JP2008170399A - Method for fixing fluorescent derivative substance to solid substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a solid substrate capable of detecting a specific substance at a high speed with high accuracy, without requiring labeling treatment due to a fluorescent substance by merely allowing a specimen to flow on the solid substrate. <P>SOLUTION: The specific substance in the specimen is detected at a high speed with high accuracy, using the fluorescence emitted from the substance, formed on the solid substrate by the condensation reaction of a fluorescent derived substance, which forms a substance having fluorescence with the detection wavelength by the condensation reaction with the specific substance in the specimen, with the specific substance by fixing the fluorescent derived substance by the direct chemical bond to the surface of the solid substrate or the chemical bond with a connection molecule via the chemical bond of the surface of the solid substrate with a joining molecule. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for immobilizing a fluorescent derivative substance on the surface of a fixed substrate, and more particularly to a method for immobilizing a fluorescent derivative substance for obtaining a solid substrate useful as a biochip for detecting a specific substance.

Among substances secreted in vivo, detection of chemical substances (bioactive amines) such as catecholamines and hydroxyindoleamines that have an aromatic ring or cyclic hydrocarbon and primary or secondary amine in their structure, It is carried out by subjecting a specimen to fluorescence derivatization with a fluorescent derivatized substance, etc., and then separating the substance by liquid chromatography, electrophoresis, etc., and specifying the substance by fluorescence (see Non-Patent Documents 1 to 3, Patent Document 1). .
However, these conventional methods use liquid chromatography or electrophoresis after the reaction between the fluorescent derivatized substance and a specific substance in the sample, so that detection is not only time consuming but also can be measured at once. There is a problem that the number is limited.

As a method for detecting a specific substance such as a protein, a substance that specifically binds to the specific substance to be detected, such as an antigen or an antibody, is directly or indirectly immobilized on the surface of a solid substrate. ing. (See Patent Documents 2 to 4).
However, these methods have a problem that, in order to detect a specific substance, a process such as labeling a specimen with a fluorescent substance is required separately, and cannot be immediately detected using a sensor.

On the other hand, Patent Document 5 describes that a specific fluorescent substance is dispersed and fixed in a porous filter to form a fluorescence generation layer. However, the technology relates to an element for measuring oxygen concentration using a “fluorescence disappearance phenomenon” in which fluorescence emitted by photoexcitation decreases in intensity according to the concentration of oxygen present.
“Fluorescent products of the reaction for the determination of catecholamines with 1,2-diphenylethylenediamine”, H. Nohta et al., Anal. Chem. 1992, 267, 137-139. “Aromaticglycinonitriles and methylamines as pre-column fluorescence derivatizationreagents for catecholamines”, H. Nohta et al., Anal. Chim. Acta 1997, 344,233-240. “Simultaneousdetermination of urinary catecholamines and 5-hidroxyindoleamines by high-performance liquid chromatography with fluorescence detection”, M. Yamaguchi et al., Analyst 1998, 123, 307-311. Japanese Patent Laid-Open No. 5-223413 JP 2005-241389 A JP 2001-247580 A International Publication No. 2002/012891 Pamphlet JP 2001-194304 A

  The present invention has been made in view of the circumstances as described above. A specific substance can be detected at a high speed and with a high degree of accuracy without requiring a labeling process with a fluorescent substance simply by flowing a specimen over the specimen. An object of the present invention is to provide a solid substrate.

  As a result of intensive research to achieve the above-mentioned object, the inventors have made a fluorescent derivative that does not emit fluorescence at the detection wavelength but forms a substance having fluorescence at the detection wavelength by a condensation reaction with a specific substance in the specimen. It has been found that by chemically immobilizing a chemical substance on the surface of a solid substrate, it becomes easy to create a biochemical chip for detecting a specific substance, and the detection system can be simplified. That is, a fluorescent substance is formed on the surface of a solid substrate on which a fluorescent derivatized substance is chemically immobilized by a condensation reaction between the analyte to be detected and the fluorescent derivatized substance, and the fluorescence is detected. As a result, it has been found that a specific substance in a specimen can be detected at high speed and with high accuracy.

The present invention has been completed based on these findings, and is as follows.
(1) A fluorescent derivatized substance that forms a substance having fluorescence at a detection wavelength by a condensation reaction with a specific substance in a specimen is directly bonded to the surface of the solid substrate or between the solid substrate surface and the connecting molecule. A method of solidifying a fluorescent derivatized substance on a solid substrate surface, wherein the fluorescent derivatized substance is immobilized by chemical bonding with a connecting molecule through chemical bonding.
(2) The connecting molecule can be chemically bonded to the surface of the solid substrate and can be chemically bonded to the fluorescent derivatized substance, or can be chemically bonded to the surface of the solid substrate and chemically bonded to other connecting molecules. Can be chemically bonded to other connecting molecules and can be chemically bonded to the fluorescent derivatized substance, or can be chemically bonded to other connecting molecules and can be chemically bonded to another connecting molecule. (1) The immobilization method according to (1) above.
(3) The specific substance is catecholamines or hydroxyindoles, and chemically binds to the fluorescent derivatized substance in the presence of an oxidizing agent or oxygen. Immobilization method.
(4) The fluorescent derivatized substance is either a benzylamine derivative, a phenylglycinonitrile derivative, or a diphenylethylenediamine derivative, and forms a phosphor by chemically bonding with the specific substance in the presence of an oxidizing agent or oxygen. The immobilization method according to any one of (1) to (3) above, wherein
(5) The immobilization method according to any one of (1) to (4), wherein the solid substrate is formed mainly of glass, plastic, metal, metal oxide, or silicon.
(6) The above-mentioned (1), wherein the specific substance does not exhibit fluorescence at the detection wavelength before chemically bonding with the fluorescent derivatized substance immobilized on the surface of the solid substrate, and exhibits fluorescence after chemical bonding. The immobilization method according to any one of 1) to (5).

  In the conventional fluorescence detection method, the detection pretreatment and the detection time such as liquid chromatography or electrophoresis require 20 to 60 minutes, but by using the solid substrate obtained in the method of the present invention, the surface can be obtained. Fluorescence detection becomes possible within 5 minutes just by passing a sample through the tube.

The solid substrate in the present invention serves as a support for ensuring the mechanical strength required for a chemical biosensor.
The shape of the substrate is not particularly limited, but is preferably formed in a card shape or a disk shape.
The material for forming the solid substrate is not particularly limited as long as it can function as a support for a fluorescence detection type chemical biosensor. For example, glass, quartz, ceramics, plastics, metal, metal oxide Alternatively, it can be formed of a material containing silicon as a main component. Plastics include polycarbonate resin, olefin resin, acrylic resin, epoxy resin, polystyrene resin, polyethylene resin, polypropylene resin, silicon resin, fluorine resin, ABS resin, urethane resin, phenol resin, polymethyl methacrylate resin, polyisobutyl methacrylate. Examples thereof include resins and polyimide resins.

  The fluorescent derivatized substance used in the present invention itself does not emit fluorescence at the detection wavelength, but forms a substance having fluorescence at the detection wavelength by condensation reaction with a specific substance in the specimen. As such fluorescent derivatized substances, those already well known in the above-described conventional liquid chromatography and catecholamine detection using electrophoresis are used, and in particular, benzylamine derivatives, phenylglycinonitrile derivatives. Or, such as a diphenylethylenediamine derivative, which forms a phosphor by chemically bonding with a specific substance in the presence of an oxidizing agent or oxygen is preferably used. An example is shown below.

In the present invention, the fluorescent derivatized substance is immobilized by a chemical bond with a connecting molecule through a chemical bond directly to the surface of the solid substrate or a chemical bond between the solid substrate surface and the connecting molecule. .
The connecting molecule used in the present invention can be (I) a chemical bond with a solid substrate surface and a chemical bond with the fluorescent derivatized substance, or (II) a chemical bond with the solid substrate surface. And those that can chemically bond with other connecting molecules, or (III) those that can chemically bond with other connecting molecules and can chemically bond with the fluorescent derivatized substance, or (IV) with other connecting molecules It is selected from those capable of chemical bonding and capable of chemical bonding with another connecting molecule.
Examples of these are described below.

ガラスなどの表面に化学結合し、且つ前記蛍光誘導体化物質であるベンジルアミン誘導体の上記（化３）で示されるｐ−キシリレンジアミン（ｐＸＤＡ）と化学結合する。
「４−メルカプト安息香酸」

メルカプト基が化学反応によって金・銀などの表面に化学結合し、且つ他方末端のカルボキシル基が前記蛍光誘導体化物質であるベンジルアミン誘導体のｐＸＤＡのアミノ基と化学結合する。 (I) Examples of those capable of being chemically bonded to the surface of a solid substrate and capable of being chemically bonded to the fluorescent derivatized substance “Di (N-succinimidyl) carbonate (DSC)”

It chemically bonds to the surface of glass or the like, and chemically bonds to p-xylylenediamine (pXDA) represented by the above (Chemical Formula 3) of the benzylamine derivative that is the fluorescent derivatized substance.
“4-Mercaptobenzoic acid”

The mercapto group is chemically bonded to the surface of gold, silver or the like by a chemical reaction, and the carboxyl group at the other end is chemically bonded to the amino group of pXDA of the benzylamine derivative which is the fluorescent derivatizing substance.

「３−（２−アミノエチルアミノ）プロピルトリメトキシシラン」

これらの（化７）及び（化８）は、一方の末端（アルコキシル基）がガラス基板表面へ化学結合し、他方の末端（アミノ基）がカルボキシル基、スクシンイミジル基あるいはイソチオシアネート基を有する他の接続分子と化学結合する。
「Ｎ−スクシンイミジル−４−マレイミドブチラート」

「Ｎ−スクシンイミジル−６−マレイミドヘキサノエート」

これらの（化９）及び（化１０）は、一方の末端（スクシンイミジル基）がガラス基板表面へ化学結合し、他方の末端（マレイミド基）がメルカプト基を有する他の接続分子と化学結合する。 (II) Examples of those capable of being chemically bonded to the surface of the solid substrate and capable of being chemically bonded to other connecting molecules “3-aminopropyltriethoxysilane (APS)”

“3- (2-Aminoethylamino) propyltrimethoxysilane”

In these (Chemical Formula 7) and (Chemical Formula 8), one terminal (alkoxyl group) is chemically bonded to the glass substrate surface, and the other terminal (amino group) is a carboxyl group, succinimidyl group or isothiocyanate group. Chemically bonds with connecting molecules.
“N-succinimidyl-4-maleimidobutyrate”

“N-succinimidyl-6-maleimidohexanoate”

In these (Chemical Formula 9) and (Chemical Formula 10), one terminal (succinimidyl group) is chemically bonded to the glass substrate surface, and the other terminal (maleimide group) is chemically bonded to another connecting molecule having a mercapto group.

一方の末端（イソチオシアネート基）が他の接続分子のアミノ基と化学結合し、他方の末端（イソチオシアネート基）が蛍光誘導体化物質のベンジルアミン誘導体であるＸＤＡのアミノ基と化学結合する。
「上記（化６）で示される４−メルカプト安息香酸」
一方の末端（メルカプト基）が他の接続分子のマレイミド基と化学結合し、他方の末端（カルボキシル基）が蛍光誘導体化物質のベンジルアミン誘導体であるＸＤＡのアミノ基と化学結合する。
「テレフタル酸」

「アジピン酸」

これらの（化１２）及び（化１３）は、一方の末端（カルボキシル基）が他の接続分子のアミノ基と化学結合し、他方の末端（カルボキシル基）が蛍光誘導体化物質のベンジルアミン誘導体であるＸＤＡのアミノ基と化学結合する。 (III) Examples of those capable of chemically bonding with other connecting molecules and capable of chemically bonding with the fluorescent derivatized substance "1,4-phenylene diisothiocyanate (PDIC)"

One end (isothiocyanate group) is chemically bonded to the amino group of the other connecting molecule, and the other end (isothiocyanate group) is chemically bonded to the amino group of XDA, which is a benzylamine derivative of the fluorescent derivatizing substance.
“4-mercaptobenzoic acid represented by the above (Chemical Formula 6)”
One end (mercapto group) is chemically bonded to the maleimide group of the other connecting molecule, and the other end (carboxyl group) is chemically bonded to the amino group of XDA, which is a benzylamine derivative of the fluorescent derivatizing substance.
"Terephthalic acid"

"Adipic acid"

In these (Chemical Formula 12) and (Chemical Formula 13), one terminal (carboxyl group) is chemically bonded to the amino group of the other connecting molecule, and the other terminal (carboxyl group) is a benzylamine derivative that is a fluorescent derivatization substance. It chemically bonds to the amino group of some XDA.

一方の末端（アミノ基）が他の接続分子のカルボキシル基、イソチオシアネート基あるいはスクシンイミジル基と化学結合し、他方の末端（アミノ基）が別の接続分子のカルボキシル基、イソチオシアネート基あるいはスクシンイミジル基と化学結合する。
「上記（化１２）で示されるテレフタル酸」又は「上記（化１３）で示されるアジピン酸」
「上記（化１１）で示されるＰＤＩＣ」 (IV) Examples of compounds that can be chemically bonded to other connecting molecules and can be chemically bonded to other connecting molecules "1,4-diaminobutane"

One end (amino group) is chemically bonded to the carboxyl group, isothiocyanate group or succinimidyl group of the other connecting molecule, and the other end (amino group) is connected to the carboxyl group, isothiocyanate group or succinimidyl group of another connecting molecule. Chemically bond.
"Terephthalic acid represented by (Chemical Formula 12)" or "Adipic acid represented by (Chemical Formula 13)"
"PDIC shown in (Chemical 11) above"

  As the specific substance that can be detected using the solid substrate on which the fluorescent derivatizing substance of the present invention is immobilized, any substance can be used as long as it forms a substance having fluorescence at the detection wavelength by a condensation reaction with the fluorescent derivatizing substance. Specifically, it is a substance secreted in the body, and has an aromatic ring or cyclic hydrocarbon and a primary or secondary amine in its structure. As described above, specific substances having such characteristics are already known, but in particular, such as catecholamines or hydroxyindoles, chemically bond with the fluorescent derivatized substance in the presence of an oxidizing agent or oxygen. Those are preferably used. An example is shown below.

５−ヒドロキシインドール−３−酢酸

5-hydroxyindole-3-acetic acid

Next, a detection method using a solid substrate obtained by the method of the present invention will be described.
A sample liquid is flowed on a solid substrate obtained by immobilizing a fluorescent derivatized substance obtained by the method of the present invention, and has fluorescence at a detection wavelength by a condensation reaction between the specific substance in the specimen and the fluorescent derivatized substance. By forming the substance and detecting the fluorescence, the presence or absence of the specific substance in the sample can be detected.
The sample solution is prepared using a solvent that can sufficiently dissolve the specific substance to be detected, and is prepared so that the pH is about 8 using a buffer solution. After flowing the specimen liquid thus prepared on the solid substrate, the specimen liquid is allowed to stand for a while to sufficiently perform a condensation reaction between the specific substance in the specimen and the fluorescent derivatized substance. Rinse and dry.

The solid substrate surface on which the fluorescent derivatized substance is immobilized by the method of the present invention is irradiated with excitation light, and is generated from a substance formed by a condensation reaction between the specific substance in the specimen and the fluorescent derivatized substance. Detect fluorescence.
For example, in the case of detecting epinephrine using p-xylerindiamine (pXDA) as a benzylamine derivative as a fluorescent derivatizing substance, the presence or absence of fluorescence having a peak at around 470 nm when irradiated with 375 nm light as excitation light Is detected.

In the above detection method, the excitation light used for fluorescence detection may be irradiated on the surface on which the fluorescent derivatization substance on the solid substrate is immobilized, or may be irradiated on the side surface thereof. When the solid substrate is made of a material that transmits excitation light, the opposite surface may be irradiated.
The fluorescence is preferably detected from a direction perpendicular to the incident light since the influence of transmitted light, reflected light, stray light, etc. is minimal.
Furthermore, when a solid substrate having a fluorescent derivatized substance immobilized on the surface thereof by the method of the present invention is used, the fluorescence spectrum of the substance formed by the condensation reaction between the specific substance in the specimen and the fluorescent derivatized substance Therefore, it is possible to specify the type of the specific substance present in the specimen.

EXAMPLES Next, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these Examples.
(Example 1)
The fluorescent derivatized material was immobilized on the surface of the synthetic quartz substrate as follows. 1 to 3 schematically show the procedure.
A synthetic quartz substrate washed with water, ethanol or the like is vacuum-dried and then treated with ozone to hydrophilize the surface, and then the connecting molecule 3-aminopropyltriethoxysilane (APS) (the above (Chemical Formula 7) )) The synthetic quartz substrate was immersed in an ethanol solution containing 10 mM at 50 ° C. for 15 hours. Thereafter, the substrate was taken out, rinsed with ethanol, dried, and allowed to stand at 100 ° C. for 1 hour to chemically bond APS to the surface of the synthetic quartz substrate. (FIG. 1).

Next, the substrate is immersed in a toluene solution containing 50 mM 1,4-phenylene diisothiocyanate (PDIC) (the above (chemical formula 11)) as a connecting molecule for 2 hours at 50 ° C., and the surface is rinsed with toluene. And dried (Figure 2).
Furthermore, it is immersed in a 50 mM borate buffer solution (pH 8.5) containing 50 mM of p-xylylenediamine (XDA) (the above (Chemical Formula 3)), which is a fluorescent derivatizing substance benzylamine derivative, at room temperature for 2 hours, and then is made into a fluorescent derivatization. Chemical bonding was performed so that the site of the substance benzylamine emerged at the terminal (FIG. 3).

  In order to investigate whether the fluorescent derivatized substrate benzylamine chemically immobilized on the substrate surface binds to catecholamines to form a phosphor, epinephrine (a chemical formula 15) 500 μM, which is a kind of catecholamines, is used. A synthetic quartz substrate having a benzylamine moiety chemically immobilized on its surface was immersed in a mixed solution of a 50 mM borate buffer solution and a 50 mM potassium ferricyanide solution containing the solution at 50 ° C. for 5 minutes.

Using this, fluorescence spectrum measurement was performed using 375 nm as excitation light, and a fluorescence peak due to fluorescence generated by the reaction of benzylamine and epinephrine on the sensor surface was confirmed near 470 nm. FIG. 4 is a diagram showing a fluorescence spectrum. In the figure, the dotted line represents the spectrum before the epinephrine reaction, and the solid line represents the spectrum after the epinephrine reaction.
From this result, it can be inferred that epinephrine was fluorescently derivatized with a benzylamine moiety chemically immobilized on the surface of the solid substrate as schematically shown in FIG.

(Example 2)
The fluorescent derivatized material was immobilized on the surface of the synthetic quartz substrate as follows. 6 and 7 schematically show the procedure.
A synthetic quartz substrate washed with water, ethanol or the like is vacuum-dried and then subjected to ozone irradiation treatment to hydrophilize the surface. Next, di (N-succinimidyl) carbonate (DSC) (connecting molecule) ) Reference) 15 ml of an acetone solution containing 5 mM was prepared, and 15 ml of a pyridine solution containing 1.25 ml of triethylamine was mixed therewith. The DSC was chemically bonded to the surface of the synthetic quartz substrate by immersing the synthetic quartz substrate hydrophilized in this mixed solution at room temperature for 15 hours (FIG. 6).
Next, after rinsing the substrate surface with ethanol and drying, 30 ml of a 1/15 M phosphate buffer solution (pH 7.4) containing 1 mM XDA which is a fluorescent derivatizing substance benzylamine derivative is prepared, and this is carried out at room temperature for 2 hours. It was immersed and chemically bonded so that the benzylamine moiety appeared at the end (FIG. 7).

(Example 3)
The fluorescent derivatized material was immobilized on the surface of the synthetic quartz substrate as follows. 1 and 8 schematically show the procedure.
A synthetic quartz substrate washed with water, ethanol or the like is vacuum-dried and then subjected to ozone irradiation treatment to make the surface hydrophilic, and then an ethanol solution containing 10 mM 3-aminopropyltriethoxysilane (APS) as a connecting molecule The synthetic quartz substrate was immersed in it at 50 ° C. for 15 hours. After taking out, rinsing the substrate surface with ethanol, drying, and leaving it to stand at 100 ° C. for 1 hour, APS was chemically bonded to the synthetic quartz substrate surface (FIG. 1).
Next, 10 ml of a phosphate buffer (pH 7.4) containing 500 μM of 4-aminomethylbenzoic acid (the above (Chemical Formula 4)) and 500 μM of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) A solution in which 10 ml of a phosphate buffer (pH 7.4) containing 10 ml was mixed, and a substrate having APS bound to the surface was immersed at room temperature for 1 hour to chemically immobilize the benzylamine moiety at the terminal ( FIG. 8).

  By chemically immobilizing a fluorescent derivatized substance on the surface of a solid substrate by the method of the present invention, fluorescent substances mainly targeted to specific substances, particularly catecholamines of neurotransmitters secreted by a load such as stress. Since it can be used as a biochemical chip for detection, it can be expected to be used in fields such as preventive medicine and the environment, and can also be expected to be applied to devices used in ubiquitous medicine (medicine that anyone can enjoy anywhere, anytime).

The figure which shows typically a mode that 3-aminopropyl triethoxysilane (APS) was combined with the synthetic quartz board | substrate surface which carried out the hydrophilic process. The figure which shows typically a mode that 1, 4- phenylene diisothiocyanate (PDIC) was couple | bonded through the thiourea bond with the board | substrate which the surface became active by the amino group. The figure which shows typically a mode that the synthetic quartz board | substrate surface in which benzylamine exists in the terminal was produced by couple | bonding p-xylylenediamine (pXDA) through a thiourea coupling | bonding. The figure which shows the fluorescence spectrum of the fluorescent substance produced | generated by reaction of benzylamine and epinephrine. The figure which shows typically the fluorescent substance produced | generated by reaction of benzylamine and epinephrine on a synthetic quartz substrate. The figure which shows typically a mode that di (N-succinimidyl) carbonate (DSC) was combined with the synthetic quartz board | substrate surface which carried out the hydrophilic process. The figure which shows typically a mode that the synthetic quartz board | substrate surface in which benzylamine exists in the terminal was produced by combining p-xylylenediamine (pXDA). The figure which shows typically a mode that the synthetic quartz substrate surface in which benzylamine exists in the terminal was produced.

Claims (6)

  A fluorescent derivatized substance that forms a substance having fluorescence at the detection wavelength by a condensation reaction with a specific substance in the sample is directly bonded to the surface of the solid substrate or chemically bonded to the connecting molecule. A method for solidifying a fluorescent derivatized substance onto a solid substrate surface, wherein the fluorescent derivatized substance is immobilized by chemical bonding with a connecting molecule through a connecting molecule.   The connecting molecule can be chemically bonded to the surface of a solid substrate and can be chemically bonded to the fluorescent derivatized substance, or can be chemically bonded to the surface of the solid substrate and can be chemically bonded to another connecting molecule. Alternatively, it can be chemically bonded to another connecting molecule and can be chemically bonded to the fluorescent derivatized substance, or can be chemically bonded to another connecting molecule and can be chemically bonded to another connecting molecule. The immobilization method according to claim 1.   The immobilization method according to claim 1 or 2, wherein the specific substance is catecholamines or hydroxyindoles, and chemically binds to the fluorescent derivatized substance in the presence of an oxidizing agent or oxygen.   The fluorescent derivatized substance is any one of a benzylamine derivative, a phenylglycinonitrile derivative, and a diphenylethylenediamine derivative, and forms a phosphor by chemically bonding with the specific substance in the presence of an oxidizing agent or oxygen. The immobilization method according to any one of claims 1 to 3.   The immobilization method according to claim 1, wherein the solid substrate is formed of glass, plastic, metal, metal oxide, or silicon as a main component.   6. The specific substance does not exhibit fluorescence at a detection wavelength before being chemically bonded to the fluorescent derivatized substance immobilized on the surface of a solid substrate, and exhibits fluorescence after chemical bonding. The immobilization method according to any of the above.

Images