JP3095902B2 - Method for producing optical fiber having carboxyl group introduced - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing an optical fiber having a carboxyl group introduced therein. More specifically, the present invention relates to a method for producing an optical fiber that can be used for measurement of a physiologically active substance by an immunoassay method, and particularly to an optical fiber for immobilizing an immunological substance having a stable carboxyl group even after long-term storage.

[0002]

2. Description of the Related Art Conventionally, in the measurement of a physiologically active substance by an immunoassay, a method of performing a fluorescent immunoassay using an optical fiber in which an immunological substance such as an antigen or an antibody is immobilized on the optical fiber. It has been known. In this case, a method of introducing a cross-linking agent that easily reacts with an amino group in an antigen or an antibody into the surface of the optical fiber is used so that the immunological substance is easily immobilized on the surface of the optical fiber. For example, in WO 90/13029,
To make it easier for the immune substance to be immobilized on the surface of the optical fiber,
The optical fiber reacts with dialdehydes such as glutaraldehyde and succinaldehyde to introduce a formyl group on the core surface of the optical fiber, and reacts and binds this formyl group with an amino group in a biologically active component such as an antigen or antibody.
This is a method of immobilizing an immunological substance such as a physiologically active component on an optical fiber. That is, in WO 90/13029, a resin optical fiber is used as a carrier for immobilizing an immunological substance such as a physiologically active component, and a formyl group is introduced into the core surface of a resin optical fiber mainly composed of a resin having an ester structure. For this purpose, the formyl group is introduced by immersing the core surface of the resin optical fiber in a 50-100 mM ethanol solvent KOH solution, an ethanol solvent NiSO ₄ solution and a mixed solution of glutaraldehyde and succinaldehyde and reacting them. . Then, the formyl group introduced on the surface of the core of the optical fiber and the amino group in the immunological substance are bonded to prepare an optical fiber made of an immunological substance-immobilized resin.

However, since formyl groups are chemically active functional groups and lack stability when formyl groups are introduced, it is not preferable to preserve optical fibers with formyl groups introduced for long periods of time. The problem has been pointed out. Accordingly, an object of the present invention is to provide a method for producing an optical fiber in which a carboxyl group is introduced on the core surface of an optical fiber as a functional group suitable for stable long-term storage, instead of a chemically unstable formyl group. is there.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems. As a result, by using a carboxyl group as a functional group that becomes a binding reaction functional group with an immune substance such as a physiologically active component, an optical fiber that is stable even under an atmosphere such as oxidation-reduction or high temperature and suitable for long-term storage is manufactured. The inventors have found that the present invention can be performed and completed the present invention.

That is, the gist of the present invention is to (1) introduce a carboxyl group on the core surface of a resin optical fiber by oxidizing a formyl group present on the core surface of the resin optical fiber with an oxidizing agent. And (2) reacting a resin optical fiber having a formyl group on the core surface with a compound having an amino group and a carboxyl group to form a carboxyl group on the core surface of the resin optical fiber. The present invention relates to a method for producing an optical fiber, characterized by introducing

The optical fiber used in the present invention is a resin optical fiber. The resin constituting the resin optical fiber is not particularly limited, but is required to be a material that does not adsorb the immunity substance and has a good translucency, such as polystyrene, polyacrylate, and polyester. , Polyacrylamide,
Polyvinyl alcohol, polyethylene terephthalate,
Polycarbonate or a copolymer thereof is exemplified. Among them, polyacrylates are preferred.
Polyacrylate is an acrylic resin having an ester structure and is, for example, a synthetic resin made of a polymer of an ester derivative such as acrylic acid and methacrylic acid, and specifically, methyl acrylate, acrylic acid Polymers such as ethyl and methyl methacrylate are exemplified. Among these polyacrylates, one particularly preferably used in the present invention is polymethyl methacrylate. This is because polymethyl methacrylate has particularly good translucency compared to other resins. The resin optical fiber used in the present invention may be, for example, a copolymer of a monomer such as methyl acrylate, ethyl acrylate, methyl methacrylate and a monomer such as styrene.

When a carboxyl group is introduced into the surface of such an optical fiber, it is desirable to peel off the cladding layer of the optical fiber to expose the core surface. The reason is that the diameter of the optical fiber is usually 1 mm and the diameter of the core cross section is only about 0.97 mm (the cross-sectional area is 0.739 mm ² ).
This is because it is necessary to increase the core surface area by peeling the clad layer. Further, it is desirable that the end face of the optical fiber is polished, and it is preferable that the polishing be performed using alcohol as a lubricant.

In the present invention, in order to introduce a carboxyl group on the core surface of a resin optical fiber, a method of first forming a formyl group on the core surface of the resin optical fiber and converting this to a carboxyl group is employed.

In order to introduce a formyl group onto the core surface of a resin optical fiber, a method using a dialdehyde such as glutaraldehyde or succinaldehyde as a compound having a formyl group, or a monoaldehyde such as glyceraldehyde is used. Method. (1) A method using a dialdehyde: a treatment solution obtained by mixing an alkali metal hydroxide solution using at least a lower alcohol as a solvent and a compound having a formyl group such as glutaraldehyde and succinaldehyde. The concentration of the alkali metal hydroxide is 0.5 to 40 mM
This is a method in which a formyl group is introduced by immersing the core surface of a resin optical fiber in a treatment solution as described above to cause a reaction.

[0010] As the lower alcohol used here, at least one alcohol selected from the group consisting of methanol, ethanol, propanol and butanol is used, and ethanol is preferred. In the present invention, an alkali metal hydroxide solution using such a lower alcohol as a solvent is used. As the alkali metal hydroxide, at least one selected from the group consisting of potassium hydroxide, sodium hydroxide and lithium hydroxide is used, and any one is preferable, but potassium hydroxide is preferable. The concentration of the alkali metal hydroxide is 0.5 to 40.
mM, preferably 13-27 mM. When the amount is less than 0.5 mM, the amount of formyl group introduced is drastically reduced. When the amount exceeds 40 mM, the degree of polymerization of a compound having a formyl group such as glutaraldehyde tends to proceed too much. Absent.

The treatment solution in the present invention is prepared by using at least the lower alcohol described above as a solvent in an amount of 0.5 to 40 m.
It is obtained by mixing an alkali metal hydroxide solution of M and a compound having a formyl group, but is preferably used by further mixing a Ni salt solution. The Ni salt solution used in the present invention is an N salt solution such as NiSO ₄ or NiCl _2.
A solution obtained by dissolving the i-salt in the lower alcohol as described above and saturated is used, and preferably a NiSO ₄ solution. The lower alcohol is not particularly limited, but ethanol is preferably used.

The concentration of the compound having a formyl group in the treatment solution of the present invention is usually 0.2 to 1M. The reason is that when the concentration is lower than 0.2 M, the amount of formyl groups that can be introduced is small, and when the concentration is higher than 1 M, the surface becomes cloudy due to the polymerization reaction. Although the Ni salt does not need to be mixed as described above, the concentration when mixed is usually 0.9 mM or less, and preferably at least about 0.01 mM or more.

In the present invention, the degree of polymerization of the compound having a formyl group such as glutaraldehyde is substantially constant in the prepared treatment solution. An optical fiber into which a formyl group has been introduced can be manufactured with a small loss and a small change in the amount of formyl group introduced into the core surface of the optical fiber. In this case, usually 45
It is performed by performing a heat treatment in a temperature range of ６０60 ° C. for usually 5 to 20 minutes. After the heat treatment, washing is usually performed with dilute hydrochloric acid and phosphate buffered saline.

(2) Method using monoaldehyde: When a formyl group is introduced into the core surface of a resin optical fiber, the optical fiber is converted into a vic-aldehyde such as glyceraldehyde.
Reaction with monoaldehyde having diol group
-A method of introducing a diol group and then oxidizing to introduce a formyl group. To react the optical fiber with glyceraldehyde, glyceraldehyde is added to a 50-100 mM ethanol solvent KOH solution at 0.1-1%.
M, and the optical fiber is immersed in the solution and reacted at 45 to 60 ° C. for 5 to 20 minutes. After the reaction, it is washed with distilled water, ethanol and the like. Thereby, a vic-diol group can be introduced into the core surface of the resin optical fiber.

Next, the thus obtained vic-
By oxidizing the diol group, it can be converted to a formyl group. Specific examples of the oxidizing agent used here include metaperiodic acid and alkali metal salts thereof, and lead tetraacetate, and preferably sodium metaperiodate. The oxidation reaction is, for example, 0.1 to 5 M
Performed by immersing the fiber in an aqueous solution of sodium metaperiodate for 1 to 6 hours under ice cooling,
After the reaction, it is washed with distilled water or the like.

There are two modes for introducing a carboxyl group onto the core surface by using the thus obtained optical fiber having a formyl group. (1) A method of oxidizing an optical fiber having a formyl group using an oxidizing agent, specifically, a method of oxidizing the optical fiber by immersing the optical fiber in a solution containing chromium trioxide, potassium permanganate, or hydrogen peroxide, and ( 2) A method of reacting an optical fiber having a formyl group with a compound having an amino group and a carboxyl group, specifically, a method of immersing the optical fiber in a solution containing β-alanine, glutamic acid, aspartic acid, or 4-aminobutyric acid. Is mentioned.

As a first embodiment, when chromium trioxide or potassium permanganate is used, a 0.1 to 10% solution of chromium trioxide or potassium permanganate using 0.1 to 10 N sulfuric acid as a solvent is used. An optical fiber having a formyl group is immersed in the mixture and reacted at 4 ° C. to room temperature for 30 minutes to 5 hours, and then 0.02 N sulfuric acid, distilled water and PBS
Wash with. When hydrogen peroxide is used, 5 to 3
An optical fiber having a formyl group is immersed in 0% hydrogen peroxide solution, treated at room temperature for 1 hour to 10 hours, and then washed with distilled water and phosphate buffered saline (PBS). By performing such a treatment, the formyl group on the optical fiber can be oxidized to introduce a carboxyl group on the core surface.

As a second embodiment, to react an optical fiber having a formyl group with a compound having an amino group and a carboxyl group, formyl is added to a 1 M aqueous solution of β-alanine, glutamic acid, aspartic acid, or 4-aminobutyric acid. Immersion of optical fiber with base
After standing for ~ 12 hours, reduce with sodium borohydride and wash with water and PBS. By performing such a treatment, a carboxyl group can be introduced onto the core surface of the optical fiber.

The surface of the optical fiber into which the carboxyl group has been introduced as described above is immersed in an aqueous solution containing an amino group-containing antigen or antibody and a condensing agent such as carbodiimide, and left at 4 to 25 ° C. to obtain the antigen. Alternatively, the antibody is bound by an amide bond, and an immunological substance necessary for the measurement of an antigen or an antibody is immobilized on the surface of the optical fiber, and is used for fluorescent immunoassay of a physiologically active substance or the like.

[0020]

The present invention will be described in more detail with reference to the following Examples and Comparative Examples, but the present invention is not limited to these Examples and the like.

Example 1 (1) A resin optical fiber made of polymethyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.) was cut into 3 cm pieces, and both end faces were polished with a polishing film using ethanol as a lubricant. (2) 10 mg of NiSO ₄ was dissolved in 0.5 ml of water, and then 2.5 ml of ethanol was added. The precipitate formed at this time is removed by centrifugation, and the collected supernatant is
-Ethanol solution. Next, 0.1 ml of a Ni-ethanol solution and 50 μl of 50% glutaraldehyde were added to 0.4 ml of a 20 mM KOH solution using ethanol as a solvent, and mixed to obtain a treatment solution.

(3) One surface of the optical fiber of (1) was immersed in the treatment solution of (2) at 50 ° C. for 10 minutes. Next, the substrate was washed with 20 mM hydrochloric acid and then with phosphate buffered saline (PBS) to introduce a formyl group on the surface of the core portion of the optical fiber. In addition, this optical fiber
A carboxyl group was introduced by immersion in a 3% chromium trioxide solution (0.3 N sulfuric acid solvent) at room temperature for 3 hours.

(4) The optical fiber subjected to the treatment of (3) was washed with 0.2N sulfuric acid, distilled water and PBS.
Next, the plate was immersed in a 2 mg / ml human pancreatic amylase solution, 20 mg of water-soluble carbodiimide (CHMC) was added thereto, and the mixture was allowed to stand at 4 ° C. overnight. After the reaction, 0.05% Tween
After washing with n-containing PBS (Tween PBS), a human pancreatic amylase-immobilized sensor was used as a detection unit.

(5) 4 mg of Na ₂ C in 100 μl of water
O ₃ and was dissolved biotin of 10mg. Then, 1.8
The mixture was mixed with 2 ml of a μM chitosan solution, 100 mg of water-soluble carbodiimide was added, and the mixture was reacted at room temperature overnight. Then 0.2 g / ml Na ₂ CO ₃ and 0.1 g / ml N
4 ml of a mixed solution of aCl is added to the mixture, and biotinylated chitosan (b
-C) was precipitated. After collecting the precipitate by centrifugation, the precipitate was washed with 0.1 g / ml of Na ₂ CO ₃ and 0.3 g / ml.
The precipitate was washed twice with a mixture of g / ml of NaCl, suspended in 2 ml of 10 mM phosphate buffer (pH 7), and dialyzed against 500 ml of the same buffer at 4 ° C. overnight.
After the dialysis, the dialysate was collected to obtain a bc suspension.

(6) 100 μg of a goat-derived anti-human Ig antibody was added to the bc suspension of the above (5), and 10 mg of water-soluble carbodiimide was further added, followed by reaction at 4 ° C. for 6 hours. After completion of the reaction, unreacted substances were removed using an anion exchange column to obtain bc to which an anti-human Ig antibody was bound. (7) Avidin 1 mg and triethylamine 0.2 m
was dissolved in 1 ml of ethanol. Next, NK1160 (a kind of cyanine dye) (manufactured by Nippon Kogaku Dye Laboratories) was added and sufficiently dissolved, and 0.3 ml of dicyclohexylcarbodiimide was added, followed by reaction at room temperature overnight. (8) After collecting and collecting the precipitate of avidin by centrifugation, the precipitate was washed twice with ethanol, and after collecting by centrifugation, the ethanol remaining in the precipitate was removed under reduced pressure using an aspirator. This residue was dissolved in 20 mM acetate buffer (pH 6.5),
Avidin modified with K1160 was obtained.

(9) The detection part of (4) was immersed in a human pancreatic amylase antibody solution of each concentration for 20 minutes. Then 0.2
After washing with a 1 M aqueous potassium thiocyanate solution containing 20% Tween 20 (Tween KSCN), anti-human Ig
It was immersed in the solution of bc to which the antibody was bound for 10 minutes. Further, the detection unit of (4) was immersed in the avidin solution modified with NK1160 of (8) for 5 minutes. (10) After further washing with Tween KSCN, the fluorescence was measured by excitation with a helium-neon laser system using the apparatus shown in FIG. 1, and it was possible to measure up to 3 ng / ml.

Example 2 In (1) of Example 1, after the treatment with chromium trioxide, the sample was allowed to stand at room temperature for one week, and the same operation as in Example 1 could be performed with good reproducibility up to 3 ng / ml. .

Example 3 The procedure of Example 1 (3) was repeated, except that a 0.5% potassium permanganate solution was used instead of the chromium trioxide treatment. Was.

Example 4 (1) A formyl group was introduced into the core surface of an optical fiber in the same manner as in (1) to (3) of Example 1. Further, this optical fiber is immersed in 8% hydrogen peroxide solution, and
Left for hours. (2) Next, the fiber was washed with water and PBS to obtain an optical fiber into which a carboxyl group was introduced. (3) Hereinafter, the human pancreatic amylase antibody was measured in the same manner as in (4) to (10) of Example 1, and the result was 4 ng / ml.
Could be measured up to

Example 5 (1) A formyl group was introduced into the surface of the core of an optical fiber in the same manner as in (1) to (3) of Example 1. Further, this optical fiber was immersed in a 1 M β-alanine solution using a 0.1% aqueous solution of sodium carbonate as a solvent, and left at room temperature for 6 hours. (2) After 6 hours, the fiber is taken out from the β-alanine solution and added to a 2% aqueous sodium borohydride solution at room temperature for 3 hours.
Dipped for 0 minutes. The fiber was sufficiently washed with water and PBS to obtain an optical fiber into which a carboxyl group had been introduced. (3) Hereinafter, the human pancreatic amylase antibody was measured in the same manner as in (4) to (10) of Example 1, and the result was 2 ng / ml.
It was measured until.

Example 6 The procedure of Example 5 (1) was repeated except that glutamic acid was used instead of β-alanine to obtain 2 ng / ml.
It was able to measure well up to reproducibility.

Example 7 The procedure of Example 5 (1) was repeated, except that aspartic acid was used instead of β-alanine, to obtain 2 ng / m 2.
1 could be measured.

Example 8 In Example 1 (1), 4-β-alanine was used instead of β-alanine.
2 ng / m 2 except that aminobutyric acid was used.
1 could be measured.

Comparative Example 1 In Example 1, (3), the chromium triacid value was not treated.
After standing at room temperature for one week, the measurement was performed using a detection unit prepared by immersing an optical fiber having a formyl group introduced into a human-derived pancreatic amylase solution, and only up to 2 mg / ml was measured, and the reproducibility was low.

[0035]

The carboxyl group introduced on the core surface of the optical fiber obtained by the present invention is stable even under an atmosphere such as redox or high temperature, and can withstand long-term storage. In addition, since the optical fiber chip does not have a formyl group, the optical fiber chip does not have to worry about white turbidity even at the time of manufacture, and can maintain the light transmittance of the optical fiber at 100% at the time of measurement.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a fluorescence immunoassay apparatus using a laser.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Optical fiber 2 Laser 3 Guide rail for optical axis alignment 4 Detector 5 Filter 6 Fluorescence detector 7 Half mirror

────────────────────────────────────────────────── ─── Continuation of the front page (56) References International Publication 90/13029 (WO, A1) Chemical Engineering, Vol. 35, No. 11, (1990), pp. 26-30 (58) Fields investigated ( Int.Cl. ⁷ , DB name) G02B 6/00-6/54 G01N 33/533 G01N 33/543

Claims (4)

(57) [Claims] 1. A method for producing an optical fiber, comprising oxidizing a formyl group present on the core surface of a resin optical fiber with an oxidizing agent to introduce a carboxyl group onto the core surface of the resin optical fiber. 2. The method according to claim 1, wherein the oxidizing agent used for the oxidation is chromium trioxide, potassium permanganate or hydrogen peroxide. 3. A resin optical fiber having a formyl group on the core surface is reacted with a compound having an amino group and a carboxyl group to introduce a carboxyl group on the core surface of the resin optical fiber. Optical fiber manufacturing method. 4. A compound having an amino group and a carboxyl group, wherein β-alanine, glutamic acid, aspartic acid,
The method according to claim 3, which is a compound selected from the group consisting of and 4-aminobutyric acid.