JPS6056254A - Selective transmission film for polarographic analysis - Google Patents

Abstract

PURPOSE:To increase mechanical properties of a selective transmission film, by constructing a selective transmission film of polarograph electrode combined with an enzyme film with a porous basic material film perforated with fine holes and a selective transmission film covering the basic material film. CONSTITUTION:A enzyme electrode 1 houses a polarograph electrode 6 and in a bottom opening of a case 2, a laminated layer 10 containing the selective transmission film is fixed. This layer 10 consists of selective transmission film 11, enzyme film 12 and porous film 13 and the selective transmission film 11 is composed of a basic material film 11a and a selective transmission substance 11b. When the specimen liquid is brought into contact with the bottom surface of the laminated layer 10 of enzyme electrode 1 thus constructed, then the special substance subjected to be analyzed found in the liquid specimen, such as alcohol, reacts with alcohol-oxidase as an enzyme in the layer film 10 and the polarograph electrode approaching the laminated layer 10 detects it.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical field to which the invention pertains] When analyzing a specific substance such as glucose using a so-called enzyme electrode, in combination with an enzyme membrane, the enzyme in the enzyme membrane is used to analyze a specific substance such as glucose. The present invention relates to a selectively permeable membrane that selectively allows a reaction product, such as hydrogen peroxide, produced by the reaction to permeate and transmit it to a polarographic electrode.

[Prior art and its problems]

The enzyme electrode mentioned above, especially one that detects hydrogen peroxide produced by an enzyme reaction using a polarographic electrode and analyzes or quantifies a specific substance that reacts with the enzyme, is known as the so-called Clark's black hydrogen oxide enzyme electrode. ,
In recent years, it has been increasingly used in medical analysis as it enables the determination of glucose and cholesterol in the blood, which were previously extremely difficult to analyze and measure. The first core of this unique analysis technology is to immobilize glucose oni-1 sidase, an enzyme that acts only on a specific substance to be analyzed, such as glucose, in the I-element membrane, and to detect a specific substance that can be detected with a polarographic electrode. Reaction products such as hydrogen peroxide and gluconic acid are produced, but even if such detectable reaction products are produced, there are usually many other substances in the sample solution to be analyzed in addition to the specific substance. Since other components are included, it is necessary to prevent these other components from acting on the polarographic electrode and falsifying the measurement results.As a means to remove or eliminate such interfering components on the measurement A porous membrane is interposed between the test solution and the enzyme membrane to prevent high molecular weight substances in the sample solution from reaching the enzyme membrane. Such a porous membrane cannot solve the problem if an interfering component with a molecular weight similar to that of the specific substance to be detected is contained.An example of this type of interfering component is uric acid when analyzing the amount of glucose in blood. In addition, when analyzing the alcohol content of sake, various components that exist in sake and are known as "kogu" may interfere with the analysis of the alcohol content. .

Therefore, as the second core of the technology, a means is needed to prevent such interfering components from reaching the polarographic electrode, or, conversely, to selectively allow only the reaction products to be detected by the polarographic electrode to pass through. , this is the permselective membrane that is the object of the present invention. Conventionally, there are two main methods for obtaining selective permselectivity.One is to immobilize an enzyme in an enzyme membrane and at the same time impart a selective permselectivity function to the immobilized enzyme membrane itself. be. In order to provide such a function, it is possible to use a substance that originally has selective and observable properties as the membrane material (Principle 12); Even if it does not become easier, there are large variations in selective permeation performance, and performance management is not easy despite many efforts.

In addition, as a second means, for example,
As disclosed in Publication No. 1, oxygen is immobilized between two polymer material membranes, and the membrane on the test liquid side of the two membranes is made a porous membrane and a high molecular weight substance is injected into the membrane. With tears in my eyes,
There is a method of making the other membrane on the polarographic electrode side a homogeneous selectively permeable membrane made of a substance that selectively permeates a specific reaction product.

This second method has been put into practical use, and while the enzyme electrode using this homogeneous selectively permeable membrane has the advantage of a high equilibrium speed until accurate measurement results are obtained, it is not suitable for selectively permeable membranes. It is necessary to produce an ultra-thin film of 2 microns or less, which requires a high degree of skill to form the film, and the process of combining it with the immobilized enzyme layer is complicated, making performance management difficult.

[Purpose of the invention]

The present invention solves the above-mentioned problems of the prior art and provides a mechanically stable selectively permeable membrane for polarographic analysis that has a simple manufacturing process and is easy to handle when assembled into an enzyme electrode. The purpose is to

[Key points of the invention]

In order to achieve the above-mentioned objective, in the present invention, a permselective membrane is formed separately from an immobilized enzyme membrane, but instead of a conventional homogeneous permselective membrane, a material having permselectivity for a specific reaction product is used. is deposited on a relatively thick porous substrate membrane to form a selectively permeable membrane. As such a base film, a porous polycarbonate film or a polyester film having micropores is suitable, and 1'% of the film is 5 microns, preferably 7 to 10 microns, and the conventional 2 microns is used. Among the selectively permeable membranes listed below, one with a convenient mechanical strength is selected. Good results can be obtained with a fine pore diameter of about 1 to 12 microns. Depending on the reaction product to be selectively permeated, acetyl cellulose, albumin, or acrylamide gel can be used, and in particular, selective permeability to hydrogen peroxide can be obtained with these materials. Adhesion can be easily achieved by dissolving the permselective substance in a volatile solvent and applying it onto the base film by means such as casting, as described below, and then evaporating the solvent. The formation of a selectively permeable membrane does not require particularly skilled skills, and the selectively permeable membrane that can be obtained has much higher mechanical strength than conventional membranes and is easier to handle when assembled into an enzyme electrode. .

[Embodiments of the invention]

The present invention will be described in detail below with reference to the drawings.

FIG. 1 shows an example of the structure of pole 1, a probe for polarographic analysis in which the selectively permeable membrane according to the present invention is used.
A laminated membrane 10 including a permselective membrane according to the present invention is incorporated into the enzyme 1B and pole 1 as shown in the lower part of the figure, and the test liquid comes into contact with the lower surface of the M-layer membrane in the direction of arrow P. be done.

The enzyme electrode 1 has a case 2 made of a cylindrical insulator such as plastic or glass, and the upper opening of the case in the figure is sealed with a lid 3. A lead 7 is embedded through the gap 8 and its extended columnar portion 8a, and is electrically connected to a polarographic electrode 6 made of platinum or the like attached to the lower end surface of the columnar portion 8a. The above-mentioned laminated film 10 is hermetically attached by the annular holder 4 and osu/glue 5 so as to close the opening 1 at the bottom of the case 20, and the upper surface of the laminated film 100 is is arranged in close proximity to the polarographic electrode 6 of. The electrolytic solution 9 is introduced into the annular space between the inner surface of the case 2 whose upper and lower openings are sealed and the outer surface of the columnar part 3a of the lid 8. As the electrolytic solution 9, a buffer solution is used, which is a well-known solution in which salts such as carbonates are dissolved in a solvent such as water and a buffer is added thereto. In addition to the above-mentioned polarographic electrode 6, the electrodes immersed in the electrolytic solution 9 include a reference electrode 8, whose leads 8a penetrate through each electrode 3 and are led out of the enzyme electrode 1.

When the lower surface of the R4 membrane 10 is brought into contact with the test liquid as described above, a specific substance to be analyzed in the plugging liquid, such as alcohol, reacts with alcohol oxidase as an enzyme contained in the R4 membrane 10. This is detected by the polarographic electrode 6 near the laminated film 0, and an electromotive force is generated between the leads 7.8a, with the reference electrode 8 as the cathode and the polarographic electrode 6 as the anode. It is read by a measuring device (not shown) as a polarogram output signal.

FIG. 2 is a cross-sectional view showing the details of the laminated membrane 10, which includes the permselective membrane 11 according to the present invention, the oxygen membrane 12, and the porous membrane 1.
It has a three-layer structure consisting of 3. The permselective membrane 11 is a base membrane 1 having a large number of micropores 11C as shown in the configuration.
1a and a permselective material 11b deposited thereon. The thickness of these layer films is, for example, 7 to 7 for the selectively permeable film 11.
10 microns, enzyme membrane 12 is 1 micron, porous membrane 13
is 5 microns. This internally porous membrane 13 comes into contact with the test liquid and guides only molecules of specific substances to be analyzed in the test liquid, such as glucose and ethyl alcohol, to the enzyme membrane 12. Other components in the test solution are enzyme membrane 1
A filtering action is performed to prevent the value from reaching 2. For this reason, the porous membrane 18 is also made of a porous glass material such as polycarbonate having a large number of micropores.

Enzyme membrane 12 An enzyme that acts only on the specific substance to be analyzed.
For example, in the case of glucose analysis, glucose oxitase is fixed in plastic or adhesive using various known means to form a film or layer, in which M+
The enzyme reacts with the specific substance that has passed through the porous membrane 13 mentioned above, and a reaction product, such as hydrogen peroxide or acetic acid molecules, which is detected by the polarographic electrode 6, is produced. In addition, as can be easily understood from the above explanation, if the combined test solution contains a substance that has the same molecular weight as the specific substance, such as uric acid, such a substance can be easily removed. However, the selectively permeable membrane 11 according to the present invention prevents the above-mentioned specific reaction products or molecules from reaching the polar side surface of the polarograph 1. only that reaches the polarographic electrode and is detected by it.

Next, an example of preparing the selectively permeable membrane 11 according to the present invention will be described. First, as the base film 11a, a circular plastic film, such as a porous polycarbonate film, having a large number of micropores or bores of appropriate pore size and an outer diameter of about 5 om'm is immersed in a solvent such as acetone. Prepare the material so that the permselective material to be applied later is well absorbed.

The pore diameter of the micropores is preferably from several microns to ten-odd microns, and it is not necessarily necessary to have pores with uniform pore diameters. It's okay. For example, a filter material 8 called "Nuclebore Filter" manufactured by General Electric Co., Ltd. in the United States has fine pores with a pore size suitable for the base membrane. Further, a thin porous Ueno 1 made of an inorganic ceramic material may be used as long as it has micropores of this size. The thickness of the base film should be one that has enough mechanical strength to be easily handled and is as thin as possible.A minimum thickness of 5 microns is required for handling.
Preferably it is between 7 and 10 microns. If the thickness of the base film becomes too large, the equilibration time during analysis becomes longer, which is not practical.

Next is acetone 100 as a solvent! Acetylcellulose as a selectively permselective material 5! Add j and stir thoroughly for about 24 hours using a magnetic stirrer or the like to dissolve or disperse acetylcellulose as uniformly as possible in the solvent. Next, the base film prepared by immersing it in acetone as described above is taken out from the acetone, and O-touble of the above-mentioned acetylcellulose solution is cast on its surface at room temperature. Thereafter, this sample is dried for several minutes in an atmosphere of dry nitrogen gas at 20 degrees Celsius to form a thin film of acetyl cellulose on the base film. As a result, acetylcellulose completely covers one surface of the base film, and
A part of it is filled into the micropores. Note that the acetylcellulose may be applied not only to one side but also to both sides of the base film. Through the above steps, the preparation of the selectively permeable membrane according to the present invention is completed.

FIG. 8 shows the test results of the permselective membrane according to the present invention prepared as described above. The polarogram shown in the figure was obtained when only the selectively permeable membrane 11 was attached instead of the laminated membrane 10 in the electrode arrangement shown in Figure 1 in order to purely test the performance of the selectively permeable membrane. This is the result. In the figure, the horizontal axis represents time in minutes, and the vertical axis represents the output signal value from the polarographic electrode. At time A in the figure, the anti-electrode surface of the selectively permeable membrane was brought into contact with an aqueous solution containing 1.2 p, p, m of hydrogen peroxide to be detected by the polarographic electrode. The polarogram shown by B in the figure shows the reaction of the polarographic electrode to this hydrogen peroxide. Hydrogen peroxide then passes through the selectively permeable membrane prepared as described above, reaches the polarographic electrode, and reaches a predetermined level. It can be seen that an output signal was generated. It is understood from this reaction curve that the equilibrium time is approximately 30 seconds, which is sufficient for practical purposes. Next, at point C, the contact of the hydrogen peroxide solution to the selectively permeable membrane was cut off, so
The signal from the electrode returned to its initial value within 30 seconds of equilibration time, as seen in the polarogram in D. Furthermore, at time E, the selectively permeable membrane was brought into contact with sake containing a considerable amount of components that interfere with the selectively permeable membrane.

As shown in the following polarogram F, no noticeable signal was generated from the electrodes, indicating that the selectively permeable membrane was effectively blocking interfering components in the sake. Although sake naturally contains ethyl alcohol, the above test was conducted without using an oxygen membrane containing alcohol oxidase, so no polarogram for alcohol appears.

The permselective membrane according to the present invention as described above is put to practical use in combination with an oxygen membrane 12 and a porous membrane 18 as shown in FIG. In order to prepare the laminated membrane 10 having such a three-layer structure, first, an enzyme compatible with a specific substance, such as crucose oxidase or alcohol oxidase, and an enzyme compatible with a specific substance, such as crucose oxidase or alcohol oxidase, are added to the surface of the porous membrane 13 such as porous polycarbonate. A mixture of an active protein such as bovine serum albumin and a crosslinking reagent such as geltaraldehyde is applied and dried to form an enzyme layer or membrane. Next, the permselective membrane according to the present invention prepared as described above is placed and lightly pressed against the enzyme layer, so that the enzyme layer acts as an adhesive and a three-layer laminated membrane 11 is formed. The attachment to the enzyme electrode 1 may be the same as in the case of conventional laminated membranes. In addition, when the selectively transparent substance is coated on only one side of the substrate membrane, the coated side is usually arranged on the polarographic electrode side.

This concludes the detailed explanation of the present invention, but the gist of the present invention is not limited to such embodiments,
It goes without saying that the type, shape, and usage of the base membrane and permselective substance should be selected depending on the specific substance to be analyzed, and the means for preparing the permselective membrane should be selected as appropriate within the scope of the present invention.

〔Effect of the invention〕

As explained above, in the present invention, in combination with the r1'f elementary membrane, a selectively permeable membrane is used, which transmits only the reaction product to be detected by the polarographic electrode and blocks substances that may interfere with the analysis. A porous base film having micropores, and a selective permeable membrane 11f adhered to the surface of the base film.
Since it is made of a material having ^ properties, it is possible to provide a selectively permeable membrane that has far superior mechanical strength to conventional selectively permeable membranes and is of uniform quality. Furthermore, according to the present invention, particularly excellent skills are not required when adjusting the permselective membrane itself or when combining it with an enzyme membrane or the like to prepare a laminated membrane, making quality control easier and more efficient. Suitable for mass production. That is, according to the conventional preparation method, an extremely thin film with a thickness of 2 microns or less, preferably about 1 micron, is formed on the flat surface of a liquid such as water, and this is scooped up by an appropriate body. There is no need to rely on the skill of a craftsman to remove the layer after forming a laminated film on top of that. However, the polarograph analyzer using the selectively permeable membrane according to the present invention is developing a wide range of uses for health management such as blood glucose and cholesterol, and for clinical chemistry analysis in medical treatment, and the present invention is applicable to such fields. It has great significance in that it can provide a highly reliable analytical device with consistent performance.

[Brief explanation of the drawing]

FIG. 1 is a sectional view showing the structure of an enzyme electrode in which a selectively permeable membrane according to the present invention is used, and FIG. 2 is a laminated membrane in which a selectively permeable membrane 11, an enzyme membrane 12, and a porous membrane 18 according to the present invention are laminated.
FIG. 8 is a partial sectional view showing the structure of O, and a polarogram showing the test results of the selectivity of the permselective membrane according to the present invention. In the figure, 1: enzyme electrode, 6: polarographic electrode, 1o: laminated membrane, iM permselective membrane, lla: base membrane, 11b = permselective substance, 11c: base membrane, micropores in 11a, 12: Enzyme membrane. 1st cause 2nd cause 3rd figure

Claims (1)

[Claims] l) Selection of combination with an enzyme membrane containing an enzyme that reacts with a specific substance to be analyzed, and selectively transmitting a specific reaction product produced by the enzyme membrane to the polarographic electrode. A permselective membrane for polarographic analysis, characterized in that the membrane is made of a porous base membrane having micropores, and a substance having selective permselectivity for the specific reaction product is adhered to the surface of the membrane. film. 2. In the selectively permeable membrane according to claim 1,
A selectively permeable membrane for polarographic analysis, characterized in that the thickness of the base membrane is 5 microns or more. 3) A selectively permeable membrane for polarographic analysis according to claim 2, wherein the thickness of the base membrane is between 7 and 10 microns. 4) In the selectively permeable membrane according to claim 1,
A permselective membrane for polarographic analysis, characterized in that the base membrane has micropores with a diameter between 1 and 12 microns. 5) In the selectively permeable membrane according to claim 1,
1. A selectively permeable membrane for polarographic analysis, characterized in that acetyl cellulose is adhered to a base membrane as a substance having selective permselectivity to hydrogen peroxide as a specific reaction product. 6) In the selectively permeable membrane according to claim 1,
A selectively permeable membrane for polarographic analysis 0, characterized in that a substance selectively permeable to a specific reaction product is adhered to the polarographic electrode side of the base membrane.