpoint of spatial resolution. Although the number of emitters is not particularly limited, for example, 1000 or more can be provided, and it is preferable to provide 10,000 or more from the viewpoint of measurement performance. The size of the measurement target sample that can be measured and the spatial resolution of the mass information distribution of the measurement target sample are determined by the number of emitters and the spacing between the emitters.

  In the configuration shown in FIG. 2, the sample is disposed in close contact with the upper end of the emitter 204, and the counter electrode 207 is disposed near the upper portion of the sample. For example, when the analyte itself is a non-conductive organic thin film, the sample can be placed in close contact with the emitters 204 and 206 as they are. At that time, the thin film is preferably thin. When the thickness of the thin film to be measured is thick, it can be used by thinning it appropriately using a microtome or the like. The thickness of the thin film sample can be set to 1 mm or less, preferably 100 μm or less, more preferably 10 μm or less, and particularly preferably 1 μm or less. Even a biological sample such as a biological tissue can be measured by placing it at the tip of the emitter by making it thin.

  A thin film sample prepared using an insulating thin film such as polyimide can also be measured. For example, the surface of a sample can be brought into contact with an insulating thin film, molecules to be analyzed can be transferred and fixed on the thin film, and the obtained thin film sample can be placed on the emitters 204 and 206 for measurement. In this case, the analysis target substance can be transferred and fixed by modifying the surface of the insulating thin film. Specifically, a substitution active functional group capable of chemically adsorbing or binding the analyte molecule to the surface of the insulating thin film can be introduced. For example, an amino group is introduced and reacted with succinic anhydride to introduce a carboxyl group, converted to an N-hydroxysuccinimide ester, or a maleimide group. As a material for the insulating thin film, polymers such as nylon, polyacrylamide, agarose, and protein can be used in addition to polyimide. In place of such a method, the surface of the two-dimensionally arranged emitter can be directly modified with the above-described substitutional active functional group to form an additional covalent molecular layer. . Through this molecular layer, the analyte molecule can be transferred and fixed on the emitter surface.

  FIG. 4A schematically shows the entire mass spectrometer according to the present invention. FIG. 4B is a schematic cross-sectional view in which the ionization unit 400 is partially enlarged, and corresponds to FIG. Here, for convenience of explanation, a portion denoted by reference numeral 400 is referred to as an ionization portion, and another electrode 101 is assigned to the counter electrode.

  For example, as shown in FIG. 4A, the mass analyzing unit 401 can use a double-focusing type part that mass-separates ions generated by ionization. Mass spectrometers having double-focusing mass separators have become mainstream as soft ionization methods develop. In the ionization chamber 402, the sample is arranged on the ionization unit 400 as described above, and the ionized sample molecules are accelerated by the acceleration voltage and guided into the sector magnet 104 to which a fan-shaped magnetic field is uniformly applied, Ions moving in a plane perpendicular to the magnetic field are observed. The sample molecular ions pass through the magnetic field sequentially from the ions with the smallest mass by making the acceleration voltage constant and increasing the magnetic flux density of the magnetic field applied by the sector magnet 104 sequentially from 0, through the slit 105, and at the detector 106. Detected, resulting in a mass spectrum.

  On the other hand, a mass spectrometer having a time-of-flight analysis type mass separator that has recently advanced utilizes the fact that ions that have received an acceleration voltage fly in a time corresponding to the mass. In this mass analyzer, the amount of ions reached as a function of time is received by a detector placed at a certain distance, and is observed as a mass spectrum, so that measurement with high sensitivity and high resolution is possible.

  In FIG. 4, a mass analysis unit having a double-focusing type mass separation unit and a detection unit (detector 106) is shown, but instead, a time-of-flight analysis type, a quadrupole type, an ion trap type, a tandem type, A mass spectrometer having an ion cyclone type mass separator and a detector may be used.

  In the mass spectrometer according to the present invention, as shown in FIG. 2, the ionization section (ion source) has a plurality of emitters, and these emitters are arranged in a plane, and can be driven independently by, for example, matrix driving. Can be reliably operated. Therefore, as shown in FIG. 5, the sample arranged in a plane with respect to the emitter is locally desorbed and ionized near each emitter. Then, in accordance with the driving of the emitter, ions are introduced into the mass analyzer and mass analysis is performed. As a result, the position of the locally driven emitter and the mass spectrum information obtained at that position are obtained. By acquiring information and constructing an image in the same manner for individual emitters arranged two-dimensionally in a plane, it is possible to image a two-dimensional distribution of mass information of organic matter components contained in the sample.

  Hereinafter, the present invention will be described more specifically with reference to examples.

As shown in FIG. 2, the ionization section in this embodiment is composed of a Spindt type emitter group. FIG. 6 shows a planar layout when the emitter group of the ionization unit is viewed from above. The emitter groups represented by the emitters 204 and 206 shown in FIG. 2 are arranged in a two-dimensional matrix as shown in FIG. As shown in FIG. 2, each emitter is electrically connected to the anode electrode 201, provided in the pore 203 of the insulating film 202, and exposed from the opening of the pore. The anode electrode 201 is provided on an insulating film formed on the silicon semiconductor substrate, and is formed from tungsten silicide (WSi ₂ ). The emitter is made of molybdenum (Mo). The diameter of the bottom of the emitter is about 0.7 to 1.0 μm. The distance between adjacent emitters is about 10 μm.

The ionization part can be produced as follows. After the anode electrode 201 is formed on the silicon substrate 200 having an insulating film formed on the surface, an insulating layer 202 made of SiO _{2 is} deposited by a CVD method. Next, the pores 203 penetrating from the insulating layer 202 to the anode portion 201 are formed by using a photolithography technique and a dry etching technique. At this time, the anode electrode 201 is exposed at the bottom of the pore. Next, after obliquely depositing aluminum, molybdenum is vertically deposited to form a conical emitter 204 made of molybdenum on the anode electrode 201 in the pores. Thereafter, molybdenum and aluminum deposited on the insulating layer 202 are removed by a lift-off method using an alkaline solution.

  As the measurement sample, for example, a biological tissue that has been sliced after freezing and dried under vacuum is used. As shown in FIG. 5, the obtained sample flakes are placed in close contact on the emitter group of the ionization section. As shown in FIG. 4, the ionization unit 400 is accommodated in the ionization chamber 402 and placed under a high vacuum.

  The emitters arranged in a two-dimensional plane of the ionization unit 400 are sequentially and independently driven to generate an electric field, locally ionize the biological tissue slices arranged on the ionization unit 400, and mass analysis of the generated ions The mass is separated by the unit 401 and detected. For example, by generating a high electric field in one emitter of the emitter group and locally ionizing a corresponding portion of the sample flake, a mass spectrum as shown in FIG. 7 is obtained. For example, several mass peaks 1000 and 1001 are obtained. , 1002 are observed. In FIG. 7, the horizontal axis represents the molecular weight of molecular ions divided by the charge, and the vertical axis represents the peak intensity. Similarly, by generating a high electric field in the other emitters of the emitter group and locally ionizing corresponding portions of the sample flakes, a mass spectrum as shown in FIG. 8 is obtained. For example, several mass peaks 1000 are obtained. , 1001, 1002 are observed. By performing the same operation for all the emitters, respective mass spectra can be obtained with respect to the emitter position coordinates (FIG. 9). Then, a peak corresponding to a specific mass number of interest, for example, peak 1000 in FIG. 7, is selected and reconstructed as an image image according to the position coordinates of the emitter. As a result, as shown in FIG. 10, the image information of the two-dimensional distribution state of the specific component contained in the biological tissue slice can be obtained from the molecular weight information and the position information. In FIG. 10, the area of the circular dot corresponds to the intensity of the peak 1000.

  According to the present invention, it is possible to directly image distribution information of components contained in a sample using mass information without adding an additive such as a matrix to the sample.

The schematic diagram of a general field desorption ionization mass spectrometer. The figure which showed typically an example of the ionization part structure suitable for implementation of this invention. The figure which showed typically the other example of the ionization part structure suitable for implementation of this invention. The figure which showed typically an example of the structure of the mass spectrometer suitable for implementation of this invention. The figure which showed typically the state which has arrange | positioned the sample to the ionization part suitable for implementation of this invention. The top view shown typically corresponding to the structure shown in FIG. FIG. 3 is a mass spectrum diagram obtained when an electric field is generated in a specific emitter 204 having the configuration shown in FIG. 2. FIG. 3 is a mass spectrum diagram obtained when an electric field is generated in another specific emitter 206 having the configuration shown in FIG. 2. The figure which showed the position coordinate of the emitter group arrange | positioned planarly in the structure shown in FIG. The figure (imaging figure) showing the peak intensity distribution of the specific molecular weight component obtained by the mass spectrometry by this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Emitter 101 Counter electrode 102 Focusing electrode 103 Slit 104 Magnet 105 Slit 106 Detection part 107 Slit 108 Emitter support part 200 Substrate 201 Anode electrode 202 Insulating film 203, 205 Pore 204, 206, 209 Emitter 207 Counter electrode 208 Carbon nanotube 400 Ionization Part 401 mass spectrometry part 402 ionization chamber 500 sample

Claims (5)

A mass spectrometry method in which a sample is ionized by a field desorption ionization method using a plurality of emitters arranged in a plane, and mass analysis is performed on the generated ions.
A step of associating a measurement surface of the sample with a plurality of emitters arranged in a plane, and placing the sample so that a corresponding portion of the sample is disposed on each emitter; and
Driving the emitters independently, sequentially generating an electric field, and ionizing portions corresponding to the emitters of the sample in response thereto;
A mass spectrometry method comprising a step of mass-analyzing ions generated by the ionization in correspondence with an emitter driven to generate the ions.   The mass spectrometry method according to claim 1, wherein two-dimensional distribution information of a measurement component on a measurement surface of a sample is obtained from a position of each emitter and a mass analysis result of ions corresponding to each emitter.   3. The mass spectrometry method according to claim 1, wherein the plurality of emitters are arranged in a matrix with the intervals between adjacent emitters being equal. An ionization unit including a plurality of emitters arranged in a plane, each emitter can be driven independently, and performs field desorption ionization of a sample;
A mass spectrometer having a mass spectrometer that mass-separates ions generated in the ionization unit and detects mass-separated ions.   The mass spectrometer according to claim 4, wherein the plurality of emitters are arranged in a matrix in which the intervals between adjacent emitters are equal.

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