JPH102862A - Immobilizing reagent for detecting fluorescence of catechol compound and vanillyl compound, and detecting method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enhance the sensitivity of detection by bringing a catechol compound or a vanillyl compound into reaction with 1,2-diarylethylenediamine held to a solid phase bonded hydrophobically under the presence of an oxidizing agent to produce a benzoxazole derivative, and quantitatively determining the fluorescence. SOLUTION: 1,2-Diarylethylenediamine is fixed by hydrophobic bonding to a hydrophobic bonding layer 2 on the surface of an immobilizing reagent. The aryl part of 1,2-diarylethylenediamine is deeply bonded to and held by the hydrophobic bonding layer 2, and the diamine part is held on the outside from the surface. A catechol compound or vanillyl compound forms a benzoxazole derivative with 1,2-diarylethylenediamine under the presence of an oxidizing agent. For the measurement of fluorescence of the benzoxazole derivative, various fluorescence measuring devices are usable. On the basis of the comparison with the fluorescence of a sample having a known concentration, the concentration of a matter to be detected in the sample can be also measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for immobilizing catechol and vanillyl compound for fluorescence detection, and a method for detecting catechol and vanillyl compound using the reagent.

[0002]

2. Description of the Related Art As a method for detecting a catechol compound and a vanillyl compound, an electrochemical detection method or a fluorescence method is known. Although the electrochemical detection method has high sensitivity, the electrode surface easily adsorbs impurities, and it is difficult to obtain reproducible results. Also, batch processing of many samples is difficult.
As a method for determining the fluorescence of a catechol compound, there are a method using ethylenediamine and a method using 1,2-diarylethylenediamine. In the former, the reagent is unstable and the sensitivity is not sufficient. The latter is highly sensitive and the reagents themselves are relatively stable, but are unstable in solution and are preferably prepared at the time of use.

[0003] Furthermore, in the fluorometric method for determination of vanillyl compounds, periodate oxidation in post-column fluorescent derivatization high-performance liquid chromatography (HPLC) is performed.
Only a method of reacting 1,2-diphenylethylenediamine is reported, and it is difficult to treat many samples. In addition, fluorescent derivatization is performed using a fluorescent reagent, other reagents,
The target compound in the sample is diluted to add a buffer or the like. Therefore, for high sensitivity, concentration by extraction, solvent removal, or the like is necessary.

[0004]

SUMMARY OF THE INVENTION The present invention provides a fluorescent reagent which does not have the drawbacks of the conventional detection methods described above and which is easy to use and has good storage stability for detecting catechol compounds and vanillyl compounds. Provided is a method capable of detecting with high sensitivity without concentrating a reaction solution.

[0005]

The detection method according to the present invention comprises:
A catechol compound or an o-methyl compound thereof (that is, a vanillyl compound), which is a compound to be detected, is reacted with 1,2-diarylethylenediamine, which is hydrophobically held on a specific solid phase, in the presence of an oxidizing agent to form a benzoxazole derivative. It is to determine the fluorescence of the benzoxazole derivative formed and obtained.

More specifically, the present invention provides a solid-phase reagent containing 1,2-diarylethylenediamine.

The aryl portion of the 1,2-diarylethylenediamine is phenyl, alkylphenyl, alkoxyphenyl, chlorophenyl, naphthyl,
It is intended to provide a solid-phased reagent characterized by being any of biphenyl.

Further, the present invention relates to a solid-phased reagent characterized in that the surface of the solid-phased reagent has a hydrophobic binding layer.

Further, the present invention relates to a solid-phased reagent, wherein the immobilized reagent has a shape of a thin plate, filter paper, or particles.

Further, the present invention relates to a method for reacting a catechol compound and a vanillyl compound together with an oxidizing agent with a solid-phased reagent containing 1,2-diarylethylenediamine to form a benzoxazole derivative, The present invention provides a method for detecting a catechol compound and a vanillyl compound by measuring the following.

[0011] The present invention also provides a method for detecting a catechol compound and a vanillyl compound, wherein the oxidizing agent is any of potassium ferricyanide.

Hereinafter, the immobilized reagent according to the present invention, its preparation, usage, and the like will be described in detail.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION

(Immobilization Reagent) The immobilization reagent according to the present invention is obtained by immobilizing 1,2-diarylethylenediamine on the surface of a hydrophobic binding layer provided on a suitable carrier (see FIG. 1).

Further, the immobilizing reagent according to the present invention may further include a layer for other uses in addition to the hydrophobic binding layer. For example, a layer containing another chemical reagent, a layer for filtering unnecessary substances in a sample, a reflective layer for more efficiently measuring the obtained fluorescence, and the like. Further, these layers may be a combination of a plurality.

The hydrophobic bonding layer according to the present invention is not particularly limited as long as it can hold 1,2-diarylethylenediamine based on hydrophobic interaction, and has hydrophobic interaction. May be selected as a group already known as Particularly preferably, reversed-phase partitioning (adsorption)
And various long-chain alkyl (C8-C18) groups developed for use in chromatography. Further, whether or not the hydrophobicity is sufficient can be easily determined by examining the adsorptivity to the actually used diarylethylenediamine. For example, when an octadecyl group is selected as the hydrophobic binding layer, the adsorbed 1,2-diphenylethylenediamine is not extracted not only for water (100%) but also for 50% acetonitrile. .

The hydrophobic binding layer according to the present invention can be provided on a suitable carrier, and the state is shown in FIG. This carrier is not particularly limited, and can take various forms depending on the purpose of use of the immobilized reagent according to the present invention. For example, when the carrier is made of silica gel, silicate bonding is possible.

Further, the immobilized reagent according to the present invention is shown in FIG.
As shown in (1), 1,2-diarylethylenediamine is immobilized on a hydrophobic binding layer on the surface of the immobilizing reagent by hydrophobic bonding. Therefore, 1,2-diarylethylenediamine needs to have an aryl moiety capable of sufficiently providing the above-mentioned hydrophobic bond. For example, phenyl, alkylphenyl, alkoxyphenyl, chlorophenyl, naphthyl, biphenyl and the like are preferably used. . In this case, the aryl moiety will be held deeply bound to the hydrophobic binding layer, and the diamine moiety will be held out of the surface due to its hydrophilicity.

(Shape of Immobilized Reagent) The shape of the immobilized reagent according to the present invention is not particularly limited, and the shape can be arbitrarily selected depending on the purpose of use. For example,
It is in the form of a plate or filter paper, granular, or powdery.
For example, a plate-like material has a shape suitable for use by directly immersing it in a sample solution together with an appropriate oxidizing agent, and in the case of a granular or powdery material, an immobilized reagent is filled in an appropriate column or the like. After the sample is poured and the sample solution is simultaneously concentrated to separate the component to be detected from the sample, the benzoxazole derivative described below can be formed.

If desired, the immobilized reagent according to the present invention may be powdered on a specific carrier (for example, glass,
It is also possible to form a layer on plastic (plastic) and overlay a layer containing a necessary oxidizing agent on or under the layer, and use the layer for dry chemistry.

(Formation of Fluorescent Benzoxazole Derivative) As shown in FIG. 3, a catechol compound or a vanillyl compound is composed of 1,2-diarylethylenediamine,
A benzoxazole derivative is formed in the presence of a suitable oxidizing agent. The resulting benzoxazole derivative has strong fluorescence at about 440 to 500 nm. The oxidizing agent mainly depends on the type of the catechol compound or vanillyl compound to be detected and the type of the 1,2-diarylethylenediamine used. Therefore, it is easy to select a suitable oxidizing agent from commonly known oxidizing agents,
Particularly preferred are potassium ferricyanide, sodium periodate, sodium iodate, peroxidase-hydrogen peroxide and the like. Also, not only chemical oxidation but also electrochemical oxidation can be used.

Furthermore, the formed benzoxazole derivative becomes more hydrophobic than the diamine moiety, and more strongly binds to the hydrophobic binding layer.

The conditions for the above-mentioned fluorescent benzoxazole formation reaction are not particularly limited, and various conditions (eg, type of buffer, type and concentration of oxidizing agent, concentration, concentration of sample, pretreatment) (Depending on the degree of the reaction) and the like, and a benzoxazole-forming reaction is performed on the surface by contacting the immobilization reagent. At this time, if necessary,
The reaction is promoted by stirring or shaking. As explained above, the fluorescent product is more hydrophobic than the reagent and is therefore retained in the carrier under normal reaction conditions.

The formed fluorescent benzoxazole derivative is stably retained on the surface of the immobilized reagent, but can be separated from the immobilized reagent by a specific means for the subsequent fluorescence measurement. (Eg, extraction with an organic solvent).

(Fluorescence Measurement) The method for measuring the fluorescence of the fluorescent benzoxazole derivative formed on the surface of the immobilized reagent or the fluorescence of the benzoxazole derivative separated by solvent extraction or the like is not particularly limited. A fluorimeter can be used. For example, a method in which fluorescence is directly observed on the surface of the carrier by irradiation of excitation light is possible. It is also possible to measure the concentration of an analyte in a sample based on comparison with fluorescence from a sample having a known concentration.

[0025]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist.

Example 1 Preparation of Immobilized Reagent A reverse phase partition type thin layer chromatography (50-100 mM 1,2-diphenylethylenediamine aqueous solution (dissolved in 0.1N hydrochloric acid at pH 4-6)) was used. TLC)
A plate (HPTLC RP18, manufactured by Merck) is immersed and left for 10 to 60 minutes in a light-shielded state.

The HPTLC plate is taken out, the surface is washed with distilled water, the water content on the surface is removed with a filter paper or the like, and the plate is dried in a desiccator.

When other 1,2-diallylethylenediamine is hardly soluble in water, it can be dissolved by adding 5-30% of alcohol.

(Example 2) Detection of catechol compound To 2 ml of 50 mM bicine buffer containing 0.1-0.6 M glycine, 1 ml of a sample and 0.1 ml of 0.05-0.2% potassium ferricyanide are added. Example 1 was added to this mixture.
Add the immobilization reagent prepared in the above, and add 10-6 at room temperature to 60 ° C.
Incubate for 0 minutes. Thereafter, the immobilized reagent was washed with distilled water, excess water was absorbed with filter paper, and the fluorescence of the surface was measured (measurement device; RF5000, manufactured by Shimadzu Corporation, measurement wavelength; 330 nm on the excitation side, 440 nm on the emission side). .

(Example 3) Detection of catechol and vanillyl compound A sample solution was adjusted to be weakly acidic (pH 4-5), and 1 ml of the solution was adjusted.
0.1 ml of 20 mM sodium periodate was added to
Leave for 10-20 minutes. (In this reaction, the catechol and the vanillyl compound are converted into o-quinones.) The same operation is performed by using 1 ml of this in place of the sample of Example 2.

(Example 4) A small amount of sample A filter paper (a small piece obtained by punching) immersed in the bicine buffer of Example 2 was placed on the immobilization reagent prepared in Example, and a sample, potassium ferricyanide was further placed thereon. The solution is dropped and reacted on the surface of the immobilized reagent. After the reaction, the surface is washed with distilled water, and the fluorescent spot generated on the surface is measured.

(Example 5) Filter paper blood sample Filter paper blood on the above-mentioned immobilized reagent (a piece obtained by punching the blood on the filter paper and punching the portion; used in clinical examination)
, And a bicine buffer and potassium ferricyanide are added dropwise thereto. After the reaction, the plate is washed with distilled water and measured for fluorescence.

Example 6 Precolumn Fluorescence Derivatization by HPLC Using an Immobilized Reagent Using an ODS Cartridge as a Carrier (FIG. 4) Using a commercially available ODS cartridge for pretreatment, the same procedure as in Example 1 was performed. 1,2-diallylethylenediamine is retained by an appropriate method. A sample is added thereto, pre-treated, and then a bicine buffer in which 0.002% potassium ferricyanide is dissolved is added to cause a fluorescence reaction. The fluorescent derivative is eluted with alcohol and used as a sample for HPLC.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing an immobilization reagent according to the present invention, which is a diagram in which a hydrophobic binding layer is provided on a support, and a diarylethylenediamine is adsorbed to a hydrophobic portion thereof. .

FIG. 2 shows a possible use of the immobilizing reagent according to the present invention, wherein (a) comprises a hydrophobic binding layer (including diarylethylenediamine) provided on a carrier,
(b) is a view showing a structure in which an oxide layer having an oxidizing agent is further provided in (a).

FIG. 3 is a diagram showing a reaction according to the present invention in which a benzoxazole derivative is generated by a reaction between 1,2-diarylethylenediamine, a catechol compound and a vanillyl compound.

FIG. 4 shows an example of use of the immobilized reagent according to the present invention (Example 6).
FIG. 2 is a diagram showing on-line derivatization using HPLC as a cartridge.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Carrier, 2 ... Hydrophobic bonding layer, 3 ... Containing oxidizing agent layer, 4 ...
Column, 5: sample solution, 6: oxidizing agent solution, 7: derivative

Claims (6)

[Claims] 1. A solid-phased reagent containing 1,2-diarylethylenediamine. 2. The solid-phased reagent according to claim 1, wherein the aryl portion of the 1,2-diarylethylenediamine is any of phenyl, alkylphenyl, alkoxyphenyl, chlorophenyl, naphthyl, and biphenyl. . 3. The solid-phased reagent according to claim 1, wherein the surface of the solid-phased reagent has a hydrophobic binding layer. 4. The immobilized reagent according to claim 3, wherein the immobilized reagent has a shape of any of a thin plate, filter paper, and particles. 5. A method comprising reacting a catechol compound and a vanillyl compound with an immobilized reagent containing 1,2-diarylethylenediamine together with an oxidizing agent to form a benzoxazole derivative, and measuring the fluorescence of the benzoxazole derivative. A method for detecting a catechol compound and a vanillyl compound. 6. The method for detecting a catechol compound and a vanillyl compound according to claim 5, wherein the oxidizing agent is any one of potassium ferricyanide, sodium periodate, and peroxidase-hydrogen peroxide.