JPH06336509A - Reagent-bonded polymer and method for bonding reagent - Google Patents

Abstract

PURPOSE:To obtain a polymer which has one or more reagents tenaciously bonded thereto and is applicable to various optical sensors, etc., by bonding the reagents to a copolymer of specific compounds at a specific group in each of the repeating units of the copolymer. CONSTITUTION:A reagent is bonded to an insoluble carrier which is a copolymer of a halogenoalkylated styrene having a structure represented by formula I (wherein R1 is a 1-4C alkyl and X is a halogen) with a compound having a structure represented by formula II (where in R2 is H or CH3 and Y is CN or phenyl) at the R1 in each of the repeating units of the copolymer through or without a spacer to thereby obtain the objective polymer. An example of this polymer is obtained by using an amine compound as the spacer and has a reagent-bonded part which comprises a quaternary group derived from the amine compound and a reagent electrostatically bonded thereto and is represented by formula III (wherein R1 and X are each as defined above; R3 to R5 each is H or a 1-4C hydroxyalkyl; A<+> or A<-> is a positively or negatively charged reagent moiety; and... indicates an electrostatic bond).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a polymer bound with a reagent for measuring at least one specific component in a liquid sample. The reagent-bound polymer can also be applied to simple liquid sample analysis and various optical sensor means for continuously measuring a specific component in a biological fluid sample in vivo.

[0002]

2. Description of the Related Art Conventionally, physical sensors for measuring physical quantities have been widely used in the fields of industrial processes, environmental measurement, medical treatment, etc. Although various attempts have been made, few biosensors have been put to practical use, which are intended to measure biologically relevant substances by utilizing the reaction specificity of enzymes and the like. This is because it is difficult to combine specific target substances such as oxygen, carbon dioxide, and hydrogen ion concentrations, but it is difficult to manufacture them, and sensor elements that selectively measure target substances are manufactured with good reproducibility. It is difficult to do. However, the number of objects that are required to be measured is remarkably increasing, and there is an increasing demand for the development of sensors that measure these rapidly and continuously. On the other hand, in the field of clinical tests and the like, so-called dry chemistry using test pieces has long been developed as a means for analyzing components in liquid samples, and various components in body fluid samples such as urine and blood have been analyzed. Came. It can be said that these are mainly simple diagnostic methods for semi-quantitative determination, and such test pieces are manufactured by impregnating an absorbent carrier such as filter paper with a reagent for detecting a specific component and drying. However, when these test pieces come into contact with a liquid sample, the reagent for component analysis elutes into the liquid sample, and therefore cannot be used for in-vivo measurement, and there is a problem of contamination due to contact. Furthermore, continuous measurement cannot be performed because it is premised on each disposable. Therefore, development of a highly safe system for continuously measuring a specific component in a biological sample in vivo is desired, and for this purpose, problems such as promptness, stability, continuity, and safety are solved. Is desired.

As one of the attempts, an electrochemical method using an electrode using glass or carbon, a semiconductor typified by an ion-sensitive field effect transistor (ISFET), a thermistor, or the like has been conventionally performed. According to this method, it is possible to detect sugars (glucose), lipids (cholesterol, triglyceride, etc.), various enzymes, pH, oxygen, carbon dioxide, or immunologically active components in liquid samples, especially biological samples such as urine and blood. However, their concentrations can be monitored. However, these electrochemical methods have technical obstacles such as poor sensitivity and response speed, influence of external electromagnetic noise, and miniaturization of the device itself.

In recent years, various optical biosensors using a functional polymer film have been developed in place of these electrochemical methods in view of such circumstances. For example, a biosensor using an optrode in which a chemical reagent is immobilized on a vinylimidazole polymer or copolymer film and attached to the tip of an optical fiber (Japanese Patent Laid-Open No. 3-65639) is known. This is p
It is said that the pH value of the liquid sample can be measured in a short time of less than 30 seconds in the range of H3 to 9. However, here, in order to carry the chemical reagent, since the positive charge existing on the imidazole unit in the polymer is mainly used, it is effective for the chemical reagent having a sulfonic acid functional group, Other reagents cannot be fixed stably. In addition, in order to graft this polymer onto an optical fiber, the process of activating the end of the optical fiber is indispensable, so it is not easy to manufacture, and when replacing after use, it is necessary to replace the entire optrode. However, the usage scene is limited. Also,
Disclosed is a technique in which a sensor element can be formed extremely easily by using a composite material in which a polyamide carrier having a carboxyl group and an amino group specifically bound to peroxidase and glucose oxidase is further immobilized with luminol. 3-11759). This complex is characterized in that it can support peroxidase and glucose oxidase on the carrier without modifying the polyamide carrier. However, this product is not versatile because it uses a unique polyamide carrier and the enzymes that can be immobilized are limited to peroxidase and glucose oxidase. Although it can be used as a test piece for a specific purpose, it is assumed that the accuracy and the like are not sufficient, and it is difficult to use it as a sensor because the test piece is disposable and the reaction is not reversible.

[0005]

Although a technique for rapidly, continuously and simply measuring a biological substance is being realized, the types of target detection elements are limited and the production of the elements is not easy. However, there is still a problem in the uniformity of performance, and at the present time, a highly practical sensor element has not been developed. The present invention is intended to solve the above-mentioned problems of the prior art. That is, a reagent that is easy to prepare, has high accuracy, enables measurement of multiple components, and has general requirements for biosensor elements, such as rapidity, stability, continuous measurement, and clinical safety. It is intended to provide a binding polymer.

In order to solve such a problem, the applicant of the present application has already proposed a reagent-bonded polymer using a copolymer of a halogenated alkylstyrene derivative and acrylic acid (Japanese Patent Laid-Open No. 4-330298). ). The present invention is a further development of this technology. Specifically, one of the objects of the present invention is to further expand the range of application by using a novel structure in the polymer portion serving as a carrier of the reagent-bound polymer.

[0007]

The present invention is a copolymer of a halogenated alkylstyrene having a structure represented by the following general formula (I) and a compound having a structure represented by the following general formula (II). Is used as an insoluble carrier, and a reagent is bound to R1 in the repeating unit of the copolymer with or without a spacer, or a reagent binding polymer, or a reagent binding method.

[0008]

[Chemical 6]

[Chemical 7] (In the formula, R1 represents an alkylene group having 1 to 4 carbon atoms that can take any position on the benzene ring, X represents halogen, R2 represents a hydrogen atom, and an alkyl group having 1 to 4 carbon atoms. Y represents a cyan group. Represents a phenyl group.)

According to the present invention, a polymer as a carrier (hereinafter referred to as a carrier polymer) can be easily prepared in a desired shape, and various reagents can be reliably incorporated into the polymer without special pretreatment. Can be fixed. The obtained reagent-bound polymer has excellent performance as a biosensor using optrode, and has versatility in that the aspect as a sensor can be used in a wide variety of usage situations and is highly practical. The present invention is described in detail below.

The reagent-bonded polymer of the present invention has a technical significance in that a desired reagent is directly immobilized on a polymer which can be processed into a desired shape. Direct immobilization means that a reagent component is chemically bound to the structure of a carrier polymer, for example, a carrier is provided with a cavity to absorb the reagent, or a reagent is supported in the structure as a multilayer structure. It is different from the fixed method. Therefore, the reaction speed between the reagent and the substance to be measured is remarkably fast, and quick measurement can be realized.

Further, the carrier polymer according to the present invention is extremely excellent in processability. Although the structure itself of this polymer is publicly known, the finding that it can exert a unique effect in relation to a certain kind of reagent has never been known.
The fact that various reagents can be reliably immobilized and the target sensor element can be stably produced within the design range was a surprising thing that could not be expected from the prior art.

Further, in the prior art, only one kind of reagent can be immobilized on the polymer under a certain control, but
In the present invention, the carrier polymer is capable of imparting multifunctionality capable of immobilizing a plurality of reagents in a predetermined quantitative ratio, in addition to processability. The reagent-bonded polymer having the above characteristics is suitable as a biosensor element using optrode or a test piece that is structurally simple and can be easily measured. However, by effectively utilizing this reagent and selecting an appropriate reagent, it can be applied to other uses. For example, it is a carrier for immobilization of immune components used for protein separation, purification, and various chromatography carriers.
Although the following description is based on the assumption of a biosensor element, it is obvious that the biosensor element can be applied to other applications without departing from the technical scope of the present invention.

The carrier polymer according to the present invention comprises a copolymer of the halogenated alkylstyrene represented by the general formula (I) and the compound represented by the general formula (II).
R1 of halogenated alkylstyrene includes methylene group, ethylene group, trimethylene group, tetramethylene group,
A linear or branched alkylene group having 1 to 4 carbon atoms such as a propylene group is preferable. A polymer having a relatively small number of carbons has higher reactivity in the polymerization reaction, and this portion serves as a reagent binding site. Therefore, a polymer having a relatively small number of carbons has a higher reaction efficiency. A methylene group is preferred from the viewpoints of easiness of reaction and practicality. A halogen atom X is substituted on R1. As X, Cl, Br, I or the like is preferable.
Cl is highly reactive. The halogen substitution position on R1 is not particularly limited. Although one halogen atom on R1 is basically used, a plurality of halogen atoms may be substituted depending on R1. The substitution position of R1 can be bonded at each of the para, meta and ortho positions, but is preferably the para position. This is because, when the reagent is immobilized after the copolymer is formed, the influence of structural obstacles is small.
Typical examples of the halogenated alkylstyrene include p-chloromethylstyrene, m-chloromethylstyrene and o-.
Examples thereof include chloromethylstyrene.

R2 of the general formula (II) is preferably a hydrogen atom or a methyl group, and Y is a cyan group,
A phenyl group and the like are preferable. Specifically, acrylonitrile, methacrylonitrile, styrene, etc. can be mentioned. The copolymer may contain one or more of each of the halogenated alkylstyrene and the compound of the general formula (II), and in some cases, the copolymer may be formed as a mixed system of various kinds.

The molar ratio of the halogenated alkylstyrene monomer to the compound of the general formula (II) in the polymerization reaction may be appropriately set depending on the intended use of the reagent-bound polymer. In principle, if the former molar ratio is increased, the hydrophilicity of the polymer becomes stronger when the spacer is introduced, the affinity with the water-soluble component becomes higher, and the measurement sensitivity of biologically relevant substances can be improved. Elution may occur during measurement, and safety may be a problem in the field of clinical testing. Moreover, since the reagent component is bound to the former R1 site, it is generally preferable if the molar ratio is increased because the reagent concentration in the polymer can be increased. Formula (I
I) The presence of the compound is essential. However, the general formula (I
I) When the molar ratio of the compound monomer is increased, the hydrophobic tendency becomes strong, and the wettability with the analyte decreases, so that the measurement sensitivity may deteriorate. Further, since this compound does not have a reagent binding site, if this molar ratio increases, the reagent concentration will decrease, which may lead to a decrease in measurement sensitivity. Further, when the molar ratio of the halogenated alkylstyrene monomer increases, thickening may occur depending on the type of the spacer, particularly when the reagent is immobilized using the spacer.
The thickening may hinder the subsequent film formation process, reagent immobilization process, and the like. Therefore, the molar ratio of the halogenated alkylstyrene monomer to the compound of the general formula (II) is preferably 3: 1 to 1: 3. More preferably, it is 2: 1 to 1: 2. Since the charged molar ratio does not always match the composition of the copolymer as it is, it is usually practical to polymerize at a molar ratio of 1: 1 in consideration of the range of reactivity of each monomer.

The copolymerization reaction of each monomer can be carried out by a general method for producing a polymer, but a nonionic type such as a peroxide or an azo compound is used as a polymerization initiator. Particularly preferred are hydrogen peroxide, t-butyl hydroperoxide and azobisisobutyronitrile. The reaction temperature is 25 to 100 ° C., preferably about 50 to 80 ° C. The reaction time is not particularly limited, but generally 3 to 48 hours is suitable. Further, it is usually desirable to carry out the reaction in an inert gas atmosphere.
The obtained carrier polymer is not significantly affected by the degree of polymerization and functions as a reagent-bound insoluble carrier. Next, a reagent is bound to R1 by using an appropriate spacer if necessary.

Reagents that bind to the carrier polymer are classified into two types depending on the binding mode. One is for electrostatically binding a reagent component to a quaternized product using an amine compound as a spacer, and the other is for covalent bonding with or without a spacer. Since this carrier polymer can immobilize the reagent in two ways, various functional organic compounds can be selected in a very wide range. For example, various chromogens, fluorescent dyes, redox compounds, etc. can be used as the dye. In addition to synthetic substances, natural products having physiological activity such as enzymes, proteins, antigens and antibodies can be immobilized as reagents. The great advantage of being able to immobilize different reagents with different properties using a common carrier polymer makes it possible to immobilize the respective polymers separately and then easily mix and integrate the respective polymers. This is because the carrier polymers are common and can be bound uniformly, and the reagents do not dissociate even after mixing in the mixing operation.

Therefore, if the dyes and enzymes, the dyes and enzymes, the fluorescent dyes, etc. are fixed to the carrier polymer individually and securely, and then they are mixed and integrated, the components become uniform. A dispersed functional film can be formed, and it is not necessary to add other components for measurement. This is an extremely effective feature as a sensor, and has never been realized by the conventional technology. However, each component may be fixed to the carrier polymer together as long as each component has the same level of binding property to the polymer. In the prior art, no means other than a method of forming a multilayer structure or forming a composite was known. If the multi-layer structure is used, the reaction rate between the reagent and the target component decreases, and it is difficult to control the amount of reagent carrier even in the complex, and the reaction rate is not fast. In view of such circumstances, the utility of the present invention in the field of sensors is extremely high. That is, the reagent-immobilized membrane obtained by the reagent-bonded polymer according to the present invention is a liquid as a sensor due to a highly efficient chemical reaction (catalytic reaction, photochemical reaction, redox reaction, etc.) or physical reaction performed by these functional compositions. It is extremely useful for analyzing specific components in a sample. The method of forming the film will be described later.

Next, the electrostatic coupling of the first aspect will be described. As a conventional technique, for example, in the above-mentioned Japanese Patent Laid-Open No. 3-65639, the negative charge of an indicator having a sulfonic acid group or a sulfone is mainly used to ionically bond to a carrier. In this method, since the functional group cannot be bonded unless it is strongly negatively charged, the usable indicator is limited and the processing method is also limited. However, in the present invention, any substance that can be electrostatically bound, that is, one that functions as an acid-base indicator, an enzyme, or the like can be supported regardless of the type of the functional group, the magnitude of the charge, and whether it is water-soluble or sparingly water-soluble.

The reason for this is not clear because the mechanism of reagent binding has not been clarified. However, in this embodiment, since a quaternary ammonium compound is required as a spacer and an acid-base indicator or an enzyme that easily coordinates with the quaternary ammonium compound is mainly used as a reagent, some electrostatic bond occurs. It is speculated that Since a carrier polymer with a spacer can basically function as an anion exchange resin, it is generally considered that a reagent is fixed to a quaternary ammonium compound by a coordinate bond by an ion exchange reaction. In some cases, it may become an anion or a cation depending on the pH, and the strength of the coordination bond should differ depending on the reagent, and the reagent binding reaction can also occur in an organic solvent in which water does not intervene. Therefore, it is difficult to uniformly consider the binding mechanism.

Presumably, the molecular structure of the carrier polymer, the size of the molecule, the affinity for the reagent, the abundance of the spacer, etc. act intricately to capture the reagent.
In any case, it exerts an effect beyond the expectation that the reagent is extremely stable after the reagent is fixed and the reagent is hardly eluted. Therefore, the term "electrostatically bound" here means to widely bind to the quaternary ammonium compound bound to R1 by utilizing electrostatic action, and simply means a binding mode by an ionic bond or a coordinate bond. do not do. The reagent binding part is considered to be represented by the following formula.

[0022]

[Chemical 8] (In the formula, R1 and X are the same as in the general formula (I), R3 and R
4 and R5 are each independently a hydrogen atom and a carbon number of 1 to 4.
Represents a hydroxyalkyl group having 1 to 4 carbon atoms, A ± represents a positively or negatively charged reagent moiety, ... Represents an electrostatic bond. )

In the formula, X is derived from the carrier polymer.
That is, when the carrier polymer is treated with a tertiary amine or the like, the tertiary amine reacts with R1 to covalently bond, quaternize, and is introduced as a quaternary ammonium compound. This serves as a spacer for reagent binding. The spacer portion exhibits cationicity, and its structure is the same as that of the strongly basic anion exchange resin. In fact, the carrier polymer having the quaternary ammonium compound introduced therein can function as the anion exchange resin. Four
The introduction of the primary ammonium compound is carried out by, for example, dissolving the carrier polymer in acetone or the like, adding a tertiary amine thereto, and adding 1
The reaction may be carried out for about 48 hours, which can be carried out based on a known technique. In the above formula, R3, R4 and R5 are the same or different and each independently represent a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group or a tert-butyl group. And an alkyl group having 1 to 4 carbon atoms such as a group and a hydroxyalkyl group having 1 to 4 carbon atoms such as a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, and a hydroxybutyl group. Specifically, trimethylamine,
Tertiary amines such as triethylamine, n-propyldimethylamine, dimethyl-n-butylamine, tri-n-butylamine, dimethylethanolamine and diethylethanolamine are used.

Next, the binding of the reagent based on the covalent bond, which is the second aspect, will be described. Reagents which can be used are amino group-containing reagents represented by NH ₂ -B (B is a main component) and carboxyl group-containing reagents represented by COOH-D (D is a main part). That is, it is bound to R1 via an amino group or a carboxyl group, and basically any reagent having an amino group or a carboxyl group can be bound. If configured as a sensor, an enzyme active substance, an immunologically active substance, a dye or the like can be used. Specifically, the enzyme is glucose oxidase, cholesterol oxidase, peroxidase, alkaline phosphatase, etc., the immune component is an antibody or fragment thereof, the antigen, etc., the dye is o-tolidine, 2,7-
Examples thereof include diaminofluorene, 3,3'-5,5'-tetraalkylbenzidine, or salts thereof.

A product obtained by binding these reagents to R1 is represented by the following formula. -R1-Sp1 = NB or -R1-NH-B (In the formula, R1 is the same as in the general formula (I), Sp1 is a spacer, and a structure obtained by the reaction of ammonia or a diamine with a dioxo compound. -R1-Sp2-CO-D (In the formula, R1 is the same as in the general formula (I), Sp2 is a spacer, and shows a structure obtained by the reaction of ammonia or a diamine with a carboxyl group-containing reagent. .) reagent NH ₂ -B binds to the carrier polymer in a covalent bond,
In this case, Sp1 is not essential even if it does not exist because the amino group of the reagent forms a covalent bond with R1.
However, the use of a spacer increases the reactivity, so that the reagent can be stably immobilized on the carrier polymer. Therefore, it is preferable to use a compound containing an amino group as a reactive functional group as a spacer in order to connect the carrier polymer and the reagent. This does not mean that the amino group of the spacer is bonded to the amino group of the reagent, but as the first step, the amino group of the spacer is bonded to the carbonyl group of another second spacer, and this carbonyl group is used to The attachment of amino groups and consequently immobilization of the reagent on the carrier polymer.

According to this method, the first amino group-containing spacer having high reactivity is first immobilized on the polymer, and then the second carbonyl group-containing spacer is bonded, so that the spacer can be easily introduced. Also, the second spacer has two carbonyl groups, and the double bond of the carbonyl group not bound to the first spacer is highly reactive and can bind the reagent. That is, as the spacer, a reaction product of ammonia or a diamine and a dioxo compound can be preferably used. Examples of the diamines include ethylenediamine, tetramethylenediamine and hexamethylenediamine, and examples of the dioxo compound include glutaraldehyde and hexamethyleneisocyanate. Glutaraldehyde is particularly preferred because of its high reactivity.

On the other hand, the reagent COOH-D is a spacer Sp.
It is covalently bound to the carrier polymer via 2, but in this case a spacer is required. Ammonia or diamine is used as the spacer, and this is bonded to R1 to form a carrier polymer having an amino group at the terminal. The amino group and the carboxyl group of the reagent are bonded to form a kind of acid amide bond (-CO-NH-), and as a result, the reagent is immobilized on the carrier polymer. In this reaction, a carbodiimide reagent generally used for peptide synthesis, a condensation reagent such as Woodward's reagent K, and the like are used to perform a bond between an amino group and a carboxyl group. Specifically, as the carbodiimide reagent, dicyclohexylcarbodiimide,
There are 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimidometh-p-toluenesulfonate, etc., and Woodward reagent K (N-ethyl-S-phenyl Isoxazolium-3'-sulfonate), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, or the like is used.

As described above, the carrier polymer can bind a reagent by using either the first mode or the second mode, but two methods are conceivable for implementation. All of these have the advantage of being able to immobilize the reagents required for analysis with a simple operation, and because they can be immobilized under mild conditions without the need for special atmospheres or high temperature conditions, enzymes or physiologically active substances can be immobilized. It is also possible to immobilize, although it is not very stable, as in. Moreover, since the reagents necessary for the analysis are firmly bound via the spacer, no elution at the time of measurement is observed. One is a method in which a carrier polymer with a spacer is molded into a predetermined shape and then immersed in a reagent-containing solution to bond the reagent to the spacer. The other is a method in which a carrier polymer with a spacer is dissolved in an organic solvent, a reagent is added thereto, the reagent is bound to the spacer in the solvent, and then the spacer is molded into a predetermined shape. The reagent can be firmly fixed by either method. As for the form, in addition to the one formed into a film itself, another form of the liquid-absorbent carrier (second carrier) used and contained therein can be used. This is because the carrier polymer for immobilizing the reagent has a highly reactive substituent for supporting the reagent in the repeating unit, as well as the properties of the polymer itself (reactivity, compatibility, film-forming property, It is because of excellent plasticity). In particular, in order to use it as a sensor after immobilizing a reagent, it is important that it can be processed into various shapes in order to expand the application range, that the immobilized reagent does not elute, the response speed is fast, It is also necessary that the reaction be continuously and reproducibly performed. The reagent-bound polymer membranes of the present invention fully meet such requirements.

Next, the film forming method of the reagent-bonded polymer according to the present invention will be described. Examples of the film forming method and its form are as follows. In order to analyze a specific component in a liquid sample, the immobilized carrier as described above must be processed into a shape suitable for analysis and used. In this case, it is desirable to process by a method generally called cast coating. Cast coating is a method of forming a dry coating film by bringing a wet coating film into contact with a smooth surface, and coating a paper with a pigment dispersion coating agent, a vinyl dispersion coating agent, a lacquer and a latex film, and a woven fabric. It is a coating method that is often used when coating foil and paper. Here, for example, a carrier polymer to which a spacer is bound in advance is dissolved in an organic solvent to form a film by cast coating, and then a composition required for analysis is immobilized, or after all components are mixed, A film suitable for analysis can be obtained by the film formation method. As the smooth substance to be cast-coated, a transparent or opaque support, for example, a film of polyvinyl chloride, polyethylene, polycarbonate, polystyrene or the like is preferably used, and a non-adhesive substance such as Teflon is also used. In this case, it is also possible to use after peeling after forming a film.

As another form, a liquid-absorbent second
It is also possible to use it after absorbing it in the carrier. Second
The carrier is preferably a porous material such as filter paper, glass fiber, membrane filter, woven fabric, non-woven fabric, wood, sponge material. In this case as well, both the method of binding the carrier polymer and the spacer and then absorbing the second carrier to immobilize the composition necessary for the analysis later, and the method of absorbing all components after mixing them are also used. Can be used. As described above, in the present invention, the above-described two forms can be adopted, and therefore, in the optical measurement, a form suitable for the intended analysis form can be selected for both transmitted light and reflected light. Further, since the shape can be freely changed, it can be applied to various analysis methods. As described above, since the reagent-bonded polymer of the present invention itself can be used as a film to form a sensor element, the contact area between the reagent and the object to be inspected, the frequency is remarkably high, the response reaction is fast and accurate. In addition, film formation and impregnation are very easy. Further, the measurement component in the test object is not limited to one kind, and several kinds of corresponding reagents can be uniformly fixed to the carrier polymer so as to measure a plurality of things. Moreover, since a plurality of reagents such as an enzyme and a chromogen can be fixed together in the polymer, it is not necessary to add a color former or the like to the test object. To fix a plurality of reagents in a polymer, each reagent may be fixed to the polymer, and a method of film-forming them by combining them may be used, or a method of fixing all reagents to the polymer at one time to form a film, Further, any method such as a method of preliminarily forming a film of a carrier polymer and then fixing a reagent, or a method of combining these may be used.

[0031]

[Function] The reagent-bound polymer used in the present invention has both a chemical property of reliably fixing a reagent with or without various spacers and a physical property of film-forming property and processability. Has the effect of causing. In short, this method is easier and more uniform than the method of enclosing and fixing the reagent in a gel lattice or a microcapsule, or fixing the one adsorbed to the bead-shaped ion exchange resin in the cellulose acetate membrane. It is possible to form a simple film, it is possible to limit the amount of the reagent, and further, since the reagent is directly fixed to the polymer to form a film, the reaction rate is remarkably increased. Hereinafter, the present invention will be described with reference to examples.

[0032]

【Example】

Example 1 Synthesis of Carrier Polymer Purified p-chloromethylstyrene and acrylonitrile monomers were added in equimolar amounts to methylethylketone, t-butylhydroperoxide was added, and the reaction vessel was replaced with nitrogen at 80 ° C. And reacted for 24 hours. After completion of the reaction, the mixture was added to a large amount of methanol to obtain a p-chloromethylstyrene / acrylonitrile copolymer.

Example 2: Synthesis of carrier polymer Purified p-chloromethylstyrene and each monomer of styrene were added to toluene in equimolar amounts, and 1% (weight ratio) of azobisisobutyronitrile was added. . The above mixture was placed in a reaction vessel sufficiently purged with nitrogen and polymerized at 70 ° C. for 10 hours, and after completion of the reaction, added to a large amount of methanol to obtain a p-chloromethylstyrene / styrene copolymer.

Example 3 Introduction of Spacer The copolymer obtained in Example 1 was dissolved in acetone to give 10
% Solution, 30% of triethylamine based on the copolymer
In addition, the mixture was reacted at room temperature for 24 hours. After completion of the reaction, the precipitate was separated by filtration and dissolved in an appropriate amount of methanol, and this solution was added to a large amount of acetone to obtain a carrier polymer with a spacer having a property of a strongly basic ion exchange resin.

Example 4 Introduction of Spacer The copolymer obtained in Example 1 was dissolved in acetone so as to have a concentration of 7%, 2% of ammonia water was added thereto and reacted at room temperature for 8 hours, After that, the amino group-introduced carrier polymer precipitated by adding to a large amount of methanol was separated by filtration.

Example 5: Binding of reagent and its performance (1) 1 g of the carrier polymer obtained in Example 3 was dissolved in 100 ml of methanol, and 20 mg of bromthymol blue was further added to dissolve it to electrostatically support the carrier. It was attached to the polymer. This solution was formed into a film on polyethylene by a cast coating method to obtain a bromthymol blue immobilized film. This film was cut into a size of 10 mmφ without peeling it from polyethylene to prepare a sensor element. This device was dipped in various buffer solutions adjusted to pH 4 to 9, taken out at the time when it became stable to a certain color change, and reflected light measurement was carried out with a spectroscopic color difference meter (SZ-Σ90, manufactured by Nippon Denshoku). The results are shown in Figure 1.
A change in reflection spectrum was observed depending on pH, and it was found that the relationship between pH and reflectance can be made into a calibration curve by selecting an appropriate wavelength.

Example 6: Binding of reagent and its performance (2) 0.5 g of the carrier polymer obtained in Example 4 was added to 50 parts of acetone.
Dissolve it in 1 ml and impregnate it with a filter paper cut into 1 cm squares to 60
It was dried at ° C for 1 hour. Glutaraldehyde was added to 0.1M phosphate buffer (pH 7) to make a 2% solution, and filter paper impregnated with a carrier polymer was put therein to react at room temperature for 1 hour, and then glucose oxidase (50 U / ml) and peroxidase (50 U / 50 U / ml) were added. ml) was dissolved in 0.1M phosphate buffer and reacted at 4 ° C. for 24 hours. The obtained enzyme-immobilized filter paper was thoroughly washed to remove unreacted substances. Meanwhile, 4-aminoantipyrine 20 mg, TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium) 20 m
g, glucose 10mg 0.1M phosphate buffer (pH 7)
It was dissolved in 50 ml to prepare an enzyme detection solution. Two 3 ml of phosphate buffers were prepared, and enzyme-immobilized filter paper was placed in each and preheated at 37 ° C., and then the enzyme-immobilized filter paper was taken out from one side. When 3 ml of the detection solution was added thereto and reacted at 37 ° C., the solution containing the filter paper exhibited a purple color, while the solution without the filter paper showed no coloration. From the above results, it was confirmed that both enzymes were immobilized on this filter paper, and it was found that elution was not observed. Further, even if the same enzyme-immobilized filter paper was put in the detection solution and reacted several times, a similar color was obtained, and it was found that the enzyme-immobilized filter paper can be repeatedly used.

Example 7: Binding of reagent and its performance (3) 0.5 g of the polymer obtained in Example 3 was dissolved in 20 ml of methanol, and 0.5 g of the polymer obtained in Example 4 was added,
Bromthymol blue 10 mg and acetone 30 ml were added and dissolved. Glass fibers were impregnated with this solution and dried at 80 ° C. for 15 minutes. Then, add glucose oxidase (100 U / ml) and 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimidometh-p-toluenesulfonate (appropriate amount) in 0.1M phosphate buffer (pH 6) at 4 ° C. The reaction was carried out for 24 hours. The obtained enzyme and chromogen-immobilized glass fiber were thoroughly washed, unreacted substances were removed, and then dried. By this operation, bromthymol blue is electrostatically bound to the polymer of Example 3, and a reagent-bound polymer in which the carboxyl group of glucose oxidase is covalently bound to the spacer introduced into the polymer of Example 4 can be obtained. Glucose solutions having different concentrations of 10 μl were added dropwise to the obtained reagent-bound polymer and reacted for 5 minutes, and the resulting coloration was measured by reflected light with a color analyzer. As shown in FIG. 2, the result showed a change in reflectance according to the glucose concentration, and it was confirmed that glucose could be quantitatively detected.

[0039]

Since the reagent-bonded polymer of the present invention has high processability, it can be directly formed into a film or impregnated into another carrier. In addition, the shape itself can be processed arbitrarily,
Its application range as an immobilization carrier is wide. In addition, electrostatic binding or covalent binding can be selected for the binding mode of the reagent, and the type of immobilizable reagent is a chemical indicator component such as pH indicator or redox indicator, or an organism such as an enzyme or an immunoactive substance. It has a wide range of biological components and is highly versatile. Further, since it is possible to bind a plurality of reagents at the same time, it is possible to carry out the measurement without adding other components simply by placing the membrane on which all the reagents necessary for the measurement are immobilized in the sample to be measured. In addition, when binding reagents, a wide range of methods can be selected, such as a method in which a plurality of reagents are individually bound in an arbitrary binding mode and then mixed, or a method in which reagents having different binding modes are simultaneously bound can be selected. It is an advantage. Furthermore, when the reagent-bound polymer according to the present invention is applied to a sensor, the reagent is directly immobilized on the polymer, and therefore the response speed and sensitivity are excellent. Further, the reagent-bonded polymer of the present invention can stably fix a reagent, and thus can provide a highly safe sensor without elution. In addition, the optical sensor is known to have a transmitted light measuring method, a reflected light measuring method, and the like, but the reagent-bound polymer of the present invention is excellent in processability and thus can be applied to a wide variety of optical sensors. .

[Brief description of drawings]

1 is a reflected light spectrum obtained in Example 5. FIG. The vertical axis is the reflected light intensity (%), and the horizontal axis is the measurement wavelength (n
m) is shown. The numbers in the figure are pH values.

FIG. 2 is a reflected light spectrum obtained in Example 7. The vertical axis is the reflected light intensity (%), and the horizontal axis is the measurement wavelength (n
m) is shown. The numbers in the figure are glucose concentrations (mg / dl).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location G01N 33/547 8310-2J

Claims (11)

[Reference Number] P-000278 [Claims] 1. A copolymer of a halogenated alkylstyrene having a structure represented by the following general formula (I) and a compound having a structure represented by the following general formula (II) is used as an insoluble carrier. A reagent-bonded polymer in which a reagent is bonded to R1 in the repeating unit of the combination with or without a spacer. [Chemical 1] [Chemical 2] (In the formula, R1 is an alkylene group having 1 to 4 carbon atoms which can take any position on the benzene ring, X is halogen, R2 is a hydrogen atom or a methyl group, and Y is a cyan group or a phenyl group.) 2. An amine compound is used as a spacer.
The reagent-bonding polymer according to claim 1, which has a reagent-bonding portion represented by the following formula, which is obtained by electrostatically bonding a reagent to a graded product. [Chemical 3] (In the formula, R1 and X are the same as in the general formula (I), R3 and R
4 and R5 are each independently a hydrogen atom and a carbon number of 1 to 4.
Represents a hydroxyalkyl group having 1 to 4 carbon atoms, A ± represents a positively or negatively charged reagent moiety, ... Represents an electrostatic bond. ) 3. The reagent-bonding polymer according to claim 1, which has a reagent-bonding moiety represented by the following formula, which is formed by covalently bonding an amino-group-containing reagent represented by NH ₂ -B (B is a main component). -R1-Sp1 = NB or -R1-NH-B (In the formula, R1 is the same as in the general formula (I), Sp1 is a spacer, and a structure obtained by the reaction of ammonia or a diamine with a dioxo compound. Is shown.) 4. The reagent-bonding polymer according to claim 1, which has a reagent-bonding portion represented by the following formula, which is obtained by covalently bonding a carboxyl group-containing reagent represented by COOH-D (D is a main part). -R1-Sp2-CO-D (In the formula, R1 is the same as in the general formula (I), Sp2 is a spacer, and shows a structure obtained by the reaction of ammonia or a diamine with a carboxyl group-containing reagent.) 5. A copolymer obtained in a molar ratio of the monomer represented by the general formula (I) to the monomer represented by the general formula (II) in the range of 3: 1 to 1: 3. The reagent-bound polymer according to claim 1, wherein 6. The electrostatically bound reagent is an enzyme active substance,
The reagent-bonded polymer according to claim 2, which is selected from immunologically active substances and acid-base indicators. 7. The reagent-bound polymer according to claim 3, wherein the amino group-containing reagent is selected from enzyme active substances, immunologically active substances and dyes. 8. The reagent-bound polymer according to claim 4, wherein the carboxyl group-containing reagent is selected from an enzyme active substance and an immunoactive substance. 9. A reagent-bound polymer film obtained by forming a film of the reagent-bound polymer according to claim 1. 10. A reagent-bound polymer support obtained by impregnating the reagent-bound polymer according to claim 1 with another liquid-absorbent carrier. 11. A copolymer of a halogenated alkylstyrene having a structure represented by the following general formula (I) and a compound having a structure represented by the following general formula (II) is used as an insoluble carrier, A method for binding a reagent in which a reagent is bound to R1 in a repeating unit of a unit with or without a spacer. [Chemical 4] [Chemical 5] (In the formula, R1 is an alkylene group having 1 to 4 carbon atoms which can take any position on the benzene ring, X is halogen, R2 is a hydrogen atom or a methyl group, and Y is a cyan group or a phenyl group.)