JP2016197041A - Molecular imprinting film, manufacturing method of the same, mold compound, and detection method of steroid hormone compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a molecular imprinting film allowing detection of a steroid hormone compound at a sensitivity higher than that in a conventional MI method, a manufacturing method of the molecular imprinting film, a mold compound used for manufacturing the molecular imprinting film, and a method capable of detecting the steroid hormone compound at a high sensitivity using the molecular imprinting film.SOLUTION: The molecular imprinting film of the present invention is a molecular imprinting film of a steroid hormone compound. The steroid hormone compound has a specific chemical structure and a lamination structure in which a polymer layer is stacked on a self-organization monomolecular film. β-cyclodextrin is bonded to the surface of the self organization monomolecular film, and a singular recognition space for the steroid hormone compound is formed on the polymer layer. The β-cyclodextrin exists at one end of the singular recognition space, and an aminooxy group exists at the other end of the singular recognition space.SELECTED DRAWING: None

Description

  The present invention relates to a molecular imprinting film having extremely high sensitivity to a steroid hormone compound, a method for producing the molecular imprinting film, a template compound used for producing the molecular imprinting film, and a steroid hormone using the molecular imprinting film. The present invention relates to a method for detecting a compound.

  In vivo information transmission substances mediate the transmission of information between cells and contribute to the maintenance of homeostasis. For example, cortisol, which is a steroid hormone, plays an important role in regulating blood pressure and blood glucose level, and is known to be secreted in response to stress. Therefore, recently, research for detecting cortisol with high sensitivity and ease as a stress marker has been conducted for the prevention of mental illness and the like.

  As a main method for detecting in vivo molecules, ELISA (Enzyme-linked immunosorbent assay) can be mentioned. ELISA immobilizes an antibody specific to the in vivo molecule to be detected, adds a sample, then binds the labeled secondary antibody to the in vivo molecule bound to the antibody, and antibody-target in vivo molecule-enzyme label This is a method for detecting a complex of antibodies. In addition, a sensor in which a gold microelectrode is modified with a self-assembled monolayer (SAM) and an anti-cortisol antibody is bound to the surface has been developed (Non-patent Documents 1 and 2). In any case, conventionally, antibodies and enzymes have been mainly used for detecting in vivo molecules.

  However, antibodies and enzymes are expensive to produce and isolate, and are unstable, so that they are difficult to store and difficult to commercialize.

  Therefore, a molecular imprinting method (MI method), which is a method for creating an artificial molecular recognition material that mimics the precise molecular recognition ability of a living body, has attracted attention. The MI method is based on forming a recognition space specific to a target molecule by polymerizing monomers in the presence of the target molecule to be detected and removing the target molecule from the obtained polymer.

  For example, in Patent Document 1, by polymerizing a monomer having two or more functional groups that interact with the steroid hormone such as itaconic acid in the presence of the steroid hormone, the steroid hormone is removed from the obtained polymer. A molecular imprinting polymer to be produced is disclosed, and Patent Document 2 discloses fine particles made of the same polymer.

  Patent Document 3 discloses a molecular imprinting polymer sensor device including a core having a space for selectively receiving and binding a substituted molecule having a molecular structure similar to a target molecule to be detected and a colorimetric indicator. Discloses a method of detecting the presence of a target molecule in a sample solution by bringing the sample solution into contact with the molecular imprinting polymer and changing the color of the sample solution.

  Patent Document 4 discloses a sensor for detecting a cortisol molecule having a molecular imprinting polymer containing a chromophore bonded to cortisol. The polymer is produced by polymerizing a ligand that binds to cortisol and has a vinyl group in the presence of cortisol.

International Publication No. 2013/046826 Pamphlet JP 2014-219353 A US Patent Application Publication No. 2012/0288944 US Pat. No. 6,833,274

Sunil K. Arya et al., Biosens Bioelectron, 2010, 25 (10), pp. 2296-2301 Cruz AF et al., Biosens Bioelectron, 2014, 62, pp. 249-254

  As described above, the MI method for forming a recognition space specific to the target molecule to be detected in the polymer and used for detecting the target molecule is already known. However, the selectivity and sensitivity of the conventional MI method are not always sufficient.

  Accordingly, the present invention provides a molecular imprinting film that enables detection of a steroid hormone compound with higher sensitivity than the conventional MI method, a method for producing the molecular imprinting film, and a template used for producing the molecular imprinting film. It is an object of the present invention to provide a compound and a method capable of detecting a steroid hormone compound with high sensitivity using the molecular imprinting film.

  The inventors of the present invention have made extensive studies to solve the above-mentioned problems. First, a template compound in which a vinyl group is introduced into a steroid hormone, which is a target compound to be detected, via an oxime group, and an adamantyl group is further introduced. Designed. By allowing the adamantyl group of the template compound to interact with β-cyclodextrin bound to the surface of the self-assembled monolayer, the template compound is oriented on the self-assembled monolayer, and the vinyl group and A vinyl monomer was copolymerized to form a polymer. Next, the template compound was removed by hydrolyzing the oxime group, thereby succeeding in obtaining a molecular imprinting film in which a specific recognition space for the target compound was oriented.

  Conventionally, a molecular imprinting polymer is produced by copolymerizing a target compound, a mixture of a vinyl compound having a vinyl group and an affinity with the target compound, and a vinyl monomer. The orientation of the recognition space was not considered at all. As a result, it was difficult to make the characteristics of the molecular imprinting polymer uniform. Specifically, some template compounds existing in a disordered state in the polymer cannot be removed, and in this case, a specific recognition space is not partially formed. In addition, since the formed specific recognition space is also formed in a disordered state in the polymer, a specific recognition space inhibits the interaction of the target compound with another specific recognition space, or the specific recognition space. It is conceivable that the selectivity for the target compound is impaired due to the duplication of the number.

  On the other hand, in the molecular imprinting film according to the present invention, the specific recognition spaces are regularly oriented, so that each specific recognition space probably acts effectively on the target compound, and the conventional molecular imprinting The sensitivity to the polymer is significantly increased and the quality is uniform.

  Hereinafter, the present invention will be described.

[1] A molecular imprinting film of a steroid hormone compound,
The steroid hormone compound is represented by the following formula (I):

[Where:
R ¹ represents a methyl group, a hydroxymethyl group or a formyl group;
R ² represents a hydrogen atom, a hydroxyl group or an oxo group,
R ³ represents a methyl group, a hydroxymethyl group or a formyl group,
R ⁴ represents a C _1-10 alkyl group substituted with one or more substituents α, or a hydroxyl group,
R ⁵ represents a hydrogen atom or a hydroxyl group,
R ⁴ and R ⁵ together may form an oxo group,
The substituent α represents a substituent selected from a hydroxyl group and an oxo group.
Having a laminated structure in which a polymer layer is laminated on a self-assembled monolayer;
Β-cyclodextrin is bonded to the surface of the self-assembled monolayer,
A specific recognition space for the steroid hormone compound is formed in the polymer layer,
A molecular imprinting membrane, wherein the β-cyclodextrin is present at one end of the specific recognition space, and an aminooxy group is present at the other end of the specific recognition space.

  [2] A template compound for forming a specific recognition space for a molecular imprinting polymer, which is represented by the following formula (II):

[Where:
X and Y independently represent a linker group,
R ¹ represents a methyl group, a hydroxymethyl group or a formyl group;
R ² represents a hydrogen atom, a hydroxyl group or an oxo group,
R ³ represents a methyl group, a hydroxymethyl group or a formyl group,
R ⁵ represents a hydrogen atom or a hydroxyl group,
R ⁶ represents a hydrogen atom or a methyl group]

[3] A method for producing a molecularly imprinted film of a steroid hormone compound,
Forming a self-assembled monolayer on a metal substrate;
Binding β-cyclodextrin to the surface of the self-assembled monolayer;
A step of allowing the adamantyl group of the template compound according to [2] to interact with the β-cyclodextrin,
Adding a vinyl monomer and copolymerizing with the vinyl group of the template compound,
A method comprising hydrolyzing an oxime group of the template compound and removing a steroid moiety of the template compound.

[4] A method for detecting a steroid hormone compound in a sample,
The steroid hormone compound is represented by the formula (I) defined in [1] above,
Contacting the sample with the molecular imprinting film according to [1] in a solvent; and
A method comprising measuring a change in a reaction system due to contact between the molecular imprinting film and the sample.

[5] Furthermore, before the contacting step, including a step of inserting a pseudosteroid hormone compound having a labeling group into the specific recognition space of the molecular imprinting film,
The method according to [4] above, wherein in the measurement step, the concentration change of the labeling group in the solution of the reaction system is measured.

  [6] The method according to [4] above, wherein in the measurement step, the change in the reaction system is measured by a surface plasmon resonance measurement method, a quartz resonator microbalance method, or an electrochemical impedance method.

In the present invention, the “C _1-10 alkyl group” means a linear or branched monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, n-hexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, 1,5- Dimethylhexyl, n-nonanyl, n-decanyl and the like.

The “linker group” has functions such as improving the reactivity and interaction ability of the compound (II) by increasing the degree of freedom of the vinyl group and adamantyl group, and facilitating the synthesis of the compound (II). It has a relatively stable structure. For example, C _1-6 alkylene group, C _6-12 arylene group, amino group (—NH—), ether group (—O—), thioether group (—S—), carbonyl group (—C (═O) — ), Thionyl group (—C (═S) —), ester group (—O—C (═O) — or —C (═O) —O—), amide group (—NH—C (═O) — Or —C (═O) —NH—), a sulfoxide group (—S (═O) —), a sulfonyl group (—S (═O) ₂ —), and a group in which two or more of these are bonded. . However, since the linker group according to the present invention needs to be stable to some extent, the above-mentioned “group having two or more bonded” includes unstable structures such as a peroxide group (—O—O—). Absent. Such unstable structures and structures that cannot be synthesized are known to those skilled in the art. In addition, if the linker group is excessively long, the selectivity of the specific recognition space for the steroid hormone compound to be detected may decrease, so when the linker group is a group in which two or more of the above groups are bound, The number of bonded groups is preferably 15 or less, more preferably 10 or less, further preferably 8 or less, and still more preferably 5 or less.

  In the steroid hormone compound (I) and the template compound (II), the dotted line in the A ring of the steroid skeleton indicates a single bond or no bond. That is, the A ring may be cyclohexane or 4,5-cyclohexene.

  In the molecular imprinting film according to the present invention, the direction and position of the specific recognition space for a specific steroid hormone compound are aligned, and the orientation is high. Therefore, it is considered that the selectivity for the target steroid hormone compound in each specific recognition space is uniform, and each can be used effectively. In addition, the specific recognition space includes a carbonyl group present at the 3rd position of the A ring of the steroid hormone compound, an oxime group, and an aminooxy group capable of forming a hydrogen bond, and the basic skeleton of the steroid hormone compound. There is also a β-cyclodextrin structure capable of hydrophobic interaction with the A to D rings. Therefore, the molecular imprinting film according to the present invention has an advantage that the detection sensitivity of a specific steroid hormone compound is extremely high as compared with the conventional molecular imprinting polymer.

FIG. 1 is a view schematically showing a method for producing a molecular imprinting film according to the present invention. FIG. 2 shows the concentration of cortisol added in the presence of a gold / glass plate on which a molecular imprinting film specific to cortisol according to the present invention is formed, and a gold / glass plate on which other polymer layers are formed. It is a graph which shows the relationship with the relative fluorescence intensity variation | change_quantity of the reaction solution depending on the density | concentration of the pseudo steroid hormone compound which moves into each reaction solution from each polymer layer by cortisol. FIG. 3 is a graph showing the results of a similar experiment performed using the other steroid hormone compounds in addition to cortisol in the experiment whose results are shown in FIG. FIG. 4 is a graph showing the results of a similar experiment performed using the uncrosslinked molecular imprinting film and the crosslinked molecular imprinting film according to the present invention in the experiment whose results are shown in FIG. FIG. 5 shows the results of a similar experiment conducted using the cross-linked molecular imprinting membrane according to the present invention and other steroid hormone compounds in addition to cortisol in the experiment shown in FIG. It is a graph.

  Hereinafter, a method for producing a template compound (II) used for forming a specific recognition space of the molecular imprinting film according to the present invention will be described first.

1. Method for Producing Template Compound (II) Since the molecular imprinting membrane according to the present invention exhibits selectivity for the steroid hormone compound (I), the template compound (II) for forming the specific recognition space is The steroid hormone compound (I) can be used as a raw material compound.

  The steroid hormone compound (I) is not particularly limited as long as it can evaluate a human disease, symptom, condition or the like by measuring the presence / absence or concentration thereof. For example, 17α-hydroxyprogesterone, 21-deoxycortisol, cortisol, cortisone, progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone, aldosterone, testosterone, 19-hydroxytestosterone, 19-oxotestosterone, Examples include androstenedione, 19-hydroxyandrostenedione, 19-oxoandrostenedione, 11β-hydroxyandrostenedione, adrenosterone, androstanedione, androsterone.

  The template compound (II) can be synthesized from the steroid hormone compound (I) according to X and Y which are linker groups.

For example, an adamantyl group is used to form a linker group Y after converting the hydroxyl group or oxo group in R ⁴ and / or R ⁵ of the steroid hormone compound (I) to a functional group if necessary. It can be introduced by reacting with a derivative. Further, when the reaction selectivity of the functional group in R ⁴ and / or R ⁵ is the same or poor with respect to the other functional group, the other functional group may be selectively protected. Those skilled in the art can easily perform these functional group conversion, linker group Y formation reaction, selective protection reaction and deprotection reaction. For example, when a primary hydroxyl group in R ⁴ and 1-adamantanecarboxylic acid are reacted with a dehydration condensing agent, an adamantyl group can be introduced by an ester bond.

  The vinyl group is formed by reacting a hydroxyamine derivative into which a vinyl group has been introduced via a linker group X with the 3-position carbonyl group of the A ring of the steroid hormone compound (I) to form an oxime group. Can be introduced. Alternatively, after reacting the hydroxylamine derivative having a functional group for forming the linker group X with the 3-position carbonyl group of the A ring of the steroid hormone compound (I) to form an oxime group, the vinyl group and the linker group A vinyl group may be introduced by further reacting a compound having a functional group for forming X.

  Next, a method for producing the molecular imprinting film according to the present invention using the template compound (II) will be described. In addition, the scheme which showed the said manufacturing method typically is shown as FIG.

2. Manufacturing method of molecular imprinting film (2-1) Formation process of self-assembled monolayer In this step, a self-assembled monolayer (SAM) is formed on a metal substrate. The SAM is stable and uniform because it is chemically bonded to the metal substrate and molecules are closely and regularly aligned on the metal substrate by intermolecular forces.

  Gold is widely used as the metal constituting the metal substrate, but silver, copper, platinum, palladium, and the like can also be used. Moreover, what formed the thin film of these metals on the glass substrate and the Teflon (trademark) board | substrate can be utilized as a metal substrate.

  As a molecule for forming SAM, linear alkyl thiol having 8 or more carbon atoms, disulfide, thiol acetate thiol and the like can be usually used. -A compound having a functional group for introducing a cyclodextrin group is used. In addition, molecules having a functional group other than the functional group for introducing the β-cyclodextrin group may be used in combination as long as the long chain portion is the same or similar to form a strong SAM. Examples of the other functional group include a group having a polymerization initiating action of a vinyl monomer.

  The formation of SAM may be according to a conventional method. For example, when a SAM-forming molecule is dissolved in ethanol or the like and the metal substrate is immersed in the obtained solution for 30 minutes to 24 hours at room temperature, the molecule binds to the surface of the metal substrate via a thioether bond, and the intermolecular The SAM is formed by densely gathering while being oriented by force. Next, excess SAM-forming molecules are removed by washing and then dried.

  In the next step, β-cyclodextrin is bound to the surface of the SAM. For example, when the SAM is very dense, such binding may be difficult. In such a case, in order to facilitate the binding of β-cyclodextrin, a linker group may be once introduced on the surface of the SAM as shown in FIG.

(2-2) β-cyclodextrin binding step In this step, β-cyclodextrin is bonded to the SAM formed on the metal substrate by the above step (2-1) or the linker group introduced on the surface of the SAM. (Hereinafter abbreviated as “β-CD”).

  β-CD is a cyclic oligosaccharide in which seven D-glucoses are cyclically bonded by α (1 → 4) glucoside bonds, and shows a characteristic that adamantane is selectively included due to the size of the cavity. In the present invention, such a characteristic is used.

  The binding of β-CD may be according to a conventional method. Specifically, the terminal functional group of the SAM-forming molecule or the terminal functional group of the linker group bonded to the SAM surface may be reacted with the reactive functional group introduced into β-CD.

(2-3) Template Compound (II) Binding Step In this step, the adamantyl group of the template compound (II) is allowed to interact with the β-CD. In this case, since the adamantyl group is included in β-CD, the orientation of the template compound (II) is uniform and oriented on the surface of the SAM.

(2-4) Copolymerization step In this step, a polymer layer containing the template compound (II) is added by adding a vinyl monomer and copolymerizing with a vinyl group in the template compound (II) oriented on the SAM surface. Form.

  The vinyl monomer to be added is not particularly limited as long as it has a vinyl group structure copolymerizable with the vinyl group in the template compound (II), and can be appropriately selected, but 2-methacryloyloxyethyl phosphorylcholine is preferable. Can be used. Since 2-methacryloyloxyethyl phosphorylcholine has a structure similar to that of phospholipid and the polymer is excellent in affinity to a living body, there is an advantage that it is suitable for analysis of biological samples such as blood.

  The polymerization conditions may be set according to a conventional method. For example, since 2-methacryloyloxyethyl phosphorylcholine is water-soluble, a polymerization initiator is added to an aqueous solvent by adding at least the substrate having undergone the above steps (2-1) to (2-3) and 2-methacryloyloxyethyl phosphorylcholine. The polymerization may be started by (which may be bonded to the SAM surface). In order to perform living radical polymerization in the reaction solution, a monovalent copper compound or a combination of a divalent copper compound and a reducing agent such as ascorbic acid may be added. The polymerization temperature may be normal temperature, or about 0 ° C. to 120 ° C., and the polymerization time may be about 10 minutes to 50 hours. Specific polymerization conditions may reduce the selectivity of the specific recognition space formed to the target compound if the polymerization is not sufficient. On the other hand, if it is excessively polymerized, the template compound cannot be removed in the next step. Since there is a possibility of fear, it may be determined by a preliminary experiment or the like.

  After the polymerization reaction, it is preferable to wash thoroughly with the solvent used in order to remove excess reagents.

(2-5) Template compound removal step In this step, the oxime group of the template compound (II) incorporated into the polymer is hydrolyzed by copolymerization of vinyl groups, and the steroid part of the template compound (II) is removed. To do. As a result, a recognition space specific to the removed steroid moiety is formed in the polymer. In addition, the said steroid part means the steroid frame | skeleton part which the adamantyl group couple | bonded through the linker group among template compound (II).

  The hydrolysis of the oxime group may be performed according to a conventional method. For example, what is necessary is just to immerse the board | substrate which passed through the said process (2-1)-(2-4) for about 2 hours or more and about 50 hours or less in acidic aqueous solutions, such as hydrochloric acid about 20 to 60 degreeC. In order to sufficiently remove the steroid moiety, it is preferable to sufficiently wash the substrate with a solvent capable of dissolving the steroid moiety.

  In the molecular imprinting film according to the present invention obtained as described above, a specific recognition space having the same three-dimensional structure as the steroid hormone compound (I) is formed. Moreover, the direction and position of this specific recognition space are uniform, and the orientation is high. The specific recognition space contains an aminooxy group capable of interacting with the 3-position carbonyl group of the A ring of the steroid hormone compound (I), and the other end of the basic skeleton of the steroid hormone compound (I). There is also a β-cyclodextrin structure capable of hydrophobic interaction with the A to D rings. Therefore, the molecular imprinting membrane according to the present invention has extremely high affinity for the steroid hormone compound (I), and its selective detection sensitivity is extremely high.

  Hereinafter, the detection method of the steroid hormone compound (I) using the molecular imprinting film according to the present invention will be described.

  The method for detecting a steroid hormone compound (I) according to the present invention comprises a step of bringing the molecular imprinting membrane according to the present invention into contact with a sample in a solvent (contacting step), and a contact between the molecular imprinting membrane and the sample. Including a step of measuring a change in the reaction system due to (measurement step).

  The sample to be measured is not particularly limited as long as the presence or absence of steroid hormone (I) should be confirmed and the concentration of steroid hormone (I) should be measured. Examples thereof include human or animal blood, serum, plasma, urine, saliva and the like.

  The solvent to be used is not particularly limited as long as it can dissolve the steroid hormone (I) in an amount contained in the sample. For example, a buffer solution that can appropriately dissolve the steroid hormone (I) to be detected can be used. When the sample is a liquid, the sample itself may be used as a solvent, or the sample may be diluted.

  In order to bring the molecular imprinting film of the present invention into contact with the sample, the molecular imprinting film may be immersed in the sample or a mixture of the solvent and the sample. Since the measurement result may vary depending on the temperature, the temperature at that time is preferably constant, for example, 20 ° C. or higher and 30 ° C. or lower, particularly 25 ° C. In addition, the immersion time can be set to a time sufficient for the target steroid hormone (I) to be sufficiently taken into the specific recognition space, for example, about 5 minutes to 5 hours.

  In the method for detecting a steroid hormone compound (I) according to the present invention, the reaction system changes due to contact between the molecular imprinting film and the sample, that is, the steroid hormone compound (I) incorporated into the specific recognition space is measured. Thus, the presence / absence and concentration of the target steroid hormone compound (I) are determined.

  Examples of the method for measuring the change in the reaction solution include a surface plasmon resonance measurement method, a quartz resonator microbalance method, and an electrochemical impedance method.

When the molecular imprinting film according to the present invention is formed on a metal film, it can be used as it is in the surface plasmon resonance measurement method. In other words, when a metal film with a molecular imprinting film is attached to a prism and irradiated so that light is totally reflected from the prism side to the metal film, surface plasmon resonance (SPR) occurs, resulting in a decrease in reflected light intensity. There is. The angle at this time changes sharply according to the amount of steroid hormone compound (I) incorporated into the molecular imprinting film formed on the metal, that is, mass change. The mass change after the addition can be displayed as measurement data (resonance unit: 1 RU = 1 pg / mm ² ) from the reflected light intensity.

  The quartz crystal microbalance method and the electrochemical impedance method also determine the presence and amount of the steroid hormone compound (I) incorporated into the specific recognition space of the molecular imprinting film by adding the sample, respectively. And change in impedance. Conventional methods can be applied to the specific conditions of these methods.

  Alternatively, the change in the reaction system before and after the addition of the sample can be measured by a change in the amount of the labeled compound in the reaction solution. Specifically, a pseudo steroid hormone compound having a labeling group is inserted into a specific recognition space of the molecular imprinting film before addition of the sample, that is, before the contact step. Since the specific recognition space has high specificity for the steroid hormone compound (I) to be measured, in the contact step, the pseudo steroid hormone compound existing in the specific recognition space is a steroid hormone compound ( I) is expelled and the concentration of the pseudosteroid hormone compound in the reaction solution increases. Therefore, in the measurement step, it is possible to determine the presence and concentration of the steroid hormone compound (I) in the sample by measuring the concentration change of the labeling group in the reaction system solution.

  The pseudo steroid hormone compound has a chemical structure similar to that of the steroid hormone compound (I) to be measured and can be taken into a specific recognition space, but compared with the steroid hormone compound (I). The affinity for the specific recognition space needs to be low. Examples of such pseudosteroid hormone compounds include bisphenol compounds that are known to have a three-dimensional structure close to that of the steroid skeleton and have a labeling group.

  Examples of the labeling group of the pseudosteroid hormone compound include a fluorescent coloring group.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Example 1: Production of molecular imprinting polymer (MIP) (1) Preparation of a template compound

(1-1) Preparation of adamantanecarboxylic acid ester 1-adamantanecarboxylic acid (270 mg, 1.5 mmol), EDC (285 mg, 1.5 mmol), and DMAP (183 mg, 1.5 mmol) were dissolved in dichloromethane and ice-cooled. Stirred under. Cortisol (362 mg, 1.0 mmol) was added thereto and stirred overnight. The reaction mixture was cloudy at the beginning of the reaction, but after stirring overnight, it became a pale yellow solution. Regarding the spot of thin layer chromatography (developing solution-hexane: ethyl acetate = 1: 1), the Rf value of DMAP is 0, the Rf value of cortisol is 0.125, and the Rf value of the target compound is 0.375. Since the spot of cortisol, which is a raw material compound, did not disappear even after stirring overnight, 1-adamantanecarboxylic acid (135 mg, 0.75 mmol), EDC (143 mg, 0.75 mmol), and DMAP (92 mg, 0.75 mmol) were newly added. ) Was added. After 3 hours, the progress of the reaction was confirmed by thin layer chromatography. However, since the raw material spots still did not disappear, the reaction was terminated, and the reaction solution was washed with saturated citric acid, saturated sodium hydrogen carbonate and saturated brine, The residue was purified by column chromatography (eluent-hexane: ethyl acetate = 1: 1). Thereafter, dehydration was performed with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and vacuum drying was performed to obtain the target compound as a white solid (yield: 212 mg, yield: 40.3%).
¹ H-NMR (300.40 MHz, CDCl ₃ ): δ = 5.68 (s, 1H, 4-position H), 2.03 (s, 3H, adamantane), 1.91 (s, 6H, adamantane), 1.72 (t, 6H, Adamantane), 2.8-1.4 (m, cortisol)
MALDI-TOF-MS m / z = 525.6 [M + H ⁺ ], 547.7 [M + Na ⁺ ]

(1-2) Oximation The adamantanecarboxylic acid ester (131 mg, 0.25 mmol) obtained in the above (1-1) was dissolved in a mixed solvent of methanol: dichloromethane = 1: 1, and pyrrolidine (124 μL, 1) was added thereto. 0.5 mmol) was added. After 20 minutes, it was confirmed that the solution had turned yellow, carboxymethoxyamine · 0.5 hydrochloride (54.5 mg, 0.5 mmol) was added, and the mixture was stirred at room temperature for 5 hours. Regarding the spot of thin layer chromatography (developing liquid-ethyl acetate) at the end of the reaction, the Rf value of pyrrolidine is 0, the Rf value of the target compound is 0.1, and the Rf value of the starting compound is 0.725. The disappearance was confirmed. The solvent was distilled off under reduced pressure, pure water was added to the residue, pH was adjusted to 2.0 with 1M HCl, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dehydrated with anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure, followed by vacuum drying to obtain the target compound as a pale yellow solid (yield: 149 mg, yield: 99.7). %).
¹ H-NMR (300.40 MHz, DMSO-d6): δ = 1.99 (s, 3H, adamantane), 1.82 (s, 6H, adamantane), 1.68 (t, 6H, adamantane), 2.5-1.5 (m, cortisol)
MALDI-TOF-MS m / z = 598.8 [M + H ⁺ ], 620.8 [M + Na ⁺ ]

(1-3) Coupling of monomer The oxime compound (149 mg, 0.25 mmol) obtained in (1-2) above was dissolved in methanol, and 4-aminostyrene (44.5 mg, 0.375 mmol) and ice-cooled. DMT-MM (138 mg, 0.5 mmol) was added. The ice bath was removed and the reaction was allowed to proceed overnight at room temperature. Regarding the spot of thin layer chromatography (developing solution-hexane: ethyl acetate = 1: 1) at the end of the reaction, the Rf value of the target compound is 0.35, and the disappearance of the spot of Rf value = 0.725 of the raw material compound It was confirmed. The solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed with saturated aqueous citric acid solution and brine, and purified by silica gel column chromatography (eluent-hexane: ethyl acetate = 1: 2). Then, after dehydrating with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, followed by vacuum drying to obtain the target compound as a white solid (yield: 61.0 mg, yield: 34.9%).
¹ H-NMR (300.40 MHz, CDCl ₃ ): δ = 8.02 (d, 1H, NH), 7.50 (d, 2H, benzene ring), 7.38 (d, 2H, benzene ring), 2.02 (s, 3H, adamantane ), 1.97 (s, 6H, adamantane), 1.73 (t, 6H, adamantane), 2.8-1.5 (m, cortisol)
MALDI-TOF-MS m / z = 699.1 [M + H ⁺ ], 721.3 [M + Na ⁺ ]

  (2) Preparation of monoamino-β-cyclodextrin

(2-1) Tosylation β-cyclodextrin (2.0 g, 1.75 mmol) was dissolved in 0.4 M aqueous sodium hydroxide solution (40 mL) under ice cooling, and p-toluenesulfonyl chloride (2.0 g, 6 eq) was added and stirred for 4 hours. The target spot (Rf value = 0.6) and disubstituted product (Rf value = 0.65) were confirmed by thin layer chromatography (developing solution-1-butanol: methanol: water = 5: 4: 3). Thus, unreacted p-toluenesulfonyl chloride was removed by filtration, the filtrate was neutralized with an aqueous hydrochloric acid solution, and then allowed to stand overnight, and the grown crystals were collected by vacuum filtration and dried in vacuo. 6-tosyl-β-cyclodextrin was obtained (yield: 830 mg, yield: 36.8%).
¹ H-NMR (300.40 MHz, DMSO-d6): δ = 7.76 (d, 2H, phenyl), 7.43 (d, 2H, phenyl), 5.89-5.57 (m, 14H, OH), 4.91-4.73 (m, 7H, CD), 4.52-4.31 (m, 7H, CD), 3.77 (m, 42H, CD), 2.43 (s, 3H, Methyl)
MALDI-TOF-MS m / z = 1311 [M + Na ⁺ ]

(2-2) Azide 6-tosyl-β-cyclodextrin (830 mg, 0.644 mmol) obtained in (2-1) above was suspended in water at 80 ° C., and sodium azide (455 mg, 7 mmol) was added. And allowed to react overnight in an oil bath at 80 ° C. After the completion of the reaction, the solution was slowly cooled to room temperature, and then this solution was poured into acetone (60 mL), and the resulting white precipitate was collected by vacuum filtration and vacuum-dried to obtain the target compound 6-azido-β-cyclodextrin. (Yield: 548 mg, Yield: 73.4%).
¹ H-NMR (300.40 MHz, DMSO-d6): δ = 5.74-5.63 (m, 14H, OH), 4.82 (m, 7H, CD), 4.52-4.46 (m, 6H, CD), 3.62-3.57 ( m, 42H, CD)
MALDI-TOF-MS m / z = 1182 [M + Na ⁺ ]

(2-3) Amination 6-Azido-β-cyclodextrin (548 mg, 0.472 mmol) and triphenylphosphine (262 mg, 1.0 mmol) obtained in (2-2) above were dissolved in DMF, and at room temperature. After stirring for 1 hour, ultrapure water (Milli-Q water, 1 mL) was added to the reaction solution, and the mixture was reacted overnight in an oil bath at 90 ° C. Thin layer chromatography (developing solution-1-butanol: methanol: water = 5: 4: 3) confirmed the target spot (Rf value = 0.075), and the starting compound spot (Rf value = 0.55). The reaction was terminated after the reaction was completed, and after slowly cooling to room temperature, the solution was poured into acetone (60 mL), and the resulting white precipitate was collected by vacuum filtration and dried in vacuo. The compound 6-amino-β-cyclodextrin was obtained (yield: 430 mg, yield: 80.4%).
¹ H-NMR (300.40 MHz, DMSO-d6): δ = 5.76-5.65 (m, 14H, OH), 4.82 (m, 7H, CD), 4.46 (b, 6H, CD), 3.63-3.55 (m, 28H, CD)
MALDI-TOF-MS m / z = 1156 [M + Na ⁺ ]

(3) Manufacture of molecular imprinting polymer (MIP) (3-1) Formation of composite self-assembled monolayer (composite SAM film) Gold / glass substrate (manufactured by JASCO Corporation) was thoroughly washed with water and ethanol Then, nitrogen gas was blown and dried. After the dried gold surface was subjected to Ar etching treatment, an ethanol solution containing 0.5 mM bis [2- (2-bromoisobutyryloxy) undecyl] disulfide + 0.5 mM 11-amino-1-undecanethiol hydrochloride (5 mL) For 24 hours at 25 ° C. The 2-bromoisobutyryl group exhibits a polymerization initiating action.

(3-2) Activation of Composite SAM Film The composite SAM film is immersed in a DMF solution (10 mL) containing 5 mM NHS-dPEG ₄ -NHS ester for 1 hour at 25 ° C. An NHS-activated carboxy group was introduced into the terminal amino group of via an PEG chain.

(3-3) Immobilization of β-cyclodextrin Monoamino-β-cyclodextrin (20.4 mg, 18 μmol) prepared in (2) above was dissolved in DMF (6 mL) to prepare a 3 mM solution. Next, β-cyclodextrin was immobilized by immersing the gold / glass substrate into which the NHS-activated carboxy group had been introduced in the solution at 25 ° C. for 3 hours.

(3-4) Immobilization of template compound After dissolving the template compound (3.5 mg, 5 μmol) prepared in (1) above in a small amount of DMSO, water (5 mL) was added to prepare a 1 mM solution. The above-mentioned gold / glass substrate on which β-cyclodextrin was immobilized was immersed in the solution at 25 ° C. for 24 hours to immobilize the template compound.

(3-5) Polymerization reaction In a reaction vessel, water (5 mL), the above-mentioned gold / glass substrate on which a template compound is immobilized, 2-methacryloyloxyethyl phosphorylcholine (MPC, 88.6 mg, 300 μmol), copper bromide ( II) (1.34 mg, 6 μmol), N, N, N ′, N ″, N ″ -pentamethyldiethylenetriamine (PMDETA, 1.25 μL, 6 μmol) was added, and the gas phase in the reaction vessel was replaced with nitrogen gas. . Next, a 0.5 mM solution in which ascorbic acid (0.52 mg, 3 μmol) was dissolved in water (1 mL) was poured into the reaction vessel, and a polymerization reaction was performed at 25 ° C. for 30 minutes.

(3-6) Removal of template compound After the polymerization reaction, the gold / glass substrate was separated and washed with water. Subsequently, in order to remove the remaining copper ion (II), it was immersed in 1M EDTA-4Na aqueous solution at 25 ° C. for 12 hours.

  Next, in order to remove the template compound by hydrolyzing the oxime group, the gold / glass substrate was immersed in 100 mM hydrochloric acid at 40 ° C. for 24 hours, and then thoroughly washed with pure water and ethanol, thereby cortisol. A gold / glass substrate on which an MPC thin film having a specific recognition space was formed was obtained.

Comparative Example 1: Preparation of a substrate on which only β-cyclodextrin was immobilized The “immobilization of a temple compound” and “removal of a template compound” in Examples 1 (3-4) to (3-6) were not performed. Except for the above, a gold / glass substrate was prepared in the same manner as in Example 1 except that a polymer thin film in which only β-cyclodextrin was immobilized was formed.

Comparative Example 2: Preparation of substrate having only oxime binding site (1) Formation of self-assembled monolayer (SAM film) Gold / glass substrate (manufactured by JASCO) was thoroughly washed with water and ethanol, and then nitrogen Gas was blown to dry. The dried gold surface was subjected to Ar etching treatment. Separately, bis [2- (2-bromoisobutyryloxy) undecyl] disulfide (1.8 μL, 3 μmol) was dissolved in ethanol (6 mL) to prepare a 0.5 mM solution. The gold / glass substrate was immersed in the solution at 25 ° C. for 24 hours.

(2) Polymerization reaction In a reaction vessel, water (3.00 mL), DMSO (1 mL), the gold / glass substrate obtained in (1) above, 2-methacryloyloxyethyl phosphorylcholine (MPC, 66.5 mg, 225 μmol), The template compound obtained in Example 1 (1) (3.15 mg, 4.5 μmol), copper (II) bromide (1.00 mg, 4.5 μmol), N, N, N ′, N ″, N ″ -Pentamethyldiethylenetriamine (PMDETA, 0.94 μL, 4.5 μmol) was added, and the gas phase in the reaction vessel was replaced with nitrogen gas. Next, a 0.5 mM solution in which ascorbic acid (0.39 mg, 2.25 μmol) was dissolved in water (0.5 mL) was injected into the reaction vessel, and a polymerization reaction was performed at 25 ° C. for 30 minutes.

(3) Removal of template compound After the polymerization reaction, the gold / glass substrate was separated and washed with water. Subsequently, in order to remove the remaining copper ion (II), it was immersed in 1M EDTA-4Na aqueous solution at 25 ° C. for 12 hours.

  Next, in order to remove the template compound by hydrolyzing the oxime group, the gold / glass substrate is immersed in 100 mM hydrochloric acid at 40 ° C. for 24 hours to form an MPC thin film having a specific recognition space for cortisol. A metal / glass substrate was obtained. However, since β-cyclodextrin is not immobilized unlike Example 1, the direction of the specific recognition space is random.

Example 2: Detection of Cortisol by Fluorescence Measurement Fluorescein isothiocyanate-bisphenol A (FITC-BPA) was dissolved in 10 mM phosphate buffer (pH 7.4) to prepare a 100 nM FITC-BPA solution. A gold / glass substrate (Example 1) on which a MIP film for cortisol was formed was immersed in the solution at 25 ° C. for 1 hour, and then a solution in which cortisol was dissolved in 10 mM phosphate buffer (pH 7.4) was added. Then, the fluorescence of the supernatant was measured. At this time, since steric conformation of cortisol and bisphenol A is similar, bisphenol A is trapped by the MIP membrane for cortisol, but is replaced by the addition of cortisol having higher affinity for the MIP membrane and leaks into the solution. To do. The excitation wavelength in the fluorescence measurement was 495 nm, and the measurement fluorescence wavelength was 515 nm. FIG. 2 shows the relationship between the relative fluorescence intensity variation [(I−I ₀ ) / I ₀ ] and the concentration of cortisol added to the solution in which the MIP gold / glass substrate is immersed. Moreover, the measurement result was processed with the graph preparation software (Nippon Pola digital "DeltaGraph"), and the coupling constant K of MIP gold / glass substrate and cortisol was calculated. The results are shown in Table 1.

  For comparison, measurement was similarly performed using a MIP gold / glass substrate (Comparative Example 1) on which only β-cyclodextrin was immobilized and a MIP gold / glass substrate having only an oxime binding site (Comparative Example 2). Went. The results are shown in FIG.

  As shown in FIG. 2 and Table 1, the MIP membrane having a cortisol-specific recognition space having both β-cyclodextrin and an oxime binding site is more selective for cortisol than the MIP membrane having only one of these. It has been proved that the affinity is high.

Example 3 Selectivity Test for Cortisol In Example 2 above, in addition to cortisol, 17-β-estradiol, cholesterol, testosterone and progesterone having similar structures were used in the same manner as in the MIP membrane according to the present invention. Affinity was measured. The structure of each compound is shown below, and the measurement results are shown in FIG. In FIG. 3, the measurement results for cortisol are indicated by “♦”, and the measurement results for other compounds are indicated by “■”.

  As shown in the results shown in FIG. 3, the MIP membrane according to the present invention was found to have selective affinity for cortisol even compared to other steroid compounds having a structure very similar to that of cortisol.

Example 4: Production of cross-linked MIP membrane and selective affinity test In Example 1 (3-5) above, water (6 mL), 2-methacryloyloxyethyl phosphorylcholine (MPC, 70.9 mg, 240 μmol), and cross-linking agent N, N′-methylenebisacrylamide (MBAAm, 9.25 mg, 60 μmol), copper (I) bromide (1.34 mg, 6 μmol), N, N, N ′, N ″, N ″ -pentamethyldiethylenetriamine ( A crosslinked MIP film was formed on a gold / glass substrate in the same manner as in Example 1 except that PMDETA, 1.25 μL, 6 μmol) and ascorbic acid (0.52 mg, 3 μmol) were used.

Using the obtained crosslinked MIP membrane and the uncrosslinked MIP membrane obtained in Example 1, selective affinity for cortisol was tested under the same conditions as in Example 2. The results are shown in FIG. Moreover, when the binding constant K with respect to the cortisol of the said bridge | crosslinking MIP film | membrane was computed from the obtained measurement result, the binding constant K was 2.30 * 10 < ¹¹ > M < ^-1 >. From these results, it can be seen that the cross-linked MIP film has a smaller amount of relative fluorescence intensity change than the non-cross-linked MIP film, but has a larger binding constant to cortisol. Therefore, it is considered that a specific recognition space for cortisol is more firmly constructed by cross-linking, so that the binding constant increases, and the non-specific adsorption site decreases, so that the amount of change in relative fluorescence intensity decreases. .

  Furthermore, in addition to cortisol, the amount of change in relative fluorescence intensity was measured using 17-β-estradiol, cholesterol, testosterone and progesterone. The results are shown in FIG. In FIG. 5, the measurement results for cortisol are indicated by “♦”, and the measurement results for other compounds are indicated by “■”.

  As shown in FIG. 5, when the cross-linked MIP film is used, the difference in relative fluorescence intensity change between cortisol and other steroid compounds is larger than when the non-cross-linked MIP film is used. It can be said that the selective affinity of the crosslinked MIP membrane for cortisol is even higher.

Claims (6)

A molecular imprinting film of a steroid hormone compound,
The steroid hormone compound is represented by the following formula (I):

[Where:
R ¹ represents a methyl group, a hydroxymethyl group or a formyl group;
R ² represents a hydrogen atom, a hydroxyl group or an oxo group,
R ³ represents a methyl group, a hydroxymethyl group or a formyl group,
R ⁴ represents a C _1-10 alkyl group substituted with one or more substituents α, or a hydroxyl group,
R ⁵ represents a hydrogen atom or a hydroxyl group,
R ⁴ and R ⁵ together may form an oxo group,
The substituent α represents a substituent selected from a hydroxyl group and an oxo group.
Having a laminated structure in which a polymer layer is laminated on a self-assembled monolayer;
Β-cyclodextrin is bonded to the surface of the self-assembled monolayer,
A specific recognition space for the steroid hormone compound is formed in the polymer layer,
A molecular imprinting membrane, wherein the β-cyclodextrin is present at one end of the specific recognition space, and an aminooxy group is present at the other end of the specific recognition space. A template compound for forming a specific recognition space for a molecular imprinting polymer, which is represented by the following formula (II):

[Where:
X and Y independently represent a linker group,
R ¹ represents a methyl group, a hydroxymethyl group or a formyl group;
R ² represents a hydrogen atom, a hydroxyl group or an oxo group,
R ³ represents a methyl group, a hydroxymethyl group or a formyl group,
R ⁵ represents a hydrogen atom or a hydroxyl group,
R ⁶ represents a hydrogen atom or a methyl group] A method for producing a molecularly imprinted membrane of a steroid hormone compound comprising:
Forming a self-assembled monolayer on a metal substrate;
Binding β-cyclodextrin to the surface of the self-assembled monolayer;
A step of causing the adamantyl group of the template compound according to claim 2 to interact with the β-cyclodextrin,
Adding a vinyl monomer and copolymerizing with the vinyl group of the template compound,
A method comprising hydrolyzing an oxime group of the template compound and removing a steroid moiety of the template compound. A method for detecting a steroid hormone compound in a sample, comprising:
The steroid hormone compound is represented by the formula (I) defined in claim 1,
Contacting the molecular imprinting film according to claim 1 with the sample in a solvent; and
A method comprising measuring a change in a reaction system due to contact between the molecular imprinting film and the sample. Furthermore, before the contacting step, including a step of inserting a pseudosteroid hormone compound having a labeling group into the specific recognition space of the molecular imprinting film,
The method according to claim 4, wherein in the measurement step, a change in the concentration of the labeling group in the reaction system solution is measured.   5. The method according to claim 4, wherein in the measurement step, the change in the reaction system is measured by a surface plasmon resonance measurement method, a quartz crystal microbalance method, or an electrochemical impedance method.