JPH11255948A - Information transmission type molecule recognizing polymer, and preparation and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an information transmission type molecule recognizing polymer, capable of being preserved for a long period of time, and further, capable of detecting a target molecule simply as an object for measurement not requiring a special device. SOLUTION: This information transmission type molecule recognizing polymer is obtained by isolating and removing a target molecule from an organic polymer obtained by polymerizing in the co-presence of the target molecule and an organic molecule having an interaction with the target molecule, and has a molecular shape structure of the target molecule as a multiply cellular material. In this case, when the target molecule and the polymer interact each other, a change in an optical characteristics of the polymer occurs.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring a substance utilizing specific binding, which is an alternative to the measurement of a substance utilizing, for example, an enzyme substrate reaction or an antigen-antibody reaction.
The present invention relates to an information-transmitting type molecular recognition polymer using a molecular recognition substance (polymer) having a target molecule and a specific binding site, a method for preparing and using the same.

[0002]

2. Description of the Related Art In a living body, a chemical reaction is extremely highly specific and highly selective due to its excellent molecular recognition ability. For example, an enzyme strictly recognizes a specific substance from a series of compounds having slightly different structures, causes a chemical reaction to occur only at a specific position within the specific substance, and selectively generates the target product. ing. Various biosensor elements have been developed by imitating such sophisticated in vivo molecular recognition.

[0003] These biosensor elements are, for example,
In the case of a device utilizing an enzyme-substrate reaction, high substrate selectivity can be obtained. Further, a sensor element utilizing an antigen-antibody reaction has been developed, and is widely used for detecting a substance contained in a trace amount in a measurement sample due to the extremely high specificity characteristic of an antibody.

On the other hand, attempts have been made to imitate a specific reaction in a living body by an artificial technique. In recent years, a technique called molecular imprinting has been developed, and artificial molecular recognition substances that mimic natural antibodies can be easily prepared (Wuff, G .: Angew. Chem.
Int. Ed. Engl. , 34, 1812-1832
(1995)). The molecular recognizing substance obtained by the molecular imprinting method includes a target molecule, a polymerizable organic molecule (functional monomer) having an interaction with the target molecule, and a cross-linking reaction between the target molecule and the functional monomer. The organic polymer obtained by polymerization in the presence of a crosslinkable monomer that can be polymerized is characterized by having the molecular shape structure of the target molecule as a porous body, obtained by free removal of the target molecule. . The production method and use of this artificial molecular recognizing substance (artificial antibody) have already been disclosed in Japanese Patent Publication No. Hei 8-506320. There is also a description about the use of this artificial antibody, which describes a detection method using an immunoassay, particularly a radioimmunoassay.

The method comprises reacting a known amount of a labeled target molecule and an unknown amount of a target molecule in a sample with an artificial molecular recognition substance (polymer) in a competitive manner to obtain a free labeled substance in the supernatant. This is an assay method for measuring either the labeled target molecule or the labeled target molecule bound to the polymer.

Further, an assay method using the molecular imprinting polymer is disclosed in
No. 15160 discloses a method using a detection method based on a change in the frequency of a quartz oscillator or a change in the resonance angle of light using surface plasmon resonance as detection of a musty odor substance.

[0007]

In the case of the above-described biosensor element, a high substrate selectivity based on the enzyme reaction can be obtained in the case of an element utilizing an enzyme-substrate reaction, but a special technique for immobilizing the enzyme on the sensor is required. Requires immobilization technology. In addition, enzymes are substances derived from living organisms and therefore lack stability, so that most of them must be handled carefully and cannot be stored for a long time.

[0008] Further, although high specificity can be obtained with a sensor element utilizing an antigen-antibody reaction, the reagent is composed of a substance derived from a living organism, like an enzyme.
The handling of the reagent requires great care. In addition, in order to obtain a specific antibody or the like, it is necessary to perform a complicated purification step from antiserum. As described above, it is considered that the use of the antibody is troublesome in handling and expensive. Also,
There is also a problem that the stability due to storage is poor, and the performance deteriorates when stored for a long period of time.

[0009] In recent years, in order to solve the above-mentioned problems, there has been disclosed a method relating to an assay or a sensor using an artificial molecular recognition substance by a high molecular weight polymer imitating a natural antibody by a molecular imprinting method.

Japanese Patent Publication No. Hei 8-506320 discloses a competitive binding assay between a target molecule and a reagent in which a target molecule to be measured is labeled. However, this method requires the preparation of a separately labeled reagent, which complicates the composition of the reagent. Furthermore, for detection of the target molecule, it is necessary to separate and measure the free labeled molecule and the label / target molecule complex bound to the artificial antibody, which causes a problem that the detection operation becomes complicated.

Next, in the method disclosed in Japanese Patent Application Laid-Open No. H8-15160, it is necessary to immobilize a molecular imprint polymer in a layer on a quartz oscillator or a metal thin film. A technique is required, and the detection involves a problem that an expensive special device such as a quartz oscillator or a surface plasmon resonance device is required. In particular, when a surface plasmon resonance device is used, there is a problem that the device becomes very expensive because an extremely special optical parameter such as an evanescent wave near the thin film is used.

An object of the present invention is to provide a molecule recognizing substance which can be stored for a long period of time, does not require a special apparatus, and can easily detect a target molecule to be measured, and a method for preparing the same. And It is another object of the present invention to provide a method for detecting a target molecule using the same.

[0013]

As a result of intensive studies to solve these problems, the present inventors have found that a molecular recognition polymer (molecularly imprinting polymer (MIP)) obtained by the molecular imprinting method has been developed. Focusing on the fact that the molecule is physically stable, and that when a certain molecule binds to a target molecule, the optical characteristics of the molecule change, the molecules having such optical characteristics are identified in advance by M
It has been found that the target molecule can be easily detected by incorporating it into the IP.

That is, the information-transmitting molecule-recognizing polymer of the present invention releases the target molecule from an organic polymer obtained by polymerization in the presence of a target molecule and an organic molecule interacting with the target molecule. A polymer having a molecular shape structure of the target molecule as a porous body obtained by removing the polymer, and when the target molecule and the polymer form a complex by specific interaction, the optical characteristics of the polymer It is characterized by a change.

Further, the method of using the information-transmitting type molecule-recognizing polymer of the present invention is characterized in that an organic polymer obtained by polymerization in the presence of a target molecule and an organic molecule having an interaction with the target molecule is obtained by A polymer having a molecular structure of the target molecule as a porous body obtained by free removal of the target molecule, wherein information transmission occurs in which a change in optical properties of the polymer occurs when the target molecule interacts with the polymer. The method is characterized in that a type molecule recognizing polymer is mixed with a sample containing a target molecule, and a change in optical properties of the mixture is measured.

Further, the method for preparing the information-transmitting type molecule-recognizing polymer of the present invention is characterized in that the polymer is polymerized in the coexistence of a target molecule and an organic molecule whose optical property changes when interacting with the target molecule. The target molecule is obtained by free removal of the target molecule from the organic polymer obtained by the above method.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The interaction referred to in the present invention means an ionic bond (electrostatic interaction), a covalent bond, a coordinate bond, a metal bond, an intermolecular bond (hydrogen bond, van der Waals force).
Is mentioned.

By the way, a reactant that specifically binds to a specific molecule, such as the relationship between avidin and biotin, is known and has been studied as host-guest chemistry. This assumes that one molecule recognizes the other molecule apart from reactions that occur in the organism, such as an antigen-antibody reaction, an enzymatic reaction, or a ligand-receptor reaction, and treats each molecule as one organic reaction. This is a chemical area treated as a functional material.

Although there are various kinds of such functional substances, it has been known that when specific binding occurs between certain different molecules in a solution, the optical properties of those molecules are changed. Is being done. For example, it is known that the fluorescence intensity is enhanced by the bond between pyrene-modified γ-cyclodextrin and cholic acid, and that the fluorescence wavelength is shifted to a longer wavelength side by the bond between the double-stranded nucleic acid and ethidium bromide. ing. This is probably because the electronic state of the molecule changes before and after the binding due to the specific binding between different molecules. Also, cryptant and AT
It is also known that when P forms a complex, the adenine residue of ATP and the acridine residue of the host overlap to induce a π-electron interaction, thereby increasing the emission intensity of the acridine residue.

The change in optical characteristics means a change in optical parameters such as absorbance, fluorescence, phosphorescence, scattered light, and optical rotation. Further, the shift of the maximum absorption wavelength and the fluorescence wavelength are also included in the change in the optical characteristics of the present invention.

In preparing the information-transmitting molecule-recognizing polymer of the present invention, an organic molecule whose optical properties change when interacting with a target molecule is used as a functional monomer.
Such molecules include porphyrins, cyclodextrins, crown ethers, calixarenes, spherands, cryptands, and the like. Edited by Hiroshi Tsukibe: Molecular Recognition Chemistry -Approach to Supramolecules- Various compounds are described in Sankyo Shuppan (1997). When these molecules (host molecules) bind to molecules that specifically bind (guest molecules), their optical properties change.

In order to polymerize these molecules, a polymerizable functional group such as a vinyl group or an allyl group is required in the molecule, and a polymerizable functional group such as porphyrin is used in the molecule. If not, the polymerizable functional group may be introduced by an appropriate method for derivatization. For example, in porphyrin, a vinyl group can be introduced by reacting with methacryloyl chloride.

In the polymerization, a crosslinkable monomer can be used. There is no particular limitation as long as it can be used to prepare MIP. For example, ethylene glycol dimethacrylate is suitably used.

To prepare MIP, a mixture of a target molecule (guest molecule), a functional monomer that specifically binds to the target molecule (host molecule), and a crosslinkable monomer, for example, 2,2-azobis (2, The polymerization is started by adding a suitable polymerization initiator such as 4-dimethyl-valeronitrile). The polymerization method can be performed according to known means. After the polymerization, the target molecule is removed by washing with a suitable solvent.

For example, using a porphyrin derivative and ethylene glycol dimethacrylate, for example, a 9-ethyladenine molecule as a nucleic acid derivative is polymerized with monomers (porphyrin derivative and ethylene glycol dimethacrylate) collected around the molecule. By removing 9-ethyladenine, a MIP in which the three-dimensional molecular structure of 9-ethyladenine is stored can be produced. The prepared MIP has a specific binding ability to a target molecule, 9-ethyladenine molecule, and the binding causes a shift in the maximum absorption wavelength depending on the concentration, for example.

Therefore, the target substance can be detected by measuring the maximum absorption wavelength of the solution obtained by mixing the prepared MIP and the unknown sample.

[0027]

EXAMPLES Example 1 Production of Porphyrin Derivative First, a porphyrin derivative used in an imprint polymer was produced by the following steps.

1. Synthesis of porphyrin propionic acid 300
ml, p-isopropylbenzaldehyde (p-iso
4.20 g of propylbenzaldehyde)
p-hydroxybenzaldehyde (p-hydroxy
Benzaldehyde (1.16 g), pyrrole (p
2.7 ml were mixed, refluxed on an oil bath at 150 ° C. for 2 hours, and then cooled at room temperature. After removing the acidic solvent by distillation under reduced pressure, an appropriate amount of chloroform was added. The reaction solution was neutralized by adding a sodium hydrogen carbonate solution little by little. The reaction solution was extracted with chloroform, dried over sodium sulfate, and added with Florisil (Wako Pure Chemical Industries, Ltd.) to dryness. The reaction solution is subjected to column chromatography (adsorbent Florisil, developing solvent dichloromethane), silica gel chromatography (developing solvent 3:
1 chloroform: petroleum ether, and chloroform). The above-mentioned chromatography operation was repeated several times, and 5- (4-hydroxyphenyl) -10,1
5,20-tris (4-isopropylphenyl) porphyrin (5- (4-hydroxyphenyl) -1
0,15,20-tris (4-isopropylp
henyl) porphyrin) was obtained.

TLC (silica gel, chloroform): R
f = 0.073-0.2441H-NMR (CDCl
3): σ-2.77 (2H, s, NH), 1.52-
1.54 (18H, m, Pr-Me), 3.17-3.
31 (3H, septet, Pr-H), 7.17-
_{7.20 (2H, d, C 6} H 4 OH), 7.57-7.
_{59 (6H, d, C 6} H 4 Pr), 8.05-8.07
_{(2H, d, C 6 H} 4 OH), 8.10-8.13 (6
H, d, C ₆ H ₄ Pr), 8.86 (8H, s, β-
H)

2. Zinc complexation of porphyrin 5- (4-hydroxyphenyl) -10,15,20-
Tris (4-isopropylphenyl) porphyrin (5
-(4-hydroxyphenyl) -10,15,
20-tris (4-isopropyphenyly
l) Porphyrin) 118 mg in chloroform 2
After adding to 0 ml and refluxing, 5 ml of a saturated methanol solution containing 650 mg of zinc acetate was added dropwise and refluxed for 3 hours. After washing the reaction solution with distilled water, the organic layer was extracted and dried over sodium sulfate. The reaction solution was confirmed by silica gel column chromatography (developing solvent 19: 1 chloroform: ethyl acetate) and absorbance measurement.

[0031] Absorbance: λmax (nm) of starting material: 419, 45
0,518,554,650 λmax (nm) of product: 423,551 1H-NMR (CDCl ₃ ): σ1.52-1.54
(18H, m, Pr-Me), 3.15-3.24 (3
H, septet, Pr-H), 7.10-7.14.
_{(2H, d, C 6 H} 4 OH), 7.51-7.54 (6
_{H, d, C 6 H 4} pr), 7.99-8.00 (2H,
_{d, C 6 H 4 OH)} , 8.02-8.07 (6H, d,
C ₆ H ₄ pr), 8.90 (8H, s, β-H)

3. Derivatization of porphyrin zinc complex 558.0 mg of porphyrin zinc complex and 454.5 μl of triethylamine were added to 30 ml of diethyl ether solution. In this solution, methacryloyl chloride (methacryloyl chloride) is added.
30 ml of a diethyl ether solution containing 318.9 μl of yl chloride was added dropwise, and the mixture was stirred for 8 hours. After washing the reaction solution with distilled water, the organic layer was dried over sodium sulfate and concentrated. Purification by fractional TLC (adsorbent: silica gel, developing solvent: chloroform) gave 515.0 mg of product (porphyrin zinc complex derivative).

1H-NMR (CDCl ₃ ): σ1.51
−1.55 (18H, m, Pr—Me), 2.42 (3
_{H, s, C 6 H 4} OCO-CH 3), 3.21-3.3
0 (3H, septet, Pr-H), 6.53-6.
_{89 (2H, d, C 6} H 4 OCOC = CH 2), 7.4
_{1-7.43 (2H, d, C 6} H 4 -O), 7.57-
_{7.60 (6H, d, C 6} H 4 Pr), 8.23-8.
_{24 (2H, d, C 6} H 4 -O), 8.10-8.13
(6H, d, C ₆ H ₄ Pr), 8.98 (8H, s, β
-H)

Example 2: Preparation of MIP Derivative 2 of porphyrin zinc complex prepared in Example 1
46 mg, 49 mg of 9-ethyladenine, 2.81 g of ethylene glycol dimethacrylate, and 15 mg of 2,2-azobis (2,4-dimethyl-valeronitrile) were each added to 7.5 ml of chloroform. The mixture was transferred to a glass test tube, replaced with nitrogen gas for 5 minutes, covered, and subjected to thermal polymerization in a 45 ° C water bath for 13 hours. The solidified polymer is crushed and sieved to a particle size of 32 to
It was adjusted to 106 μm and washed with methanol to remove 9-ethyladenine as a target molecule.

Example 3 Spectroscopic Evaluation 1 mg of the 9-ethyladenine imprinted polymer prepared in Example 2 was added to 0 μM, 1 μM, 10 μM, and 100 μM.
It was added to 5 ml of a 9-ethyladenine solution of μM, 1 mM, 10 mM, and 100 mM, incubated, and the visible absorption spectrum was measured by a spectrophotometer.

As the concentration of 9-ethyladenine increased, a shift of the maximum absorption wavelength to longer wavelengths was observed. Therefore, it was confirmed that 9-ethyladenine can be quantified by determining the absorption wavelength.

[0037]

The information-transmitting molecular recognition polymer (polymer) according to the present invention can be combined with a chemical substance, for example, 9-ethyladenine, so that the optical properties of the polymer, for example, the maximum absorption wavelength according to the concentration. Can change and detect target chemicals. By applying the information-transmitting molecule-recognizing polymer according to the present invention to various optical sensors and the like, it becomes possible to detect a target substance simply and with high sensitivity.

According to the present invention, JP-A-8-506320
No complicated operation, such as labeling a target molecule to be measured as disclosed in the above publication, is required. The optical parameters used in the present invention may be highly versatile parameters such as absorbance and fluorescence, and do not require a special labeling substance or an expensive dedicated device. Further, when this optical parameter change occurs in the visible region, visual detection and quantification become possible.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a method (molecular imprinting method) for producing an information-transmitting molecular recognition polymer of the present invention.

FIG. 2 is a view showing a reaction formula of Example 1 of the present invention.

FIG. 3 is a diagram showing measurement results of Example 3 of the present invention.

Claims (8)

[Claims] 1. A molecule of the target molecule obtained by free-removing the target molecule from an organic polymer obtained by polymerization in the presence of a target molecule and an organic molecule having an interaction with the target molecule. An information-transmitting molecule-recognizing polymer, which is a polymer having a shape structure as a porous body, wherein a change in optical properties of the polymer occurs when the target molecule interacts with the polymer. 2. The organic molecule according to claim 1, wherein the organic molecule is a host molecule, the target molecule is a guest molecule, and the optical property of the molecule changes when a host-guest molecule complex is formed. Information transmission type molecular recognition polymer. 3. The information-transmitting molecule-recognizing polymer according to claim 2, wherein the organic molecule is selected from the group consisting of porphyrins, cyclodextrins, crown ethers, calixarenes, spherands, and cryptands. 4. The information-transmitting molecular recognition polymer according to claim 1, wherein said organic molecule is polymerized by a polymerizable functional group selected from a vinyl group and an allyl group. 5. The method according to claim 1, wherein the change in the optical property is absorbance, fluorescence,
5. The information-transmitting molecular recognition polymer according to claim 1, wherein the change is an optical parameter selected from the group consisting of phosphorescence, scattered light, and optical rotation. 6. A molecule of the target molecule obtained by freeing and removing the target molecule from an organic polymer obtained by polymerization in the presence of a target molecule and an organic molecule having an interaction with the target molecule. A polymer having a shape structure as a porous body, and an information-transmitting type molecule-recognizing polymer in which a change in optical characteristics of the polymer occurs when the target molecule interacts with the polymer, is mixed with a sample containing the target molecule. And a method for measuring the change in optical properties of the mixture. 7. The method according to claim 7, wherein the change in the optical property is absorbance, fluorescence,
7. The method according to claim 6, wherein the change is an optical parameter selected from the group consisting of phosphorescence, scattered light, and optical rotation. 8. An organic polymer obtained by polymerizing in the coexistence of a target molecule and an organic molecule whose optical properties change when interacting with the target molecule is freed of the target molecule. A method for preparing an information-transmitting molecule-recognizing polymer, characterized by being obtained by: