JP2617745B2 - Immobilization method of biocatalyst in small and narrow areas - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Summary] The present invention relates to a method for immobilizing a biocatalyst in a fine and narrow area, and can immobilize the biocatalyst only in a predetermined fine and narrow area. The purpose of the present invention is to provide a method for immobilizing a biocatalyst in a fine and narrow area that can improve utilization efficiency, and to fix a biocatalyst on the surface of a hydrophilic substrate having a back surface coated with a water-repellent film. Forming a concave portion in the region, forming a film of a polymer compound that is hardly dissolved by exposure on the surface of the substrate, and using a photomask having a light-transmitting region in a region other than a predetermined region for fixing the biocatalyst; After exposing the film of the polymer compound, developing, this substrate is immersed in an aqueous solution containing a biocatalyst and an immobilization carrier, then pulled up, and the aqueous solution containing the biocatalyst and the immobilization carrier is only in the concave portion. Choice And fixing the biocatalyst in the recess.

[Industrial Application Field] The present invention relates to a method for immobilizing a biocatalyst in a fine and narrow area.

[Prior art] Enzymes, microorganisms, cells, antigens, antibodies and other substances having biological activity or bioreactivity (hereinafter referred to as biocatalysts)
The so-called biosensor, which utilizes the property of reacting specifically with a specific substance, detects material changes and physical changes that occur at that time, converts it into an electric signal, etc., and detects and quantifies it,
In the fields of clinical medicine, clinical examination, food production plants, agriculture, and the like, they are widely used as measurement devices, diagnostic devices, food analysis devices, plant process control means, water quality management means, and the like. Normally, the biocatalyst portion of the biosensor is a portion that converts the amount of change based on the specific reaction into an electric signal,
So-called integrated with the transducer,
It is arranged in the vicinity thereof and is often used in the form of a film.

There can be various transducers depending on the reaction of the biocatalyst. (A) An oxygen electrode that generates an electric signal in response to a change in oxygen ion concentration, (b) a carbon dioxide gas concentration,
A carbon dioxide electrode that generates an electric signal in response to a change in the concentration of carbonate ions, (c) a hydrogen peroxide electrode that generates an electric signal in response to a change in the concentration of hydrogen peroxide, (d) hydrogen ions in a solution A hydrogen-ion-sensitive field-effect transistor (pH-ISFET) that generates an electric signal in accordance with the concentration is known.

Immobilization of a biocatalyst is widely used as one method for miniaturizing or highly integrating a biosensor because the immobilization stabilizes the biocatalyst and enables high density.

As a conventional immobilization method, a suspension is applied to a biocatalyst carrier, and the suspension is applied or dropped on a transducer portion, for example, an ISFET gate portion.
Thereafter, a general method is to form a biocatalyst immobilized film by advancing a crosslinking reaction.

However, with such a conventional immobilization method, it is difficult to selectively immobilize a biocatalyst such as an enzyme or a microorganism in a fine and narrow place, so that integration is difficult, and Unnecessary portions are coated with the biocatalyst, and there is a disadvantage that the use efficiency of the expensive biocatalyst is poor.

As an improvement of the conventional method, an attempt has been made to apply a lift-off method often used in a process of a semiconductor or the like to immobilization, and is actually applied to an integrated ISFET biosensor or the like.

[Problems to be solved by the present invention] The above-mentioned drawbacks of the conventional method seem to have been partially solved by using the lift-off method for immobilization, but this method still wastes enzymes and microorganisms. Although the improved method is suitable for immobilization on the ISFET sensitive part, for example, the immobilization of the sensitive part of the small oxygen electrode on the permeable film is not suitable from the viewpoint of adhesion. There is a problem of application difficulty.

An object of the present invention is to eliminate the drawbacks of the conventional method and the improved method for immobilization, so that the biocatalyst can be immobilized only in a predetermined fine and narrow area, and the utilization efficiency of the biocatalyst can be improved. To provide a method for immobilizing a biocatalyst in a fine and narrow area that can improve the biocatalyst, in other words, to provide a method for immobilizing a biocatalyst that can be applied to substrates other than ISFET. It is in.

[Means for Solving the Problems] Means taken by the present invention to achieve the above object are:
It has the following configuration.

 1a to 1f are principle views of the manufacturing process of the present invention.

A concave portion 3 is formed in a predetermined region for fixing a biocatalyst on the surface of a hydrophilic substrate 1 such as a glass plate, and the surface of the substrate 1 (see FIG. 1a) whose back surface is covered with a water-repellent film 7 is hardly melted by exposure. Is formed (see FIG. 1b), and the polymer compound film 2 is exposed using a photomask 4 having a light-transmitting region other than a predetermined region where the biocatalyst is fixed. (See Fig.1c)
As shown in the figure, a substrate 1 in which a portion excluding the concave portion 3 is covered with the polymer compound film 2 is manufactured.

After the substrate 1 is immersed in an aqueous solution 5 containing a biocatalyst and an immobilized carrier, the substrate 1 is pulled up, and the aqueous solution 5 containing the biocatalyst and the immobilized carrier is selectively retained only in the concave portion 3 (see FIG. 1e). The biocatalyst is immobilized in the concave portion 3 by performing a treatment corresponding to an immobilized cross-linking agent or the like, for example, irradiation with ultraviolet light such as mercury lamp light, or vapor deposition of a cross-linking agent. 6 (see FIG. 1f).

The concave portion 3 for fixing the biocatalyst of the substrate 1 is formed in advance by etching using a photolithography method or the like. That is, a polymer compound which is solubilized or hardly dissolved by exposure is applied to the substrate 1 to form a film of the polymer compound, and the polymer compound film is exposed and developed using an exposure mask, and etched. Then, the substrate 1 on which the concave portion 3 is formed as shown in FIG. 1a is used.

As the substrate 1, a substrate whose back surface is previously coated with the water-repellent film 7 is used. This film is formed, for example, by applying a negative photoresist on the entire back surface and performing pre-baking.

As a substrate that can be used, a substrate in which at least the concave portion forming region is easily compatible with water is preferable. For example, glass, acrylic, or the like is preferable, but the range of selection is considerably wide.

The polymer compound that forms the film covering the portion excluding the concave portion is, for example, if a water-repellent compound such as a negative photoresist is used, an aqueous solution containing a biocatalyst or an immobilized carrier is used except when the concentration is high, It is completely repelled from the film surface of the coating polymer compound. Therefore, when such a substrate formed by coating the coating polymer compound in a portion other than the concave portion is immersed in an aqueous solution containing a biocatalyst and an immobilization carrier and slowly pulled up, the aqueous solution remains only in the concave portion and remains in the concave portion. Does not remain in the part.

The substrate having the immobilized biocatalyst manufactured in this manner can be used as a biosensor by using it in close proximity to a sensitive part such as an ISFET, a small oxygen electrode, or a carbon dioxide electrode.

As a matter of course, it is necessary to use a substrate, a cross-linking agent, and a coating polymer compound that do not deactivate the biocatalyst to be immobilized.

[Function] The present invention provides a method of immobilizing a biocatalyst on a substrate in a predetermined fine and narrow area, wherein at least the predetermined area for immobilizing the biocatalyst is formed in a substrate having a hydrophilic portion, and further comprising the concave portion. Is coated with a water-repellent polymer compound and immersed in an aqueous solution containing a biocatalyst and an immobilized carrier.
Therefore, since the aqueous solution stays only in the concave portion of the substrate, the aqueous solution is fixed without waste.

Example Hereinafter, a method for immobilizing a biocatalyst according to an example of the present invention will be described with reference to the drawings, in the case where glucose oxidase is used as the biocatalyst.

Example A biocatalyst is immobilized on a substrate as follows.

(1) After applying, for example, a negative photoresist (OMR-83 manufactured by Tokyo Ohka) as a polymer compound to one surface of the glass substrate 1, an area other than the area where the biocatalyst is immobilized is exposed,
The exposed negative photoresist is developed and pre-baked to form a resist mask (not shown) having an opening in a region where the biocatalyst is immobilized. The size of the opening of the resist mask (not shown) is about 0.5 mm × 1 mm. At this time, the same photoresist is applied to the back surface, and post-baking is performed.

(2) The substrate 1 treated as described above is immersed in a solution containing a 50% aqueous hydrofluoric acid solution and a 50% ammonium fluoride aqueous solution in a ratio of 1: 6 (v / v), and the glass substrate 1 Etch. The depth shall be about 0.3-0.5mm. In this way,
The recess 3 shown in FIG. 1a is formed.

(3) In the same manner as in (1) above, a negative photoresist 2 is applied to the substrate 1 (see FIG. 1b), and exposed and developed using a photomask 4 having a light-transmitting region other than the concave portion 3. (See FIG. 1c), the polymer film 2 of the negative photoresist remains only around the recess 3 (see FIG. 1d).

(4) A, B and C solutions prepared as follows
Mix at a ratio of 1: 2: 4 (v / v), and add about 10 mg of glucose oxidase to 700 μl of this mixture.

Solution A is an aqueous solution obtained by dissolving 30 g of acrylamide and 0.8 g of N, N'-methylenebisacrylamide in 100 ml of pure water. Liquid B is an aqueous solution obtained by dissolving 4 mg of riboflavin in 100 ml of pure water. Solution C is an aqueous solution obtained by dissolving 0.23 ml of N, N, N ', N'-tetramethylenediamine in 100 ml of pure water.

(5) A glass substrate manufactured as described in (3), in which a concave portion 3 is formed in a region where the biocatalyst is fixed, and a film 2 such as a negative photoresist is formed around the concave portion 3. 1 is immersed in an aqueous solution 5 containing the biocatalyst and the immobilized carrier prepared in the step shown in (4), and slowly pulled up, and the biocatalyst and the immobilized carrier are contained only in the concave portion 3 of the glass substrate 1. Retain the aqueous solution 5 (No.
1e).

(6) The aqueous solution 5 containing the biocatalyst and the immobilized carrier retained in the concave portion 3 is irradiated with ultraviolet light or the like to immobilize the enzyme and convert it into the immobilized biocatalyst 6 (see FIG. 1f).

A substrate having the enzyme immobilized on the surface is disposed close to the sensitive part of a pH-ISFET or a small oxygen electrode to complete a biosensor.

[Effects of the present invention] As described above, when the method for immobilizing a biocatalyst of the present invention is used, the periphery is covered with a water-repellent film, and the concave portion having a hydrophilic inside is formed in a predetermined fine and narrow area. When the substrate is immersed in an aqueous solution containing a biocatalyst and an immobilizing carrier, the aqueous solution containing the biocatalyst and the carrier stays only in the concave portion, and the biocatalyst is fixed only in a predetermined narrow area. Becomes possible. Therefore, the biosensor can be highly integrated, and further, there is an advantage that the waste of the biocatalyst is reduced and the economic burden is reduced.

In addition, if desired, the biocatalyst-immobilized portion can be set in a region other than the sensitive portion such as a transducer, so that it can be used for manufacturing various biosensors.

[Brief description of the drawings]

1a to 1f are explanatory views of the principle of the manufacturing process of the present invention. In the figure, 1: glass substrate (hydrophilic substrate), 2: polymer compound film (negative photoresist film), 3: concave, 4: photomask,
5: an aqueous solution containing a biocatalyst and an immobilized carrier, 6: an immobilized biocatalyst, 7: a water-repellent membrane.

 ──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Naomi Kojima 1015 Ueodanaka, Nakahara-ku, Kawasaki-shi, Kanagawa Fujitsu Limited (56) References JP-A-62-32351 (JP, A) JP-A-62-144060 (JP, A) JP-A-62-54155 (JP, A)

Claims (3)

(57) [Claims] 1. A concave portion (3) is formed in a predetermined area for fixing a biocatalyst on a surface of a hydrophilic substrate (1) having a back surface covered with a water-repellent film (7), and a concave portion is formed on the surface of the substrate (1). A film (2) of a polymer compound that is hardly dissolved by exposure is formed, and the film of the polymer compound is formed using a photomask (4) having a light-transmitting region in a region other than a predetermined region for fixing a biocatalyst. After exposing 2), the developed substrate (1) is immersed in an aqueous solution (5) containing a biocatalyst and an immobilized carrier, and then lifted up. ) Is selectively retained only in the concave portion (3), and the biocatalyst is immobilized in the concave portion (3). 2. The method for immobilizing a biocatalyst on a fine and narrow area according to claim 1, wherein the polymer compound which is hardly dissolved by the exposure is a negative photoresist. 3. The method for immobilizing a biocatalyst on a fine and narrow area according to claim 1, wherein the immobilization carrier is a polyacrylamide gel.