JPH0763393B2 - Flow injection analysis method and flow injection analysis device - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a flow injection analysis method and a flow injection analysis device.

[0002]

2. Description of the Related Art Conventionally, as an example of flow injection analysis, there is one using a flow injection device equipped with a reaction section in which glass beads or chitosan beads on which a biocatalyst is immobilized are packed in a tubular column. This device continuously sends a substrate to a tubular column using a carrier fluid to cause a catalytic reaction in the column, and the reaction product, that is, a reaction product or a reaction consumption product is measured by a detection section provided separately. It is a system called. However, in this device, beads are usually packed in a tubular column having a small diameter of about 1 to 2 mm, so that the operation of immobilizing the biocatalyst is difficult and extremely complicated.
Further, in this apparatus, once air bubbles enter the column, it is difficult to remove them, and therefore the column must always be kept in a wet state, which makes conditioning difficult. Further, when the inside of the column was always kept in a wet state, it was apt to be affected by foreign matters in water and the activity of the catalyst was apt to decrease, so that it could not be stored for a long period of time. Furthermore, impurities such as proteins and bacteria in the substrate during the measurement tend to cause clogging, resulting in a decrease in catalytic function.

Further, as an analyzer utilizing a functional material such as a catalyst, an enzyme-immobilized membrane is mounted on a sensitive membrane of an electrode,
There is an enzyme sensor in which this is covered with a cellulose membrane and fixed with an O-ring. This enzyme sensor is a combination of an enzyme-immobilized membrane that acts as a molecular identification element and an electrode that acts as a transducer. By bringing the substrate into contact with the enzyme-immobilized membrane of the enzyme sensor, the enzyme-immobilized membrane is The system is such that the substrate and the enzyme are reacted with each other and the reaction product, that is, the reaction product or the reaction consumption product is measured by the electrode. In this enzyme sensor, since the above-mentioned immobilized enzyme membrane is in direct contact with the surface of the electrode, it is susceptible to deterioration due to oxidation, which is unstable and has a short service life. The mounting and demounting on the surface was complicated. Further, in order to obtain a stable output response with this enzyme sensor, for example, in the case of glucose measurement, the surface of the membrane is covered with ascorbic acid or the like so that an inhibitor such as ascorbic acid that causes a potential shift does not reach the electrode surface. It has to be made into a dense structure that allows glucose to pass through, but it is technically difficult to produce such a membrane structure, and mass production is difficult.

Further, although different fields are used, a sheet holding a catalyst is used, and the sheet is arranged in a spiral or multi-stage in the reactor, and the substrate is passed through the reactor so that the substrate and the catalyst are separated. A bioreactor that can be used repeatedly has been proposed. In the case of this bioreactor, since a sheet-shaped carrier for immobilization is used and the catalyst is immobilized thereon, there is an advantage that a large amount of catalyst can be immobilized on the sheet. However, this bioreactor is suitable for mass production of useful substances because it is necessary to pass the substrate through the entire reactor to bring the substrate into contact with the catalyst. It was not suitable for measurement using a very small amount of sample. In addition, as with the enzyme sensor, continuous measurement is not possible. Therefore, when measuring a large number of samples over multiple times, it is necessary to use a large amount of substrate for that number of times, which is very uneconomical. Is.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances and is capable of performing continuous measurement and highly efficient measurement operation, as well as catalysts and the like. An object of the present invention is to provide a flow injection analysis method and a flow injection analysis device capable of contributing to a reaction in a state where the functional material does not deteriorate in function.

[0006]

In order to achieve the above object, in the invention according to claim 1, "a sheet having a continuous porous structure in which a functional material such as a catalyst is held, Flow-in analysis, characterized in that the reaction product is detected by bringing the substrate and the functional material into contact with each other by causing them to be forcedly sent and moving in the longitudinal direction in the same sheet catalyst. Method"
Was the gist. Further, in the invention according to claim 2, "a reactor part, a means for transporting a carrier fluid for forcibly feeding the substrate into the reactor part, an injection part for injecting the substrate into the carrier fluid, and a reactor part" In the flow injection analyzer, which comprises a detection unit for detecting the reaction product reacted in, the reactor unit includes a sheet having a continuous porous structure in which a functional material such as a catalyst is held, and this sheet The flow injection analyzer is characterized in that the injection part is arranged on one end side in the longitudinal direction and the detection part is arranged on the other end side. Further, in the invention according to claim 3,
The gist is "a flow injection analyzer characterized in that the sheet is a non-woven fabric in which a catalyst is entrapped and fixed in a silk fibroin layer formed on the surface of constituent fibers".

The flow injection analysis method and the flow injection analysis apparatus of the present invention will be described in detail below. First, the flow injection analysis method according to claim 1 will be described. The analysis method of the present invention uses a sheet having a continuous porous structure in which a functional material such as a catalyst is retained, forcibly feeding the substrate into this sheet, and moving the sheet in the longitudinal direction to obtain the substrate. It is characterized in that it reacts with a functional material by contacting it.

The sheet uses a fiber sheet having a continuous porous structure such as non-woven fabric, felt, woven fabric, knitted fabric or a composite thereof, or a synthetic resin sheet having a continuous porous structure as a carrier, on which a functional material such as a catalyst is added. It is carried. As the functional material, a metal catalyst such as platinum, a substance having a catalytic function such as an enzyme, a microbial cell, an organelle, a biocatalyst such as a tissue piece of a plant, and a reagent such as a color former can be used. In particular, since the biocatalyst has high selectivity and high sensitivity, it enables quantitative analysis of a trace amount of a substrate from a mixture containing a plurality of substances.
In addition, microbial cells having multiple catalysts (enzymes) inside,
When a biocatalyst such as organelle or plant tissue piece is used, there is an advantage that multi-stage or plural kinds of reactions can be carried out at once by one sheet. It should be noted that the sheet may carry a plurality of types of functional materials, and in this case, a multi-step or a plurality of types of reactions can be caused in the sheet as in the case of microbial cells and organelles. As a method of fixing the functional material to the sheet, there are a method of using a binder and a method of kneading a functional material such as a catalyst into a constituent (for example, fiber) of the sheet. Among them, as a method for immobilizing biocatalyst such as enzyme, covalent bond to carrier, ionic bond, physical adsorption, carrier binding method for immobilizing catalyst itself by biochemical affinity, silk fibroin with network structure, etc. An immobilization method such as an encapsulation method in which the catalyst is confined and immobilized in the lattice of the molecular gel or in the resin film can be applied. In particular, silk fibroin is desirable as a means for immobilizing a biocatalyst because it has a property that it does not allow large molecules such as proteins to pass but small molecules such as substrates. The substrate is forcibly fed into the sheet thus configured.

When the substrate is forcibly sent to the sheet, a buffer solution such as a phosphate buffer solution or a borate buffer solution and a carrier fluid such as an inert gas are sent by a pump or the like from one end side in the longitudinal direction of the sheet, and this sheet is sent. The carrier fluid that has passed through the inside is made to flow out to the other end side in the longitudinal direction of the sheet, so that a flow from one end to the other end in the longitudinal direction of the sheet is created.

The flow velocity of the carrier fluid is not particularly limited, but there is a relation between the flow velocity of the carrier fluid, the volume of the sheet and the space velocity, that is, space velocity = carrier flow velocity / sheet volume. , Sheet size, reaction rate of functional materials such as catalysts, peak intensity of substrate response curve, etc. must be taken into consideration. If the carrier flow rate is 0.5 ml / min, the width of the sheet is 0.3 to 1.0 cm, the length is 2 to 10 cm, and the thickness is 0.
It is desirable to be in the range of 1 to 1.5 mm. This is because the peak intensity of the substrate response curve is high, the response speed is fast, and the space velocity condition is 30 to 3000 h ^-1 , preferably 200 to 400 h ^-1 .

Substrate is then injected into this carrier fluid flow. Examples of the substrate and the functional material that reacts with the substrate include glucose-glucose oxidase, ethanol-alcohol oxidase, uric acid-uricase, L-amino acid-L amino acid oxidase, L-asparagine-asparaginase, urea-urease. , Cholesterol-cholesterol esterase, triglyceride-lipase, glucose-Pseudomonas fluorescens, ammonia-digesting bacteria, BOD-tricosporone cataneum (Tr
ichosporon cutaneum) and the like. Syringe the substrate into the carrier fluid flow described above,
It is injected by an injector such as a loop injector or a valve injector, and the substrate is forcibly moved together with the carrier fluid from one end side to the other end side in the longitudinal direction of the sheet. In the process of this transfer, the substrate and the functional material come into contact with each other, and the reaction occurs there. In this way, since the substrate is moved in the longitudinal direction in the sheet having a porous structure, the probability of contact with the functional material is increased, and the reaction efficiency is dramatically improved. It is also possible to mix a predetermined amount of the substrate and the carrier fluid in advance and directly feed this to the sheet with an injector or the like.

Next, the reaction product generated on the sheet, that is, the reaction product or the reaction consumption product is detected. The detection method is not particularly limited, and for example, luminol and red blood salt are added to hydrogen peroxide generated by catalytic reaction with glucose when glucose oxidase is used as a catalyst to cause luminescence, and this luminescence amount is measured by a photoelectric counter. There is a method of measuring, a method of measuring hydrogen peroxide produced by a catalytic reaction with cholesterol when cholesterol esterase is used as a catalyst, and a method of measuring dissolved oxygen consumed by this catalytic reaction with a hydrogen peroxide electrode or an oxygen electrode. .

Next, a flow injection analyzer according to claim 2 will be described. The analyzer of the present invention is
It comprises an injection part, a means for transporting a carrier fluid, a reactor part and a detection part. The means for transporting the carrier fluid is such that a buffer fluid such as a phosphate buffer solution or a borate buffer solution or a carrier fluid such as an inert gas is forcibly sent to the reactor section to create a flow by the carrier fluid, and is usually a pump. Is used. The injection part has a function of injecting the substrate into the flow, and specifically, an injector such as a syringe, a loop injector, a valve injector or the like is used. In addition, the carrier fluid and the substrate are mixed in advance,
It is also possible to forcibly feed this into the reactor section by an injector, and to create a flow by the carrier fluid and forcibly feed the substrate at the same time.

The reactor section is provided with a sheet having a continuous porous structure in which a functional material such as a catalyst is held, and the injection section is arranged at one end side in the longitudinal direction of the sheet, and On the other end side in the longitudinal direction, a detection unit for detecting the reaction product is arranged. Then, means for transporting the carrier fluid and the carrier fluid and the substrate sent by the injection unit flow in from one end side of the sheet, and the carrier fluid and the substrate move to the other end side in the longitudinal direction of the sheet. It has become.
During the process in which the substrate moves in the longitudinal direction inside the sheet, the substrate and the functional material in the sheet come into contact with each other so that a reaction takes place there.

As a concrete example of this reactor part, FIG. 1 has an inflow part 2 and an outflow part 3, and a seat 5 is housed between the inflow part 2 and the outflow part 3 in the longitudinal direction. A plastic cell 1 provided with a portion 4 is used, a transport means 6 such as a pump and an injection portion 7 are connected to the inflow portion 2 of the cell 1, and a detection portion 8 is connected to the outflow portion 3 to store the cell 1 in the storage portion 4 It is shown that the carrier fluid and the substrate are moved in the longitudinal direction of the sheet 5 by containing the sheet 5 therein. According to this reactor section,
The carrier fluid and the substrate flow into the cell 1 through the inflow portion 2 of the plastic cell 1, and the carrier fluid and the substrate move in the sheet 5 of the storage portion 4 provided in the cell 1 in the longitudinal direction, A reaction occurs when the substrate and the functional material come into contact with each other, and the reaction product by this reaction is also the cell 1
It is designed to flow out to the detection section through the outflow section 3 of the.

The sheet of the reactor section has already been described in the description of the flow injection analysis method according to the first aspect of the present invention, so a specific example will be described here. The sheet is mainly composed of hydrophilic fibers such as rayon, using a nonwoven fabric three-dimensionally entangled with these fibers by hydroentanglement, to form a thin gel layer of silk fibroin on the surface of the constituent fibers of this nonwoven fabric, It is preferable that the silk fibroin layer has a catalyst entrapped and fixed. This is because the silk fibroin gel layer has a heterogeneous structure in which the crystal structure region is concentrated on the surface and there are few such regions inside because of the large catalyst molecules such as enzymes. This is because substrates and reactants, which are low molecular weight compounds, can freely enter and exit. In addition, since the degree of freedom of the catalyst such as the enzyme is large in the gel layer, it is in a state where it is easy to react. Therefore, the activity of the catalyst in the layer is high, and it is easy to contact the specific chemical substance with the catalyst. can do. Furthermore, since the silk fibroin gel layer is formed on the surface of the constituent fibers of the non-woven fabric composed mainly of hydrophilic fibers, the affinity between the fibers and the gel layer is high, so that the gel layer does not easily fall off the fiber surface. Become. The above-mentioned sheet is obtained by impregnating a hydro-entangled nonwoven fabric mainly composed of hydrophilic fibers with a silk fibroin aqueous solution containing a catalyst to adhere silk fibroin to the surface of the constituent fibers of the nonwoven fabric, drying it, and then insolubilizing it with alcohol. Can be obtained in a process.

The detection unit detects the reaction product or reaction consumption product generated on the sheet, and the detection means is not particularly limited, and a known one may be selected and used.

[0018]

【Example】

Example 1 A fibrous web made of domesticated silkworm refined silk was entangled by a hydroentangling method to obtain a nonwoven fabric having a thickness of 0.5 mm and an apparent density of 0.18 g / cm ³ . On the other hand, a 3% silk fibroin aqueous solution was prepared, and glucose oxidase (E
C. 1.1.3.4 Aspergillus nig
er, 241 units / mg) was added to the silk fibroin at a ratio of 3% to prepare a solution. The above non-woven fabric is impregnated with this solution, and it is used at 20 ° C. and 50% relative temperature for
After air-drying for an hour, it was immersed in an 80% aqueous methanol solution for 30 seconds to insolubilize the silk fibroin. When the obtained sheet was observed with a microscope, the surface of the silk fibers was uniformly covered with a thin gel layer of silk fibroin, and the voids between the fibers were not blocked, leaving continuous voids.
Further, even when immersed in a phosphate buffer of pH 7 at room temperature for 1 month, the enzyme was hardly eluted and it was stable.

The above sheet was cut into a rectangle having a width of 5 mm and a length of 50 mm, and a thickness of 0.5 was placed between two acrylic plates.
A mm-thick silicone sheet was filled as a spacer to prepare a flat plate-shaped reactor part. A pump for creating a flow by the carrier fluid by forcibly feeding the carrier fluid into the sheet is arranged at one end side of the reactor in the longitudinal direction of the sheet, and a loop injector for injecting the substrate into the flow is provided. A biosensor was prepared by installing an oxygen electrode as a detection unit on the other end side.

A phosphate buffer solution having a pH of 7 as a carrier fluid is forcibly fed into the sheet by a double plunger type pump at a rate of 0.5 ml / min, and 10 times as a substrate.
Injecting μl of glucose (glucose) aqueous solution into the flow of carrier fluid with a loop injector, and catalytically reacting with glucose oxidase in the reactor section,
The dissolved oxygen concentration was measured by the oxygen electrode at the outflow portion of the reactor. The measurement of the dissolved oxygen concentration is based on the fact that glucose oxidase consumes oxygen when it oxidizes glucose.

This measured value was obtained as the substrate response curve shown in FIG. 2, and the curve showed a peak type. The glucose concentration is changed in the range of 0.05 to 2.00%, and the measurement is performed.
When the reciprocal of the glucose concentration and the reciprocal of the peak intensity were plotted, a linear calibration curve showing a proportional relationship was obtained as shown in FIG. Therefore, according to the biosensor of Example 1, the concentration of glucose contained in an unknown sample can be measured using this calibration curve.

Comparative Example 1 A disc-shaped cell designed to allow a carrier fluid to flow in a direction perpendicular to the sheet surface was fitted with the disc obtained by punching out the sheet obtained in Example 1 into a disc having a diameter of 22 mm. Then, the reactor part was created. When the dissolved oxygen concentration was measured in the same manner as in Example 1 except that the reactor part was changed to the above reactor part, the substrate responsiveness was extremely low, and the peak intensity was 1 of the peak intensity detected in Example 1.
When the content was 0% or less, the sensitivity was low and the product could not be used.

Comparative Example 2 Commercially available porous chitosan beads (Chitopearl BCW2603 manufactured by Fuji Boseki Co., Ltd.) were used as a carrier for immobilizing an enzyme, immersed in a 1% aqueous solution of glucose oxidase for 1 hour, insolubilized with glutaraldehyde and washed with water. . The beads were filled in a Teflon tube having an inner diameter of 2 mm so that the layer length was 40 mm to prepare a reactor part. The dissolved oxygen concentration was measured in the same manner as in Example 1 except that the reactor part was changed to the above reactor part. As a result, the substrate responsiveness was low, and the peak intensity was about 50% of the peak intensity detected in Example 1. Therefore, sufficient detection sensitivity was not obtained.

Example 2 Ascorbate oxidase (EC.
1.10.3.3, Cucurbita sp. , 31
A sheet was obtained in the same manner as in Example 1 except that 4 units / mg) was used. The addition ratio of ascorbate oxidase to silk fibroin was 1%. Using this sheet, a reactor part was prepared in the same manner as in Example 1, and a biosensor was prepared using this reactor part.

A phosphate buffer solution having a pH of 7 as a carrier fluid was forcibly fed into the sheet at a rate of 0.5 ml / min by a double plunger type pump, and 10 μl of an aqueous solution of L-ascorbic acid (JI) was used as a substrate in this flow.
S special grade reagent) was injected by a loop injector to cause a catalytic reaction with ascorbate oxidase in the reactor part, and the dissolved oxygen concentration was measured by an oxygen electrode at the outflow part of the reactor. The measurement of the dissolved oxygen concentration is based on that ascorbate oxidase consumes oxygen when oxidizing L-ascorbic acid.

The measured values were obtained as a substrate response curve, which showed a peak shape. The L-ascorbic acid concentration was changed in the range of 0.05 to 2.00% for measurement,
When the reciprocal of the L-ascorbic acid concentration and the reciprocal of the peak intensity were plotted, a linear calibration curve showing a proportional relationship was obtained as shown in FIG. Therefore, according to the biosensor of Example 1, it is possible to measure the concentration of L-ascorbic acid contained in an unknown sample using this calibration curve.

The glucose and L-ascorbic acid contained in the commercially available apple juice containing 10% fruit juice were quantified using the biosensors of Examples 1 and 2, and the glucose concentration was 5.4%. The L-ascorbic acid concentration was 0.06%. As a comparison, when a similar sample was measured by high performance liquid chromatography, values of glucose 5.6% and L-ascorbic acid 0.06% were obtained, and the values almost coincide with the values measured by the biosensor of the present invention. Was obtained.

Example 3 A fibrous web made of viscose rayon having a fineness of 1.5 denier and a fiber length of 51 mm was entangled by a hydroentangling method to give a nonwoven fabric having a thickness of 0.5 mm and an apparent density of 0.20 g / cm ³ . . On the other hand, a 3% silk fibroin aqueous solution was prepared, and uricase (EC 1.7.3.
3, Candida sp. , 3.6 units / m
g) was added to silk fibroin at a ratio of 10% to prepare a solution. The solution is impregnated into the above non-woven fabric,
After air-drying at a relative temperature of 50 ° C. and 50 ° C. for 2 hours, the silk fibroin was insolubilized by immersion in an 80% aqueous methanol solution for 30 seconds. When the obtained sheet was observed with a microscope, the surface of the rayon fiber was uniformly covered with a thin gel layer of silk fibroin, and the voids between the fibers were not blocked, leaving continuous voids. Further, even when immersed in a 50 mM borate buffer solution having a pH of 8.5 at room temperature for one month, the enzyme was hardly eluted and it was stable. Using this sheet, a reactor part was prepared in the same manner as in Example 1, and a biosensor was prepared using this reactor part.

50 m of pH 8.5 as carrier fluid
0.5 ml of M borate buffer (containing 1 mM EDTA and a small amount of surfactant) with a double plunger type pump
While forcibly feeding into the sheet at a rate of / min, 20 μl of uric acid aqueous solution as a substrate was injected into the flow by a loop injector to cause a catalytic reaction with uricase in the reactor section, and the dissolved oxygen concentration was adjusted by the oxygen electrode at the reactor outlet section. It was measured. The measurement of the dissolved oxygen concentration is based on the fact that uricase consumes oxygen when oxidizing uric acid.

The measured values were obtained as a substrate response curve, which showed a peak shape. Uric acid concentration of 8 × 10 ^-4 to 40
The measurement was carried out by changing in the range of × 10 ^-4 %, and the reciprocal of the uric acid concentration and the reciprocal of the peak intensity were plotted.
A linear calibration curve showing a proportional relationship was obtained as shown in FIG.
Therefore, according to the biosensor of Example 1, it is possible to measure the concentration of uric acid contained in an unknown sample using this calibration curve.

[0031]

According to the present invention, the above-mentioned structure is provided.
In the flow injection analysis method described, the substrate is forcibly fed into a sheet having a continuous porous structure that holds a functional material such as a catalyst, and is moved in the longitudinal direction within the sheet to obtain the functionality of the substrate. Since it is configured to react by contacting with a material, continuous measurement is possible, a large amount of functional materials can be immobilized, and a highly efficient reaction on a sheet is realized. Therefore, the amount of the substrate and the functional material required for the measurement can be minimized.

Further, in the flow injection analyzer according to the second aspect, the substrate is sent to the sheet having a continuous porous structure in the reactor portion, and the substrate is transferred to the substrate in the process of moving in the longitudinal direction inside the sheet. Since the reaction with the functional material is performed, a highly efficient reaction can be realized. Further, in this device, the reactor section and the detection section are separated, and when the function of the functional material of the reactor section deteriorates, only the reactor section needs to be replaced, and the cost can be reduced accordingly. Moreover, the replacement operation can be performed very easily. In addition, in this device, since the reactor part is provided with a sheet having a porous structure, it is easy to remove air bubbles, and it is not necessary to always keep a wet state, and the carrier fluid can be flowed for several minutes. Can be conditioned. Also, because it can be stored in a dry state,
It is unlikely to be affected by contaminants in water, and the contaminants are less likely to reduce the activity of the functional material, which enables long-term storage. Also, in this device, since the sheet of the reactor part has a porous structure in which large voids are continuous, clogging is less likely to occur due to contaminants such as proteins and microorganisms, and the function of the functional material due to clogging of the sheet It is possible to prevent the deterioration. Further, in this device, a plurality of sheets having different functional materials fixed thereon are arranged in parallel, and a multi-item measurement can be performed by feeding a substrate into these sheets, and a plurality of sheets having different functional materials fixed can be measured. By arranging the sheets in series, it is possible to measure by a multistep reaction.

In the flow injection analyzer according to claim 3, the silk fibroin gel layer formed on the surface of the constituent fibers of the non-woven fabric used as the sheet is separated from the gel layer because the catalyst molecules such as enzymes are large. It has a property that substrates and reactants, which are low-molecular weight compounds, can freely move in and out, while it cannot easily go in and out, and this gel layer has a high degree of freedom of the catalyst, so it is highly efficient. A catalytic reaction can be realized.

[Brief description of drawings]

FIG. 1 is a front view schematically showing a flow injection analyzer according to claim 2.

FIG. 2 is a substrate response curve obtained as a result of the measurement in Example 1.

FIG. 3 is a graph showing the measurement results of Example 1 and Example 2 as a calibration curve.

FIG. 4 is a graph showing the measurement results of Example 3 as a calibration curve.

[Explanation of symbols]

1 ... Plastic cell 2 ... Inflow part 3 ...
Outflow section 4 ... Storage section 5 ... Sheet 6 ... Transportation means 7 ... Injection section 8 ... Detection section

Claims (3)

[Claims] 1. A substrate and a functional material are forcibly fed into a sheet having a continuous porous structure in which a functional material such as a catalyst is held, and the substrate and the functional material are moved in the longitudinal direction. A flow injection analysis method, which comprises contacting and reacting to detect the reaction product. 2. A reactor section, a means for transporting a carrier fluid for forcing a substrate into the reactor section, an injection section for injecting the substrate into the carrier fluid, and detection for detecting a reaction product reacted in the reactor section. In the flow injection analyzer, the reactor section is provided with a sheet having a continuous porous structure in which a functional material such as a catalyst is held, and the injection is provided at one end side in the longitudinal direction of the sheet. A flow injection analysis device, characterized in that a detector is provided on the other end side of the flow injection analyzer. 3. The flow injection analyzer according to claim 2, wherein the sheet is a non-woven fabric in which a catalyst is entrapped and fixed in a silk fibroin layer formed on the surface of the constituent fibers.