JPS61155850A - Enzyme sensor - Google Patents

Abstract

PURPOSE:To maintain the specified thickness of an enzyme-immobilized film and to improve the durability of an enzyme sensor by coating a porous or hydrophilic hollow yarn-like film immobilized with enzyme to the sensitive part of a slender superminiature electrode. CONSTITUTION:A source electrode and drain electrode 1 of a semiconductor ion sensor are provided in a catheter 2 and a lead wire 3 is connected thereto. The top end of a sensor 1 is used as the sensitive part (gate region) 4 and is projected from the catheter 2. The part 4 is coated with the porous hollow yarn-like film 6 immobilized with the enzyme and a resin 7 is sealed into the aperture at the top end thereof. The circumference of the electrode 1 is fixed with an electrical insulating layer 8. For example, the porous hollow yarn-like film which is made of PP or PVA and is immobilized with urease, glucose oxidase, etc. is used for the film. The hollow yarn-like film immobilized with the enzyme is used for the enzyme sensor and therefore the durability and performance of the sensor are improved.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an enzyme sensor. The present invention particularly relates to a small, flat, water-resistant enzyme sensor suitable for biochemical and medical applications.

(Prior technology) Conventionally, the sensitive part of the 02 and fi20□ electrodes using the principle of polarography is coated with an enzyme-immobilized membrane by dip coating. Enzyme sensors are known that measure the concentration of a substrate by using a sensor to detect changes in the concentration of a substance that increase or decrease due to the reaction between the substrate and the enzyme in the membrane. (For example, JP-A-53-1
(No. 49397, etc.) (Problems to be Solved by the Invention) However, enzyme sensors in which the sensitive part of the electrode is coated with an enzyme-immobilized membrane by dip coating do not exhibit WI during use.
There has been a problem in that the enzyme concentration in the sample solution cannot be stably measured over a long period of time due to a decrease in summer temperature or drift. The reason for this is that even if an attempt is made to form a uniform film on the sensitive part of an ultra-small electrode by dip coating, it is difficult to prevent strain from occurring at the tip of the needle, causing pinholes and cracks. When a pinhole occurs, substrates and substances to be changed by the enzyme reaction may enter through this portion without passing through the enzyme membrane, which is thought to cause measurement errors.

In addition, many miniaturized enzyme sensors are often used as multiplex sensors in combination with other enzyme sensors or ion sensors, but they are very close! In practice, it is extremely difficult to accurately coat different types of enzyme-immobilized membranes or ion-sensitive membranes on the sensitive parts of the poles.

(Means for Solving the Problems) The present invention was achieved as a result of intensive studies to solve the above-mentioned problems. That is, the enzyme sensor is characterized in that the sensitive part of the elongated chemical substance sensitive sensor is coated with a porous or hydrophilic hollow fiber membrane on which an enzyme is immobilized.

(Function) In the present invention, an enzyme-immobilized membrane is used as a coating method for an enzyme-immobilized membrane, which was thought to have no choice but to be coated with a dip coat to coat the sensitive part of an elongated ultra-small electrode. It is characterized by being coated with a fixed porous or hydrophilic hollow fiber membrane.

The reason why the enzyme concentration in the test liquid can be stably measured over a long period of time due to such characteristics is presumed to be as follows.

■Since the enzyme-immobilized membrane is preformed into a hollow fiber shape, it is possible to obtain a membrane with a constant thickness and without pinholes or cracks.

■The sensitive part of the elongated electrode is inserted and fixed into the hollow part of the hollow fiber, so there is no peeling of the membrane.

(Example) Next, an example of the enzyme sensor of the present invention will be described with reference to the drawings. Figures 1 and 2 are examples of electrodes, for example, Special Publication No. 55-1.
3544 is a cross-sectional view of an enzyme sensor using an ion sensor (hereinafter referred to as I81.T +!-) that utilizes the electric field effect of a semiconductor, and shows the source electrode and drain l! electrode of an elongated ISI"ETI. After connecting lead wire 3 to
A sensing portion 4 (gate region) accommodated in the catheter 2 and provided at the tip of the 18FET is fixed within the catheter by an electrically insulating resin 8 sealed within the catheter so as to protrude from the tip of the catheter. The tip sensitive portion 4 of this I 8 FET is coated with a hollow fiber membrane 6 on which an enzyme is immobilized. Further, the opening at the tip of the hollow fiber membrane is sealed with resin 7. FIG. 2 shows an example of an enzyme sensor in which an 18FET 1 is placed inside an opening made in the side wall of a catheter 2, and a hollow fiber membrane 6 on which an enzyme is immobilized is coated on the catheter so as to close the opening. .

The enzyme-immobilized hollow membrane coated on the catheter 2 must be a hydrophilic or hydrophobic porous membrane in order for the substrate or product to reach the sensor surface. Here, the hydrophilic membrane refers to a membrane whose weight increases by 30% or more when it is immersed in water at 37°C. 30% weight increase
If it is lower, the permeability of water and the substrate will decrease, resulting in a decrease in sensitivity and response speed. Examples of such hydrophilic hollow fiber membranes include cellulose, cellulose derivatives of carboxymethylcellulose, polyamino acids, gelatin, hydroxyethyl methacrylate, and crosslinked polyvinyl alcohol. Further, a hydrophobic porous membrane is a membrane having continuous pores in its cross section, and has a porosity of 30% or more.

The porosity can be determined from the specific gravity of the substance constituting the hollow fibers and the specific gravity of the hollow fibers. In addition, the pore size is 0.035μ to the maximum probability in the pore size distribution curve.
Preferably, the thickness is 15μ. Examples of such hydrophobic porous hollow fiber membranes include hollow fibers made of polypropylene, polyacrylonitrile triacetylcellulose, and polysulfone.

As the method for immobilizing the enzyme on the hollow fiber membrane, any of the covalent bonding method, crosslinking method, entrapment method, etc. may be used.

Commercially available hollow fiber membranes can be used as the hollow fiber membranes mentioned above, but when hollow fibers of a special diameter are required or when a small amount of hollow fibers is to be produced, a wire-like core with the same inner diameter as the required one is used. After coating with the polymer, the required polymer can be obtained by grinding or dissolving the core. The diameter of the hollow fiber is determined by the diameter of the electric machine, but those with an inner diameter of 1 mg or less are usually used. In addition, if the wall thickness of the hollow membrane is too large, the response will be slow, and if it is too small, the mechanical strength will decrease, making it difficult to fabricate the sensor, so it is usually from 200μ to 0μ.
are used.

In order to immobilize the enzyme on the hollow fiber membrane, either the enzyme and the polymer, if necessary, a crosslinking agent such as glutaraldehyde, are added and molded into a hollow fiber shape, or
Alternatively, the enzyme may be immobilized after the hollow fiber membrane is prepared or after the hollow fiber membrane is coated on the sensitive part of the electrode. An example of the former is to dissolve cellulose acetate, # element, and glutaraldehyde in methylene chloride, coat it on a wire, mold it into a hollow fiber shape, and then remove it from the wire.
ζmari can be obtained. An example of it is obtained by activating a cellulose hollow fiber for an artificial kidney with a cyanogen bromide solution of PN 11-pH 12 and immersing it in an enzyme solution.

The hollow fiber membrane with the enzyme immobilized thus obtained is
As shown in FIG. 1, the tip 7 of the hollow fiber membrane 6 is sealed, and the 18F
The sensitive part 4 of the ET is coated. The purpose of sealing the tips of the hollow fiber membranes is to prevent the eK of the sensor from being impaired due to the substrate and enzyme reaction products diffusing from the tips and reaching the sensitive part of the electrode. Therefore, the opening at the tip of the hollow membrane is sealed. 3 and 4 are examples of multiple sensors using the sensor of the present invention. I81 described in Figure 5 No. 13544! 'ET has the same structure. This sensor has a common drain and three gates (□□□
Heading areas 4(a), 4(b), and 4(cle) are arranged at appropriate intervals in the vertical direction. channel stopper)
and separate each gate.

Although the spacing between these gates can be made as small as desired,
If it is too small, it will be difficult to bring the boundary of the sensitive film between the gates, so this distance should be 0.5 to 3 m+.
is preferred. [8] is arranged at the other end of the 18PET to connect a lead wire for taking out the output. In Fig. 5, the drain electrode 10 common to each 181'ET, the substrate electrode Mll and each 18FET (7)
7-7.11j 12 (81,12(bl, tz(c
Five electrodes of 1 are provided. The multiple sensor shown in FIG. 5 has a length of 12 m+, a width of 0.5 MIn% and a thickness of 150 microns. The smaller the size of the sensor, the better from the viewpoint of use, but if it is too small, the element may break during processing, so it is usually 5 to 20 rto in length and 0.3 to 1 in width.
0 rrtm and a thickness of 100 to 3OOμ are preferred.

Although the above I8FET was fabricated on a silicon wafer, it is also possible to fabricate it on an insulating substrate such as sapphire.

This sensor is embedded in the catheter 2 by bonding the lead wire 3 to the electrode and fixing it to the support 5, leaving the gate part as shown in FIG. is filled with insulating resin 8,
Make sure that the bonding part does not short-circuit due to the measurement liquid.

As shown in Fig. 3 (bl), the sensor processed in this way is covered with a hollow fiber membrane 6 (C) on which an enzyme is immobilized on the gate part (0) at the root of the group. Method for covering the hollow fiber membrane. is the same as the single sensor shown in Fig. 1, but the opening at the tip of the hollow fiber membrane covers another hollow fiber membrane, so there is no need to seal it.Next, unlike the hollow fiber membrane 6 (C) The enzyme-immobilized hollow fiber membrane 6(b) is coated on the second gate portion 4(b) as shown in FIG. 3(C).The outer diameter of each hollow fiber membrane may be different, but the same It is preferable to use the outer diameter because the shape becomes smooth.Next, by sealing the tip opening of the hollow membrane with adhesive 6 as described above, a multiple enzyme sensor capable of measuring three types of enzyme concentrations can be obtained.

Furthermore, in addition to 18NET, a polarographic 02 sensor or H2O2 sensor may be fabricated on the silicon wafer 1 and coated with an enzyme immobilization hollow system.

The multisensor fabricated in this manner is elongated and has excellent water resistance, so it is suitable for monitoring blood or body fluids by inserting it into the bloodstream, catheter, or tissue using an indwelling needle.

Furthermore, a plurality of enzyme sensors of the present invention can be arranged side by side. Figure 4 is an example of a horizontal multiple sensor.
Note: 2° They are formed side by side on a single silicon wafer. At this time, the bonding parts are arranged as one piece, but the gate sensitive parts are manufactured separately. By covering each of these 18FETs with hollow fiber membranes 6(a), 6(b), and 6(C), a multiple sensor can be made. This sensor is primarily suitable as a flow-through cell type sensor,
Furthermore, by bringing the gate portion of this sensor into direct contact with the measuring liquid, it is possible to measure the concentration of a chemical substance in a nominal amount of a sample.

Example 1 A porous polypropylene hollow fiber membrane (Mitsubishi Rayon KPF360A) with an inner diameter of 300μ and an outer diameter of 360μ was immersed in 1% urease phosphate buffer H (pH 6,86) for 30 minutes to absorb urease, and then washed with water. Dry. The opening at the tip of this hollow fiber membrane was sealed with epoxy resin, and the gate area of the l8NET pH sensor was coated as shown in FIG. C sensor o, oIM phosphor a! FIG. 6 shows the source potential when immersed in a urea solution dissolved in a buffer solution. As shown in Figure 6, urea # degree 10-2 to 10-4y/rnll
shows a good correlation. Moreover, the 50% response time of the sensor was 10 seconds or less in this range, indicating a quick response.

Example 2 8 kG of polyvinyl alcohol with an average degree of strength of 1700,
4 kg of polyethylene glycol with a molecular weight of 311,000, 160 g of porium, and 30 g of vinegar were dissolved in 506 mm hot water. This bath liquid is poured into a ring nozzle using caustic soda 80 Q/
1 and discharged into a coagulation bath containing 230 g/l of mirabilite to obtain a hollow fiber membrane. Then glutaraldehyde 0.5y/l, hydrochloric acid 3
Crosslinking treatment was performed at 50° C. for 2 hours in a bath of Q/l, and then heat treated in hot water at 100° C. for 1 hour, followed by washing with water. The polyvinyl alcohol hollow fiber membrane thus obtained had an inner diameter of 700 μm and an outer diameter of 1,000 μm, and the membrane wall had a homogeneous porous structure. The mode pore size determined from the pore size distribution curve using a mercury porosimeter is 0.26μ, and the porosity determined from the apparent specific gravity of the hollow fiber membrane is 57%.
Met. In addition, the permeability of bovine serum albumin is 100%.
The transmittance of bovine gamma globulin G was 97%.

This polyvinyl alcohol hollow fiber membrane was immersed in a bath solution consisting of 5% aminoacetaldehyde dimethyl acetal and 10% sulfuric acid at 80°C for 5 hours, washed with running water for 5 hours, and then washed with 1% sulfuric acid at 15°C. Glucose oxidase was immersed in a phosphate buffer solution for 1 hour, and then immersed in a phosphate buffer solution for 1 hour.
I washed it in the evening.

This hollow fiber membrane is used as a Clark-type oxygen sensor for venous blood measurement (
Manufactured by Kontron (In5travascular PO
2monitor636)) diameter 0.65m PO2
A glucose sensor was produced by coating the sensitive part. 3 This sensor showed good correlation between glucose concentrations of 0 to 10011Kildi.

Example 3 A multiplex 18FflT having three gates shown in Fig. 5 at 1.2 inch intervals was fabricated, and this element was attached to a nylon 11 catheter (inner diameter 0.6 m+, outer diameter 1.05 mm) as a support. It was inserted as shown in Figure 3 (al) together with a stainless steel wire with a diameter of 0.3 wm and fixed with epoxy resin.The two gate parts 4 of this sensor (a).

4(b) was coated with a hollow fiber membrane on which urease and glucose oxidase had been immobilized, prepared by the method of Example 2, to produce a sensor shown in FIG. 3 (C1). When the difference in source potential was measured by immersing it in showed that.

(Effects) As described above, the enzyme sensor of the present invention is extremely useful in practice since single and multiple sensors with good durability and uniform performance can be obtained at a good yield.

[Brief explanation of drawings]

Figures 1 and 2 are cross-sectional views of the enzyme sensor of the present invention, Figure 3 is a cross-sectional view of the multiplex sensor at each step to explain the manufacturing method of the multiplex enzyme sensor, and Figure 6 is a cross-sectional view of the enzyme sensor of the present invention. 3 is a graph showing the relationship between concentration and source potential. 1...18FET 2...Catheter 3
... Lead wire 4 ... Sensing part 6 ... Enzyme-immobilized hollow fiber membrane 7 ... Resin 8 ... Electrical insulating resin

Claims (1)

[Claims] An enzyme sensor characterized in that a sensitive part of an elongated chemical substance sensitive sensor is coated with a porous or hydrophilic hollow fiber membrane on which an enzyme is immobilized.