Hitherto, the molecular weight of a bio-substance (e.g., protein, peptide, saccharide, or oligonucleotide), a polymer, or a synthetic polymer has been known to be correctly determined through laser desorption ionization mass spectrometry (LDI-MS) by means of a measurement device therefor.
In the analytical method, individual molecules of the sample are required to be ionized through laser radiation without decomposing the molecules. In a procedure typically employed to avoid decomposition of sample molecules, a sample is applied onto a medium which absorbs laser light, or a mixture of a sample and a medium which absorbs laser light is fed to a spectrometer. This ionization technique without causing decomposition of the sample is called “soft LDI-MS.” One known technique among soft LDI-MS techniques is a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In mass spectrometry, a sample is fragmented by means of a mass-separation section of a TOF (time-of-flight) type, a quadrupole type, an ion-trap type, a sector type, a Fourier-transformation type, or a combination thereof, the thus-separated fragment ions are detected by means of a detector, and the mass numbers of the ions are determined. Among such techniques, a method employing a time-of-flight mass separation section is frequently employed, since no particular limitation is imposed on the principally measureable mass range. In matrix-assisted laser desorption ionization mass spectrometry, a variety of matrices are used.
Meanwhile, in MALDI-MS, a low-molecular-weight organic compound is used as an ionizing agent, and interfering ions are generated from the agent. The thus-generated interfering ions have molecular weights almost equivalent to the molecular weight of the organic compound (mass number: ≦500), and these interfering ions form clusters having total molecular weights of 103 or higher (as mass number). All these ions mask target ions to be detected, generally making mass spectrometry of a sample molecule of interest difficult. In addition, in MALDI-MS, the type of a matrix agent suitable for ionization varies depending on the type of sample molecule. Therefore, an analysis sample must be prepared with an appropriately selected matrix agent, and such selection is carried out on a trial and error basis, which is inconvenient. In order to overcome the drawback, there is proposed soft LDI-MS employing an inorganic compound in micropowder form serving as an ionizing agent.
As disclosed in Patent Documents 1 and 2 (in “Background Art” section), examples of the micropowder-form inorganic compound include cobalt micropowder, titanium oxide micropowder, graphite powder, carbon nanotubes, and solid carbon black (mean particle size: ≦100 nm, PVC blackness: ≦50). Also known is a technique for suppressing locational variation in sensitivity and resolution in which a support substrate having a carbon-containing surface layer is used, to thereby uniformly and minutely crystallize sample molecules. In the micropowder method, since a suspension of the liquid sample and micropowder is applied onto a sample substrate for mass spectrometry, difficulty is encountered in uniformly applying the sample. Particularly in the case of high-sensitivity mass spectrometry, failure to attain uniform application is generally problematic. In addition, an ionization medium is scattered in an ion source chamber through laser light radiation, resulting in problematic contamination.
In order to overcome the above problem, there is proposed a soft LDI-MS technique employing a porous silicon substrate as a sample substrate, the method being called a desorption/ionization-mass spectrometry on porous silicon (DIOS-MS). In DIOS-MS, a sample solution is applied onto the surface of a porous silicon substrate having nano-order micropores and dried. The thus-treated substrate is placed in an ion source in the mass spectrometer, and subjected to the same subsequent procedure as performed in MALDI-MS. The surface of the sample is irradiated with laser light, whereby mass spectrometry is performed. Although the principle of ionization in DIOS-MS has not been elucidated in detail, one conceivable mechanism for ionizing a sample is as follows. The silicon nano-structure is rapidly heated via high-efficiency absorption of laser light, whereby sample molecules instantaneously dissociate. Also, components bounded to or adsorbed on porous silicon are ionized, to thereby transfer electric charge to the sample molecules.
There have also been proposed other sample substrates. Examples include a silicon nano-wire substrate formed of a silicon substrate on which silicon nano-wire has been grown on gold microparticles deposited on the silicon substrate, a plastic substrate having a grooved surface, a substrate coated with metal membrane, an etched silicon substrate, and a chip-like substrate employing a sponge-form substance.
DIOS-MS is an advantageous technique in that a sample substrate itself is employed as an ionization medium, to thereby facilitate uniform application of a sample and prevent problematic generation of interference peaks which would otherwise occurs in MALDI-MS. However, the ionization efficiency varies considerably depending on the conditions under which porous silicon has been formed, and great difficulty is encountered in production of sample substrates having the same porous structure at high reproducibility. Thus, at present, DIOS-MS cannot necessarily be employed as a reliable mass spectrometric technique. Furthermore, since a large portion of the applied sample are incorporated into the porous structure, most of the sample molecules are not ionized and remain in the porous structure. Such unionized molecules interfere with high-sensitivity measurement and make washing of the sample substrate after measurement difficult. Failure in washing causes prevention of generation of peaks attributed to a precedent sample. Thus, DIOS-MS is not necessarily a suitable technique for repeated sample measurement.
In the nano-wire method, gold microparticles serving as a nano-wire material are mechanically bonded to the silicon substrate in an unstable state. Therefore, the nano-wire-gold microparticle structure is prone to be broken by laser light irradiation in measurement or in a sample substrate washing step performed after measurement. Thus, the nano-wire method is not necessarily a suitable technique for repeated sample measurement.
Under such circumstances, the present inventors previously developed techniques disclosed in Patent Documents 1 and 2. The technique disclosed in Patent Document 1 employs, as an ionization medium for absorbing laser light, a pyroelectric (e.g., ferroelectric) crystal substrate having a flat surface. In this technique, pyroelectric crystals are instantaneously polarized by photoenergy of laser light, and sample molecules are ionized by utilizing surface charge or electric field generated by the polarization. One problem involved in the technique is a small specific surface area of the flat substrate onto which a sample is applied, resulting in low sensitivity. Accordingly, in order to enhance sensitivity, the present inventors developed the technique disclosed in Patent Document 2. In the technique disclosed in Patent Document 2, sensitivity is enhanced by forming a large number of fine protruded dots (i.e., quantum dots having a diameter of 20 nm to 100 nm) made of semiconductor on the surface of a flat semiconductor substrate, to thereby increase the specific surface area of the substrate. Since the substrate employs quantum dots, washing of the substrate is easier as compared with the aforementioned porous silicon substrate.
Meanwhile, graphite has a laminar structure in which carbon atoms are hexagonally bonded together and each plane is formed of the hexagons arranged in a plane. The π-electron orbitals of the carbon atoms extend in a direction normal to the plane direction, whereby an unordinary electric field is provided near the graphite surface. Graphite is also known to efficiently absorb laser light employed in laser desorption ionization mass spectrometry. Thus, studies have been conducted on graphite powder as a matrix employed in LDI-MS (Non-Patent Document 1). However, as described in the sections of “Background Art” and “Examples” in Patent Document 3, when graphite powder which has been subjected to no further treatment is employed as a matrix, graphite scatters to generate interference peaks, and the scattered graphite contaminates the ion source chamber, which is considerably problematic. Due to conductivity, graphite as a contaminant provides electric discharge upon application of high voltage thereto for ion acceleration by the ion source chamber, and the electric discharge may give severe damage to relevant devices. Therefore, an LDI-MS technique employing graphite powder is applied to a limited range of research. One alternative substrate for overcoming the drawback is produced by dispersing graphite powder in water-ethanol solvent, to thereby form thin film, and thermally fixing the thin film onto OHP film (Patent Document 3). Although scattering of graphite is prevented in the production procedure, the particle size, amount, and film thickness and graphite powder forming the graphite thin film cannot be controlled precisely or uniformly.
At present, there has never been provided a sample substrate for laser desorption ionization mass spectrometry which substrate can be used to carry samples having a wide range of molecular weights, can gain sufficient sensitivity without noise, and has locational uniformity in sensitivity. Therefore, there is keen demand for a sample substrate which enables uniform application of a sample solution thereto, which does not generate interference peaks upon irradiation of the sample-coated substrate surface with laser light, which can be easily washed after measurement, which can be applied to analysis of various samples, and which attains high-sensitivity measurement. In addition, there is demand for realization of the method and device in laser desorption ionization mass spectrometry employing the substrate having such characteristics to prevent generation of interference peaks, with respect to mass spectrometry of various substances.
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