JP2017053830A - Probe for raman spectroscopic analysis, and imaging method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stimuli-responsive probe for issuing a signal only when the probe exists at a desired timing and in a desired place, and an imaging method using the probe.SOLUTION: The present invention provides: a probe for photoactivation-type Raman spectroscopic analysis that is made of a complex of a copolymer containing, as repeating units, at least a photoactivation-type Raman label unit and a hydrophilic unit and metal particles having a surface enhanced Raman scattering activity; and a method of Raman-imaging a sample using the probe. In the present invention, in a living body and in a biological sample such as a molecule inside or outside a cell, a cell, a cell organelle, or a composition, a chronological/spatial behaviour of a target can be traced by Raman spectroscopic analysis.SELECTED DRAWING: None

Description

  The present invention relates to a probe for photo-activated Raman spectroscopy and a Raman imaging method using the probe.

  In recent years, molecular imaging based on Raman spectroscopy (Raman imaging) has attracted attention. Raman imaging is based on a technique for detecting and visualizing intrinsic vibrations of the molecule, and imaging of unorganized organelles without staining and labeling is possible by using the vibration of localized molecules as an index.

  Raman imaging can also selectively image only a target molecule using a molecular skeleton that does not originally exist in a living body as a tag. As such tags, for example, molecules having carbon-carbon or carbon-nitrogen triple bonds such as acetylene and cyanide, metal nanoparticles, and the like are known, and methods combining these have been reported so far (non-patented). Literatures 1-3).

Journal of American Chemical Society, 136, 2014, 13558-13561 Chemical Communications, 47, 2011, 3156-3158 Nanoscale, 2, 2010, 1413-1416

  On the other hand, since any tag introduced into a sample such as a cell always emits a signal, it is difficult to track the temporal and spatial behavior of the analysis target, and the information obtained from Raman imaging is limited. It was.

  Therefore, an object of the present invention is to provide a stimulus-responsive probe that emits a signal only when it exists at a desired location at a desired timing, and an imaging method using the same.

  The inventors of the present invention have disclosed a photo-activated Raman spectroscopic analysis of a complex of a polymer in which a plurality of hydrophilic sites and a photoactivation site containing an alkyne protected by a cyclopropenone skeleton are introduced, and a metal nanoparticle. Has been found to function as a probe. The inventors of the present invention have also confirmed that an alkyne signal generated from the inside of a cell can be detected with a Raman microscope after the light-activated Raman spectroscopic analysis probe is actually introduced into a living cell and irradiated with light. The present invention is based on these findings.

（上記式中、
Ｒ^１は水素原子またはＣ_１−４アルキルを表し、
Ｒ^２は式（ｉ）：

（上記式中、Ｒ^１１〜Ｒ^２０は、同一または異なっていてもよく、水素原子、Ｃ_１−６アルキル基、Ｃ_１−６アルコキシ基、アミノ基、ニトロ基、アゾ基、メルカプト基、カルボキシル基、フェニル基、不飽和の５〜７員の単環複素環式基または９〜１１員の不飽和の二環複素環式基を表し、Ｒ^１１〜Ｒ^２０の隣り合う二つの基はそれらが結合している炭素原子と一緒になって５〜７員の飽和または不飽和の単環炭素環または単環複素環あるいは９〜１１員の飽和または不飽和の二環炭素環または二環複素環を形成していてもよく、Ｒ^１５およびＲ^１６は一緒になってＣ_１−４アルキレン基を表していてもよく、但し、Ｒ^１１〜Ｒ^２０、Ｒ^１１〜Ｒ^２０の隣り合う二つの基が一緒になって形成する炭素環または複素環上の水素原子並びにＲ^１５およびＲ^１６が一緒になって表すＣ_１−４アルキレン基上の水素原子のいずれか一つは式（ａ）との結合を表す。）
を表す）
で表される、上記（１）に記載の光活性化型ラマン分光分析用プローブ。
（３）親水性ユニットが式（ｂ）：

（上記式中、
Ｒ^３は水素原子またはＣ_１−４アルキルを表し、
Ｒ^４は水素原子、水酸基またはＣ_１−６アルキル基（このアルキル基は１個または２個以上の水酸基、カルボキシル基および／またはアミノ基により置換されていてもよい）を表す）
で表される、上記（１）または（２）に記載の光活性化型ラマン分光分析用プローブ。
（４）共重合体がアクリル系共重合体またはメタクリル系共重合体である、上記（１）〜（３）のいずれかに記載の光活性化型ラマン分光分析用プローブ。
（５）共重合体の数平均分子量が４，０００〜８００，０００である、上記（１）〜（４）のいずれかに記載の光活性化型ラマン分光分析用プローブ。
（６）共重合体が末端に金属粒子表面修飾ユニットをさらに含む、上記（１）〜（５）のいずれかに記載の光活性化型ラマン分光分析用プローブ。
（７）金属粒子表面修飾ユニットが式（ｃ）：

（上記式中、Ｚは−Ｃ（＝Ｓ）−または−ＣＨ（−ＳＨ）−を表し、Ｒ^２１はフェニルなどの芳香族基、基−Ｓ−Ｒ^２２（Ｒ^２２は炭素数１〜１４のアルキル基、好ましくは炭素数１２のアルキル基、を表す）、基−Ｏ−Ｒ^２３（Ｒ^２３はＣ_１−６アルキル基、好ましくはエチル基、を表す）または基−Ｎ（−Ｒ^２４）（−Ｒ^２５）（Ｒ^２４はＣ_１−６アルキル基、好ましくはメチル基、を表し、Ｒ^２５はフェニルなどの芳香族基を表す）を表す。）
で表される、上記（６）に記載の光活性化型ラマン分光分析用プローブ。
（８）共重合体が末端に標的結合ユニットをさらに含む、上記（１）〜（７）のいずれかに記載の光活性化型ラマン分光分析用プローブ。
（９）標的結合ユニットが式（ｄ）：

（上記式中、Ｒ^３１、Ｒ^３２およびＲ^３３はそれぞれ独立して
水素原子、
シアノ、カルボキシル基、Ｎ−ヒドロキシコハク酸イミドエステル基、パラニトロフェニルエステル基、アミノ基、−ＣＨ_２ＮＨ_２、チオール基、−Ｎ−Ｃ（＝ＮＨ_２ ^＋Ｃｌ⁻）ＣＨ_２ＣＨ_２ＣＨ_２ＳＨ、マレイミド基、ヨードアセトアミド基、アジド基、アルキン基、ジベンゾシクロオクチン基、ケトン基、アルデヒド基、ヒドラジン基、ヒドロキシアミン基、ジアジリン基、ベンゾフェノン基、ビオチン基、イミノビオチン基、ハロゲン化アルキル基、シトシニルメチルフェニル基およびグアニルメチルフェニル基からなる群（以下「官能基群Ａ」という）から選択される官能基、
Ｃ_１−６アルキル基（このアルキル基は同一または異なっていてもよく前記官能基群Ａから選択される１種または２種以上の官能基により置換されていてもよい）、または
−Ｑ１−Ｑ２−Ｃ_１−６アルキル（Ｑ１は結合またはＣ_１−６アルキレンであり、Ｑ２は−ＮＨＣＯ−または−ＣＯＮＨ−であり、Ｃ_１−６アルキル基は、同一または異なっていてもよく前記官能基群Ａから選択される１種または２種以上の官能基により置換されていてもよい）を表し、
Ｒ^３１、Ｒ^３２およびＲ^３３の少なくとも一つが前記官能基群Ａから選択される官能基または官能基群Ａから選択される官能基により置換されたＣ_１−６アルキル基あるいは前記官能基群Ａから選択される官能基により置換された基−Ｑ１−Ｑ２−Ｃ_１−６アルキルを表す。）
で表される、上記（８）に記載の光活性化型ラマン分光分析用プローブ。
（１０）共重合体が、上記（２）に記載の式（ａ）の光活性化型ラマン標識ユニットと、上記（３）に記載の式（ｂ）の親水性ユニットとを繰り返し単位として少なくとも含んでなるものである、上記（１）に記載の光活性化型ラマン分光分析用プローブ。
（１１）共重合体が、下記式（Ｉａ）：

（上記式中、Ｒ^１〜Ｒ^４は上記（２）に記載の式（ａ）および上記（３）に記載の式（ｂ）において定義された内容と同義であり、ｍおよびｎは各繰り返し単位の比を表す０より大きい数であり、ｍが１のとき、ｎは１〜１０００である。）
で表される共重合体である、上記（１）または（１０）に記載の光活性化型ラマン分光分析用プローブ。
（１２）共重合体が、その一方の末端に上記（７）に記載の式（ｃ）の金属粒子表面修飾ユニットを有し、もう一方の末端に上記（９）に記載の式（ｄ）の標的結合ユニットを有するものである、上記（１０）または（１１）に記載の光活性化型ラマン分光分析用プローブ。
（１３）共重合体が、下記式（Ｉ）：

（上記式中、Ｒ^１〜Ｒ^４、Ｒ^２１、Ｒ^３１、Ｒ^３２およびＲ^３３は上記（２）に記載の式（ａ）、上記（３）に記載の式（ｂ）、上記（７）に記載の式（ｃ）および上記（９）に記載の式（ｄ）において定義された内容と同義であり、ｍおよびｎは各繰り返し単位の比を表す０より大きい数であり、ｍが１のとき、ｎは１〜１０００である。）
で表される共重合体である、上記（１）または（１２）に記載の光活性化型ラマン分光分析用プローブ。
（１４）（ａ）上記（１）〜（１３）のいずれか一項に記載の光活性化型ラマン分光分析用プローブを試料と接触させる工程と、
（ｂ）前記試料に光照射し、試料からの光活性化ラマンスペクトルを検出する工程を含んでなる、試料をイメージングする方法。
（１５）試料が細胞を含有し、細胞内および／または細胞外の分析対象の存在をイメージングする、上記（１４）に記載の方法。
（１６）試料が細胞を含有し、細胞および／または細胞小器官をイメージングする、上記（１４）または（１５）に記載の方法。
（１７）工程（ａ）の前に、（ｃ）細胞内移行物質および／または分析対象と相互作用する物質を光活性化型ラマン分光分析用プローブに連結する工程をさらに含んでなる、上記（１４）〜（１６）のいずれかに記載の方法。
（１８）光照射およびラマンスペクトルの検出をレーザーラマン顕微鏡を用いて行う、上記（１４）〜（１７）のいずれかに記載の方法。 According to the present invention, the following inventions are provided.
(1) Photo-activated Raman spectroscopic analysis comprising a complex of a copolymer comprising at least a photo-activated Raman labeling unit and a hydrophilic unit as repeating units and metal particles having surface-enhanced Raman scattering activity Probe.
(2) The photoactivated Raman labeling unit is represented by the formula (a):

(In the above formula,
R ¹ represents a hydrogen atom or C _1-4 alkyl;
R ² represents formula (i):

(In the above formula, R ^{11 to} R ²⁰ may be the same or different, and are a hydrogen atom, a C _1-6 alkyl group, a C _1-6 alkoxy group, an amino group, a nitro group, an azo group, a mercapto group, a carboxyl group. Represents a group, a phenyl group, an unsaturated 5- to 7-membered monocyclic heterocyclic group or a 9- to 11-membered unsaturated bicyclic heterocyclic group, and the two adjacent groups of R ^{11 to} R ²⁰ are 5-7 membered saturated or unsaturated monocyclic carbocyclic or monocyclic heterocyclic ring or 9-11 membered saturated or unsaturated bicyclic carbocyclic or bicyclic heterocyclic together with the carbon atom to which A ring may be formed, and R ¹⁵ and R ¹⁶ together may represent a C _1-4 alkylene group, provided that two adjacent R ^{11 to} R ²⁰ and R ^{11 to} R ²⁰ are adjacent to each other. Hydrogen atoms on carbocyclic or heterocyclic rings formed by groups together And one of the hydrogen atoms on _{C 1-4} alkylene ^{group R 15} and ^{R 16} represent together represent a bond with formula (a).)
Represents)
The probe for photoactivated Raman spectroscopic analysis according to (1) above, represented by:
(3) The hydrophilic unit is represented by the formula (b):

(In the above formula,
R ³ represents a hydrogen atom or C _1-4 alkyl;
R ⁴ represents a hydrogen atom, a hydroxyl group or a C _1-6 alkyl group (this alkyl group may be substituted with one or two or more hydroxyl groups, a carboxyl group and / or an amino group).
The probe for photoactivation Raman spectroscopic analysis according to (1) or (2), which is represented by:
(4) The probe for photoactivation type Raman spectroscopic analysis according to any one of (1) to (3), wherein the copolymer is an acrylic copolymer or a methacrylic copolymer.
(5) The probe for photoactivation Raman spectroscopic analysis according to any one of (1) to (4), wherein the copolymer has a number average molecular weight of 4,000 to 800,000.
(6) The probe for photoactivatable Raman spectroscopic analysis according to any one of (1) to (5), wherein the copolymer further comprises a metal particle surface modification unit at the terminal.
(7) The metal particle surface modification unit is represented by the formula (c):

(In the above formula, Z represents —C (═S) — or —CH (—SH) —, R ²¹ represents an aromatic group such as phenyl, and the group —S—R ²² (R ²² represents 1 to 14 carbon atoms). An alkyl group, preferably an alkyl group having 12 carbon atoms), a group —O—R ²³ (wherein R ²³ represents a C _1-6 alkyl group, preferably an ethyl group) or a group —N (—R ²⁴ ) (— R ²⁵ ) (R ²⁴ represents a C _1-6 alkyl group, preferably a methyl group, and R ²⁵ represents an aromatic group such as phenyl).
The probe for photoactivation type Raman spectroscopic analysis according to (6) above, represented by:
(8) The probe for photoactivated Raman spectroscopic analysis according to any one of (1) to (7), wherein the copolymer further comprises a target binding unit at the terminal.
(9) The target binding unit is of formula (d):

(In the above formula, R ³¹ , R ³² and R ³³ are each independently a hydrogen atom,
Cyano, carboxyl group, N-hydroxysuccinimide ester group, paranitrophenyl ester group, amino group, —CH ₂ NH ₂ , thiol group, —N—C (═NH ₂ ⁺ Cl ⁻ ) CH ₂ CH ₂ CH ₂ SH, maleimide group, iodoacetamide group, azide group, alkyne group, dibenzocyclooctyne group, ketone group, aldehyde group, hydrazine group, hydroxyamine group, diazirine group, benzophenone group, biotin group, iminobiotin group, halogenated alkyl group , A functional group selected from the group consisting of a cytosynylmethylphenyl group and a guanylmethylphenyl group (hereinafter referred to as “functional group group A”),
A C _1-6 alkyl group (the alkyl groups may be the same or different and may be substituted with one or more functional groups selected from the functional group A), or -Q1-Q2 -C _1-6 alkyl (Q1 is a bond or C _1-6 alkylene, Q2 is -NHCO- or -CONH-, and the C _1-6 alkyl groups may be the same or different, and Which may be substituted by one or more functional groups selected from A),
At least one of R ³¹ , R ³² and R ³³ is a functional group selected from the functional group A or a C _1-6 alkyl group substituted by a functional group selected from the functional group A or the functional group A It represents the radicals _{-Q1-Q2-C 1-6} alkyl substituted by a functional group selected from. )
The probe for photoactivation Raman spectroscopic analysis as described in said (8) represented by these.
(10) The copolymer comprises at least a photoactivated Raman labeling unit of the formula (a) described in (2) above and a hydrophilic unit of the formula (b) described in (3) above as repeating units. The probe for photoactivatable Raman spectroscopic analysis according to (1) above, comprising:
(11) The copolymer has the following formula (Ia):

(In the above formula, R ^{1 to} R ⁴ have the same meanings as defined in the formula (a) described in the above (2) and the formula (b) described in the above (3), and m and n are each repeated. (It is a number greater than 0 representing the unit ratio, and when m is 1, n is 1-1000.)
The probe for photoactivated Raman spectroscopic analysis according to (1) or (10) above, which is a copolymer represented by the formula:
(12) The copolymer has a metal particle surface modification unit of the formula (c) described in the above (7) at one end thereof, and the formula (d) described in the above (9) at the other end. The probe for photoactivated Raman spectroscopic analysis according to the above (10) or (11), which has a target binding unit.
(13) The copolymer has the following formula (I):

(In the above formula, R ^{1 to} R ⁴ , R ²¹ , R ³¹ , R ³² and R ³³ are the formula (a) described in the above (2), the formula (b) described in the above (3), the above (7 ) And formula (d) described in (9) above, and m and n are numbers greater than 0 representing the ratio of each repeating unit, and m is When n is 1, n is 1-1000.)
The probe for photoactivated Raman spectroscopic analysis according to (1) or (12) above, which is a copolymer represented by the formula:
(14) (a) contacting the sample with the probe for photoactivated Raman spectroscopy according to any one of (1) to (13) above;
(B) A method for imaging a sample, comprising the steps of irradiating the sample with light and detecting a photoactivated Raman spectrum from the sample.
(15) The method according to (14) above, wherein the sample contains cells and images the presence of intracellular and / or extracellular analytes.
(16) The method according to (14) or (15) above, wherein the sample contains cells and images cells and / or organelles.
(17) Before the step (a), the method further comprises (c) a step of linking a substance that interacts with an intracellular migration substance and / or an analyte to a probe for photo-activated Raman spectroscopic analysis ( The method according to any one of 14) to (16).
(18) The method according to any one of the above (14) to (17), wherein light irradiation and detection of a Raman spectrum are performed using a laser Raman microscope.

  The probe for Raman spectroscopic analysis of the present invention can emit a strong Raman signal suitable for imaging a biological sample such as a living body, a molecule inside or outside a cell, a cell, an organelle, or a tissue in response to light irradiation. Therefore, according to the probe for Raman spectroscopic analysis of the present invention, it is possible to image a target existing at a desired location at a desired timing, and to track the temporal and spatial behavior of the target. It is advantageous.

It is the figure which showed the Raman spectrum before and behind light irradiation of the PBS solution (2 mM) of the compound 9. A: Before light irradiation, B: After light irradiation. The horizontal axis represents the wave number (1 / cm ⁻¹ ), and the vertical axis represents the Raman intensity. It is the figure which showed the Raman spectrum before and behind light irradiation of the PBS solution (200 micromol) of compound 9. A: Before light irradiation, B: After light irradiation. The horizontal axis represents the wave number (1 / cm ⁻¹ ), and the vertical axis represents the Raman intensity. It is the figure which showed the Raman spectrum before and behind light irradiation of the composite solution of the compound 9 and a gold nanoparticle. A: Before light irradiation, B: After light irradiation. It is the figure which showed the result of having observed the HeLa cell into which the probe for photoactivation type | mold Raman spectroscopic analysis of this invention was introduce | transduced on the conditions with or without light irradiation. A: photomicrograph of cells after light irradiation, B: photomicrograph of cells before light irradiation, C: Raman spectrum at the intersection of microscopic images of A, D: Raman spectrum at the intersection of microscopic images of B. It is a figure which shows the result of the principal component analysis of the whole spectrum of the Raman imaging image obtained by scanning the area | region containing the whole cell. A: Analysis results of cells after light irradiation, B: Analysis results of cells before light irradiation.

Detailed description of the invention

Definitions In the present specification, the “C _1-6 alkyl group” represents an alkyl group having 1 to 6 carbon atoms, preferably an alkyl group having 1 to 4 carbon atoms, that is, a C _1-4 alkyl group. An “alkyl group” as a group or part of a group means a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms. Examples of “C _1-6 alkyl group” include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, neopentyl, i-pentyl. , T-pentyl, n-hexyl, i-hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

In the present specification, the “C _1-6 alkoxy group” represents an alkoxy group having 1 to 6 carbon atoms, preferably an alkoxy group having 1 to 4 carbon atoms, that is, a C _1-4 alkoxy group. The “alkoxy group” as a group or a part of the group means a C 1-6 alkoxy group in which the alkyl portion is linear, branched or cyclic. Examples of “C _1-6 alkoxy group” include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy and the like.

  In the present specification, “unsaturated carbocycle” and “unsaturated heterocycle” mean a carbocycle and a heterocycle having one or more unsaturated bonds such as a double bond.

  In the present specification, the “5- to 7-membered monocyclic carbocycle” means a monocyclic hydrocarbon having 5 to 7 ring members. Examples of 5-7 membered monocyclic carbocyclic groups include phenyl, cyclopentyl, cyclohexyl and cycloheptyl.

  As used herein, “9 to 11-membered bicyclic carbocycle” means a bicyclic hydrocarbon having 9 to 11 ring members. An example of a 9-11 membered bicyclic carbocyclic group is naphthyl.

  In the present specification, the “5- to 7-membered monocyclic heterocycle” means a monocyclic heterocycle having 5 to 7 ring members, and includes 1 to 3, preferably 1 or 2 heteroatoms. It can be a monocyclic heterocycle containing as ring member atoms and the remaining ring member atoms being carbon atoms. Here, the “heteroatom” is selected from an oxygen atom, a nitrogen atom and a sulfur atom, and when the heterocycle contains a plurality of heteroatoms, the heteroatoms may be the same or different. Examples of 5- to 7-membered heterocyclic groups include pyridyl, furyl, thienyl, pyrrolyl, pyranyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, morpholino, isoxazolyl, oxazolyl, thiazolyl, imidazolyl, isothiazolyl And pyrazyl.

  As used herein, “9 to 11-membered bicyclic heterocycle” means a bicyclic heterocycle having 9 to 11 ring atoms, and includes 1 to 4, preferably 1 or 2 heteroatoms. It can be a bicyclic heterocycle containing as ring member atoms and the remaining ring member atoms being carbon atoms. Examples of 9-11 membered bicyclic heterocyclic groups include naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl and phthalazinyl.

  As used herein, “optionally substituted alkyl group” refers to an alkyl group in which one or more hydrogen atoms on the alkyl group are substituted by one or more substituents (which may be the same or different). Group and unsubstituted alkyl group. It will be apparent to those skilled in the art that the maximum number of substituents can be determined depending on the number of substitutable hydrogen atoms on the alkyl.

  In the present specification, the “halogen atom” is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

  In the present specification, the “photoactivated Raman spectroscopic probe” means a probe capable of emitting a signal suitable for Raman spectroscopic analysis in response to light irradiation. A specific configuration will be described below.

Probe for Photo-Activated Raman Spectroscopy The probe for photo-activated Raman spectroscopy of the present invention (hereinafter sometimes referred to as “probe of the present invention”) includes a photo-activated Raman label unit, a hydrophilic unit, And a composite of a metal particle having surface enhanced Raman scattering activity.

The copolymer constituting the probe of the present invention can be an acrylic copolymer or a methacrylic copolymer. When the copolymer constituting the probe of the present invention is an acrylic copolymer, the repeating unit constituting the copolymer is a repeating unit derived from a monomer such as acrylic acid, acrylic ester, and acrylic amide. Can be. When the copolymer constituting the probe of the present invention is a methacrylic copolymer, the repeating unit constituting the copolymer is a repeating unit derived from a monomer such as methacrylic acid, methacrylic acid ester or methacrylic acid amide. Can be. These repeating units can have a photo-activated Raman labeling moiety of the group -R ² or a hydrophilic group of the group -R ⁴ as described later.

  The weight average molecular weight of the copolymer constituting the probe of the present invention can be 5,000 to 900,000, preferably 5,000 to 200,000. In the present specification, the “weight average molecular weight” can be obtained by polystyrene conversion by a known gel permeation chromatography (GPC) method.

  The number average molecular weight of the copolymer constituting the probe of the present invention can be 4,000 to 800,000, preferably 4,000 to 200,000. In the present specification, the “number average molecular weight” can be obtained by polystyrene conversion by a known GPC method.

  The copolymer constituting the probe of the present invention comprises at least a photoactivatable Raman label unit and a hydrophilic unit. The photoactivatable Raman label unit and the hydrophilic unit in the copolymer may be the same or different.

  The molar ratio of the photoactivatable Raman labeling unit to the hydrophilic unit is not particularly limited as long as Raman spectroscopic analysis can be performed on a sample obtained from a living body. For example, the molar ratio is set to 1: 0.1 to 1: 100. Preferably, it is 1: 1-1: 100, More preferably, it is 1: 1-1: 20.

  In the copolymer constituting the probe of the present invention, the arrangement order of each unit is not particularly limited, and may be randomly arranged (random copolymer) or alternately arranged (alternate copolymer). May be arranged in a block form (block copolymer). The copolymer constituting the probe of the present invention is preferably a random copolymer.

  The photo-activated Raman labeling unit is a unit capable of emitting a signal suitable for Raman spectroscopic analysis in response to light irradiation. The photoactivatable Raman labeling unit is a repeating unit constituting the copolymer, and may be a repeating unit having the photoactivatable Raman labeling moiety of the formula (i), preferably the formula (a ).

In formula (a), R ¹ is preferably a hydrogen atom or a methyl group.

R ² in the formula (a) represents the formula (i). R ^{11 to} R ¹⁴ and R ^{17 to} R ^{20 in} formula (i) may preferably be the same or different, and are a hydrogen atom, a C _1-4 alkyl group, a C _1-4 alkoxy group, an amino group, Represents a nitro group, an azo group, a mercapto group, a carboxyl group, a phenyl group or an unsaturated 5- to 7-membered heterocyclic group, and R ¹⁵ and R ¹⁶ together represent an alkylene group having 2 carbon atoms, provided that Any one of hydrogen atoms on the alkylene group having 2 carbon atoms represented by R ^{11 to} R ¹⁴ and R ^{17 to} R ²⁰ and R ¹⁵ and R ¹⁶ together represents a bond with the formula (a), Preferably, one selected from R ^{11 to} R ¹⁴ and one selected from R ^{17 to} R ²⁰ may be the same or different and each represents a C _1-4 alkoxy group (particularly preferably methoxy), with the remaining being hydrogen original Represents a child, and R ¹⁵ and R ¹⁶ together represent an alkylene group having 2 carbon atoms, provided that R ^{11 to} R ¹⁴ and R ^{17 to} R ²⁰ and R ¹⁵ and R ¹⁶ represent together Any one of the hydrogen atoms on the alkylene group of 2 represents a bond with the formula (a). In the above case, preferably one hydrogen atom on the alkylene group having 2 carbon atoms represents a bond with the group of the formula (a).

R ^{11 to} R ²⁰ in formula (i) may also be the same or different, and may be a hydrogen atom, a C _1-4 alkyl group, a C _1-4 alkoxy group, an amino group, a nitro group, or an azo group. , A mercapto group, a carboxyl group, a phenyl group or an unsaturated 5- to 7-membered heterocyclic group, provided that any one of R ^{11 to} R ²⁰ represents a bond with the formula (a), more preferably , One selected from R ^{11 to} R ¹⁵ and one selected from R ^{16 to} R ²⁰ may be the same or different and each represents a C _1-4 alkoxy group (particularly preferably methoxy), and the remaining represents a hydrogen atom. Any one of R ^{11 to} R ²⁰ represents a bond with formula (a).

（上記式中、Ｒ^１１〜Ｒ^１４およびＲ^１７〜Ｒ^２０は式（ｉ）において定義された内容と同義であり、Ｒ^１０１およびＲ^１０２は同一または異なっていてもよく、水素原子、Ｃ_１−６アルキル基、Ｃ_１−６アルコキシ基、アミノ基、ニトロ基、アゾ基、メルカプト基、カルボキシル基、フェニル基、不飽和の５〜７員の単環複素環式基または９〜１１員の不飽和の二環複素環式基を表し、但し、Ｒ^１１〜Ｒ^１４、Ｒ^１７〜Ｒ^２０、Ｒ^１０１およびＲ^１０２のいずれか一つが式（ａ）との結合を表す。） R ² in the formula (a) preferably represents the following formula (ii).

(In the above formula, R ^{11 to} R ¹⁴ and R ^{17 to} R ²⁰ have the same meanings as defined in formula (i), R ¹⁰¹ and R ¹⁰² may be the same or different, and may be a hydrogen atom, C _{1 -6} alkyl group, C _1-6 alkoxy group, amino group, nitro group, azo group, mercapto group, carboxyl group, phenyl group, unsaturated 5-7 membered monocyclic heterocyclic group or 9-11 membered Represents an unsaturated bicyclic heterocyclic group, provided that any one of R ^{11 to} R ¹⁴ , R ^{17 to} R ²⁰ , R ¹⁰¹ and R ¹⁰² represents a bond with formula (a).

R ^{11 to} R ¹⁴ and R ^{17 to} R ²⁰ in the above formula (ii) are preferably the same or different and may be a hydrogen atom, a C _1-4 alkyl group, a C _1-4 alkoxy group, an amino group, Represents a nitro group, an azo group, a mercapto group, a carboxyl group, a phenyl group or an unsaturated 5- to 7-membered heterocyclic group, wherein R ¹⁰¹ and R ¹⁰² represent a hydrogen atom, provided that R ^{11 to} R ¹⁴ , R Any one of ^{17 to} R ²⁰ , R ¹⁰¹ and R ¹⁰² represents a bond with formula (a), and more preferably one selected from R ^{11 to} R ¹⁴ and one selected from R ^{17 to} R ^20. One is (particularly preferably methoxy) well _{C 1-4} alkoxy group optionally the same or different represents the remainder represents a hydrogen ^{atom, R 101} and ^{R 102} represents a hydrogen atom, provided ^{that, R} 11 to R ¹ , One of ^R 17 ^{~R 20, R 101} and ^{R 102} represent a bond with the formula (a). In the above formula (ii), preferably, any one of R ¹⁰¹ and R ¹⁰² represents a bond with the formula (a), and the other represents a hydrogen atom.

Two adjacent groups of R ^{11 to} R ²⁰ in formula (i) and formula (ii) are taken together with the carbon atom to which they are attached to form a 5- to 7-membered saturated or unsaturated monocyclic carbocycle Alternatively, it may form a monocyclic heterocyclic ring or a 9-11 membered saturated or unsaturated bicyclic carbocyclic ring or bicyclic heterocyclic ring. Here, the “two adjacent groups” are R ¹¹ and R ¹² , R ¹² and R ¹³ , R ¹³ and R ¹⁴ , R ¹⁴ and R ¹⁵ , R ¹⁶ and R ¹⁷ , R ^{17 in} the formula (i). And R ¹⁸ , R ¹⁸ and R ^19, and R ¹⁹ and R ²⁰ are meant. In the formula (ii), R ¹¹ and R ¹² , R ¹² and R ¹³ , R ¹³ and R ¹⁴ , R ¹⁷ and R ¹⁸ , R ¹⁸ and R ^19, and R ¹⁹ and R ²⁰ are meant. When two adjacent groups in the formula (i) form a carbocyclic or heterocyclic ring, it is preferably selected from a pair of two adjacent groups selected from R ^{11 to} R ¹⁵ and R ^{16 to} R ²⁰ Any one or both of a set of two adjacent groups can form a carbocyclic or heterocyclic ring. R ^{11 to} R ²⁰ that do not form a carbocycle or a heterocycle may preferably be the same or different and each represents a hydrogen atom or a C _1-4 alkoxy group (particularly preferably methoxy), more preferably a hydrogen atom. When two adjacent groups in the formula (ii) form a carbocyclic or heterocyclic ring, it is preferably selected from a pair of two adjacent groups selected from R ^{11 to} R ¹⁴ and R ^{17 to} R ²⁰ Any one or both of a set of two adjacent groups can form a carbocyclic or heterocyclic ring. R ^{11 to} R ¹⁴ and R ^{17 to} R ²⁰ that do not form a carbocyclic or heterocyclic ring may preferably be the same or different and each represents a hydrogen atom or a C _1-4 alkoxy group (particularly preferably methoxy), more preferably Represents a hydrogen atom.

  The carbocycle and heterocycle formed by two adjacent groups include benzene, pyridine, pyrimidine, furan, thiophene, pyrrole, pyran, pyridazine, pyrrolidine, piperidine, and pyridine. Examples include a perazine ring, a morpholine ring, an isoxazole ring, an oxazole ring, a thiazole ring, an imidazole ring, an isothiazole ring, and a pyrazine ring.

In a preferred embodiment of the photoactivatable Raman labeling unit, R ¹ represents a hydrogen atom or a methyl group, and R ² represents a group selected from formula (i) (wherein R ^{11 to} R ^{15 are} selected from one group) A group in which one group selected from R ^{16 to} R ²⁰ represents methoxy, and the remainder represents a hydrogen atom, provided that any one of R ^{11 to} R ²⁰ represents a bond with formula (a)) The repeating unit of (a) is mentioned.

In a preferred embodiment of the photoactivatable Raman labeling unit, R ¹ represents a hydrogen atom or a methyl group, and R ² represents a group selected from formula (ii) (wherein R ^{11 to} R ^{14 are} selected from one group) One group selected from R ^{17 to} R ²⁰ represents methoxy, the rest represents a hydrogen atom, one of R ¹⁰¹ and R ¹⁰² represents a bond with formula (a), and the other represents a hydrogen atom. And a repeating unit of the formula (a).

In a preferred embodiment of the photo-activated Raman labeling unit, R ¹ represents a hydrogen atom or a methyl group, and R ² is a group selected from the formula (i) (wherein R ^{11 to} R ^{15 are} selected). Two adjacent groups and one or both of a pair of adjacent two groups selected from R ^{16 to} R ²⁰ together with the carbon atom to which they are bonded together Forming a 7-membered saturated or unsaturated monocyclic carbocycle or monocyclic heterocycle or a 9-11 membered saturated or unsaturated bicyclic carbocycle or bicyclic heterocycle, the remainder representing a hydrogen atom, provided that Examples thereof include a repeating unit of the formula (a) in which any one of R ^{11 to} R ²⁰ represents a bond with the formula (a).

In a preferred embodiment of the photoactivatable Raman labeling unit, R ¹ represents a hydrogen atom or a methyl group, and R ² is a group selected from the formula (ii) (wherein R ^{11 to} R ^{14 are} selected). Two adjacent groups and one or both of a set of two adjacent groups selected from R ^{17 to} R ²⁰ together with the carbon atom to which they are bonded together to form a 7-membered saturated or bicyclic carbocyclic or bicyclic heterocyclic ring, saturated or unsaturated monocyclic carbocyclic or monocyclic heterocyclic ring or 9-11 membered unsaturated, the remaining represents a hydrogen atom, R ¹⁰¹ And any one of R ¹⁰² represents a bond with the formula (a), and the other represents a hydrogen atom).

The hydrophilic unit is a unit that can impart hydrophilicity to the copolymer. The hydrophilic unit is a repeating unit of the copolymer, and may be a repeating unit having the group -R ⁴ (hydrophilic group), and is preferably a repeating unit represented by the formula (b). .

In formula (b), R ³ is preferably a hydrogen atom or a methyl group.

In formula (b), R ⁴ is preferably a C _1-6 alkyl group substituted by one or more hydroxyl groups, more preferably C ₁ substituted by one or more hydroxyl groups. _{A -4} alkyl group, even more preferably a _C1-4 alkyl group substituted by 1 or 2 hydroxyl groups.

As a preferred embodiment of the hydrophilic unit, R ³ represents a hydrogen atom or a methyl group, and R ⁴ represents a C _1-4 alkyl group substituted by one or two hydroxyl groups. Repeat units are mentioned.

  The probe of the present invention can have a metal particle surface modification unit at the end of the copolymer. The metal particle surface modification unit is a unit capable of forming a complex with metal particles having surface enhanced Raman scattering activity. Techniques for forming complexes with metal particles are widely known in the art, and those skilled in the art can select and introduce a metal particle surface modification unit.

  Examples of the metal particle surface modification unit include a dithiophenyl ester group, a trithioalkyl carbonate group, and a thiol group. As will be described later, the copolymer of the present invention can be produced by RAFT polymerization. In that case, a copolymer having a dithiophenyl ester structure at the end can be obtained by using benzodithioate or alkyltrithiocarbonate as a RAFT agent. Copolymers and copolymers having a trithioalkyl carbonate structure can be produced. A copolymer having a thiol structure can also be produced by reducing a dithiophenyl ester group or a trithioalkyl carbonate group after polymerization.

  In addition, surface-modified gold nanoparticles intended to be complexed with intracellular probes and the like are commercially available (Cosmo Bio, etc.), and in order to complex with such surface-modified gold nanoparticles, a predetermined amount is used. A metal particle surface modification unit having a functional group or a binding substance may be introduced at the end of the copolymer.

The metal particle surface modification unit is preferably a repeating unit represented by the formula (c). Among the repeating units represented by the formula (c), preferred are
-Z represents -C (= S)-, R ²¹ represents phenyl,
-Z represents -CH (-SH)-, R ²¹ represents phenyl,
-Z represents -C (= S)-, R ²¹ represents a group -S-R ²² (R ²² represents an alkyl group having 10 to 14 carbon atoms),
· Z is -CH (-SH) - ^{represents, R 21} is a group ^{-S-R 22 (R} 22 represents an alkyl group having 10 to 14 carbon atoms) include those representing the.

Specific examples of the metal particle surface modification unit are as follows.

  Apart from the metal particle surface modification unit, the probe of the present invention can have a target binding unit at the end of the copolymer. The target binding unit is a unit that can introduce a substance that can interact with an analysis target or a transfer substance to cells or the like as necessary. As such a unit, for example, a structure having a functional group (for example, a carboxyl group, an amino group, a cyano group, etc.) capable of covalently binding a site capable of interacting with an analysis target. As will be described later, the copolymer of the present invention can be produced by RAFT polymerization. In that case, when benzodithioate or alkyltrithiocarbonate is used as the RAFT agent, a target binding unit is introduced into the ester moiety. Thus, a copolymer having a target binding unit at the terminal can be produced. Moreover, a new desired functional group can be introduced by a chemical reaction with respect to the functional group at the terminal after polymerization. Examples of such functional groups include carboxyl groups, N-hydroxysuccinimide ester groups, paranitrophenyl ester groups, amino groups, thiol groups, maleimide groups, iodoacetamide groups, azide groups, alkyne groups, and dibenzocyclooctynes. Group, ketone group, aldehyde group, hydrazine group, hydroxyamine group, diazirine group, benzophenone group, biotin group, iminobiotin group, halogenated alkyl group, cytosynylmethylphenyl group, and guanylmethylphenyl group.

The target binding unit is preferably a repeating unit represented by the formula (d). Among the repeating units represented by the formula (d), preferred are
R ³¹ , R ³² and R ³³ are each independently a hydrogen atom,
Cyano, carboxyl group, N-hydroxysuccinimide ester group, paranitrophenyl ester group, amino group, —CH ₂ NH ₂ , thiol group and —N—C (═NH ₂ ⁺ Cl ⁻ ) CH ₂ CH ₂ CH ₂ A functional group selected from the group consisting of SH (hereinafter referred to as “functional group B”);
C _1-4 alkyl group (this alkyl group may be the same or different and may be substituted with one or more functional groups selected from the functional group B), or -Q1-Q2 -C _1-6 alkyl (Q1 is a bond or C _1-4 alkylene, Q2 is -NHCO- or -CONH-, and the C _1-6 alkyl groups may be the same or different, and Which may be substituted by one or more functional groups selected from A),
At least one of R ³¹ , R ³² and R ³³ is a functional group selected from the functional group B or a C _1-4 alkyl group substituted by a functional group selected from the functional group B or the functional group A It represents the radicals _{-Q1-Q2-C 1-6} alkyl substituted by a functional group selected from the like.

Among the repeating units represented by the formula (d), more preferred are:
R ³¹ , R ³² and R ³³ are each independently a hydrogen atom,
A functional group selected from the group consisting of cyano, carboxyl group, —CH ₂ NH ₂ and —N—C (═NH ₂ ⁺ Cl ⁻ ) CH ₂ CH ₂ CH ₂ SH (hereinafter referred to as “functional group C”);
A _C1-4 alkyl group (this alkyl group may be the same or different and may be substituted with one or more functional groups selected from the functional group C), or -Q1-Q2 -C _1-6 alkyl (Q1 is a bond or C _1-4 alkylene, Q2 is -NHCO- or -CONH-, and the C _1-6 alkyl groups may be the same or different, and Which may be substituted by one or more functional groups selected from A),
At least one of R ³¹ , R ³² and R ³³ is a functional group selected from the functional group C, a C _1-4 alkyl group substituted by a functional group selected from the functional group C, or the functional group A It represents the radicals _{-Q1-Q2-C 1-6} alkyl substituted by a functional group selected from the like.

Specific examples of the target binding unit are as follows.

  A preferred embodiment of the copolymer in the present invention comprises at least the repeating unit of the formula (a) and the repeating unit of the formula (b), and a more preferred embodiment is the formula (Ia). It is a copolymer represented. In the copolymer of the formula (Ia), the photoactivatable Raman label unit and the hydrophilic unit are not particularly limited, and may be arranged randomly, arranged alternately, or arranged in blocks. In the formula (Ia), m and n represent a ratio (molar ratio) between the photoactivated Raman labeling unit and the hydrophilic unit. When m is 1, n is 1 to 1000, preferably 1 to 1. 100, more preferably 1-20.

  As a still more preferable embodiment of the copolymer in the present invention, the copolymer comprises at least the repeating unit of the formula (a) and the repeating unit of the formula (b), and the repeating unit of the formula (c) at one end thereof. In some cases, a copolymer having a repeating unit of the formula (d) at the other end may be mentioned, and a copolymer represented by the formula (I) is particularly preferable. In the copolymer of the formula (I), the photoactivatable Raman label unit and the hydrophilic unit are not particularly limited, and may be arranged randomly, arranged alternately, or arranged in blocks. In the formula (I), m and n represent the ratio (molar ratio) between the photoactivated Raman labeling unit and the hydrophilic unit. When m is 1, n is 1 to 1000, preferably 1 to 1. 100, more preferably 1-20.

  In the formula (I) and the formula (Ia), the number of repeating units (m ′) of the photoactivated Raman labeling unit and the number of repeating units (n ′) of the hydrophilic unit are, for example, m ′ = 1 to 100. N ′ = 10 to 1000, preferably m ′ = 5 to 20 and n ′ = 50 to 200.

  The weight average molecular weight of the copolymer represented by the formula (I) and the formula (Ia) can be 5,000 to 900,000, preferably 5,000 to 200,000.

  The number average molecular weight of the copolymer represented by the formula (I) and the formula (Ia) can be 4,000 to 800,000, preferably 4,000 to 200,000.

  The probe of the present invention is obtained by complexing a copolymer comprising at least a photoactivatable Raman label unit and a hydrophilic unit with metal particles having surface enhanced Raman scattering activity.

  In the present invention, the “metal particles having surface-enhanced Raman scattering activity” are not particularly limited as long as they have surface-enhanced Raman scattering activity (SERS activity). For example, metal particles such as gold, silver, and platinum (preferably Are metal nanoparticles). Among the metal particles having surface-enhanced Raman scattering activity, those capable of obtaining a stronger Raman signal include gold nanoparticles and silver nanoparticles, and most preferably gold nanoparticles.

  As a metal particle which forms a composite_body | complex with the probe of this invention, a thing with a particle size of about 5-900 nm can be used, Preferably it is a thing of about 10-100 nm. In addition, the particle size of a metal particle can be calculated | required from the observation result with a transmission electron microscope (TEM).

Production Method The probe of the present invention can be produced by producing a copolymer portion and compositing the copolymer with metal particles.

  When the copolymer constituting the probe of the present invention is an acrylic copolymer, a monomer such as acrylic acid having a vinyl bond, acrylic acid ester or acrylic acid amide is polymerized by a radical polymerization reaction or the like. Copolymers can be produced. When the copolymer constituting the probe of the present invention is a methacrylic copolymer, the monomer such as methacrylic acid, methacrylic acid ester and methacrylic acid amide having a vinyl bond is polymerized by a radical polymerization reaction or the like. Copolymers can be produced.

  A copolymer comprising at least a photoactivatable Raman labeling unit and a hydrophilic unit as repeating units is obtained by polymerizing a raw material monomer having no photoactivatable Raman labeling moiety or hydrophilic group introduced thereinto. Then, it can be produced by introducing a photo-activated Raman labeling site and a hydrophilic group into the polymer. In this case, when the side chain in the repeating unit of the polymer is a reactive functional group, a photoactivated Raman labeling site and a hydrophilic group can be introduced therein. When the functional group (for example, carboxyl group) of the side chain in the repeating unit of the polymer has an active ester structure, a photo-activated Raman labeling site and a hydrophilic group are introduced by an amide bond by nucleophilic reaction of amine. be able to. When the metal particle surface modification unit or the target binding unit is introduced into the end of the polymer, it can be carried out by carrying out a polymerization reaction using a RAFT agent having both units.

  A copolymer comprising at least a photoactivatable Raman label unit and a hydrophilic unit as a repeating unit is composed of a raw material monomer in which a photoactivatable Raman label site is introduced in advance and a raw material in which a hydrophilic group is introduced in advance. It can also be produced by preparing monomers and polymerizing them.

  The copolymer constituting the probe of the present invention can be preferably produced by RAFT polymerization reaction. Specifically, it can be produced by polymerizing raw material monomers to prepare a polymer, and then introducing a photo-activated Raman labeling site and a hydrophilic group. By using benzodithioate or alkyltrithiocarbonate as the RAFT agent as the RAFT agent used in the RAFT polymerization reaction, a polymer having a dithiophenyl ester structure or a trithioalkyl carbonate structure at the terminal can be produced. In the above case, a polymer having a target binding unit at one end can be produced by introducing a target binding unit into the ester portion of benzodithioate or alkyltrithiocarbonate.

（上記スキーム中、Ｒ^１は前記式（ａ）で定義された内容と同義であり、Ｒ^４１はペンタフルオロフェニルなどの活性エステルを表し、Ｚは−（Ｃ＝Ｓ）−を表し、Ｒ^２１は前記式（ｃ）で定義された内容と同義であり、Ｒ^３１、Ｒ^３２およびＲ^３３は前記式（ｄ）で定義された内容と同義であり、ｐは約１０〜約１０００を表す。） Synthesis of the polymer skeleton by RAFT polymerization reaction can be carried out according to the following scheme. The reaction conditions can be appropriately set according to the type of polymerization initiator used. Examples of the reaction initiator include 2,2′-azobisisobutyronitrile and 2,2′-azobis (2,4-dimethyl). Valeronitrile), 2,2′-azobi (4-methoxy2,4-dimethylvaleronitrile), 1,1′-azobis (cyclohexane-1-carbonitrile), etc. The polymerization reaction can be carried out at the reaction temperature for 4 to 72 hours.

(In the above scheme, R ¹ has the same meaning as defined in formula (a), R ⁴¹ represents an active ester such as pentafluorophenyl, Z represents — (C═S) —, R ²¹ Is synonymous with the content defined in the formula (c), R ³¹ , R ³² and R ³³ are synonymous with the content defined in the formula (d), and p represents about 10 to about 1000. )

（上記スキーム中、Ｒ^１〜Ｒ^４、Ｒ^１１〜Ｒ^１４、Ｒ^１７〜Ｒ^２０、Ｒ^２１、Ｒ^３１、Ｒ^３２、Ｒ^３３、Ｒ^４１、ｍおよびｎは前記式（ａ）、式（ｂ）、式（ｃ）、式（ｄ）、式（ｉｉ）、式（Ｉ）および前記スキームで定義された内容と同義であり、Ｚは−（Ｃ＝Ｓ）−を表し、Ｘはハロゲン原子（例えば塩素原子）を表す。） Moreover, introduction of a photo-activated Raman labeling site and a hydrophilic group into a polymer skeleton obtained by RAFT polymerization can be carried out according to the following scheme. That is, a photo-activated Raman labeling site and a hydrophilic group are introduced by an amide bond by nucleophilic reaction of an amine to an active ester to obtain a copolymer of the formula (I).

(In the above scheme, R ^{1 to} R ⁴ , R ^{11 to} R ¹⁴ , R ^{17 to} R ²⁰ , R ²¹ , R ³¹ , R ³² , R ³³ , R ⁴¹ , m and n are the same as the above formula (a), formula ( b), the formula (c), the formula (d), the formula (ii), the formula (I), and the contents defined in the above scheme, Z represents-(C = S)-, and X represents a halogen atom. Represents an atom (for example, a chlorine atom).)

The photoactivatable Raman-labeled compound of formula (ii ′) can be synthesized according to Example 1. That is, in Example 1, R ¹³ and R ¹⁸ are methoxy groups, and R ¹¹ , R ¹² , R ¹⁴ , R ¹⁷ , R ¹⁹ , R ²⁰ and R ¹⁰¹ are hydrogen atoms. Although a method for producing an activated Raman labeling site is described, the photoactivatable Raman labeling compound other than the above is prepared by using a raw material compound having a substituent or structure corresponding to the target photoactivated Raman labeling site. It will be obvious to those skilled in the art that they can be synthesized by preparing them.

  The probe of the present invention can be produced by compositing the copolymer part produced as described above with metal particles having surface enhanced Raman scattering activity.

  The formation of the complex can be performed according to a known method. For example, a complex can be formed by bringing a copolymer having a metal particle surface modification unit at the terminal into contact with metal particles having surface enhanced Raman scattering activity. When surface-modified metal particles are used, a complex can be formed by reacting or contacting with a copolymer having a predetermined functional group or binding molecule.

Imaging Method by Raman Spectroscopic Analysis The probe of the present invention can be used as a labeling agent for Raman spectral analysis, and a sample can be imaged by a Raman spectrum. Here, “imaging” means visualizing an analysis target.

  The probe of the present invention can emit a light-responsive Raman signal. In particular, the probe of the present invention having the photoactivatable Raman labeling moiety of the above formula (i) or formula (ii) in the polymer portion is irradiated with light when the carbon-carbon triple bond is protected with a cyclopropenone skeleton. As a result, a carbon-carbon triple bond is generated. Since this carbon-carbon triple bond is a molecular skeleton that does not originally exist in the living body, the probe can emit a distinct Raman signal. Therefore, by using the probe of the present invention as a photoresponsive labeling agent, an analysis target at a desired location can be labeled at a desired timing, and its presence can be detected as a Raman signal.

  For example, when a probe of the present invention capable of binding to a certain substance is injected into a cell, and light is irradiated to a desired location in the cell at a desired timing, the molecule moves from one location to another in the cell at a certain timing. Only can be selectively imaged. In addition, when the probe of the present invention is injected into a cell or an organelle and irradiated with light at a desired timing at a desired timing, a change in the spatial behavior or morphology of the cell can be imaged. Such imaging includes, for example, tracking intracellular transport of drug discovery target proteins, imaging of organelle morphology and behavior in cells involved in important biological phenomena and diseases, and in vivo imaging of specific cells during ontogeny. It can be used for tracking. For example, the probe of the present invention capable of binding to the intracellular substance of the cell is accumulated in the cell or inside the cell nucleus, laser irradiation is performed on a part of the cell nucleus to photoactivate the label, and the cell is observed with a Raman microscope. Thus, the intracellular behavior of the nuclear material can be observed. Here, “organelle” is secreted extracellularly in addition to intracellular membrane organelles (eg, nucleus, endoplasmic reticulum, Golgi apparatus, endosome, lysosome, peroxisome, mitochondria, chloroplast). It is used in the meaning including secretory vesicles (for example, exosomes).

  In the imaging method of the present invention, the “sample” is not particularly limited as long as light irradiation and detection of a Raman spectrum are possible. However, the probe of the present invention is not affected by the background signal of a biological sample, and Raman. Since a spectrum can be detected, a biological sample is preferable. Examples of biological samples include samples isolated from living organisms such as tissues, cells, blood, serum, plasma, etc. In addition to these, samples isolated from plants (eg, plant cells, plant tissues) And microorganisms.

  The probe of the present invention can image an analyte by directly binding to the analyte, or an antibody, ligand, receptor, complementary nucleic acid probe, aptamer, polysaccharide, which specifically binds to the analyte, By binding to a substance that interacts with the analysis target such as lipid, the analysis target can be imaged through these.

  When the probe of the present invention is used for imaging of an analyte inside or outside a cell, a substance that migrates to a cell or organelle and / or a substance that interacts with the analyte is linked to a probe for photo-activated Raman spectroscopy. You may implement the process to perform before the contact process of the probe of this invention and a sample. Substances that interact with the analyte and substances that accumulate in cells and organelles can be bound via various crosslinking agents and linker compounds. Crosslinking agents and linker compounds that can be used in the present invention are commonly used in the art, and those skilled in the art can appropriately select and use them.

  Analyzes that can be imaged include, but are not limited to, biomolecules. Examples of the analysis target to be subjected to the imaging method of the present invention include amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids. , Lipids, hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, metabolites, cofactors, inhibitors, drugs, formulations, nutrients, prions, toxins, poisons, explosives , Pesticides, chemical warfare substances, biological hazardous substances, radioisotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagens, narcotics, amphetamines, barbiturates, hallucinogens, waste, pollution Substances and the like.

  The probe of the present invention can also be accumulated in cells and organelles by techniques such as microinjection to image cells or organelles, or modified with antibodies, ligands, receptors, aptamers, polysaccharides, etc. By contacting the probe of the present invention with a cell or organelle, the cell or organelle can be imaged by selectively accumulating in the cell or organelle. In the latter case, the probe of the present invention can be transferred or accumulated in the cell or cytoplasm by linking a cell-permeable substance such as polyarginine peptide or a cytoplasmic substance such as polyethyleneimine. Moreover, the probe of the present invention can be accumulated in the specific cell by linking a substance that enables accumulation in the specific cell, and in this case, the specific cell can be imaged. For example, by linking a cancer cell accumulation substance such as folic acid or cyclic RGD peptide, the probe of the present invention is transferred or accumulated in the cancer cell, respectively, and the behavior of the cancer cell (eg chemotaxis or phagocytosis by immune cells). Can be tracked in space and time.

  When the probe of the present invention is used for imaging of cells or organelles, the step of linking a substance that migrates to cells or organelles to the probe for photo-activated Raman spectroscopic analysis includes the probe of the present invention and a sample. You may implement before a contact process. Substances that accumulate in cells and organelles can be bound via various crosslinking agents and linker compounds. Crosslinking agents and linker compounds that can be used in the present invention are commonly used in the art, and those skilled in the art can appropriately select and use them.

  In the imaging method of the present invention, the Raman spectroscopic analysis including the light irradiation and the detection of the Raman spectrum can be performed according to an ordinary Raman spectroscopic measurement method.

The excitation wavelength at the time of light irradiation is not particularly limited, but can be 280 to 450 nm, preferably 300 to 400 nm. The wavelength of measuring Raman spectrum is not particularly limited and may be a case of probe 450～900Cm ^-1 with photoactivatable site of the formula (i), preferably 500~800cm ^-1.

  For example, measurement of Raman spectroscopy can be performed using a commercially available Raman spectroscopy analyzer. Examples of the Raman spectroscopic analyzer include general Raman spectroscopic analyzers in general, and a laser Raman microscope is preferable.

  The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

Example 1 Production of Probe for Photo-Activated Raman Spectroscopy (1) Synthesis of N- (1,2-bis (3-methoxyphenyl) ethyl) amine (Compound 2)

Magnesium (6.4 g, 266 mmol) was added to a dried 100 mL three-necked flask, and a tetrahydrofuran (THF) solution (10 mL) containing 1,2-dibromoethane (100 μL) was added dropwise, and stirring was started. Further, a THF solution (55 mL) of 3-methoxybenzyl chloride (Compound 1) (5.50 mL, 37.9 mmol) was added dropwise over 3 hours, and the mixture was further stirred for 30 minutes. The obtained THF solution was transferred to a 200 mL two-necked flask containing 3-methoxybenzonitrile (3.3 mL, 27.0 mmol) and heated to reflux for 1 hour. Then, sodium borohydride _{(NaBH 4) (2.83g, 74.8mmol} ) was slowly added 1 hour stirring in methanol (40 mL) of. The reaction solution was concentrated by distilling off the solvent. The mixture was suspended in pure water and extracted three times with dichloromethane, and the organic phase was dried over magnesium sulfate and concentrated. When thin layer chromatography (trichloromethane: methanol = 50: 1, Rf = 0.21) was performed in the crudely purified state, coloration by ninhydrin was confirmed. Purification by silica gel column chromatography (trichloromethane: methanol = 50: 1) was performed, and fractions of the same substance were collected and the solvent was distilled off under reduced pressure to obtain Compound 2 as a pale yellow oil. The yield was 6.96 g, and the yield was 64%. Identification was performed by ¹ H NMR.
¹ H NMR (600 MHz, CDCl ₃ ): δ 1.57 (brs, 2H), 2.77 (dd, 1H), 2.97 (dd, 1H), 3.78 (s, 3H), 3.92 (S, 3H), 4.16 (dd, 1H), 6.72-6.96 (m, 6H), 7.20 (m, 2H)

(2) Synthesis of N- (1,2-bis (3-methoxyphenyl) ethyl) trifluoroacetamide (Compound 3)

Pyridine (4.1 mL) was added to a dichloromethane solution (28 mL) of compound 2 (2.85 g, 11.7 mmol). After cooling to 0 ° C., trifluoroacetic anhydride (3.0 mL, 21 mmol) was added dropwise, and the ice bath was removed and allowed to react overnight. The disappearance of the starting compound, Compound 2, was confirmed by thin layer chromatography. After quenching with water, extraction was performed twice from the aqueous phase. The extracted organic phases were combined, washed once with water, dried over magnesium sulfate and concentrated. Purification by silica gel column chromatography (ethyl acetate: hexane = 1: 5, Rf = 0.3) and concentration of the same fraction gave compound 3 as a pale yellow solid. The yield was 3.05 g, and the yield was 78%. Identification was performed by ¹ H NMR.
¹ H NMR (600 MHz, CDCl ₃ ): δ 3.11 (d, 2H), 3.72 (s, 3H), 3.78 (s, 3H), 5.21 (q, 1H), 6.49 (Brs, 1H), 6.56-6.85 (m, 6H), 7.16 (t, 1H), 7.28 (t, 1H)

(3) N- (4,9-dimethoxy-1-oxo-6,7-dihydro- 1H -dibenzo [a, e] -cyclopropane [c] cycloocten-6-yl) trifluoroacetamide (Compound 4 ) Synthesis

Aluminum chloride (4.40 g, 32.8 mmol) was added to a dried 100 mL two-necked flask, and then the atmosphere was argon. Dichloromethane (45 mL) and tetrachlorocyclopropene (1.05 mL, 8.6 mmol) were added thereto, and the mixture was stirred at room temperature for 10 minutes and then cooled to −20 ° C. Further, a dichloromethane solution (69 mL) of compound 3 (2.85 g, 8.06 mmol) was added dropwise and stirred for 2 hours. The mixture was returned to room temperature and further stirred for 1 hour. 24 mL of water was added to the reaction solution and stirred vigorously for 30 minutes. The aqueous phase was extracted 3 times with dichloromethane, dried over magnesium sulfate and concentrated. Silica gel column chromatography (acetone: hexane = 1: 1, Rf = 0.25 → 7: 3) was performed, and the same fraction was concentrated to obtain Compound 4 as a pale yellow solid. The yield was 1.58 g, and the yield was 49%. Identification was performed by ¹ H NMR.
¹ H NMR (600 MHz, DMSO-d ₆ ): δ 2.90 (d, 1H), 3.41 (dd, 1H), 3.82 (s, 6H), 4.84 (brs, 1H), 6 .94 (s, 1H), 7.09 (m, 3H), 7.89 (m, 2H), 10.46 (d, 1H)

(4) N- (4,9-dimethoxy-1-oxo-6,7-dihydro- 1H -dibenzo [a, e] -cyclopropane [c] cycloocten-6-yl) amine hydrochloride (compound 5 ) Synthesis

Compound 4 (168 mg, 0.414 mmol) was added to a 100 mL two-necked flask, and then an argon atmosphere was established. Methanol (8.6 mL) and 40% hydrochloric acid (6.6 mL) were added thereto and reacted at 40 ° C. for 24 hours. Although in suspension, the solution turned yellow. The disappearance of the starting compound 4 was confirmed by thin layer chromatography (chloroform: methanol = 9: 1). The solvent was distilled off by reducing the pressure while stirring at room temperature. After dissolving in 2-propanol, precipitation was performed by adding diethyl ether, and the solid was collected by suction filtration. The reaction proceeded quantitatively to give Compound 5 as a light yellow powder. It is thought to be in the form of hydrochloride. Identification was performed by ¹ H NMR.
¹ H NMR (600 MHz, DMSO-d ₆ ): δ 1.04 (d, 2H), 3.10 (s, 1H), 3.30 (s, 1H), 3.91 (s, 6H), 4 .42 (brs, 1H), 7.12 (m, 1H), 7.18 (s, 1H), 7.26 (s, 2H), 7.88 (brs, 1H), 7.92 (s, 1H)

(5) Synthesis of poly (pentafluorophenyl methacrylate) (compound 8)

4-Cyano-4- (phenylcarbonothioylthio) pentanoic acid (Compound 7) (54.8 mg, 0.196 mmol), azobisisobutyronitrile (AIBN) (4 0.1 mg, 0.25 mmol) was added, and after drying under reduced pressure, an argon atmosphere was obtained. Dioxane (1.0 mL) and pentafluorophenyl methacrylate (Compound 6) (1.0 g, 4.0 mmol) were further added. Freeze deaeration was performed for 6 cycles to remove oxygen. Finally, nitrogen substitution was performed. The oil bath was set at 90 ° C. and reacted for 18 hours. After the reaction, when hexane was added, precipitation occurred. The precipitate was collected by suction filtration. It was redissolved with THF, precipitated with hexane, and purified by performing the operation of collecting by suction filtration twice to obtain Compound 8 as a light red powder. The yield was 373 mg, and the conversion rate was 37%. ^From the measurement result of the ¹ H NMR spectrum, it was confirmed that the unreacted monomer was successfully removed (data not shown). The average degree of polymerization of the obtained polymer was 63 (p = 63) from the proton proton ratio of the terminal aromatic ring and the main chain, and the average molecular weight determined therefrom was M _n = 15.9 kDa. .

(6) Synthesis of compound (compound 9) in which a photoactivation site and a hydrophilic site are introduced into compound 8

Compound 8 (204.5 mg) and compound 5 (61.2 mg, 0.178 mmol) were added to a 50 mL two-necked flask, and an argon atmosphere was established. N, N-dimethylformamide (DMF) / THF (1: 1) and triethylamine (TEA) (0.13 mL) were added thereto, and the reaction was performed overnight at room temperature. Further, 1-amino-2-propanol (0.25 mL) was added and the reaction was performed for 11 hours. The reaction solution was added to diethyl ether in an ice bath, resulting in precipitation. The resulting precipitate was removed by centrifugation (10000 g, 10 minutes), and the supernatant was removed. DMF was added and redissolved there, and added to diethyl ether in an ice bath to cause precipitation. The resulting precipitate was removed by centrifugation (10000 g, 10 minutes), and the supernatant was removed. This operation was repeated until no yellow viscous compound was observed. Finally, it was washed once with diethyl ether and washed with DMF. Drying was performed overnight under reduced pressure to obtain Compound 9 as a white powder. Yield was 90 mg. Identification was performed by ¹ H NMR. A peak due to the introduction of compound 5 and 1-amino-2-propanol was observed (data not shown). From the integral ratio, it was considered that the ratio of the photoactivated site in the polymer was 10.6 mol% to 12.2 mol%. That is, it was confirmed that 6.6 to 7.7 out of the degree of polymerization 63 were converted into photoactivated sites. In addition, the average molecular weight was estimated to be 10.6 kDa to 10.8 kDa from the calculation.

(7) Complex formation of compound 9 and gold nanoparticles Gold nanoparticles (GNP) (manufactured by Sigma-Aldrich) and a citrate buffer were placed in a screw bottle and stirred at room temperature. A PBS solution (44 μL) of Compound 9 was added to the obtained GNP solution (0.032 nM, 400 μL), and the mixture was stirred at room temperature for 30 minutes to prepare a complex of Compound 9 and gold nanoparticles (Compound 9: 200 μM). , GNP: 0.03 nM). This was used as the probe for photo-activated Raman spectroscopic analysis of the present invention.

Example 2: Observation with a Raman microscope using a probe for photo-activated Raman spectroscopic analysis PBS was added to the compound 9 prepared in Example 1 (6) to prepare 2 mM and 200 μM solutions. These solutions were irradiated with light at 365 nm (2.4 mW / cm ² ) and photoactivated (excitation at 785 nm). About the solution before and behind light irradiation, the Raman spectrum measurement was performed using the inVia confocal Raman microscope (made by Renishaw).

As a result, in the case of 2 mM solution, a peak at 1850 cm ⁻¹ derived from C═O stretching of cyclopropenone was observed before light irradiation (FIG. 1A), but this peak disappeared after light irradiation, and newly Two broad peaks derived from DBCO alkyne were generated at 2138 cm ⁻¹ and 2165 cm ⁻¹ (FIG. 1B). On the other hand, in the case of the 200 μM solution, the above characteristic peak was weak or not observed before and after the light irradiation (FIGS. 2A and B). That is, it was clarified that compound 9 can detect a signal only at a high concentration of millimolar order.

Next, the composite solution prepared in Example 1 (7) was irradiated with light of 365 nm (2.4 mW / cm ² ) to perform photoactivation (excitation at 532 nm). About the solution before and behind light irradiation, the Raman spectrum measurement was performed using the inVia confocal Raman microscope (made by Renishaw).

As a result, a peak at 1850 cm ⁻¹ derived from C═O stretching of cyclopropenone was observed before the light irradiation (FIG. 3A), but after the light irradiation, this peak disappeared and was newly increased to 2142 cm ⁻¹ . Two broad peaks derived from DBCO alkyne occurred at 2168 cm ⁻¹ (FIG. 3B).

From the above results, it was confirmed that the complex produced in Example 1 (7) (the photoactivated Raman spectroscopic probe of the present invention) can detect a signal even in the micromolar order.

Example 3: Manufacture of a photo-activated Raman spectroscopic probe for introduction into cells In order to allow a photo-activated Raman spectroscopic probe to be introduced into cells by endocytosis, photoactivation A probe (a photo-activated Raman spectroscopic probe for introduction into a cell) in which a molecular device (polyarginine peptide) for introduction into a cell was introduced into a biomolecule binding unit of a probe for type Raman spectroscopy was prepared.

実施例１（６）で作製された化合物９（１３．４ｍｇ、１．２６μｍｏｌ、１ｅｑ．）とＮ，Ｎ’−ジシクロヘキシルカルボジイミド（ＤＣＣ）（０．５１ｍｇ、２．４７μｍｏｌ、１．９６ｅｑ．）を二口フラスコに入れ、アルゴン雰囲気にした。これにＤＭＦ（０．３ｍＬ）、アジドプロピルアミン（１０μＬ、７６．３μｍｏｌ、６０．６μｍｏｌ）を加え、１９時間撹拌を行った。その後、薄層クロマトグラフィー（クロロホルム：メタノール＝４：１）により、出発物質である化合物９の消失を確認した。更に、ニンヒドリン呈色によりアジド由来の呈色も確認した。そこで、反応溶液を氷冷したジエチルエーテル中に加えて遠心分離を行い、得られた再沈殿物を回収し、減圧乾燥を行い、アジド化された化合物９を得た。 (1) Synthesis of azidated compound 9

Compound 9 (13.4 mg, 1.26 μmol, 1 eq.) Prepared in Example 1 (6) and N, N′-dicyclohexylcarbodiimide (DCC) (0.51 mg, 2.47 μmol, 1.96 eq.) Were used. A two-necked flask was placed in an argon atmosphere. DMF (0.3 mL) and azidopropylamine (10 μL, 76.3 μmol, 60.6 μmol) were added thereto, and the mixture was stirred for 19 hours. Then, the disappearance of the starting compound 9 was confirmed by thin layer chromatography (chloroform: methanol = 4: 1). Furthermore, azide-derived coloration was also confirmed by ninhydrin coloration. Therefore, the reaction solution was added to ice-cooled diethyl ether and centrifuged, and the resulting re-precipitate was collected and dried under reduced pressure to obtain azide compound 9.

(2) Synthesis of Compound 9 Modified with Polyarginine Peptide Peptide (G3R15GYC (NH2-GGGRRRRRRRRRRGRYC-COOH) (SEQ ID NO: 1): Mw = 2855.3, 1.10 mg, 764 μM, 1 eq.) In PBS (0. 5 mL). To this, a DMSO solution (50 μL) of maleimide alkyne (0.21 mg, 1 eq.) Was added and allowed to react for 24 hours at room temperature under light shielding. The obtained reaction solution was subjected to argon bubbling for 15 minutes, and the azide compound 9 (4.06 mg, 1 eq.) Prepared in Example 3 (1), copper sulfate pentahydrate (0.0. 92 mg, 9.6 eq.), Tris (3-hydroxypropyltriazolylmethyl) amine (THPTA) (1.86 mg, 11.2 eq.) And sodium ascorbate (7.37 mg, 93.4 eq.) Were added. The mixture was transferred to a tube and reacted for 4 days on a shaker. The obtained reaction solution was transferred to a dialysis tube (molecular weight fraction: 3.5 kDa) and purified by dialysis for 8 hours to obtain Compound 9 modified with polyarginine peptide.

(3) Complex formation of compound 9 modified with polyarginine peptide and gold nanoparticles 1.5 mL (0.032 nM) of a solution obtained by dissolving gold nanoparticles in a citrate buffer is added to a microtube. After centrifugation (1500 g, 4 ° C., 30 minutes), the supernatant was removed. To this was added 1 mL of an aqueous solution (1.5 mg / mL) of Compound 9 modified with the polyarginine peptide prepared in Example 3 (2), and the reaction was carried out on a shaker for 4 days under light shielding. After centrifugation (1500 g, 4 ° C., 30 minutes), the supernatant was removed. MilliQ water was added to this and redispersed. After centrifugation (1500 g, 4 ° C., 30 minutes), the supernatant was removed twice, and this was used for photo-activated Raman spectroscopy for intracellular introduction. A probe was used. In addition, the photo-activated Raman spectroscopic probe for introduction into cells is stored as it is and used in Example 4 below after re-dispersing with serum-free DMEM medium (1 mL) immediately before use for cell imaging. did.

Example 4: Observation with a Raman microscope using a photo-activated Raman spectroscopic probe A quartz glass bottom dish (35 mm in diameter) was used to obtain HeLa cells (obtained from RIKEN BioResource Center) in serum-containing DMEM medium for 10 hours. Cultured. After removing the medium and washing with a PBS solution, the cell-introduced photoactivated Raman spectroscopic probe (1 mL) prepared in Example 3 (3) was added, followed by incubation at 37 ° C. for 2 hours. After culturing, Raman microscope observation and Raman spectrum using inVia confocal Raman microscope (manufactured by Renishaw) for each of light irradiation with a wavelength of 360 nm (4.3 J / cm ² ) and control without light irradiation. Measurements were made. Before observation with a Raman microscope, the medium was removed and replaced with serum-containing DMEM medium (1 mL). Excitation light of 785 nm was used for Raman microscope observation.

As a result, a polymer peak enhanced by the SERS effect was observed from the intracellular Raman spectrum irradiated with light, and a peak (2160 cm ⁻¹ ) derived from alkyne activated by light irradiation was also observed. (FIGS. 4A and C). On the other hand, from the Raman spectrum in the cells not irradiated with light, the polymer peak enhanced by the SERS effect was observed, but the peak derived from alkyne was not observed (FIGS. 4B and D).

  Next, in order to show that the above results do not show only the Raman spectrum of a convenient site in the cell, the Raman imaging image obtained by scanning the region including the entire cell is shown. All spectra were subjected to principal component analysis using Raman microscope image analysis software (WiRE4, Renishaw).

  As a result, peaks derived from alkyne were observed in the third component and the fourth component from the cells irradiated with light (FIG. 5A), but from the cells not irradiated with light, even the tenth component was examined. No peak derived from alkyne was observed (FIG. 5B). Since the fifth component and thereafter are mostly noise and a significant peak was not observed, it was omitted.

  Based on the above results, it is possible to introduce a photo-activated Raman spectroscopic probe into a cell by modifying a molecular device for introduction into the cell into the biomolecule-binding unit. It was confirmed that the alkyne signal can be detected by a Raman microscope.

Claims (18)

  A probe for photo-activated Raman spectroscopic analysis comprising a complex of a copolymer comprising at least a photo-activated Raman labeling unit and a hydrophilic unit as repeating units, and metal particles having surface-enhanced Raman scattering activity. The photoactivated Raman labeling unit is represented by the formula (a):

(In the above formula,
R ¹ represents a hydrogen atom or C _1-4 alkyl;
R ² represents formula (i):

(In the above formula, R ^{11 to} R ²⁰ may be the same or different, and are a hydrogen atom, a C _1-6 alkyl group, a C _1-6 alkoxy group, an amino group, a nitro group, an azo group, a mercapto group, a carboxyl group. Represents a group, a phenyl group, an unsaturated 5- to 7-membered monocyclic heterocyclic group or a 9- to 11-membered unsaturated bicyclic heterocyclic group, and the two adjacent groups of R ^{11 to} R ²⁰ are 5-7 membered saturated or unsaturated monocyclic carbocyclic or monocyclic heterocyclic ring or 9-11 membered saturated or unsaturated bicyclic carbocyclic or bicyclic heterocyclic together with the carbon atom to which A ring may be formed, and R ¹⁵ and R ¹⁶ together may represent a C _1-4 alkylene group, provided that two adjacent R ^{11 to} R ²⁰ and R ^{11 to} R ²⁰ are adjacent to each other. Hydrogen atoms on carbocyclic or heterocyclic rings formed by groups together And one of the hydrogen atoms on _{C 1-4} alkylene ^{group R 15} and ^{R 16} represent together represent a bond with formula (a).)
Represents)
The probe for photoactivation type | mold Raman spectroscopic analysis of Claim 1 represented by these. The hydrophilic unit is represented by the formula (b):

(In the above formula,
R ³ represents a hydrogen atom or C _1-4 alkyl;
R ⁴ represents a hydrogen atom, a hydroxyl group or a C _1-6 alkyl group (this alkyl group may be substituted with one or two or more hydroxyl groups, a carboxyl group and / or an amino group).
The probe for photoactivation type | mold Raman spectroscopic analysis of Claim 1 or 2 represented by these.   The probe for photoactivatable Raman spectroscopic analysis according to any one of claims 1 to 3, wherein the copolymer is an acrylic copolymer or a methacrylic copolymer.   The probe for photoactivatable Raman spectroscopic analysis according to any one of claims 1 to 4, wherein the copolymer has a number average molecular weight of 4,000 to 800,000.   The probe for photoactivation type Raman spectroscopic analysis according to any one of claims 1 to 5, wherein the copolymer further includes a metal particle surface modification unit at a terminal. The metal particle surface modification unit is represented by the formula (c):

(In the above formula, Z represents —C (═S) — or —CH (—SH) —, R ²¹ represents an aromatic group, and group —S—R ²² (R ²² represents an alkyl group having 1 to 14 carbon atoms). ), A group —O—R ²³ (R ²³ represents a C _1-6 alkyl group) or a group —N (—R ²⁴ ) (— R ²⁵ ) (R ²⁴ represents a C _1-6 alkyl group, R ²⁵ represents an aromatic group).
The probe for photoactivation type | mold Raman spectroscopic analysis of Claim 6 represented by these.   The probe for photoactivated Raman spectroscopic analysis according to any one of claims 1 to 7, wherein the copolymer further comprises a target binding unit at a terminal. The target binding unit is of formula (d):

(In the above formula, R ³¹ , R ³² and R ³³ are each independently a hydrogen atom,
Cyano, carboxyl group, N-hydroxysuccinimide ester group, paranitrophenyl ester group, amino group, —CH ₂ NH ₂ , thiol group, —N—C (═NH ₂ ⁺ Cl ⁻ ) CH ₂ CH ₂ CH ₂ SH, maleimide group, iodoacetamide group, azide group, alkyne group, dibenzocyclooctyne group, ketone group, aldehyde group, hydrazine group, hydroxyamine group, diazirine group, benzophenone group, biotin group, iminobiotin group, halogenated alkyl group , A functional group selected from the group consisting of a cytosynylmethylphenyl group and a guanylmethylphenyl group (hereinafter referred to as “functional group group A”),
A C _1-6 alkyl group (the alkyl groups may be the same or different and may be substituted with one or more functional groups selected from the functional group A), or -Q1-Q2 -C _1-6 alkyl (Q1 is a bond or C _1-6 alkylene, Q2 is -NHCO- or -CONH-, and the C _1-6 alkyl groups may be the same or different, and Which may be substituted by one or more functional groups selected from A),
At least one of R ³¹ , R ³² and R ³³ is a functional group selected from the functional group A or a C _1-6 alkyl group substituted by a functional group selected from the functional group A or the functional group A It represents the radicals _{-Q1-Q2-C 1-6} alkyl substituted by a functional group selected from. )
The probe for photoactivation type | mold Raman spectroscopic analysis of Claim 8 represented by these.   The copolymer comprises at least a photoactivated Raman labeling unit of the formula (a) according to claim 2 and a hydrophilic unit of the formula (b) according to claim 3 as repeating units. The probe for photo-activated Raman spectroscopic analysis according to claim 1. The copolymer is represented by the following formula (Ia):

(In the above formula, R ^{1 to} R ⁴ are synonymous with the contents defined in the formula (a) according to claim 2 and the formula (b) according to claim 3, and m and n are (It is a number greater than 0 representing the ratio, and when m is 1, n is 1-1000.)
The probe for photoactivation type | mold Raman spectroscopic analysis of Claim 1 or 10 which is a copolymer represented by these.   The copolymer has a metal particle surface modification unit of the formula (c) according to claim 7 at one end and a target binding unit of the formula (d) according to claim 9 at the other end. The probe for photoactivation type | mold Raman spectroscopic analysis of Claim 10 or 11 which has. The copolymer is represented by the following formula (I):

(In the above formula, R ^{1 to} R ⁴ , R ²¹ , R ³¹ , R ³² and R ³³ are the formula (a) according to claim 2, the formula (b) according to claim 3, and the claim 7, respectively. Wherein the formula (c) and the content defined in the formula (d) according to claim 9 are synonymous, and m and n are numbers greater than 0 representing the ratio of each repeating unit, and when m is 1, n is 1-1000.)
The probe for photoactivation type | mold Raman spectroscopic analysis of Claim 1 or 12 which is a copolymer represented by these. (A) bringing the probe for photo-activated Raman spectroscopy according to any one of claims 1 to 13 into contact with a sample;
(B) A method for imaging a sample, comprising the steps of irradiating the sample with light and detecting a photoactivated Raman spectrum from the sample.   15. The method of claim 14, wherein the sample contains cells and images the presence of intracellular and / or extracellular analytes.   16. A method according to claim 14 or 15, wherein the sample contains cells and images cells and / or organelles.   The step of (c) further comprising, before step (a), (c) linking a substance that interacts with an intracellular translocation substance and / or an analyte to a probe for photo-activated Raman spectroscopic analysis. The method as described in any one of. The method according to any one of claims 14 to 17, wherein light irradiation and detection of a Raman spectrum are performed using a laser Raman microscope.

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