JP2012207954A - Substrate for multiple analysis, manufacturing method thereof, and analytical method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate for multiple analyses carrying an analytical sample and performing a spectroscopic analysis and a mass spectrometric analysis of a target substance in the analytical sample.SOLUTION: The substrate for multiple analyses includes: a conductive transparent substrate surface-treated by a first water-soluble molecule; a metal nanorod complex that is covered with a second water-soluble molecule having an interaction necessary for binding with the first water-soluble molecule and fixed onto the conductive transparent substrate by the interaction between the first water-soluble molecule and the second water-soluble molecule; and a surface modifier that is retained by the metal nanorod complex and accelerates ionization of the target substance. The manufacturing method of the substrate for multiple analyses, and an analytical method using the substrate for the multiple analyses are provided.

Description

  The present invention relates to a composite analysis substrate for carrying both a spectroscopic analysis and a mass spectrometry by supporting a target substance, and in particular, a spectroscopic analysis using localized surface plasmon resonance (LSPR) ( LSPR spectroscopy) and surface assisted laser desorption / ionization mass spectrometry (SALDI-MS) (SALDI-MS), and using metal nanorods as markers for the LSPR spectroscopy and the SALDI The present invention relates to a composite analysis substrate used as a mass analysis support substance, a method for producing the same, and an analysis method for performing spectroscopic analysis and mass analysis using the composite analysis substrate.

  In recent years, for the purpose of medical care such as detection of influenza virus, pregnancy test, etc., diagnosis of various genetic diseases, various cancers, infectious diseases, lifestyle-related diseases, etc., decision of treatment policy, prognosis management after treatment, Or, for the purpose of environmental protection and criminal control such as inspection of environmentally regulated substances and drug tests such as narcotics, various biological substances such as genes, various proteins, peptides, polysaccharides and various chemical substances (target substances) As one of the means, LSPR spectroscopic analysis using metal fine particles as a marker is known, and in particular, many techniques for applying LSPR of gold nanorods to sensing technology have been proposed. (For example, see Non-Patent Documents 1 to 6 and Patent Documents 1 and 2).

  On the other hand, mass spectrometry (MS) is an analysis method that ionizes target substances in an analysis sample and separates them by mass, enables molecular weight measurement, and structure analysis by dissociation products (fragment ions). Is separated and detected based on the difference between the quotient (m / z) of the mass (m) and the valence (z) of the ion, and its detection sensitivity and selectivity are high. Widely used for the purpose of qualitative identification and identification of various target substances. As a method for ionizing the target substance in mass spectrometry, there is a laser desorption / ionization (LDI) method in which the target substance in the analysis sample is irradiated with laser light to ionize the target substance. . Ions ionized by the LDI method are separated at a difference of m / z by passing through a mass separator such as a time-of-flight (TOF) and observed by a detector. Further, the intensity appears at the peak depending on the molecular ion concentration at the time of detection. By analyzing the obtained mass spectrum, the structure of the sample molecule to be measured can be analyzed (LDI-MS).

However, even in this LDI-MS, efficient ionization may not be possible depending on the type of target substance, and various improved methods have been proposed for the purpose of improving this point, and one of them is SALDI mass spectrometry. is there. And about this SALDI mass spectrometry, for example, a method (Patent Document 3 and Non-Patent Document 7) using a porous silicon plate having a porous layer of submicron order as a substrate for analysis, wire-like metal, dendritic metal, A method using an analysis substrate having a metal layer made of petal-like metal (Patent Document 4), an analysis having a film made of a metal oxide such as TiO ₂ , ZnO, SnO ₂ , ZrO ₂ or metal oxide fine particles In addition to a method using a substrate for a substrate (Patent Document 5), a method using a substrate for analysis provided with nano-sized metal or metal oxide fine particles (Patent Document 6), etc., Fe, Co, And a method using an analytical substrate provided with a metal oxide layer made of an oxide of one or two or more metals selected from Cu (Patent Document 7) or coating metal fine particles such as cobalt on a substrate. A method using a clothed analysis substrate (Patent Document 8) has been proposed. Furthermore, a method using an analysis substrate in which a plurality of metal fine particles arranged on a substrate are coated with a scattering prevention film has been proposed (Patent Document 9).

  However, each of the above LSPR spectroscopic analyzes is a simple analytical method for the purpose of a screening test for whether or not a target substance may exist in an analysis sample (specimen). If an analytical sample suspected of being positive is found by this, the target material may be identified by conducting a more accurate qualitative analysis of the target material by mass spectrometry or the like. is necessary. For this reason, this LSPR spectroscopic analysis is required to be able to carry out a large number of analysis samples efficiently and in a short time. The form of a test kit provided with a test element and a labeling reagent, or as an LSPR marker on a substrate It has also been proposed to provide a gold nanorod in the form of an analysis chip in which a target substance capture substance is modified (see Patent Documents 1 and 2).

  In the SALDI mass spectrometry, the situation is the same as in the case of the LSPR spectroscopic analysis, and it is required that a large number of analysis samples can be carried out efficiently and in a short time. However, the analysis principles of SALDI mass spectrometry and LSPR spectroscopy are completely different, and the analysis substrate used in LSPR spectroscopy cannot be used as it is as an analysis substrate for SALDI mass spectrometry. When performing SALDI mass spectrometry on an analytical sample suspected of being positive by analysis, prepare an analytical sample for SALDI mass spectrometry again, and use the newly prepared analytical sample as an analytical substrate for SALDI mass spectrometry. In addition to preparing separate analysis substrates for LSPR spectroscopy and SALDI mass spectrometry, it is also necessary to prepare analysis samples in duplicate.

  By the way, Non-Patent Document 8 reports that LSPR-LDI-MS analysis is performed using gold nanorods, and a dispersion of gold nanorods treated with cetyltrimethylammonium bromide (CTAB) is used as a solution. LSPR spectroscopic analysis is performed as it is, and a dispersion of gold nanorods treated with 4-aminothiophenol (4-ATP) is dropped on the sample substrate and dried. It is described that LDI-MS is performed using the sample spot formed above.

  However, in Non-Patent Document 8, it is necessary to use a dispersion of CTAB-treated gold nanorods excellent in dispersibility in a solution when performing LSPR spectroscopic analysis using gold nanorods as a marker for LSPR. When performing LDI-MS using nanorods as a support substance, the CTAB-treated gold nanorods are converted to 4-ATP-treated gold nanorods suitable for mass spectrometry, and a dispersion of this 4-ATP-treated gold nanorods is prepared. In addition, it is necessary to prepare a sample spot by dropping a 4-ATP-treated gold nanorod dispersion onto a sample substrate and drying it. For this reason, CTAB-treated gold nanorods and 4-ATP-treated gold nanorods suitable for each analysis are prepared for each spectroscopic analysis and mass spectrometry, and an analysis sample for LSPR spectroscopy and an analysis sample for SALDI mass spectrometry Must be prepared separately, and it takes a lot of time for these analyses, and it does not meet the needs of the era of analyzing a large number of analysis samples efficiently and in a short time. It also causes a great deal of stress for subjects who provide duplicate specimens.

  As a method for synthesizing gold nanorods and silver nanorods, there have been many reports so far, such as Patent Documents 10 to 14 and Non-Patent Documents 9 to 14.

JP 2009-150,708 JP 2009-265,062 U.S. Pat.No. 6,288,390 JP 2008-107,209 JP U.S. Patent No. 7,122,792 JP 2008-204,654 JP 2010-151,727 JP-A-62-284,256 JP 2010-066,060 JP 2005-068,447 A JP 2005-097,718 JP 2005-298,891 A JP 2006-118,036 A JP 2006-169,544

I. Pastoriza-Santos, J. Perez-Juste, L. M. Liz-Marzan, Chem. Mater., 18, p2465 (2006) A. Gole, C. J. Murphy, Chem. Mater., 17, p1325 (2005) N. J. Durr, T. Larson, D. K. Smith, B. A. Korgel, K. Sokolov, A. Ben-Yakar, Nano Letters, 7, p941 (2007) A. Gole, C. J. Murphy, Langmuir, 21, p10756 (2005) K. M. Mayer, S. Lee, H. Liao, B. C. Rostro, A. Fuentes, P. T. Scully, C. L. Nehl, J. H. Hafner, ACS NANO, (2008) S. M. Marinakos, S. Chen, A. Chilkoti, Anal. Chem., 79, p5278 (2007) Wei, J. Buriak, J. Siuzdak, Nature 1999, 401, 243. Edward T. Castellana, Roberto C. Gamez, Mario E. Gomez, and David H. Russell, Langmuir 2010, 26 (8), pp6066-6070 Y-Y. Yu, S-S. Chang, C-L. Lee, C. R. C. Wang, J. Phys. Chem. B, 101, p6661 (1997) S-S. Chang, C-W. Shih, C-D. Chen, W-C. Lai, C. R. C. Wang, Langmuir, 15, 701 (1997) N. R. Jana, L. Gearheart, C. J. Murphy, J. Phys. Chem. B, 105, p4065 (2001) F. Kim, J. H. Song, P. Yang, J. Am. Chem. Soc., 124, p14316 (2002) Y. Niidome, K. Nishioka, H. Kawasaki, S. Yamada, Chem. Commun., P2376 (2003) N. R. Jana, L. Gearheart, C. J. Murphy, Chem. Commun., P617 (2001)

  Therefore, the present inventors can perform LSPR spectroscopic analysis and SALDI mass spectrometry using the same sample substrate obtained by supporting the analysis sample on the analysis substrate. In addition to the need for duplicate preparation, the preparation of the sample substrate carrying the analytical sample is only required once, and we have intensively studied the development of a composite analysis substrate that can remarkably reduce the labor involved in spectroscopic analysis and mass spectrometry. As a result, surprisingly, the metal nanorods are fixed in a uniformly dispersed state on the conductive transparent substrate, and then the surface modifier that promotes ionization of the target substance is held on the metal nanorods, thereby allowing spectroscopic analysis. The present inventors have found that a composite analysis substrate capable of performing both analysis and mass spectrometry can be prepared, and the present invention has been completed.

  Accordingly, an object of the present invention is to provide a composite analysis substrate capable of carrying out both spectroscopic analysis and mass spectrometry by supporting an analysis sample.

  Another object of the present invention is to provide a method for manufacturing a composite analysis substrate for manufacturing a composite analysis substrate capable of carrying out both spectroscopic analysis and mass spectrometry by supporting such an analysis sample. There is to do.

  Furthermore, an object of the present invention is to provide an analysis method for carrying out both spectroscopic analysis and mass spectrometry analysis by supporting an analysis sample on a composite analysis substrate capable of performing both spectroscopic analysis and mass spectrometry. There is.

That is, the present invention is a composite analysis substrate for carrying an analysis sample and performing spectroscopic analysis and mass spectrometry of a target substance in the analysis sample,
A conductive transparent substrate surface-treated with a first water-soluble molecule, coated with a second water-soluble molecule having an interaction necessary for binding to the first water-soluble molecule, and the first water-soluble molecule; A metal nanorod complex immobilized on the conductive transparent substrate by the interaction of one water-soluble molecule and a second water-soluble molecule, and held on the metal nanorod complex to promote ionization of the target substance 4 -A substrate for composite analysis, comprising a surface treatment agent such as aminothiophenol.

Further, the present invention is a method for producing a composite analysis substrate for carrying an analysis sample and performing spectroscopic analysis and mass spectrometry of a target substance in the analysis sample,
(A) preparing a surface-treated substrate by surface-treating the conductive transparent substrate with a first water-soluble molecule;
(B) A second water-soluble molecule having an interaction necessary for binding to the first water-soluble molecule is added to the dispersion of the metal nanorods, and the metal nanorod is added with the second water-soluble molecule. Coating to form a metal nanorod composite;
(C) preparing a metal nanorod-immobilized substrate in which the metal nanorod composite is fixed to the surface-treated substrate by bringing the surface-treated substrate into contact with the metal nanorod composite;
(D) a step of bringing a surface modifier into contact with the metal nanorod-immobilized substrate obtained in the step C to prepare an analysis substrate holding the surface modifier, It is a manufacturing method.

  Furthermore, the present invention is a method for analyzing a target substance in an analysis sample using the above-described composite analysis substrate. The sample substrate is prepared by supporting an analysis sample on the composite analysis substrate. Screening whether the target substance is present in the analysis sample from the absorption spectrum obtained by the spectroscopic analysis, and then, from the mass spectrum of the mass analysis performed using the same sample substrate, the target substance in the analysis sample This is an analysis method characterized by performing qualitative analysis.

  In the present invention, the conductive transparent substrate requires that at least the surface on which the metal nanorods are fixed be surface-treated with the first water-soluble molecule, and the metal nanorods described above include the first metal nanorods. It must be coated with a second water-soluble molecule that has the necessary interaction to bind to the water-soluble molecule.

  Here, as for the material of the conductive transparent substrate used in the present invention, it is sufficient if it has transparency necessary for performing spectroscopic analysis and also has conductivity necessary for performing mass spectrometry. Metal oxides such as ITO (indium-tin oxide), IZO (indium-zinc oxide), AZO (aluminum-zinc oxide), GZO (gallium-zinc oxide), TO (tin oxide), and IO (indium oxide) In view of the fact that the thermal conductivity is relatively small and the energy from the laser is not easily dissipated during mass spectrometry, and analysis is possible with a relatively low laser energy, ITO, IZO, TO, etc. are preferable. . ITO is particularly preferable because of its low resistivity. Also, the shape of the conductive transparent substrate is not particularly limited as long as it is a shape capable of performing spectroscopic analysis and mass spectrometry. Even if the entire surface is a flat plate shape, an analysis sample is placed on the surface. One having an appropriate cross-sectional shape for dropping (for example, a shape such as a parenthesis closing shape of vertical braces, a parenthesis closing shape of vertical brackets, a parenthesis closing shape of vertical turtle shell brackets, a parenthesis closing shape of vertical angle brackets, etc.) Alternatively, it may be provided with a plurality of recesses (wells).

  Further, the metal nanorods used in the present invention are nano-sized metal particles that can generate LSPR from the necessity of performing LSPR spectroscopic analysis, and the types thereof are, for example, gold (Au), silver (Ag) , Copper (Cu), platinum (Pt), aluminum (Al), and alloys thereof, etc. The major axis length is 400 nm or less, preferably 200 nm or less, and the aspect ratio is 1 or more. A large rod shape is preferable. Specifically, a metal nanorod having an aspect ratio in which the maximum absorption wavelength of LSPR is in a range of 700 to 2000 nm is used. As for the long axis length, if it exceeds 400 nm, it tends to settle, and it is difficult to secure the adsorption time in the process of adsorbing the metal nanorods to the substrate. As the metal nanorod, a gold nanorod is preferable. When gold nanorods are immobilized on a substrate, they are chemically stable and hardly oxidized. In addition, since the gold nanorods form a covalent bond with the thiol group of the surface modifier, it is possible to effectively retain the surface modifier in the gold nanorod complex.

  And, the first water-soluble molecule that surface-treats the conductive transparent substrate and the second water-soluble molecule that coats the metal nanorods only need to have an interaction necessary for bonding to each other, For example, an anionic molecule, a cationic molecule, a hydrogen bonding molecule, etc. can be mentioned, and when an anionic molecule (cationic molecule) is used as the first water-soluble molecule, the second water-soluble molecule is used. By using a cationic molecule (anionic molecule) as the molecule, the metal nanorods can be electrostatically immobilized on the conductive transparent substrate, and as the first water-soluble molecule and the second water-soluble molecule, When hydrogen bonding molecules are used, metal nanorods can be immobilized on the conductive transparent substrate via hydrogen bonding.

  Here, examples of the anionic molecule include anionic polymers such as polystyrene sulfonate (PSS), polyacrylic acid, polycarboxylic acid, and polyphosphoric acid, sodium dodecyl sulfate (SDS), cholic acid, ascorbic acid, citric acid, and the like. Anionic small molecules such as acids can be exemplified, and examples of the cationic molecules include polyallylamine hydrochloride (PAH), cationic polymers such as polyamino acids, and 3-aminopropyltriethoxysilane. (3-aminopropyltriethoxysilane (APTES), Poly (diallyldimethylammonium chloride) (PDDA), CTAB, cetyltrimethylammonium chloride (CTAC), 2-aminoethanethiol hydrochloride (2-AET), 4-aminobutanethiol hydrochloride , 6-aminohexanethiol hydrochloride, etc. Examples include thione small molecules, and examples of the hydrogen bonding molecule include nucleic acids (DNA, RNA), carbohydrates (cellulose, starch), proteins (enzymes, peptides), and the like. .

  In the present invention, metal nanorods (metal nanorod composites) fixed on a conductive transparent substrate surface-treated with a first water-soluble molecule and coated with a second water-soluble molecule are subjected to SALDI mass spectrometry. In order to carry out, it is necessary to retain a surface modifier having an action or function of promoting ionization of the target substance. As the surface modifier used for this purpose, when adsorbed in a large amount on the surface of the gold nanorod Any compound that can replace and remove CTAB, which may inhibit ionization, can be widely used. For example, it is adsorbed on metal nanorods as shown by R-SH (R: hydrocarbon, SH: thiol group). A compound in which the functional group is a thiol group can be suitably used. In the R-SH compound, the hydrocarbon R is preferably a compound having a functional group capable of supplying a proton, such as an amino group, a carboxyl group, or a hydroxy group. Specifically, 4-aminothiophenol (4-ATP), 2-aminothiophenol (2-ATP), 6-amino-1-hexanethiol, 6-carboxy-1-hexanethiol, 6-hydroxy-1 -Hexanethiol etc. can be mentioned. When the metal species of the metal nanorod is gold, the surface modifier is preferably 4-aminothiophenol, and a thiol group forms a covalent bond on the surface of the gold nanorod and electrostatically adsorbs on the gold nanorod surface. The effect of eliminating the remaining CTAB is obtained, it becomes possible to reduce CTAB that causes noise in mass spectrometry, and it is possible to effectively promote the ionization of the target substance in the vicinity of the gold nanorod. In addition, since the amino group for supplying protons has the same charge as CTAB used in the synthesis of the gold nanorods, it is possible to prevent aggregation of the gold nanorods.

  Regarding these surface modifiers, in addition to the above-mentioned surface modifiers having a thiol group, proton supply ability and sodium ions used for the purpose of proton addition and / or sodium ion addition to an analysis sample in ionization of mass spectrometry You may use the ionization adjuvant which has supply ability. For example, ionization aids having proton supply ability include trifluoroacetic acid, citric acid, monoammonium dihydrogen citrate, diammonium monohydrogen citrate, triammonium citrate, tartaric acid, malic acid, succinic acid, oxalic acid, Cinnamic acid, diglycolic acid, sebacic acid and the like can be mentioned. Examples of the ionization aid having sodium ion supply ability include sodium trifluoroacetate, sodium iodide, monosodium dihydrogen citrate, disodium monohydrogen citrate, monosodium tartrate, monosodium succinate, Examples include trisodium citrate, disodium tartrate, and disodium succinate. These may be used by mixing two or more different types.

  In the present invention, the metal nanorod composite fixed on the conductive transparent substrate captures the target substance in the analysis sample by binding to the target substance in the analysis sample together with the surface modifier, if necessary. A substance may be attached. As the capture substance used for this purpose, any substance that specifically binds to the target substance can be used. Specifically, for example, when the target substance is bithion or a bithion labeled substance, avidin is used. In addition, when the target substance is an antigen or an antigen-labeling substance, an antibody is used, and a biomolecule recognition molecule such as an aptamer can also be used. The substance that specifically binds to the target substance is preferably adsorbed on the metal nanorods. When the target substance interacts with the supplementary substance and the metal nanorods are present in the vicinity, the absorption wavelength shift of the metal nanorods becomes clear.

  The composite analysis substrate of the present invention is basically manufactured by the following steps. That is, in order to bind the first water-soluble molecules to each other in the dispersion process of the metal nanorods in the step A in which the conductive transparent substrate is surface-treated with the first water-soluble molecules to prepare the surface-treated substrate. B step of adding a second water-soluble molecule having a necessary interaction and coating the metal nanorod with the second water-soluble molecule to form a metal nanorod composite, and the surface treatment substrate and the metal nanorod composite C process for preparing a metal nanorod-immobilized substrate in which a metal nanorod complex is immobilized on a surface-treated substrate by contacting the substrate with the metal nanorod-immobilized substrate obtained in step C, and promoting ionization of a target substance And the step D for preparing the analytical substrate holding the surface modifier, and if necessary, the analytical substrate obtained in the step D. The target substance is bound to the target substance and brought into contact with the target substance to capture the target substance, and the ionization process is further promoted in the E process for preparing the analysis board holding the target substance and the analysis board obtained in the step D. In order to make it, it manufactures through F process which adds the ionization adjuvant which does not contain a thiol group.

  Here, in the above step A, the conductive transparent substrate is surface-treated with the first water-soluble molecule, and the first water-soluble molecule is attached to at least a portion of the surface of the conductive transparent substrate where the metal nanorods are fixed. Or a solution of the first water-soluble molecule is usually prepared, and using this prepared solution, application such as dipping, brushing, spraying, etc. The surface of the conductive transparent substrate may be treated by an appropriate means. For example, when the first water-soluble molecule is APTES (water-soluble low molecule), the conductive transparent substrate is dipped in a 2-propanol solution having an APTES concentration of 0.1 to 10 wt%, and then with water. APTES adsorption-conductive transparent substrate can be obtained by drying at room temperature after washing. When the first water-soluble molecule is PAH (cationic polymer), it is preferable to use PAH having a weight average molecular weight of 1,000 to 100,000, and conducting in a PAH aqueous solution having a PAH concentration of 0.1 to 10 mg / ml. The PAH adsorption-conductive transparent substrate is obtained by immersing and removing the conductive transparent substrate, then washing with water and drying at room temperature.

  Moreover, about said B process, what is necessary is just to be able to coat | cover a metal nanorod with said 2nd water-soluble molecule, for example, the method etc. which mix metal nanorod aqueous dispersion and 2nd water-soluble molecule aqueous solution etc. are illustrated. be able to.

  Specifically, for example, the metal nanorod is a gold nanorod, and the second water-soluble molecule has an electrostatic interaction with APTES (water-soluble small molecule) which is the first water-soluble molecule. The case of using PSS (anionic polymer) having a specific example will be described below. Specifically, it can be carried out by the following method.

That is, first, gold ions in an aqueous solution in which a quaternary ammonium salt [CH ₃ (CH ₂ ) _n N ⁺ (CH ₃ ) ₃ Br ⁻ (n is an integer of 1 to 15)], which is a cationic surfactant, is dissolved. To synthesize gold nanorods. As a method for synthesizing gold nanorods and silver nanorods, for example, gold nanorods and silver nanorods described in Patent Documents 10 to 14 and Non-Patent Documents 9 to 14 can be synthesized. When trimethylammonium bromide (CTAB) is used to reduce gold and silver ions in this CTAB solution by various reduction methods, CTAB is adsorbed on the surface of the resulting nanorods, and the CTAB adsorbed to the nanorods stably dispersed in water-gold Nanorod aqueous dispersion and CTAB adsorption-silver nanorods are obtained.

  About this CTAB adsorption-gold nanorod aqueous dispersion, preferably, the CTAB adsorption-gold nanorod is repeated by repeating the washing operation of adding water for redispersion after removing the supernatant by centrifuging. Excess CTAB contained in the aqueous dispersion should be removed. If CTAB is excessively removed by this washing operation, the gold nanorods aggregate and do not re-disperse in water.

  Next, when PSS having a weight average molecular weight of 200,000 or less, more preferably 1000 to 100,000 is added and stirred in the CTAB adsorption-gold nanorod aqueous dispersion prepared in this way, the negatively charged PSS is obtained. CTAB adsorption coated with positively charged CTAB-electrostatically adsorbed on the surface of gold nanorods to obtain an aqueous dispersion of PSS-coated gold nanorods (PSS-NR) (PSS-NR aqueous dispersion) . Here, the amount of PSS added is usually 0.1 mg / ml or more and 100 mg / ml or less, preferably 1 mg / ml or more and 10 mg or less with respect to the CTAB adsorption-gold nanorod aqueous dispersion having a gold content of 0.1 mg / ml. When the addition amount of PSS is less than 0.1 mg / ml, the treatment amount on the surface of the gold nanorods is insufficient and aggregation of the gold nanorods occurs. On the contrary, the addition amount of PSS If the amount is more than 100 mg / ml, an excess of PSS that is not adsorbed on the gold nanorods is generated, which is disadvantageous in cost.

  Further, with respect to the above-mentioned step C, the metal nanorod is fixed to the transparent conductive substrate by the interaction between the first water-soluble molecule on the conductive transparent substrate side and the second water-soluble molecule on the metal nanorod side. Therefore, it is sufficient that the conductive transparent substrate surface-treated with these first water-soluble molecules and the metal nanorod composite coated with the second water-soluble molecules can be brought into contact with each other. What is necessary is just to prepare the dispersion liquid of the metal nanorod composite body coat | covered with water-soluble molecule | numerator, and making it contact by appropriate means, such as application | coating, such as immersion, brush coating, and spraying, using this prepared dispersion liquid. For example, in order to fix the gold nanorods (PSS-NR) obtained in the above-mentioned B step on the surface of the APTES adsorption-conductive transparent substrate obtained in the above-mentioned A step, it was simply obtained in the B step. The PAH adsorption-conductive transparent substrate obtained in step A may be immersed in the PSS-NR aqueous dispersion, then washed with water and dried. As a result, the gold nanorods can be uniformly distributed on the surface of the PAH adsorption-conductive transparent substrate. Gold nanorod-immobilized substrate (metal nanorod-immobilized substrate) with PSS-NR) immobilized thereon is obtained.

  Furthermore, for the above step D, it is sufficient that the surface modifier can be adsorbed to the metal nanorod composite of the metal nanorod-immobilized substrate prepared as described above. It is possible to prepare a surface modifier solution by dissolving in, and immerse the metal nanorod-immobilized substrate in the prepared surface modifier solution, or brush the surface modifier solution on the metal nanorod-immobilized substrate. Alternatively, it may be applied by means such as spraying. By this D step, an analysis substrate (composite analysis substrate) in which the surface modifier is held on the metal nanorod composite of the metal nanorod-immobilized substrate is obtained.

  Here, regarding the solvent used for preparing the surface modifier solution, the type of the surface modifier used and the first and second water-soluble molecules for fixing the metal nanorods on the conductive transparent substrate. It is appropriately selected depending on the type, for example, water, alcohols such as methanol, ethanol, propanol, 2-propanol, butanol and diacetone alcohol, ketones such as acetone, 2-butanone and acetylacetone Esters such as ethyl acetate, ethers such as diethyl ether and tetrahydrofuran (THF), aliphatic hydrocarbons such as hexane and petroleum ether, aliphatic and aromatic halogenated groups such as chloroform, methylene chloride and chlorobenzene Hydrocarbons, aromatic hydrocarbons such as benzene, toluene, etc. It can be used alone or as a mixed solution of two or more.

  In addition, in the present invention, if necessary, a capture substance for capturing a target substance is brought into contact with the metal nanorod complex of the analysis substrate obtained in the above-mentioned D process by the above-mentioned E process. An analytical substrate (composite analytical substrate) holding the substance is prepared, but there is no particular limitation on the method of contacting the capture substance at this time. For example, a capture substance solution in which the capture substance is dissolved is prepared in advance. The analysis substrate obtained in the D step may be immersed in the prepared capture substance solution, and the capture substance solution may be brushed, sprayed, etc. on the analysis substrate obtained in the D step. The substrate for analysis (composite analysis substrate) in which the capture substance is held together with the surface modifier on the metal nanorod complex of the substrate immobilized with metal nanorods may be obtained.

  In addition, in the present invention, if necessary, in addition to the above-mentioned F step, the metal nanorod composite of the analysis substrate obtained in the above D step may be subjected to proton addition to the analysis sample and / or in ionization of mass spectrometry. Prepare a substrate for analysis (substrate for composite analysis) by holding a compound with proton supply ability or sodium ion supply ability used for the purpose of sodium ion addition and holding this compound. There is no particular limitation on the method to be performed, for example, a solution in which the compound is dissolved in advance is prepared, and the prepared solution may be applied to the analysis substrate obtained in the step D by means such as dropping or spraying, As a result, the metal nanorod composite of the metal nanorod-immobilized substrate has a proton supplying ability and / or a sodium ion supplying ability together with the surface modifier. That compound is a substrate for analysis, which is held (substrate composite analysis) is obtained.

  When performing both LSPR spectroscopic analysis and SALDI mass spectrometry analysis using the composite analysis substrate obtained as described above, first prepare the sample substrate by supporting the analysis sample on the composite analysis substrate. Then, LSPR spectroscopic analysis and SALDI mass spectrometry may be performed using the prepared sample substrate according to conventional methods. Based on the absorption spectrum obtained by LSPR spectroscopy analysis of this sample substrate, it is judged whether or not the target substance is present in the analytical sample (screening test), and if there is a possibility that the target substance is present in the analytical sample Then, SALDI mass spectrometry is performed using the same sample substrate, and the target substance in the analysis sample is qualitatively determined from the obtained mass spectrum.

Further, in the analysis method of the present invention, as shown in the above F step, if necessary, in the mass analysis, the analysis sample carried on the sample substrate may be irradiated with protons and / or target substances by laser irradiation. An ionization aid that supplies cations may be added, thereby obtaining higher detection sensitivity in SALDI mass spectrometry. For example, in the case of angiotensin I, which is a kind of biological substance, in experiments using trifluoroacetic acid (CF ₃ COOH) as an ionization aid, it is a practical detection sensitivity of fmol order (1 fmol = 0.001 pmol). ) And of course an amol order (1 amol = 0.001 fmol).

  According to the composite analysis substrate of the present invention, using the same sample substrate obtained by supporting the analysis sample on the analysis substrate, LSPR spectroscopic analysis and SALDI mass spectrometry can be continuously performed. In addition to the need to prepare analytical substrates and analytical samples in duplicate, the preparation of the sample substrate carrying the analytical sample can be done only once, and the labor required for spectroscopic analysis and mass spectrometry can be significantly reduced. .

FIG. 1 is a graph showing absorption spectra (a) to (e) of LSPR spectroscopic analysis measured for angiotensin I using the composite analysis substrate according to Example 1 of the present invention.

FIG. 2 is a graph showing the shift amount of the LSPR band with respect to the supported concentration of angiotensin I measured when 100 μL of an angiotensin I solution was dropped using the composite analysis substrate according to Example 1 of the present invention.

FIG. 3 is a graph showing mass spectra (a) to (f) of SALDI mass spectrometry measured for angiotensin I using the composite analysis substrate according to Example 1 of the present invention.

FIG. 4 is a graph showing a mass spectrum of SALDI mass spectrometry measured for the composite analysis substrate (sample substrate a) according to Example 2 of the present invention.

FIG. 5 is a graph showing mass spectra of SALDI mass spectrometry measured for sample substrates b to e according to Example 2 of the present invention.

FIG. 6 is a graph showing mass spectra of SALDI mass spectrometry measured for sample substrates f to k according to Example 2 of the present invention.

FIG. 7 is a graph showing mass spectra of SALDI mass spectrometry measured for sample substrates 1 to o according to Example 2 of the present invention.

  Hereinafter, preferred embodiments of the present invention will be described in more detail based on examples and test examples.

[Example 1]
[Preparation of gold nanorod aqueous dispersion]
1 ml of gold nanorod aqueous dispersion (gold content 0.3 mg / ml) synthesized in 400 mM CTAB aqueous solution was put into a centrifuge tube, and 15000 (× g) relative centrifugal acceleration (centrifugal acceleration divided by the gravitational acceleration of the earth) And the gold nanorods were allowed to settle at the bottom of the centrifuge tube, and the supernatant containing CTAB was removed. Water was added to the precipitated gold nanorods and redispersed to obtain 1 ml of a gold nanorod aqueous dispersion. This operation was repeated twice to obtain 1 ml of a gold nanorod aqueous dispersion from which excess CTAB was removed (NR aqueous dispersion, gold content 0.3 mg / ml).

[Preparation of APTES-modified ITO substrate (Step A)]
A hydrophilic substrate obtained by subjecting an ITO substrate with a size of 75 mm x 25 mm x 0.9 mm to a hydrophilic treatment at a reflux temperature of a 1: 1 (v / v) solution of 25 wt% -ammonia water-30 wt% -hydrogen peroxide solution. ITO substrate is immersed in 0.1 ~ 10wt% -2-propanol (2-propanol) solution of 3-aminopropyltriethoxy-silane (APTES) for 1 hour, and APTES modified ITO substrate (surface treatment substrate) ) Was prepared.

[Preparation of gold nanorod composite (step B)]
Gold nanorod composite coated with PSS by adding 1 ml of 3 mg / ml polystyrene sulfonate (PSS, polymerization average molecular weight 70000) aqueous solution to 1 ml of NR aqueous dispersion obtained above and stirring at 25 ° C. for 1 hour (PSS-NR) was prepared. After completion of the stirring, the mixture was centrifuged at 8000 (× g) for 10 minutes to allow PSS-NR to settle at the bottom of the centrifuge tube, thereby removing excess PSS. Water was added to the precipitated PSS-NR to obtain 1 ml of a PSS-NR aqueous dispersion in which PSS-NR was dispersed (PSS-NR aqueous dispersion, gold content 0.3 mg / ml).

[Preparation of PSS-NR fixed ITO substrate]
Next, the APTES-modified ITO substrate obtained above was immersed in a PSS-NR aqueous dispersion, the substrate was dried, and the PSS was applied to the surface of the APTES-modified ITO substrate by electrostatic interaction between APTES and PSS. -NR was fixed, and a PSS-NR fixed ITO substrate (metal nanorod fixed substrate) was prepared.

[Preparation of substrate for composite analysis]
Further, the prepared PSS-NR fixed ITO substrate was immersed in a 1-100 mM ethanol solution of 4-aminothiophenol (4-ATP), the substrate was dried, and gold was deposited on the ITO substrate. The composite analysis substrate of Example 1 in which the nanorod complex was immobilized and 4-ATP was retained was prepared.

[Preparation of sample substrate]
100 μl of an aqueous solution having angiotensin concentrations of 0 μM, 0.005 μM, 0.01 μM, 0.25 μM, and 0.5 μM dissolved in water was dropped onto the composite analysis substrate of Example 1 prepared in this manner, and an angiotensin I as an analytical sample was dropped. (ATI) -supported sample substrate (a: ATI-0mol-supported sample substrate, b: ATI-0.5 pmol) (ATI) supported at 0 mol, 0.5 pmol, 1 pmol, 25 pmol, and 50 pmol (ATI) (Support-sample substrate, c: ATI-1pmol support-sample substrate, d: ATI-25pmol support-sample substrate, and e: ATI-50pmol support-sample substrate) and an ITO substrate without a gold nanorod composite A sample substrate carrying 50 pmol of angiotensin I (f: ATI-50 pmol carrying-ITO substrate) was prepared.

[LSPR spectroscopic analysis of sample substrate]
For the sample substrates (a) to (e) prepared above, spectral characteristics (disappearance spectrum) in the wavelength region of 400 to 1200 nm using an ultraviolet-visible near-infrared spectrophotometer (V-570 manufactured by JASCO Corporation) (Extinction) was measured.
The results are shown in FIG. Moreover, the plot of the peak shift amount of the absorption spectrum with respect to the angiotensin I carrying concentration is shown in FIG.

[SALDI mass spectrometry of sample substrate]
For the sample substrates (a) to (f) prepared above, a MALDI-TOF / MS apparatus (AXIMA-CFR manufactured by Shimadzu Seisakusho Co., Ltd.) was used, and a 337 nm N ₂ pulse laser (3 ns) was used as the excitation light source. ), Time-of-flight mass spectrometry was performed, and a mass spectrum was measured.
The results are shown in FIG.

  As is clear from the results shown in FIG. 1A, in the LSPR spectroscopic analysis, the two SP bands in the absorption spectrum of ATI-0pmol-supported sample substrate a are fixed on the ITO substrate without aggregation of gold nanorods. In all cases, a peak shift due to the loading of angiotensin I is observed. Further, as is apparent from the results shown in FIG. 2, a tendency that the peak shift amount tends to increase as the concentration of angiotensin I supported increases.

  Further, as apparent from the results shown in FIG. 3, in SALDI mass spectrometry, an ITO substrate without gold nanorod complex and 50 pmol of angiotensin I supported on ATI-50 pmol-supported ITO substrate f and ATI-without angiotensin I supported On the other hand, the peak of angiotensin I was not observed in both the 0 pmol support and the sample substrate a, whereas the gold nanorod complex was fixed and the ATI-0.5 pmol support supporting the angiotensin I and the sample substrate b was 1296.5 m / z. Of ATI-1 pmol support-1298.2 m / z for sample substrate c, 1294.2 m / z for ATI-25 pmol support-sample substrate d, and 1293.2 m / z for ATI-50 pmol support-sample substrate e “Angiotensin I peak” was observed.

[Example 2]
[Preparation of sample substrate]
Sample solutions having an angiotensin I (ATI) concentration of 0 μmol, 0.005 μM, 0.01 μM, 0.25 μM, and 0.5 μM, respectively, were prepared in water, and 100 μL of each sample solution was prepared in the above Example 1. A group A (pM) which was dropped onto a composite analysis substrate prepared in the same manner as above and air-dried to carry angiotensin I (ATI) as an analysis sample at a loading of 0 mol, 0.5 pmol, 1 pmol, 25 pmol and 50 pmol. Sample substrates a to e (Angiotensin I supported-substrate for composite analysis) (a: ATI-0mol supported-sample substrate, b: ATI-0.5pmol supported-sample substrate, c: ATI-1pmol supported-sample substrate, d: ATI-25 pmol-supported sample substrate and e: ATI-50 pmol-supported sample substrate) and ATI concentrations 0 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM,. 5nM and 1nM sample solutions were prepared, and each of these sample solutions 100 μL was dropped onto a composite analysis substrate prepared in the same manner as in Example 1 above, and air-dried to carry an angiotensin I (ATI) as an analysis sample in an amount of 0 mol, 0.5 fmol, 1 fmol, 5 fmol, 10 fmol, 50 fmol. , And group B (fM) sample substrates a and fk supported at 100 fmol (angiotensin I support-composite analysis substrate) (a: ATI-0mol support-sample substrate, f: ATI-0.5 fmol support-sample substrate , G: ATI-1 fmol support-sample substrate, h: ATI-5 fmol support-sample substrate, i: ATI-10 fmol support-sample substrate, j: ATI-50 fmol support-sample substrate, and k: ATI-100 fmol support- In the same manner, sample solutions having ATI concentrations of 0 pM, 0.01 pM, 0.1 pM, 0.5 pM, and 1 pM were prepared, and 100 μL of each of these sample solutions was prepared as in Example 1 above. Drop the sample onto a composite analysis substrate prepared in the same way, let it air dry, Group C (aM) sample substrates a and l-o (angiotensin I supported-substrate for complex analysis) loaded with 0 mol, 1 amol, 10 amol, 50 amol, and 100 amol of supported I (ATI) (a: ATI-0 mol) Support-sample substrate, l: ATI-1amol support-sample substrate, m: ATI-10amol support-sample substrate, n: ATI-50amol support-sample substrate, and o: ATI-100amol support-sample substrate).

[LSPR spectroscopic analysis of sample substrate]
About each sample board | substrate (a)-(o) of each group AC prepared in this way, a wavelength 400-1200 nm is used using an ultraviolet visible near-infrared spectrophotometer (JASCO Corporation V-570). Spectral characteristics (disappearance spectrum) in the region were measured, and the peak wavelength and peak shift value in the disappearance spectrum of each of the obtained sample substrates (a) to (o) were determined.
The results are shown in Table 1.

[SALDI mass spectrometry of sample substrate]
Next, 0.1% -trifluoroacetic acid solution was prepared by dissolving trifluoroacetic acid as an ionization aid in water at a concentration of 0.1%. 100 μl each was added onto the analysis samples carried on the substrates (a) to (o) and allowed to dry naturally, and then time-of-flight mass spectrometry was performed in the same manner as in Example 1 to measure mass spectra. .

  The mass spectrum measured for the sample substrate (a) loaded with 0 mol of angiotensin I (ATI) as an analysis sample is shown in FIG. 3, and sample substrates (b) to (b) of group A (pM) loaded with angiotensin I (ATI) are shown. FIG. 5 shows the mass spectrum measured for (e), FIG. 6 shows the mass spectrum measured for the sample substrates (f) to (k) of group B (fM), and further shows the mass spectrum of group C (aM). FIG. 7 shows mass spectra measured for the sample substrates (l) to (o).

  The monoisotopic mass of angiotensin I in this measurement mode is m / z = 1296.7, which is not observed on ATI-0mol-supported sample substrate a, whereas ATI-0.01pmol-supported sample substrate 1 to ATI −0.5 μmol supported—Angiotensin I peaks were observed at all concentrations up to the sample substrate e, and were found to be detectable in the amol order.

Claims (10)

A composite analysis substrate for carrying an analysis sample and performing spectroscopic analysis and mass spectrometry of a target substance in the analysis sample,
A conductive transparent substrate surface-treated with a first water-soluble molecule, coated with a second water-soluble molecule having an interaction necessary for binding to the first water-soluble molecule, and the first water-soluble molecule; A metal nanorod complex immobilized on the conductive transparent substrate by the interaction of one water-soluble molecule and a second water-soluble molecule, and held on the metal nanorod complex to promote ionization of the target substance 4 -A substrate for composite analysis, comprising a surface modifier such as aminothiophenol.   2. The composite analysis substrate according to claim 1, wherein the metal nanorod complex fixed on the conductive transparent substrate further holds a capture substance for binding to the target substance and capturing the target substance.   The composite analysis substrate according to claim 1 or 2, wherein the metal nanorods constituting the metal nanorod composite are gold nanorods.   The substrate for complex analysis according to claim 1, wherein the surface modifier that promotes ionization of the target substance is 4-aminothiophenol. A method of manufacturing a composite analysis substrate for carrying an analysis sample and performing spectroscopic analysis and mass spectrometry of a target substance in the analysis sample,
(A) preparing a surface-treated substrate by surface-treating the conductive transparent substrate with a first water-soluble molecule;
(B) A second water-soluble molecule having an interaction necessary for binding to the first water-soluble molecule is added to the dispersion of the metal nanorods, and the metal nanorod is added with the second water-soluble molecule. Coating to form a metal nanorod composite;
(C) preparing a metal nanorod-immobilized substrate in which the metal nanorod composite is immobilized on the surface-treated substrate by bringing the surface-treated substrate into contact with the metal nanorod composite;
(D) contacting the surface of the metal nanorod-immobilized substrate obtained in step C with a surface modifier that promotes ionization of the target substance, and preparing a substrate for analysis holding the surface modifier. A method for manufacturing a composite analysis substrate.   (E) The method further comprises the step of preparing the analysis substrate holding the capture substance by further bringing the analysis substrate obtained in the step D into contact with a capture substance for capturing the target substance by binding to the target substance. Item 6. A method for producing a composite analysis substrate according to Item 5.   The method for producing a composite analysis substrate according to claim 5 or 6, wherein the metal nanorods are gold nanorods.   The method for producing a substrate for composite analysis according to claim 5, wherein the surface modifier is 4-aminothiophenol.   A method for analyzing a target substance in an analysis sample using the composite analysis substrate according to any one of claims 1 to 4, wherein the analysis sample is supported on the composite analysis substrate to prepare a sample substrate. Screening whether the target substance is present in the analysis sample from the absorption spectrum obtained by spectroscopic analysis of the sample substrate, and then in the analysis sample from the mass spectrum of the mass analysis performed using the same sample substrate An analysis method characterized by qualitating a target substance.   The analysis method according to claim 9, wherein an ionization auxiliary agent that supplies protons and / or cations to a target substance by laser irradiation is added to an analysis sample supported on a sample substrate during the mass spectrometry.

Images