JP2005134348A - Time-resolved fluorescence analyzing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a time-resolved fluorescence analyzing method simultaneously and highly sensitively analyzing a plurality of object matters to be analyzed on one microchip. <P>SOLUTION: The method includes a step in which the plurality of object matters (A) to be analyzed each being supported in the form of a dot on the microchip are brought to contact with fluorescent rare-earth metal complex inclusion core/shell type particles (B) each of which is a core/shell type particle being composed of a core portion (a) practically made of a water-insoluble high polymer compound and a shell portion (b) that is practically made of a water-soluble high polymer compound with a reactive functional group and covers the surface of the core portion so as to be like a brush. The core portion and the shell portion make up the core/shell type particle as a whole which is composed of a block copolymer of a water-insoluble high polymer and a water-soluble high polymer. In the fluorescent rare-earth metal complex inclusion core/shell type particle, a fluorescent rare-earth metal complex is encapsulated in the core portion, and a partner which can specifically bind the object matter to be analyzed, is coupled to the shell portion. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a time-resolved fluorescence analysis method in which a plurality of analytes are supported on one microchip and the analytes are analyzed using fluorescent rare earth metal complex-encapsulated core-shell type particles. “Analysis” in the present specification includes “detection” for determining the presence or absence of an analysis target substance, and “measurement” for quantitatively or semi-quantitatively determining the amount of the analysis target substance.

Many studies have been made on methods for detecting antigen-antibody reactions, ligand-receptor binding reactions, or DNA or RNA hybridization reactions, and many reports have been made in proportion thereto.
The most common and has been performed since the 1950s is the radioisotope labeling method, which has been used for all the analysis of the reaction. However, alternatives are required from the regulation of radioactive material handling and disposal, and labeling methods such as enzymes, fluorescent materials, and luminescent materials have been developed.

Peroxidase or alkaline phosphatase is frequently used as a labeling enzyme, and a chromogenic substrate, a fluorescent substrate, a luminescent substrate, or the like has been used depending on the application.
Fluorescein or rhodamine is generally used as the fluorescent material, and recently, a device has been devised to extract several pieces of information at once by using different fluorescent materials such as red, blue and yellow.
As the luminescent substance, an acridinium ester or luminol derivative is used, and after direct labeling, a luminescence initiator is added to illuminate and measured with a detector.

Recently, the concept of a DNA chip or a protein chip has started to fuss. In this method, DNA probes, antigens, or antibodies are immobilized on the surface of a slide glass or plastic chip individually in the form of dots, and the reaction solution containing the sample is applied over the entire surface to investigate the reactivity with each dot at once. To do.
As a label for this chip reaction, a fluorescent substance has been preferred and used so that dots on the chip can be directly detected by an image processing apparatus. In addition, as an attempt to increase sensitivity, a method has been studied in which an enzyme is used as a labeling object, and the signal of a dot that shines over the entire surface of the luminescent substrate is captured by a camera.

Fluorescent substances are preferably used for labeling integrated chips. This is because the fluorescent substance labeling method is easy, and after completion of the binding reaction on the chip, the liquid can be discarded and observed in a dry state. It depends on the merits of being able to obtain a lot of information by labeling a fluorescent substance having a different color tone on a sample. However, the detection sensitivity of the fluorescent substance itself is 10 to the ninth power level, and only a substance having a microgram level concentration per mL can be measured.
In order to detect DNA, the sample concentration can be amplified and observed by the polymerase chain reaction (PCR) method, so it is possible to detect signals of thousands to tens of thousands of dots on one chip in combination with a fluorescent substance label. It can be done. However, this is a limited method in which measurement is performed after the sample is amplified by the PCR method.

  On the other hand, proteins or chemical substances having physiological activity cannot be amplified, and the concentration when they are involved in living organisms is low, so that they cannot be detected by the fluorescent substance labeling chip method. Investigation of a chip system that attempts to overcome this lack of sensitivity of fluorescent substances is also progressing, and a measurement system that combines an enzyme label, a luminescent substrate, and a CCD camera has been reported. However, this method requires a system with a large restriction that the luminescent substrate is placed on the chip in a liquid state and taken with a camera from above, and the luminescent substrate shines non-specifically due to various contaminants, increasing the reliability of the measurement. The current situation is that it has been lost.

  While there are technical advances related to integrated chips, improvements to labeling substances such as fluorescent substances have also been attempted. For example, WO 02/097436 pamphlet (Patent Document 1) discloses a core-shell type particle enclosing a signal generating substance. The signal-generating substance-encapsulating core-shell type particle is substantially composed of (1) a core portion substantially composed of a water-insoluble polymer compound and (2) a water-soluble polymer compound having a reactive functional group, A core-shell type particle comprising a shell portion covering the surface of the core portion in a brush-like shape, and wherein the core portion and the shell portion as a whole are composed of a block copolymer of a water-insoluble polymer and a water-soluble polymer. In the above, a signal generating substance is enclosed in the core part, and can be used as a highly sensitive labeling substance.

  However, the pamphlet stated that CRP could actually be measured by a time-resolved fluorescence measurement method in a 96-well plate for ELISA using a europium chelate encapsulated particle-labeled anti-CRP (C-reactive protein) antibody. Although described in the examples, there is no disclosure or suggestion that the signal-generating substance-encapsulated core-shell type particles can be used for time-resolved fluorescence measurement on a microchip.

International Publication No. WO02 / 097436 Pamphlet

  An object of the present invention is to provide a time-resolved fluorescence analysis method capable of analyzing a plurality of analytes simultaneously and with high sensitivity on one microchip.

Said subject is according to the invention,
(A) a plurality of analytes supported in the form of dots on a microchip;
(B) (a) a core portion substantially composed of a water-insoluble polymer compound, and (b) a water-soluble polymer compound having a reactive functional group, and covering the surface of the core portion in a brush shape. A core-shell type particle comprising a block copolymer of a water-insoluble polymer and a water-soluble polymer as a whole. Including a step of contacting a core-shell type particle encapsulating a fluorescent rare earth metal complex in which a metal complex is encapsulated and a partner capable of specifically binding to an analyte is bound to the shell portion. It can be solved by a time-resolved fluorescence analysis method.

  According to a preferred aspect of the time-resolved fluorescence analysis method of the present invention, the substance to be analyzed supported in the form of dots on the microchip is (1) the substance to be analyzed which is immobilized in the form of dots on the microchip. By being bound to a partner capable of specifically binding to (2), it is supported in the form of dots on the microchip, or (2) directly immobilized in the form of dots on the microchip.

  According to the time-resolved fluorescence analysis method of the present invention, a plurality of analytes can be analyzed simultaneously and with high sensitivity on one microchip. That is, a highly sensitive analysis method that can obtain only one item of measurement result in one reaction can obtain information on multiple items simultaneously in one reaction according to the present invention. The number of dots can be freely adjusted as long as the detector is compatible, and different information such as protein and DNA, drug and DNA can be obtained in a single chip reaction.

  (A) a core part mainly composed of a water-insoluble polymer compound; and (b) a shell part mainly comprising a water-soluble polymer compound having a reactive functional group and covering the surface of the core part in a brush shape. The core-shell type particle, that is, the core-shell type particle itself having a water-soluble polymer compound brush having a reactive functional group on the surface layer on the surface of the core part mainly composed of a water-insoluble polymer compound, As described above in the background art, it is known. Further, a fluorescent rare earth metal complex-encapsulated core-shell type particle itself in which a fluorescent rare earth metal complex is encapsulated in the core portion and a partner capable of specifically binding to the analyte is bound in the shell portion. Are also known.

  In the present invention, the water-insoluble polymer compound that can be used to form the core portion is a polymer compound that does not dissolve in water, and when the core portion is formed, a fluorescent rare earth metal is contained therein. The polymer is not particularly limited as long as it is a polymer compound that can encapsulate the complex. Examples of the water-insoluble polymer compound include hydrophobic polymers [eg, polystyrene, polymethyl methacrylate, poly (2-hydroxyethyl methacrylate), poly (N-isopropylacrylamide) polyisoprene, polyvinyl chloride, polylactic acid. , Polylactone, polylactam, etc.], a water-soluble polymer (eg, polyvinyl alcohol, polyacrylamide, polydimethylacrylamide, polyallylamine, polyacrylic acid, etc.) cross-linked gel, or a water-insoluble natural polymer compound (eg, gelatin or Polysaccharides).

  In the present invention, the core portion is substantially formed from only one type of the water-insoluble polymer compound or substantially formed from a combination of two or more types of the water-insoluble polymer compound. be able to.

  The shape of the core part is not particularly limited, but is generally approximately spherical or approximately elliptical. Further, the dimension of the core part is not particularly limited and can be appropriately changed according to the use, but the diameter of the substantially spherical core part is generally about 5 nm to 500 nm.

  In the present invention, the water-soluble polymer compound that can be used to form the shell portion is a linear polymer compound that can be bonded to the surface of the core portion at one end (bonding end). And can have a reactive functional group at the other end (free end) or a reactive functional group can be introduced, and can be arranged in a brush shape on the surface of the core portion. As long as it is, it is not particularly limited. In this specification, the “shell part covering the surface of the core part in a brush shape” means that the shell part is composed of a number of linear water-soluble polymer compounds, and each of the linear water-soluble polymer compounds is one of them. In the reaction solution in which the other free end is reacted with at least a water-soluble polymer compound and an introduced product (for example, an antigen or an antibody), It means that it protrudes from the surface in the form of thread or rod in the system. In addition, at the free ends of these brush-like water-soluble polymer compound chains, there are reactive functional groups that can bind to an introduced product [for example, a physiologically active substance (eg, antibody, enzyme, or DNA)]. Therefore, the outermost shell of the shell portion is covered with a large number of reactive functional groups.

  Examples of the water-soluble polymer compound include polyethylene glycol (PEG), polyvinyl alcohol, polyvinyl pyrrolidone, polyamino acid, polyacrylic acid, polydimethylaminoethyl methacrylate, and polyallylamine. PEG or polyvinyl Alcohol is preferred.

  In the present invention, the water-soluble polymer compound chain constituting the shell portion is such that each water-soluble polymer compound chain is substantially formed from only one kind of the water-soluble polymer compound, or each water-soluble polymer compound chain. The compound chain may be substantially formed from a combination of two or more kinds of the water-insoluble polymer compounds different from each other. In addition, the lengths of the respective chains do not have to be the same, and even when each water-soluble polymer compound chain is formed of only one type of water-soluble polymer compound, the length of one type is different. It can be formed substantially only from a chain, or can be formed substantially from a combination of two or more types of chains. Furthermore, even when each water-soluble polymer compound chain is substantially formed from a combination of two or more different water-insoluble polymer compounds, it is also possible to select and combine different chain lengths. .

  It is preferable that the water-soluble polymer compound chain constituting the shell portion substantially completely covers the entire surface of the core portion. In addition, each water-soluble polymer compound chain constituting the shell portion does not need to have substantially the same length, but is approximately spherical or approximately elliptical depending on the free end of each water-soluble polymer compound chain. A shell partial shell surface is preferably formed.

  The ratio between the diameter of the core portion and the thickness of the shell portion can be appropriately changed according to the application. For example, the diameter of the core portion can be, for example, 5 nm to 500 nm, and the thickness of the shell portion can be, for example, 5 nm to 500 nm.

  The reactive functional group present at the free end of the water-soluble polymer compound chain is the other end (free end) before the one end (bonding end) of the water-soluble polymer compound is bonded to the core surface. Alternatively, after one end (bonding end) of the water-soluble polymer compound is bonded to the surface of the core portion, it can be introduced into the other end (free end). Any of these reactive functional groups is stable in water (or an aqueous solvent), and the introduction product [for example, the fluorescent rare earth metal complex-encapsulated core-shell type particle of the present invention is used as a labeling substance [for example, , A physiologically active substance (for example, an antibody, an enzyme, or DNA)], as long as it is a functional group capable of reacting with it, for example, an aldehyde group, a carboxyl group, a mercapto group, an amino group, a maleimide group, A vinyl sulfone group, a methanesulfonyl group, etc. can be mentioned, An aldehyde group, an amino group, a carboxyl group, or a maleimide group is preferable.

  In addition, in order to adjust the amount of introduction [for example, physiologically active substance (eg, antibody, enzyme, or DNA)] when the fluorescent rare earth metal complex-encapsulated core-shell type particle of the present invention is used as a labeling substance. And a water-soluble polymer compound chain having no reactive functional group at the free end (for example, a water-soluble polymer compound chain having a hydrogen atom, a methylene group, or a hydroxyl group at the free end) mixed in an arbitrary ratio It is also possible to do. In this case, in order not to interfere with the reaction between the introduced product and the functional group present at the free end, the length of the water-soluble polymer compound chain having no reactive functional group is changed to the water-soluble polymer compound having a reactive functional group. It is desirable to make it shorter than the chain.

As a method for preparing core-shell type particles having a water-soluble polymer compound brush having a reactive functional group on the surface layer on the surface of a core portion mainly composed of a water-insoluble polymer compound, there have been reported so far. Various known methods can be applied. As the known method, for example,
(1) (a) A block copolymer (parent-hydrophobic block copolymer) in which a hydrophilic segment having a reactive functional group and a hydrophobic segment are combined, and (b) a hydrophobic polymer are mixed. Emulsion method of preparing particles;
(2) A dispersion polymerization method in which a hydrophobic monomer is polymerized using a water-soluble polymer macromonomer having a reactive functional group as a dispersant; or (3) a method of introducing a water-soluble polymer compound brush on the surface of hydrogel particles, etc. Can be mentioned.

  Examples of the method for synthesizing the hydrophilic-hydrophobic block copolymer used in the emulsion method (1) and the method for synthesizing the water-soluble polymer macromonomer having a reactive functional group used in the dispersion polymerization method (2) include: It is possible to use a method (for example, WO96 / 33233, WO99 / 571743, JP11-322917, or JP2001-324507) already developed by the present inventor and its collaborators. it can.

［式中、Ｒ^１Ａ及びＲ^２Ａは、独立して、炭素数１〜１０のアルコキシ基、アリールオキシ基、又はアリール−（炭素数１〜３のアルキルオキシ）基であるか、あるいは、Ｒ^１Ａ及びＲ^２Ａは、一緒になって、炭素数１〜６のアルキル基で置換されていてもよい式：
−Ｏ−ＣＨ（Ｒ’）−ＣＨ−Ｏ−
（ここでＲ’は水素原子又は炭素数１〜６のアルキル基である）
で表されるエチレンジオキシであるか、あるいは、Ｒ^１Ａ及びＲ^２Ａは、一緒になって、オキシ（＝Ｏ）であり、
Ｌは、式：
−ＣＨ（Ｒ^３Ａ）−Ｏ−ＣＯ−ＣＨ（Ｒ^４Ａ）−
又は
−（ＣＨ_２）_ｒ−
で表される２価の基であり、ここで、Ｒ^３Ａ及びＲ^４Ａは、独立して、水素原子、炭素数１〜１０のアルキル基、アリール基、又はアリール−（炭素数１〜３のアルキルオキシ）基であり、ｒは２〜５の整数であり、ｍ_Ａは２〜１０，０００の整数であり、ｎ_Ａは２〜１０，０００の整数であり、ｐ_Ａは１〜５の整数であり、ｑは０又は１〜２０の整数であり、そして、Ｚ_Ａは、ｑが０であるとき、水素原子、アルカリ金属、アセチル基、アクリロイル基、メタクリロイル基、シンナモイル基、ｐ−トルエンスルホニル基、２−メルカプトプロピオニル基、２−アミノプロピオニル基、アリル基、又はビニルベンジル基であり、ｑが１〜２０の整数であるとき、炭素数１〜６のアルコキシカルボニル基、カルボキシルメルカプト基、又はアミノ基である］
で表されるヘトロテレケリックブロックコポリマーを挙げることができる。前記の式（ＩＡ）で表されるヘトロテレケリックブロックコポリマーは、例えば、ＷＯ９６／３３２３３号公報に記載の製造方法により調製することができる。 Examples of the compound that can be used as the parent-hydrophobic block copolymer or the water-soluble polymer macromonomer include, for example, the formula (IA) described in WO96 / 33233:

[Wherein, R ^1A and R ^2A are independently an alkoxy group having 1 to 10 carbon atoms, an aryloxy group, or an aryl- (alkyloxy having 1 to 3 carbon atoms) group, or R ^1A And R ^2A together may be substituted with an alkyl group having 1 to 6 carbon atoms:
-O-CH (R ')-CH-O-
(Where R ′ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
Or R ^1A and R ^{2A taken} together are oxy (═O),
L is the formula:
—CH (R ^3A ) —O—CO—CH (R ^4A ) —
Or - _{(CH 2) r} -
In which R ^3A and R ^4A are independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an aryl group, or aryl- (having 1 to 3 carbon atoms). alkyl) group, r is integer from 2 to 5, _{m a} is an integer of 2 to 10,000, _{n a} is an integer of 2 to 10,000, _{p a} is from 1 to 5 is an integer, q is 0 or an integer of 1 to 20, and, _{Z a} when q is 0, a hydrogen atom, an alkali metal, an acetyl group, an acryloyl group, methacryloyl group, cinnamoyl group, p- toluene A sulfonyl group, a 2-mercaptopropionyl group, a 2-aminopropionyl group, an allyl group, or a vinylbenzyl group, and when q is an integer of 1 to 20, an alkoxycarbonyl group having 1 to 6 carbon atoms, a carboxyl mercapto group, Or Is an amino group]
And a heterotelechelic block copolymer represented by the formula: The heterotelechelic block copolymer represented by the above formula (IA) can be prepared, for example, by the production method described in WO96 / 33233.

（式中、Ａ’及びＢ’は、相互に独立して、有機シリル型のアミノ保護基を表すか、あるいは、それらが結合する窒素原子と一緒になって、４〜７員のジシラ−アザシクロヘテロ環式環を形成することのできる有機シリル型のアミノ保護基であり、
Ｙは、水素原子、アルカリ金属、又は適当な反応によりアルカリ金属に代えて導入可能な有機基であり、
Ｒは水素原子又は炭素数１〜６のアルキル基であり、
ｎ_Ｂは１〜２０，０００の整数であり、そして、
ｍ_Ｂは０〜２０，０００の整数である）
で表されるポリオキシエチレン誘導体を挙げることができる。前記の式（ＩＢ）で表されるポリオキシエチレン誘導体は、例えば、ＷＯ９９／５７１７４３号公報に記載の製造方法により調製することができる。 Examples of the compound that can be used as the above-mentioned parent-hydrophobic block copolymer or water-soluble polymer macromonomer include the formula (IB) described in WO99 / 571743:

Wherein A ′ and B ′, independently of one another, represent an organosilyl-type amino protecting group, or together with the nitrogen atom to which they are attached, a 4-7 membered disila-aza An organosilyl-type amino protecting group capable of forming a cycloheterocyclic ring;
Y is a hydrogen atom, an alkali metal, or an organic group that can be introduced in place of the alkali metal by an appropriate reaction,
R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
n _B is an integer from 1 to 20,000, and
m _B is an integer of 0 to 20,000)
The polyoxyethylene derivative represented by these can be mentioned. The polyoxyethylene derivative represented by the formula (IB) can be prepared, for example, by the production method described in WO99 / 571743.

（式中、Ｒ^１Ｃ、Ｒ^２Ｃ、及びＲ^３Ｃは、相互に独立して、直鎖若しくは分岐のアルキル基又はアラルキル基を表し、
Ｚ_Ｃは、水素原子、アクリロイル基、メタクリロイル基、ビニルベンジル基、アリル基、パラトルエンスルホニル基、モノ−若しくはジ−低級アルキル置換アミノ基、カルボキシル基若しくはそのエステル基を有するアルキル基、アルデヒド基若しくはそのアセタール基を有するアルキル基、及びアルカリ金属からなる群より選ばれ、
ｍ_Ｃは０又は１であり、
ｎ_Ｃは０〜２０，０００の整数であり、そして、
ｐ_Ｃは正数２又は３であるが、
但し、ｍ_Ｃとｎ_Ｃは同時に０とならない）
で表される有機シリルスルフィド基含有化合物又はポリオキシエチレン誘導体を挙げることができる。前記の式（ＩＣ）で表される有機シリルスルフィド基含有化合物又はポリオキシエチレン誘導体は、例えば、特開平１１−３２２９１７号公報に記載の製造方法により調製することができる。 Examples of the compound that can be used as the above-mentioned parent-hydrophobic block copolymer or water-soluble polymer macromonomer include, for example, the formula (IC) described in JP-A-11-322917:

(Wherein R ^1C , R ^2C and R ^3C each independently represent a linear or branched alkyl group or an aralkyl group,
Z _C represents a hydrogen atom, an acryloyl group, a methacryloyl group, a vinylbenzyl group, allyl group, p-toluenesulfonyl group, mono- - or di - lower alkyl-substituted amino group, an alkyl group having a carboxyl group or its ester group, or aldehyde group Selected from the group consisting of an alkyl group having an acetal group and an alkali metal,
m _C is 0 or 1,
n _C is an integer from 0 to 20,000, and
p _C is a positive number 2 or 3,
However, m _C and n _C are not 0 at the same time)
An organic silyl sulfide group-containing compound or a polyoxyethylene derivative represented by The organic silyl sulfide group-containing compound or polyoxyethylene derivative represented by the above formula (IC) can be prepared, for example, by the production method described in JP-A-11-322917.

のユニットを表し、Ｒ^１は、水素原子又は低級アルキル基であり、Ｒ^２は、水素原子、メチル基、１−メチルエチル基、１−メチルプロピル基、２−メチルプロピル基、２−ベンジルオキシカルボニルエチル基、又はベンジルオキシカルボニルメチル基であり、Ｌ_１は、式：
−（ＣＨ_２）ｑ_Ｄ−Ｏ−
（ここで、ｑ_Ｄは１〜６の整数である）の連結基を表し、Ｌ_２は、原子価結合又は式：
−ＣＨ_２ＣＨ_２ＮＨ−
の連結基を表し、Ｌ_３は、カルボニル基、メチレン基、

の基を表し、Ｒは、水素原子又はメチル基を表し、ｍ_Ｄは１０〜２０，０００の整数であり、そして、ｎ_Ｄは０〜１０，０００の整数である］
で表されるホモポリマー又はブロックコポリマーを挙げることができる。なお、官能基Ａは、特に限定されるものではないが、例えば、アルデヒド基（−ＣＨＯ）、アミノ基（−ＮＨ_２）、メルカプト基（−ＳＨ）、水酸基（−ＯＨ）、カルボキシル基（−ＣＯＯＨ）を挙げることができる。前記の式（ＩＤ）で表されるホモポリマー又はブロックコポリマーは、例えば、特開２００１−３２４５０７号公報に記載の製造方法により調製することができる。 Furthermore, examples of the compound that can be used as the above-mentioned parent-hydrophobic block copolymer or water-soluble polymer macromonomer include, for example, the formula (ID) described in JP-A No. 2001-324507:
_{A-L 1 - (CH 2} CH 2 O) m D -L 2 - (B) n D -L 3 -CR = CH 2 (ID)
[Wherein A represents a functional group and B represents

R ¹ is a hydrogen atom or a lower alkyl group, and R ² is a hydrogen atom, a methyl group, a 1-methylethyl group, a 1-methylpropyl group, a 2-methylpropyl group, 2-benzyloxy. A carbonylethyl group or a benzyloxycarbonylmethyl group, and L ₁ is a group represented by the formula:
- _{(CH 2)} q D -O-
Wherein q _D is an integer from 1 to 6, and L ₂ is a valence bond or a formula:
—CH ₂ CH ₂ NH—
L ₃ represents a carbonyl group, a methylene group,

R represents a hydrogen atom or a methyl group, m _D is an integer of 10 to 20,000, and n _D is an integer of 0 to 10,000]
The homopolymer or block copolymer represented by these can be mentioned. The functional group A is not particularly limited. For example, the functional group A is an aldehyde group (—CHO), an amino group (—NH ₂ ), a mercapto group (—SH), a hydroxyl group (—OH), a carboxyl group (— COOH). The homopolymer or block copolymer represented by the formula (ID) can be prepared, for example, by the production method described in JP-A-2001-324507.

  According to the emulsion method (1) of core-shell type particles, each compound represented by the formula (IA), formula (IB), formula (IC), or formula (ID) (ie, parent-hydrophobic) The core-shell type particles (that is, the particles before encapsulating the fluorescent rare earth metal complex) can be prepared by mixing the type block copolymer) and the hydrophobic polymer.

  Further, according to the dispersion polymerization method (2) of the core-shell type particles, each compound represented by the formula (IA), formula (IB), formula (IC), or formula (ID) (that is, By polymerizing a hydrophobic monomer using a water-soluble polymer macromonomer) as a dispersant, core-shell type particles (that is, particles before encapsulating the fluorescent rare earth metal complex) can be prepared.

Further, according to the dispersion polymerization method (2) of the core-shell type particles, by using a water-soluble polymer macromonomer as a dispersant, and then suspending the fluorescent rare earth metal complex and the hydrophobic monomer, Encapsulation of the fluorescent rare earth metal complex and preparation of the core-shell type particles can be performed simultaneously. Further, for example, as the group Z _A in the above formula (IA), the group Y in the formula (IB), or the group Z _C in the formula (IC), a carboxyl group which is a functional group capable of forming a complex with a fluorescent rare earth metal, It is also possible to prepare a core-shell type particle by encapsulating a fluorescent rare earth metal complex in which a β-diketone ligand or a ferroin-based ligand is introduced to stabilize the fluorescent rare earth metal complex.

  The fluorescent rare earth metal complex that can be used in the present invention analyzes (including detection and measurement) the signal from the outside of the core-shell type particle even when it is enclosed in the core part of the core-shell type particle. As long as it is a substance that can be used, for example, lanthanoid (for example, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, or Lutetium), yttrium, or scandium, and the like. Lanthanoid complexes are preferable, and europium, samarium, terbium, or dysprosium complexes are more preferable.

  The diameter of the core-shell type particles encapsulating the fluorescent rare earth metal complex can also be appropriately changed according to the use, but in the case of a roughly spherical particle, it can be about 10 nm to 1 mm.

  The fluorescent rare earth metal complex-encapsulated core-shell type particles that can be used in the present invention can be prepared, for example, by a known production method (for example, the production method described in International Publication No. WO02 / 097436).

  For example, in the production method described in International Publication No. WO02 / 097436, for example, core-shell type particles (that is, particles before encapsulating a fluorescent rare earth metal complex) prepared by various known methods described above are used. In order to encapsulate the fluorescent rare earth metal complex, first, an organic solvent (for example, acetone or toluene) that swells the water-insoluble polymer compound that forms the core portion of the core-shell type particle is included in a certain ratio. The core-shell type particles and the fluorescent rare earth metal complex are immersed in the solution. By the immersion, the water-insoluble polymer compound swells, and the fluorescent rare earth metal complex is taken into the core portion with the swelling. Subsequently, when the organic solvent is removed from the mixture, the water-insoluble polymer compound contracts, but the hydrophobic fluorescent rare earth metal complex cannot escape from the core part and is encapsulated in the core part. . If desired, a fluorescent rare earth metal complex-encapsulated core-shell type particle can be obtained by removing the fluorescent rare earth metal complex that has not been incorporated into the core portion.

  In the production method, the method for removing the organic solvent from the mixture is not particularly limited. For example, the method of evaporating the organic solvent by evaporation or the like, or the method of shrinking in a non-solvent ( For example, a method in which a solution containing an organic solvent is replaced with a solution not containing the organic solvent) can be used.

  In addition, the ratio of the organic solvent in the organic solvent-containing solution used to swell the core portion may swell the water-insoluble polymer compound to such an extent that the fluorescent rare earth metal complex is incorporated into the water-insoluble polymer compound. Although it will not specifically limit as long as it is a ratio which can be performed, For example, it can be 40-60 (vol / vol)%.

  In the present invention, the reactive functional group present on the surface of the fluorescent rare earth metal complex-encapsulated core-shell type particle is further analyzed as an introduced substance (for example, a physiologically active substance) capable of reacting with the reactive functional group. A partner capable of binding specifically to the target substance can be bound.

  The partner is particularly limited as long as it can react with the reactive functional group present on the surface of the core-shell type particle encapsulating the fluorescent rare earth metal complex and can bind specifically to the analyte. Although it is not a thing, bioactive substances, for example, protein (for example, antibody or enzyme) or nucleic acid (for example, DNA or RNA) etc. can be mentioned.

  The method of bonding the introduced substance to the reactive functional group present on the surface of the core-shell type particle encapsulating the fluorescent rare earth metal complex is selected from the known bonding methods based on the kind of the introduced substance and reactive functional group. It can be appropriately selected depending on the case.

  For example, when the reactive functional group is an aldehyde group, the amino group and the Schiff base of a physiologically active substance [eg, protein (eg, antigen, antibody, receptor, etc.), peptide, low molecular hormone, DNA, etc.] By forming, they can be combined.

  Moreover, when a reactive functional group is a carboxyl group, it can couple | bond with the amino group of a bioactive substance using a condensing agent (for example, carbodiimide etc.). Alternatively, the carboxy group can be activated in advance with succinimide or maleimide or the like, and can be bonded by mixing with a physiologically active substance in that state.

  As the core-shell type particles encapsulating the fluorescent rare earth metal complex, the various particles described so far can be used. However, a large amount of the europium chelate having a long lifetime fluorescence is contained in the styrene portion as the core, and the external particle The surface has hydrophilic polyethylene glycol (PEG) in the shape of a brush and is hydrophilic, and it can bind antibodies, DNA, and physiologically active substances to the sensitive group at the PEG end while maintaining activity by chemical modification. Highly functional particles having the property of being capable of being produced are preferred.

  When this particle is irradiated with excitation light having a wavelength characteristic in the vicinity of 350 nm as a pulse, and a fluorescence having a wavelength characteristic at 615 nm is measured after a period of time in microseconds, a high S / N ratio is not affected by surrounding fluorescence. Can be detected. Further, these particles do not require a solution at the time of observation, and can be measured in a dry state like a fluorescent substance. One europium chelate has a limit in measurement sensitivity, but this reagent in which many europium chelates are encapsulated in particles can be measured with higher sensitivity than a detection system using an enzyme and a luminescent substrate. By using these particles as the labeling object of the chip system, it is possible to solve the problems of the labeling object so far.

As a microchip used in the method of the present invention, a chip generally used as a DNA chip (DNA array) or a protein chip can be used. In the method of the present invention, for example, various types of antigens and DNA are individually immobilized in a dot shape on a glass or plastic substrate and used for analysis.
In the case of immobilizing on glass, for example, a method of activating the surface with a silane coupling agent, or in the case of DNA immobilization, a method of adsorbing DNA or RNA with a polylysine coat can be used.
In the case of a plastic chip, a method of adsorbing to a styrene chip, a method of activating a carboxyl group of a carboxyl group-introduced plastic chip with carbodiimide and binding to an amino group of protein or DNA, or an amino group-containing plastic chip to glutar A method of activating with an aldehyde or the like and binding to an amino group of protein or DNA can be employed. In addition, a method for using a metal chip has been recently developed.

As the chip, either a transparent chip or an opaque chip can be used. In the case of a transparent chip, excitation light can be applied to the reaction surface, and fluorescence transmitted through the chip can be measured, or fluorescence reflected from the chip can be measured. When it is opaque, the fluorescence reflected from the chip is measured.
In addition, as the chip, for example, a completely flat chip, a chip recessed only in the reaction part, or a chip surrounded by a wall so that the reaction solution does not spill around the flat part can be used.

  An object that can be analyzed by the method of the present invention (that is, an analyte) is not particularly limited as long as a specific binding partner exists, but for example, a protein (for example, an antigen, an antibody, Or a receptor), a nucleic acid (eg, DNA or RNA), or a chemical substance having physiological activity. The test sample that can be analyzed by the method of the present invention is not particularly limited as long as it is a sample that may contain the analysis target substance. In particular, a biological sample such as blood, serum, plasma, urine, medulla Liquid, or a cell or tissue disruption liquid.

  In the method of the present invention, a plurality of analytes are immobilized on a single microchip in the form of dots, for example, directly by the binding method, or via a partner that specifically binds to the analyte. It can be supported indirectly. Indirect loading can be performed, for example, by fixing the partner in a dot shape on the microchip by the binding method and then bringing the test sample into contact with the entire dot on the chip. . The contact reaction may be a stationary reaction, or a reaction promoting method by shaking or warming may be employed.

  In order to place the substance to be analyzed or its partner in the form of dots, for example, a method of attaching to the tip of the pin and pressing it against the chip, or a method of hitting from a distance by an ink jet method can be employed. The plurality of test samples immobilized on one microchip can be one type of test sample (that is, all the same test sample), or can be two or more different types of test samples. it can.

  The contact between the analysis target substance supported in the form of dots on the microchip and the fluorescent rare earth metal complex-encapsulated core-shell type particles to which the partner for the analysis target substance is bound is, for example, a suspension containing the particles Can be carried out by placing it on the entire chip and reacting. This reaction may be performed, for example, when mixed with a test sample and performed together or after the test sample is removed, and the one that gives a specific signal is selected.

  Subsequently, after removing the label that did not contribute to the reaction (that is, the core-shell type particles encapsulating the fluorescent rare earth metal complex) by a method such as washing, the time-resolved fluorescence of the label immobilized on the chip is analyzed. (Preferably measured). When the chip is transparent, fluorescence is measured from the direction where the excitation light is applied or from the opposite side. For example, when europium chelate is used as the fluorescent rare earth metal complex, the chip surface is irradiated with excitation light having a wavelength characteristic in the vicinity of 340 nm for a short time of nanoseconds to microseconds, and after waiting for units of microseconds, the vicinity of 615 nm is irradiated. By measuring the fluorescence, the fluorescence of the europium chelate encapsulated particles can be measured without being affected by the fluorescence of the chip substrate, the fluorescence of the immobilized substance, and / or the fluorescence emitted from the measurement device side.

When analyzing time-resolved fluorescence, for example, there are a method in which excitation light is applied to each dot separately and a method in which the entire chip is applied. The method of applying each dot separately has the advantage that there is no fluorescence crosstalk and the amount of fluorescence of the dots can be accurately measured. The method applied to the entire chip has an advantage that the fluorescence amount of a large number of dots can be measured at a time. However, in this case, a device that captures fluorescence on a two-dimensional plane as an image is required.
In the embodiment as described above, a lot of information possessed by the specimen can be obtained at a time from the reaction with one chip.

As explained so far, one aspect of the method of the present invention is:
(1) A step of immobilizing a plurality of test samples possibly containing an analysis target substance in a dot shape on a microchip;
(2) Contacting the analysis target substance immobilized in the form of dots on a microchip and a fluorescent rare earth metal complex-encapsulated core-shell type particle bound with a partner capable of specifically binding to the analysis target substance The step of causing;
(3) a step of removing the analyte-unreacted fluorescent rare earth metal complex-encapsulated core-shell type particles immobilized on the microchip from the microchip; and (4) immobilized on the microchip. And analyzing the fluorescence derived from the fluorescent rare earth metal complex of the core-shell type particles encapsulating the fluorescent rare earth metal complex bound to the substance to be analyzed by time-resolved fluorescence analysis.

Another aspect of the method of the present invention is as follows:
(1) A step of immobilizing a plurality of partners capable of specifically binding to the analyte on the microchip in a dot shape;
(2) A step of bringing the partner immobilized in a dot shape on the microchip into contact with a test sample that may contain the analysis target substance;
(3) An analysis target substance bound to the partner immobilized on a microchip, and a fluorescent rare earth metal complex-encapsulated core-shell type particle to which a partner capable of specifically binding to the analysis target substance is bound Contacting with;
(4) a step of removing, from the microchip, the unreacted fluorescent rare earth metal complex-encapsulated core-shell particles bound to the partner immobilized on the microchip; and (5) the microchip Analyzing the fluorescence derived from the fluorescent rare earth metal complex of the fluorescent rare earth metal complex-encapsulated core-shell type particle bound to the analyte to be bound to the partner immobilized thereon by time-resolved fluorescence analysis Including.

  In the method of the present invention, all these steps can be constructed as one automated system.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention. Hereinafter, the core-shell type particles may be referred to as “nanoparticles”.

Example 1: Preparation of anti-human IgE antibody-conjugated europium chelate encapsulated nanoparticles for simultaneous analysis of allergen-specific antibodies As a rare earth metal complex encapsulated core-shell type particle, containing europium chelate in a central styrene polymer, Nanoparticles having a diameter of 160 nm and having an amino group at the end of the brush-like PEG on the surface (manufactured by Nanocarrier Co., Ltd.) were used. N-succinimidyl-4-N-maleimidomethylcyclohexane-1-carboxylate (SMCC) adjusted to 5 mg / mL with dimethylformamide in 0.5 mL of 10 mg / mL nanoparticles (20 mmol / L phosphate buffer pH 7.0) ) 50 μL was added, and the mixture was shaken at room temperature for 1 hour. The reaction solution was passed through a Sephadex G25 column (equilibrated with 1.5 × 12 cm, 50 mmol / L phosphate buffer pH 7.0), and only the cloudy particles were pooled.
On the other hand, 50 μL of ₂₀₀ mmol / L 2-mercaptoethylamine was added to 1 mL of 2 mg of anti-human IgE antibody F (ab ′) ₂ fraction (50 mmol / L acetate buffer pH 4.5) and subjected to a reduction reaction at 37 ° C. for 90 minutes. Subsequently, the antibody Fab ′ fractions that were eluted first were pooled through a Sephadex G25 column (1.5 × 12 cm, equilibrated with 50 mmol / L phosphate buffer pH 7.0).
4 mg of maleimide group-introduced nanoparticles and 1.5 mg of antibody Fab ′ fraction were mixed and allowed to stand at room temperature overnight.
The reaction was passed through a Sepharose CL6B column (1 × 90 cm, equilibrated with saline) to pool the initially clouded anti-human IgE antibody-bound nanoparticles.

Example 2: Preparation of a protein chip having 7 types of allergenic proteins, positive standard dots, and blank dots A chip having a 4 mm wall on a square styrene plate with a side of 12 mm was prepared as a reaction tank, and 2 μL each. Mite, wheat, rice, soybean, egg, milk, and peanut allergen-causing proteins and standard IgE serum were individually placed and allowed to adsorb at room temperature for 1 hour with nothing left as a blank.
After the liquid of each dot was removed with aspirate, 0.1% Tween 20 was further added to a 20 mmol / L phosphate buffer solution (hereinafter referred to as PBS) to which 0.15 mol / L sodium chloride was added. The entire reaction vessel was washed 3 times with a buffer solution (hereinafter referred to as T-PBS) to obtain an allergen-immobilized chip.

Example 3: Immune Reaction A sample obtained by adding 200 μL of PBS to 20 μL of sample serum suspected of allergy was placed in the reaction vessel of the allergen-immobilized chip prepared in Example 2 and allowed to stand at room temperature for 1 hour.
The reaction solution was aspirated and then washed 3 times with T-PBS. 200 μL of anti-human IgE antibody-bound nanoparticles (20 million counts / second as time-resolved fluorescence amount) prepared with PBS containing 1% albumin were placed in the reaction vessel of the chip and allowed to stand at room temperature for 1 hour.
The reaction solution was aspirated and then washed 5 times with T-PBS. This chip was placed on a chip time-resolved fluorescence measuring device (manufactured by Corona Electric Co., Ltd., a prototype), and the time-resolved fluorescence amount of 9 dots was measured.

  Table 1 shows the measurement results obtained by the method of the present invention (that is, the measured values of allergen-specific IgE antibodies in 7 items of an allergy positive sample). In addition, using 9 wells of a 96-well ELISA plate, the same protein as that used in Example 2 was immobilized separately, and the immune reaction of Example 3 was performed for each, and measured with a time-resolved fluorescence plate reader (Wallac). The results are shown as a control. The amount of allergen-causing protein, the amount of specimen serum and the dilution method, and the amount of anti-human IgE antibody-binding nanoparticles used in the control were exactly the same as the conditions in the method of the present invention.

  The method of the present invention can be applied to time-resolved fluorescence analysis.

Claims (2)

(A) a plurality of analytes supported in the form of dots on a microchip;
(B) (a) a core portion substantially composed of a water-insoluble polymer compound, and (b) a water-soluble polymer compound having a reactive functional group, and covering the surface of the core portion in a brush shape. A core-shell type particle comprising a block copolymer of a water-insoluble polymer and a water-soluble polymer as a whole. Including a step of contacting a core-shell type particle encapsulating a fluorescent rare earth metal complex in which a metal complex is encapsulated and a partner capable of specifically binding to an analyte is bound to the shell portion. A time-resolved fluorescence analysis method. The analysis target substance supported in the form of dots on the microchip binds to (1) a partner that can be specifically bound to the analysis target substance immobilized in the form of dots on the microchip. 2. The time-resolved fluorescence analysis method according to claim 1, wherein the time-resolved fluorescence analysis method is supported in the form of dots on the chip, or (2) directly fixed in the form of dots on the microchip.