JP2012207954A - Substrate for multiple analysis, manufacturing method thereof, and analytical method - Google Patents
Substrate for multiple analysis, manufacturing method thereof, and analytical method Download PDFInfo
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- JP2012207954A JP2012207954A JP2011072194A JP2011072194A JP2012207954A JP 2012207954 A JP2012207954 A JP 2012207954A JP 2011072194 A JP2011072194 A JP 2011072194A JP 2011072194 A JP2011072194 A JP 2011072194A JP 2012207954 A JP2012207954 A JP 2012207954A
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
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Abstract
Description
この発明は、標的物質を担持して分光分析と質量分析の両分析を行うための複合分析用基板に係り、特に、局在表面プラズモン共鳴(Localized Surface Plasmon Resonance:LSPR)を利用した分光分析(LSPR分光分析)と、表面支援レーザー脱離イオン化質量分析(Surface Assisted Laser Desorption/Ionization Mass Spectroscopy:SALDI-MS)(SALDI質量分析)とにおいて、金属ナノロッドを前記LSPR分光分析のマーカーとして用いると共に前記SALDI質量分析の支援物質として用いる複合分析用基板及びその製造方法、並びに、この複合分析用基板を用いて分光分析と質量分析を行う分析方法に関する。 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.
近年、インフルエンザウイルスの検出、妊娠検査等を始めとして、各種の遺伝病、各種の癌、感染症、生活習慣病等の診断、治療方針の決定、治療後の予後管理等の医療を目的に、あるいは、環境規制物質の検査や麻薬等の薬物検査等の環境保護や犯罪取締を目的に、遺伝子、各種のタンパク質、ペプチド、多糖類等の種々の生体関連物質や種々の化学物質(標的物質)の検出が頻繁に行われるようになり、その手段の一つとして、金属微粒子をマーカーとするLSPR分光分析が知られており、特に金ナノロッドのLSPRをセンシング技術に応用する技術も多数提案されている(例えば、非特許文献1〜6及び特許文献1、2参照)。
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
一方、質量分析(MS)は、分析試料中の標的物質をイオン化して質量別に分離し、分子量の測定、及び解離生成物(フラグメントイオン)による構造解析を可能とする分析法であり、標的物質をイオン化し、質量(m)とそのイオンの価数(z)の商(m/z)の差により分離・検出を行うものであって、その検出感度や選択性が高く、多くの分野で種々の標的物質の定性や同定等を目的に、幅広く利用されている。そして、このような質量分析の標的物質をイオン化する手法として、分析試料中の標的物質にレーザー光を照射してこの標的物質をイオン化するレーザー脱離イオン化法(Laser Desorption/Ionization:LDI)がある。LDI法によりイオン化されたイオンは、飛行時間型(Time-of-Flight, TOF)等の質量分離部を通ることによってm/zの差で分離され、検出器で観測される。また、検出された時の分子イオン濃度によってピークに強度が現れる。得られた質量スペクトルを解析することで測定対象とする試料分子の構造解析が行える(LDI-MS)。 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).
しかるに、このLDI−MSにおいても、標的物質の種類によっては効率的なイオン化ができない場合があり、この点の改善を目的に様々な改良方法が提案されており、その一つとしてSALDI質量分析がある。そして、このSALDI質量分析については、例えば、サブミクロンオーダーの多孔質層を備えたポーラスシリコンプレートを分析用基板として用いる方法(特許文献3及び非特許文献7)、ワイヤ状金属、樹枝状金属、花弁状金属等からなる金属層を備えた分析用基板を用いる方法(特許文献4)、TiO2、ZnO、SnO2、ZrO2等の金属酸化物からなる膜又は金属酸化物微粒子を備えた分析用基板を用いる方法(特許文献5)、ナノサイズの金属又は金属酸化物微粒子を備えた分析用基板を用いる方法(特許文献6)等が提案されているほか、基材上にFe、Co、及びCuから選ばれた1種又は2種以上の金属の酸化物からなる金属酸化物層が設けられた分析用基板を用いる方法(特許文献7)や基材上にコバルト等の金属微粒子を塗布した分析用基板を用いる方法(特許文献8)等が提案されている。更に、基板上に配置した複数の金属微粒子を、飛散防止膜で被覆した分析用基板を用いる方法(特許文献9)等が提案されている。 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).
しかしながら、上記のLSPR分光分析は、そのいずれも、あくまでも分析試料(検体)中に標的物質が存在する可能性があるか否かのスクリーニングテストを目的とする簡易分析法であり、このLSPR分光分析により陽性が疑われる分析試料が見つけ出された場合には、この陽性が疑われた分析試料について、改めて質量分析等によってより正確な標的物質の定性分析を行い、標的物質の同定を行うことが必要である。このため、このLSPR分光分析については、多数の分析試料を効率良く短時間で実施できることが求められており、検査素子及び標識用試薬を備えた検査キットの形態や、基板上にLSPRのマーカーとしての金ナノロッドを固定化すると共に標的物質の捕捉物質を修飾した分析用チップの形態で提供することも提案されている(特許文献1及び2参照)。
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
また、上記のSALDI質量分析においても、事情は上記のLSPR分光分析の場合と変わりなく、多数の分析試料を効率良く短時間で実施できることが求められている。しかしながら、このSALDI質量分析とLSPR分光分析とは、その分析原理が全く異なるものであり、LSPR分光分析で用いた分析用基板をそのままSALDI質量分析の分析用基板として用いることができず、LSPR分光分析により陽性が疑われた分析試料についてSALDI質量分析を行う際には、改めてSALDI質量分析のための分析試料を調製し、この新たに調製された分析試料をSALDI質量分析のための分析用基板に担持させる必要があり、LSPR分光分析用とSALDI質量分析用とで、それぞれ別個の分析用基板を用意しなければならないほか、分析試料についても重複して調製しなければならない。 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.
ところで、非特許文献8には、金ナノロッドを用いてLSPR−LDI−MS分析を行うことが報告されており、セチルトリメチルアンモニウムブロマイド(cethyltrimethylammonium brimide:CTAB)で処理された金ナノロッドの分散液を溶液状態のまま用いてLSPR分光分析を行い、また、4-アミノチオフェノール(4-aminothiophenol:4‐ATP)で処理された金ナノロッドの分散液をサンプル基板上に滴下して乾燥させ、このサンプル基板上に形成されたサンプルスポットを用いてLDI−MSを行うことが記載されている。
By the way, Non-Patent
しかしながら、この非特許文献8においては、金ナノロッドをLSPRのマーカーとしてLSPR分光分析を行う際には溶液中での分散性に優れたCTAB処理金ナノロッドの分散液を用いる必要があり、また、金ナノロッドを支援物質としてLDI−MSを行う際には、CTAB処理金ナノロッドを質量分析に適した4‐ATP処理金ナノロッドに変換してこの4‐ATP処理金ナノロッドの分散液を調製し、調製された4‐ATP処理金ナノロッドの分散液をサンプル基板上に滴下し乾燥させてサンプルスポットを調製する必要がある。このため、分光分析と質量分析のたびにそれぞれ各分析に適したCTAB処理金ナノロッドや4‐ATP処理金ナノロッドを準備し、また、LSPR分光分析用の分析試料とSALDI質量分析用の分析試料とを別個に重複して用意しなければならず、これらの分析に多大な手間を要し、多数の分析試料を効率良く短時間で分析するという時代の要請に応えられるものではなく、分析試料として検体を重複して提供する被験者にとっても多大なストレスの原因になる。
However, in
なお、金ナノロッドや銀ナノロッドの合成方法としては、例えば特許文献10〜14や非特許文献9〜14等、これまでに多くの報告がある。
As a method for synthesizing gold nanorods and silver nanorods, there have been many reports so far, such as
そこで、本発明者らは、分析用基板に分析試料を担持させて得られた同じ試料基板を用いてLSPR分光分析とSALDI質量分析とを行うことができ、これによって分析用基板や分析試料を重複して調製する必要がないほか、分析試料を担持した試料基板の調製も1回で済み、分光分析と質量分析を行う際の手間を顕著に軽減できる複合分析用基板の開発について鋭意検討した結果、意外なことには、導電性透明基板上に金属ナノロッドを均一に分散させた状態で固定し、その後にこの金属ナノロッドに標的物質のイオン化を促進する表面修飾剤を保持させることにより、分光分析と質量分析の両分析を行うことができる複合分析用基板を調製できることを見出し、本発明を完成した。 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.
従って、本発明の目的は、分析試料を担持させて分光分析と質量分析の両分析を行うことができる複合分析用基板を提供することにある。 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.
また、本発明の他の目的は、このような分析試料を担持させて分光分析と質量分析の両分析を行うことができる複合分析用基板を製造するための複合分析用基板の製造方法を提供することにある。 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.
更に、本発明の目的は、このような分光分析と質量分析の両分析を行うことができる複合分析用基板に分析試料を担持させ、分光分析と質量分析の両分析を行う分析方法を提供することにある。 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.
すなわち、本発明は、分析試料を担持して分析試料中の標的物質の分光分析及び質量分析を行うための複合分析用基板であり、
第一の水溶性分子で表面処理された導電性透明基板と、前記第一の水溶性分子と互いに結合するために必要な相互作用を有する第二の水溶性分子で被覆され、かつ、前記第一の水溶性分子と第二の水溶性分子との相互作用により前記導電性透明基板上に固定された金属ナノロッド複合体と、この金属ナノロッド複合体に保持され、標的物質のイオン化を促進する4‐アミノチオフェノール等の表面処理剤とを備えていることを特徴とする複合分析用基板である。
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.
また、本発明は、分析試料を担持して分析試料中の標的物質の分光分析及び質量分析を行うための複合分析用基板を製造する方法であり、
(A)導電性透明基板を第一の水溶性分子で表面処理して表面処理基板を調製する工程と、
(B)金属ナノロッドの分散液中に前記第一の水溶性分子と互いに結合するために必要な相互作用を有する第二の水溶性分子を添加し、この第二の水溶性分子で金属ナノロッドを被覆して金属ナノロッド複合体を形成する工程と、
(C)前記表面処理基板と金属ナノロッド複合体とを接触させることにより表面処理基板に金属ナノロッド複合体を固定させた金属ナノロッド固定化基板を調製する工程と、
(D)前記C工程で得られた金属ナノロッド固定化基板に表面修飾剤を接触させてこの表面修飾剤を保持した分析用基板を調製する工程と
を有することを特徴とする複合分析用基板の製造方法である。
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.
更に、本発明は、上記の複合分析用基板を用いて分析試料中の標的物質を分析する方法であり、前記複合分析用基板に分析試料を担持させて試料基板を調製し、この試料基板の分光分析で得られた吸収スペクトルから分析試料中に標的物質が存在するか否かのスクリーニングを行い、次いで、同じ試料基板を用いて行われた質量分析の質量スペクトルから前記分析試料中の標的物質の定性を行うことを特徴とする分析方法である。 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.
本発明において、前記導電性透明基板は、少なくとも金属ナノロッドが固定される表面が第一の水溶性分子で表面処理されていることが必要であり、また、上記の金属ナノロッドは、前記第一の水溶性分子と互いに結合するために必要な相互作用を有する第二の水溶性分子で被覆されていることが必要である。 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.
ここで、本発明で用いる導電性透明基板の素材については、分光分析を行う上で必要な透明性を有すると共に、質量分析を行う上で必要な導電性を有するものであればよく、例えば、ITO(indium-tin oxide)、IZO(indium-zinc oxide)、AZO(aluminum-zinc oxide)、GZO(gallium-zinc oxide)、TO(tin oxide)、IO(indium oxide)等の金属酸化物を挙げることができ、また、熱伝導率が比較的小さくて質量分析の際にレーザーからのエネルギーが散逸し難く、比較的低いレーザーエネルギーで分析できるという観点から、好ましくはITO、IZO、TO等である。特にITOは抵抗率が低く好ましい。また、この導電性透明基板の形状についても、分光分析及び質量分析を行うことができる形状であれば特に制限はなく、表面全体が平滑な平板状であっても、また、表面に分析試料を滴下するための適宜の断面形状(例えば、縦小括弧の括弧閉じ形状、縦大括弧の括弧閉じ形状、縦亀甲括弧の括弧閉じ形状、縦山括弧の括弧閉じ形状等の形状)を有する1つ又は複数の凹部(ウェル)を備えたものであってもよい。 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).
また、本発明で用いる金属ナノロッドとしては、LSPR分光分析を行う必要からLSPRが発生し得るナノ(nano)サイズの金属粒子であり、その種類については、例えば、金(Au)、銀(Ag)、銅(Cu)、白金(Pt)、アルミニウム(Al)、及びこれらの合金等を挙げることができ、その長軸の長さが400nm以下、好ましくは200nm以下であって、アスペクト比が1より大きいロッド形状であるのがよく、具体的には、LSPRの最大吸収波長が700〜2000nmの範囲内にあるアスペクト比の金属ナノロッドが用いられる。長軸長さについては、400nmを超えて長くなると、沈降し易くなる傾向があり、基板に金属ナノロッドを吸着させる工程において吸着時間の確保が難しくなる。金属ナノロッドとしては、金ナノロッドが好ましい。金ナノロッドを基板上に固定化した場合、化学的に安定で、酸化され難い。また、金ナノロッドは、表面修飾剤のチオール基と共有結合を形成するため、効果的に表面修飾剤を金ナノロッド複合体中に保持することが可能となる。 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.
そして、前記導電性透明基板を表面処理する第一の水溶性分子と前記金属ナノロッドを被覆する第二の水溶性分子とは、互いに結合するために必要な相互作用を有するものであればよく、例えば、アニオン性分子、カチオン性分子、水素結合性分子等を挙げることができ、そして、第一の水溶性分子としてアニオン性分子(カチオン性分子)を用いた場合には、第二の水溶性分子としてカチオン性分子(アニオン性分子)を用いることにより、静電気的に導電性透明基板上に金属ナノロッドを固定化することができ、また、第一の水溶性分子と第二の水溶性分子としてそれぞれ水素結合性分子を用いた場合には、水素結合を介して導電性透明基板上に金属ナノロッドを固定化することができる。 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.
ここで、前記アニオン性分子としては、例えば、ポリスチレンスルホネート(PSS)、ポリアクリル酸、ポリカルボン酸、ポリリン酸等のアニオン性高分子等やドデシル硫酸ナトリウム(SDS)、コール酸、アスコルビン酸、クエン酸等のアニオン性低分子を例示することができ、また、前記カチオン性分子としては、例えば、ポリアリルアミンハイドロクロライド(PAH)、ポリアミノ酸等のカチオン性高分子等や3-アミノプロピルトリエトキシシラン(3-aminopropyltriethoxysilane;APTES)、Poly(diallyldimethylammonium chloride) (PDDA) 、CTAB、セチルトリメチルアンモニウムクロライド(cethyltrimethylammonium chloride : CTAC)、2-アミノエタンチオールハイドロクロライド(2-AET)、4-アミノブタンチオールハイドロクロライド、6-アミノヘキサンチオールハイドロクロライド等のカチオン性低分子等を例示することができ、更に、前記水素結合性分子としては、核酸(DNA、RNA)、糖質(セルロース、デンプン)、タンパク質(酵素、ペプチド)等を例示することができる。 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. .
本発明においては、第一の水溶性分子で表面処理された導電性透明基板上に固定され、第二の水溶性分子で被覆された金属ナノロッド(金属ナノロッド複合体)については、SALDI質量分析を行う上で、標的物質のイオン化を促進する作用又は機能を有する表面修飾剤が保持されていることが必要であり、この目的で使用される表面修飾剤としては、金ナノロッド表面に大量に吸着するとイオン化を阻害する可能性があるCTABを置換除去できる化合物であれば、幅広く利用可能であり、例えばR−SH(R:炭化水素、SH:チオール基)で示されるような、金属ナノロッドに吸着する官能基がチオール基である化合物が好適に利用可能である。このR-SH化合物において、炭化水素Rとしては、プロトンを供給し得る官能基、例えばアミノ基、カルボキシル基、ヒドロキシ基を有する化合物が好ましい。具体的には、4‐アミノチオフェノール(4-ATP)、2−アミノチオフェノール(2-ATP)、6-アミノ-1-ヘキサンチオール、6-カルボキシ-1-ヘキサンチオール、6-ヒドロキシ-1-ヘキサンチオール等を挙げることができる。金属ナノロッドの金属種が金の場合の表面修飾剤としては、好ましくは4‐アミノチオフェノールであり、チオール基にて金ナノロッド表面に共有結合を形成して、金ナノロッド表面に静電気的に吸着し残存しているCTABを排除する効果が得られ、質量分析でノイズの原因となるCTABを低減することが可能となり、金ナノロッド近傍の標的物質のイオン化を効果的に促進することが可能となるばかりでなく、さらにプロトンを供給するアミノ基は金ナノロッドの合成で使用するCTABと同じ電荷であるため金ナノロッド同士の凝集を防止することが可能となる。 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.
これらの表面修飾剤については、上記チオール基を有する表面修飾剤の他に、質量分析のイオン化において、分析試料へのプロトン付加及び/又はナトリウムイオン付加の目的で使用されるプロトン供給能やナトリウムイオン供給能を有するイオン化補助剤を使用してもよい。例えば、プロトン供給能を有するイオン化補助剤としては、トリフルオロ酢酸、クエン酸、クエン酸二水素一アンモニウム、クエン酸一水素二アンモニウム、クエン酸三アンモニウム、酒石酸、リンゴ酸、コハク酸、シュウ酸、ケイ皮酸、ジグリコール酸、セバシン酸等が挙げられる。また、ナトリウムイオン供給能を有するイオン化補助剤としては、トリフルオロ酢酸ナトリウム、ヨウ化ナトリウム、クエン酸二水素一ナトリウム、クエン酸一水素二ナトリウム、酒石酸一水素一ナトリウム、コハク酸一水素一ナトリウム、クエン酸三ナトリウム、酒石酸二ナトリウム、コハク酸二ナトリウムなどが挙げられる。これらは、異なる種類のものを2以上混合して用いてもよい。 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.
本発明において、前記導電性透明基板上に固定された金属ナノロッド複合体には、必要により、前記表面修飾剤と共に、分析試料中の標的物質と結合して分析試料中の標的物質を捕捉する捕捉物質が付着されていてもよい。この目的で用いられる捕捉物質としては、標的物質と特異的に結合する物質であれば幅広く利用可能であり、具体的には、例えば、標的物質がビチオン又はビチオン標識化物質である場合にはアビジンが使用され、また、標的物質が抗原又は抗原標識物質である場合には抗体が用いられ、またアプタマーなどの生体分子認識分子も利用可能である。標的物質と特異的に結合する物質は、金属ナノロッドに吸着した状態が好ましい。標的物質が補足物質と相互作用した際に、金属ナノロッドが近傍に存在すると、金属ナノロッドの吸収波長シフトが明確となる。 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.
そして、本発明の複合分析用基板は、基本的には以下の工程により製造される。すなわち、前記導電性透明基板を第一の水溶性分子で表面処理して表面処理基板を調製するA工程と、前記金属ナノロッドの分散液中に前記第一の水溶性分子と互いに結合するために必要な相互作用を有する第二の水溶性分子を添加し、この第二の水溶性分子で金属ナノロッドを被覆して金属ナノロッド複合体を形成するB工程と、前記表面処理基板と金属ナノロッド複合体とを接触させることにより表面処理基板に金属ナノロッド複合体を固定化させた金属ナノロッド固定化基板を調製するC工程と、前記C工程で得られた金属ナノロッド固定化基板に標的物質のイオン化を促進する表面修飾剤を接触させ、この表面修飾剤を保持した分析用基板を調製するD工程とを経て製造され、更に、必要により、前記D工程で得られた分析用基板を更に標的物質と結合してこの標的物質を捕捉するための捕捉物質と接触させ、捕捉物質を保持した分析用基板を調製するE工程や前記D工程で得られた分析用基板にイオン化を更に促進させる目的でチオール基を含まないイオン化補助剤を添加するF工程を経て製造される。 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.
ここで、上記のA工程においては、導電性透明基板を第一の水溶性分子で表面処理し、導電性透明基板の表面の少なくとも金属ナノロッドが固定される部分に第一の水溶性分子を付着させ、あるいは、吸着させ、若しくは化学的に結合させればよく、通常、第一の水溶性分子の溶液を調製し、この調製された溶液を用いて、浸漬、刷毛塗りや噴霧等の塗布等の適当な手段で導電性透明基板の表面を処理すればよい。例えば、第一の水溶性分子がAPTES(水溶性低分子)である場合には、APTES濃度0.1〜10wt%の2-プロパノール溶液中に導電性透明基板を浸漬して取り出し、次いで水で洗浄した後に常温で乾燥させることにより、APTES吸着-導電性透明基板が得られる。また、第一の水溶性分子がPAH(カチオン性高分子)である場合には、好ましくは重量平均分子量1000〜100000のPAHを用い、PAH濃度0.1〜10mg/mlのPAH水溶液中に導電性透明基板を浸漬して取り出し、次いで水で洗浄した後に常温で乾燥させることにより、PAH吸着-導電性透明基板が得られる。 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.
また、上記のB工程については、前記第二の水溶性分子で金属ナノロッドを被覆することができればよく、例えば、金属ナノロッド水分散液と第二の水溶性分子水溶液を混合する方法等を例示することができる。 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.
ここで、具体的には、例えば金属ナノロッドが金ナノロッドであって、第二の水溶性分子として上記の第一の水溶性分子であるAPTES(水溶性低分子)に対して静電気的な相互作用を有するPSS(アニオン性高分子)を用いた場合を例にして説明すると、具体的には、以下に示す方法により行うことができる。 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.
すなわち、先ず、カチオン性界面活性剤である4級アンモニウム塩〔CH3(CH2)nN+(CH3)3Br-(nは1〜15の整数)〕が溶解した水溶液中で金イオンを還元して金ナノロッドを合成する。金ナノロッドや銀ナノロッドの合成方法としては、例えば、特許文献10〜14や非特許文献9〜14等に記載の金ナノロッドや銀ナノロッドの合成方法で、4級アンモニウム塩としてn=15のヘキサデシルトリメチルアンモニウムブロミド(CTAB)を用い、このCTAB溶液中で金イオンや銀イオンを各種還元方法で還元すると、生成したナノロッドの表面にCTABが吸着し、ナノロッドが水中に安定に分散したCTAB吸着-金ナノロッド水分散液やCTAB吸着-銀ナノロッドが得られる。
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
このCTAB吸着-金ナノロッド水分散液については、好ましくは、遠心分離して上澄みを除去した後に水を添加して再分散させる洗浄操作を通常1〜3回程度繰り返して行い、CTAB吸着-金ナノロッド水分散液に含まれている余剰なCTABを除去するがよい。この洗浄操作によりCTABを過剰に除去すると金ナノロッドが凝集して水に再分散しなくなる。 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.
次に、このようにして調製したCTAB吸着-金ナノロッド水分散液中に、好ましくは重量平均分子量200000以下、より好ましくは1000〜100000のPSSを添加して攪拌すると、負の電荷を持つPSSは正電荷のCTABで被覆されているCTAB吸着-金ナノロッドの表面に静電気的に吸着し、PSSで被覆された金ナノロッド(PSS‐NR)の水分散液(PSS-NR水分散液)が得られる。ここで、PSSの添加量については、金含有率0.1mg/mlのCTAB吸着-金ナノロッド水分散液に対して、通常0.1mg/ml以上100mg/ml以下、好ましくは1mg/ml以上10mg/ml以下であるのがよく、このPSSの添加量が0.1mg/mlより少ないと、金ナノロッド表面への処理量が不足して金ナノロッドの凝集が発生し、反対に、PSSの添加量が100mg/mlより多いと、金ナノロッドに吸着しないPSSの余剰分が発生し、コスト的に不利である。 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.
更に、上記のC工程については、前記導電性透明基板側の第一の水溶性分子と前記金属ナノロッド側の第二の水溶性分子との相互作用により透明導電性基板に金属ナノロッドを固定するものであるから、これら第一の水溶性分子で表面処理された導電性透明基板と第二の水溶性分子で被覆された金属ナノロッド複合体とを互いに接触させることができればよく、例えば、第二の水溶性分子で被覆された金属ナノロッド複合体の分散液を調製し、この調製された分散液を用いて、浸漬、刷毛塗りや噴霧等の塗布等の適当な手段で接触させればよい。例えば、上記のA工程で得られたAPTES吸着-導電性透明基板の表面に上記のB工程で得られた金ナノロッド(PSS‐NR)を固定させるためには、単純にB工程で得られたPSS-NR水分散液中にA工程で得られたPAH吸着-導電性透明基板を浸漬し、次いで水洗し乾燥すればよく、これによってPAH吸着-導電性透明基板の表面に均一に金ナノロッド(PSS‐NR)が固定された金ナノロッド固定化基板(金属ナノロッド固定化基板)が得られる。 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.
更にまた、上記のD工程については、以上のようにして調製された金属ナノロッド固定化基板の金属ナノロッド複合体に表面修飾剤を吸着させることができればよく、例えば、予め表面修飾剤を適当な溶剤に溶解して表面修飾剤溶液を調製し、この調製された表面修飾剤溶液中に金属ナノロッド固定化基板を浸漬してもよく、また、金属ナノロッド固定化基板上に表面修飾剤溶液を刷毛塗り、噴霧等の手段で塗布してもよい。このD工程により、金属ナノロッド固定化基板の金属ナノロッド複合体に表面修飾剤が保持された分析用基板(複合分析用基板)が得られる。 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.
ここで、前記表面修飾剤溶液を調製するために使用される溶剤については、使用する表面修飾剤の種類や、金属ナノロッドを導電性透明基板上に固定する第一及び第二の水溶性分子の種類等に応じて適宜選択されるものであり、例えば、水のほか、メタノール、エタノール、プロパノール、2-プロパノール、ブタノール、ダイアセトンアルコール等のアルコール類、アセトン、2-ブタノン、アセチルアセトン等のケトン類、酢酸エチル等のエステル類、ジエチルエーテル、テトラヒドロフラン(THF)等のエーテル類、ヘキサン、石油エーテル等の脂肪族系炭化水素類、クロロホルム、塩化メチレン、クロロベンゼン等の脂肪族系及び芳香族系ハロゲン化炭化水素類、ベンゼン、トルエン等の芳香族系炭化水素類等を挙げることができ、これらを単独で又は2種以上の混合溶液として用いることができる。 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.
また、本発明においては、必要により、更に上記のE工程により、上記のD工程で得られた分析用基板の金属ナノロッド複合体に、標的物質を捕捉するための捕捉物質を接触させてこの捕捉物質が保持された分析用基板(複合分析用基板)を調製するが、この際の捕捉物質を接触させる方法についても特に制限はなく、例えば、予め前記捕捉物質が溶解した捕捉物質溶液を調製し、この調製された捕捉物質溶液中にD工程で得られた分析用基板を浸漬してもよく、また、D工程で得られた分析用基板上に前記捕捉物質溶液を刷毛塗り、噴霧等の手段で塗布してもよく、これによって金属ナノロッド固定化基板の金属ナノロッド複合体に表面修飾剤と共に捕捉物質が保持された分析用基板(複合分析用基板)が得られる。 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.
また、本発明においては、必要により、更に上記のF工程により、上記のD工程で得られた分析用基板の金属ナノロッド複合体に、質量分析のイオン化において、分析試料へのプロトン付加および/またはナトリウムイオン付加の目的で使用されるプロトン供給能やナトリウムイオン供給能を有する化合物を接触させてこの化合物が保持された分析用基板(複合分析用基板)を調製するが、この際の化合物を接触させる方法についても特に制限はなく、例えば、予め前記化合物が溶解した溶液を調製し、この調製された溶液をD工程で得られた分析用基板に滴下、噴霧等の手段で塗布すればよく、これによって金属ナノロッド固定化基板の金属ナノロッド複合体に表面修飾剤と共にプロトン供給能、及び/又は、ナトリウムイオン供給能を有する化合物が保持された分析用基板(複合分析用基板)が得られる。 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.
そして、以上のようにして得られた複合分析用基板を用いてLSPR分光分析とSALDI質量分析の両分析を行う際には、先ず、複合分析用基板に分析試料を担持させて試料基板を調製し、この調製された試料基板を用いて、それぞれ定法に従ってLSPR分光分析とSALDI質量分析とを行えばよい。この試料基板のLSPR分光分析で得られた吸収スペクトルを基に分析試料中に標的物質が存在するか否かを判定し(スクリーニングテスト)、もし分析試料中に標的物質が存在する可能性が認められたら、引き続き同じ試料基板を用いてSALDI質量分析を行い、得られた質量スペクトルから前記分析試料中の標的物質の定性を行う。 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.
また、本発明の分析方法においては、上記F工程に示されるように、必要により、前記質量分析の際に、試料基板上に担持された分析試料に、レーザー照射により標的物質にプロトン及び/又はカチオンを供給するイオン化補助剤を添加してもよく、これによって、SALDI質量分析においてより高い検出感度を得ることができる。例えば、生体関連物質の一種であるアンギオテンシンI(angiotensin I)の場合、イオン化補助剤としてトリフルオロ酢酸(CF3COOH)を用いた実験では、実用的な検出感度であるfmolオーダー(1fmol=0.001pmol)で分析することができるのは勿論、amolオーダー(1amol=0.001fmol)でも分析可能である。 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).
本発明の複合分析用基板によれば、分析用基板に分析試料を担持させて得られた同じ試料基板を用いて、LSPR分光分析とSALDI質量分析とを連続して行うことができ、これによって分析用基板や分析試料を重複して調製する必要がないほか、分析試料を担持した試料基板の調製も1回で済み、分光分析と質量分析を行う際の手間を顕著に軽減することができる。 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. .
以下、実施例及び試験例に基づいて、本発明の好適な実施の形態をより詳細に説明する。 Hereinafter, preferred embodiments of the present invention will be described in more detail based on examples and test examples.
〔実施例1〕
〔金ナノロッド水分散液の調製〕
400mMのCTAB水溶液中で合成された金ナノロッド水分散液1ml(金含有量0.3mg/ml)を遠沈管に入れ、15000(×g)の相対遠心加速度(遠心加速度を地球の重力加速度で除したもの)で10分間遠心分離して金ナノロッドを遠沈管の底に沈降させ、CTABを含む上澄み液を除去した。沈降した金ナノロッドに水を添加して再分散させ、金ナノロッド水分散液1mlを得た。この操作を2回繰り返し、余剰のCTABを除去した金ナノロッド水分散液1mlを得た(NR水分散液、金含有量0.3mg/ml)。
[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).
〔APTES修飾ITO基板の調製(A工程)〕
75mm×25mm×0.9mmの大きさのITO基板を25wt%-アンモニア水−30wt%-過酸化水素水の1:1(v/v)溶液中還流温度下で親水処理し、得られた親水化ITO基板を3-アミノプロピルトリエトキシシラン(3-aminopropyltriethoxy-silane;APTES)の0.1〜10wt%-2-プロパノール(2-propanol)溶液中に1時間浸漬し、APTES修飾ITO基板(表面処理基板)を調製した。
[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.
〔金ナノロッド複合体の調製(B工程)〕
上で得られたNR水分散液1mlに、濃度3mg/mlのポリスチレンスルホネート(PSS、重合平均分子量70000)水溶液1mlを添加し、25℃で1時間攪拌してPSSで被覆された金ナノロッド複合体(PSS‐NR)を調製した。攪拌終了後、8000(×g)で10分間遠心分離してPSS−NRを遠沈管の底に沈降させ、余剰のPSSを除去した。沈降したPSS−NRに水を添加し、PSS−NRが分散したPSS‐NR水分散液1mlを得た(PSS‐NR水分散液、金含有量0.3mg/ml)。
[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).
〔PSS‐NR固定化ITO基板の調製〕
次に、上で得られたAPTES修飾ITO基板をPSS‐NR水分散液に浸漬後、基板を乾燥して、APTESとPSSとの間の静電相互作用により、APTES修飾ITO基板の表面にPSS−NRを固定化し、PSS‐NR固定化ITO基板(金属ナノロッド固定化基板)を調製した。
[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.
〔複合分析用基板の調製〕
更に、調製されたPSS‐NR固定化ITO基板を4-アミノチオフェノール(4-aminothiophenol;4-ATP)の1〜100mM-エタノール溶液中に浸漬し、基板を乾燥して、ITO基板上に金ナノロッド複合体が固定化され、かつ、4-ATPが保持された実施例1の複合分析用基板を調製した。
[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.
〔試料基板の調製〕
このようにして調製された実施例1の複合分析用基板に、水に溶解したアンギオテンシン濃度がそれぞれ0μM、0.005μM、0.01μM、0.25μM、0.5μMの水溶液を100μl滴下し、分析試料のアンギオテンシンI(ATI)を担持量0mol、0.5pmol、1pmol、25pmol、及び50pmolで担持した試料基板(アンギオテンシンI担持-複合分析用基板)(a:ATI-0mol担持-試料基板、b:ATI-0.5pmol担持-試料基板、c:ATI-1pmol担持-試料基板、d:ATI-25pmol担持-試料基板、及びe:ATI-50pmol担持-試料基板)を調製すると共に、金ナノロッド複合体無しのITO基板にアンギオテンシンIを50pmol担持させた試料基板(f:ATI-50pmol担持-ITO基板)を調製した。
[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.
〔試料基板のLSPR分光分析〕
上で調製された試料基板(a)〜(e)について、紫外可視近赤外分光光度計(日本分光株式会社製V-570)を用い、波長400〜1200nmの領域での分光特性(消失スペクトル(Extinction)を測定した。
結果を図1に示す。また、アンギオテンシンI担持濃度に対する吸収スペクトルのピークシフト量のプロットを図2に示す。
[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.
〔試料基板のSALDI質量分析〕
また、上で調製された試料基板(a)〜(f)について、MALDI-TOF/MS装置(島津セイサクショ株式会社製AXIMA-CFR)を用い、また、励起光源として337nmのN2パルスレーザー(3ns)を用いて照射し、飛行時間型の質量分析を行い、質量スペクトルを測定した。
結果を図3に示す。
[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.
図1中の(a)に示す結果から明らかなように、LSPR分光分析において、ATI-0pmol担持-試料基板aの吸収スペクトルにおける2つのSPバンドはITO基板上に金ナノロッドが凝集することなく固定されていることを示しており、いずれの場合もアンギオテンシンIの担持によるピークシフトが観察される。また、図2に示す結果から明らかなように、アンギオテンシンIの担持濃度が高くなると、ピークシフト量が大きくなる傾向が観察される。 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.
また、図3に示す結果から明らかなように、SALDI質量分析において、金ナノロッド複合体無しのITO基板にアンギオテンシンIを50pmol担持させたATI-50pmol担持-ITO基板fとアンギオテンシンI担持無しのATI-0pmol担持-試料基板aは共にアンギオテンシンIのピークが観察されず、これに対して、金ナノロッド複合体が固定され、アンギオテンシンIを担持したATI-0.5pmol担持-試料基板bでは1296.5m/zの、ATI-1pmol担持-試料基板cでは1298.2m/zの、ATI-25pmol担持-試料基板dでは1294.2m/zの、及びATI-50pmol担持-試料基板eでは1293.2m/zの「アンギオテンシンIのピーク」がそれぞれ観察された。 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.
〔実施例2〕
〔試料基板の調製〕
水中に分析試料のアンギオテンシンI(ATI)濃度がそれぞれ0μmol、0.005μM、0.01μM、0.25μM、及び0.5μMの試料溶液を調製し、これら各試料溶液の100μLを上記の実施例1と同様にして調製された複合分析用基板に滴下し、自然乾燥させて分析試料のアンギオテンシンI(ATI)を担持量0mol、0.5pmol、1pmol、25pmol、及び50pmolで担持したグループA(pM)の試料基板a〜e(アンギオテンシンI担持-複合分析用基板)(a:ATI-0mol担持-試料基板、b:ATI-0.5pmol担持-試料基板、c:ATI-1pmol担持-試料基板、d:ATI-25pmol担持-試料基板、及びe:ATI-50pmol担持-試料基板)を調製し、また同様にして、ATI濃度0nM、0.005nM、0.01nM、0.05nM、0.1nM、0.5nM、及び1nMの試料溶液を調製し、これら各試料溶液の100μLを上記の実施例1と同様にして調製された複合分析用基板に滴下し、自然乾燥させて分析試料のアンギオテンシンI(ATI)を担持量0mol、0.5fmol、1fmol、5fmol、10fmol、50fmol、及び100fmolで担持したグループB(fM)の試料基板a及びf〜k(アンギオテンシンI担持-複合分析用基板)(a:ATI-0mol担持-試料基板、f:ATI-0.5fmol担持-試料基板、g:ATI-1fmol担持-試料基板、h:ATI-5fmol担持-試料基板、i:ATI-10fmol担持-試料基板、j:ATI-50fmol担持-試料基板、及びk:ATI-100fmol担時-試料基板)を調製し、更に同様にして、ATI濃度0pM、0.01pM、0.1pM、0.5pM、及び1pMの試料溶液を調製し、これら各試料溶液の100μLを上記の実施例1と同様にして調製された複合分析用基板に滴下し、自然乾燥させて分析試料のアンギオテンシンI(ATI)を担持量0mol、1amol、10amol、50amol、及び100amolで担持したグループC(aM)の試料基板a及びl〜o(アンギオテンシンI担持-複合分析用基板)(a:ATI-0mol担持-試料基板、l:ATI-1amol担持-試料基板、m:ATI-10amol担持-試料基板、n:ATI-50amol担持-試料基板、及びo:ATI-100amol担持-試料基板)を調製した。
[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).
〔試料基板のLSPR分光分析〕
このようにして調製された各グループA〜Cの各試料基板(a)〜(o)について、紫外可視近赤外分光光度計(日本分光株式会社製V-570)を用い、波長400〜1200nmの領域での分光特性(消失スペクトル)を測定し、得られた各試料基板(a)〜(o)の消失スペクトルにおけるピーク波長と、ピークシフトの値を求めた。
結果を表1に示す。
[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.
〔試料基板のSALDI質量分析〕
次に、水にイオン化補助剤としてトリフルオロ酢酸を0.1%の濃度で溶解して0.1%-トリフルオロ酢酸溶液を調製し、この0.1%-トリフルオロ酢酸溶液を上で調製された各試料基板(a)〜(o)に担持された分析試料の上に100μlづつ添加して自然乾燥させ、その後に、実施例1と同様にして飛行時間型の質量分析を行い、質量スペクトルを測定した。
[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. .
分析試料であるアンギオテンシンI(ATI)担持量0molの試料基板(a)について測定された質量スペクトルを図3に、アンギオテンシンI(ATI)が担持されたグループA(pM)の試料基板(b)〜(e)について測定された質量スペクトルを図5に、また、グループB(fM)の試料基板(f)〜(k)について測定された質量スペクトルを図6に、更に、グループC(aM)の試料基板(l)〜(o)について測定された質量スペクトルを図7に示す。 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).
本測定モードにおけるアンギオテンシンIのモノアイソトピック質量はm/z=1296.7であり、ATI-0mol担持-試料基板aにおいては観察されないのに対して、ATI-0.01pmol担持-試料基板l〜ATI-0.5μmol担持-試料基板eまでの全ての濃度でアンギオテンシンIのピークが観察され、amolオーダーで検出可能であることが判明した。
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
Claims (10)
第一の水溶性分子で表面処理された導電性透明基板と、前記第一の水溶性分子と互いに結合するために必要な相互作用を有する第二の水溶性分子で被覆され、かつ、前記第一の水溶性分子と第二の水溶性分子との相互作用により前記導電性透明基板上に固定された金属ナノロッド複合体と、この金属ナノロッド複合体に保持され、標的物質のイオン化を促進する4‐アミノチオフェノール等の表面修飾剤とを備えていることを特徴とする複合分析用基板。 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.
(A)導電性透明基板を第一の水溶性分子で表面処理して表面処理基板を調製する工程と、
(B)金属ナノロッドの分散液中に前記第一の水溶性分子と互いに結合するために必要な相互作用を有する第二の水溶性分子を添加し、この第二の水溶性分子で金属ナノロッドを被覆して金属ナノロッド複合体を形成する工程と、
(C)前記表面処理基板と金属ナノロッド複合体とを接触させることにより表面処理基板に金属ナノロッド複合体を固定化させた金属ナノロッド固定化基板を調製する工程と、
(D)前記C工程で得られた金属ナノロッド固定化基板に標的物質のイオン化を促進する表面修飾剤を接触させ、この表面修飾剤を保持した分析用基板を調製する工程と
を有することを特徴とする複合分析用基板の製造方法。 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.
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