JPH0511576B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a membrane for enzyme immobilization. More specifically, the present invention relates to an enzyme immobilization membrane used in a field effect transistor urea sensor used for rapid measurement of urea concentration in body fluids. [Prior Art] In the medical field, measuring the urea concentration in body fluids is essential for understanding kidney function. A typical example of a conventionally used method for quantifying urea is an enzyme complex method. This method uses urease enzyme to decompose urea, and the generated ammonia is reacted with nicotinic acid adenine dinucleotide (NADH) and α-ketoglutaric acid in the presence of glutamate dehydrogenase (GLDH) to produce nicotinamide adenine dinucleotide. In this method, it is decomposed into dinucleotide (NAD), glutamic acid, and hydrogen peroxide, and the amount of NADH consumed at this time is measured by a colorimetric method. (NH ₂ ) ₂ CO + H ₂ O Urease −−−−−−→ 2NH ₃ +
CO ₂ NH ₃ + H ₂ O → NH ₄ ⁺ + OH ^- NADH + NH ₄ ⁺ α-ketoglutaric acid GLDH −−−−→ NAD + Glutamic acid + H ₂ O _2This method requires deproteinization of the specimen, and colored specimens are It has drawbacks such as the fact that it cannot be used as is, the pretreatment is time-consuming, the operation is complicated, and the cost is high because the enzyme can only be used once. Other methods have been developed, such as a urea sensor that combines an immobilized urease enzyme membrane with an ammonium ion electrode or an ammonia gas electrode, but in this case, it is difficult to detect coexisting ions or inhibitory substances such as volatile amines. It has the disadvantage of being affected. Additionally, the sensor is large in shape and has weak material strength, so it has the disadvantage that it cannot be used, for example, as a sensor inserted into the body. Another method is to cover the silicon nitride (Si ₃ N ₄ ) surface of a hydrogen ion-sensitive field effect transistor (ISFET) with a polymer film and immobilize urease enzyme on it to create a urea sensor. You can also see it. When the polymer membranes used in this manner are classified in terms of their thickness, they can be divided into thick films of about 1000 to 2000 μm and thin films of about 100 μm or less. The former film thickness is generally made from cellulose triacetate, polyvinyl chloride, etc. by the dip method, etc., and can immobilize a large amount of enzymes, etc., but has a slow response speed and is generally easy to peel off. It has the following drawbacks. On the other hand, films with the latter thickness are generally formed by vapor deposition and have a fast response speed, but have the drawback of not being able to immobilize large quantities of enzymes and the like. [Problems to be solved by the invention] For this reason, in the determination of urea, a smaller, more robust, simpler, faster, and less expensive means of determination is needed, taking into account future applications in internal measurements. desired. The present inventors believe that this problem can be effectively solved by using a method for immobilizing the urease enzyme on the silicon nitride surface of the ISFET by depositing an enzyme immobilization reagent on the silicon nitride surface of the ISFET. (pages 5-6 of the abstracts of the 3rd Chemical Sensor Research Conference). in particular,
An enzyme immobilization reagent is deposited on the inorganic carrier surface of the ISFET, and an amino group of the enzyme is bonded to the functional group of the enzyme immobilization reagent on the vapor deposition surface to form an immobilized enzyme film. It constitutes a urea sensor. In addition to such a field-effect transistor-urea sensor which is excellent in responsiveness, the present invention also provides the following features:
As a result of various studies in search of a thick membrane that could immobilize a large amount of enzymes and also had good peeling resistance, polybutylvinyral was the only one among various polymer membrane materials that was found to be resistant to peeling. We have found that it is possible to form an immobilization membrane with excellent properties. [Means for Solving the Problem] and [Operation] Therefore, the present invention relates to a membrane for enzyme immobilization, which comprises a mixture of polyvinyl butyral, a triamine compound, and an aldehyde compound on an inorganic carrier. It forms a membranous body. Examples of inorganic carriers include silicon nitride (Si ₃ N ₄ ), silicon oxide (SiO ₂ ), aluminum oxide (Al ₂ O ₃ ), and thallium oxide (Ta _{2 O), which are used as protective film or insulating film materials for semiconductors} . ₅ ), boron nitride (BN), magnesium oxide (MgO), beryllium oxide (BeO), boron glass, phosphorus glass, arsenic glass, tin oxide (SnO ₂ ), indium oxide (In _{2 )} used as transparent conductive film materials. O ₃ ), copper iodide (CuI), aluminum, gold, palladium, etc. used as electrode materials, and silicon nitride is preferably used. A mixture film of polyvinyl butyral, a triamine compound, and an aldehyde compound is formed on these inorganic carriers. Glutaraldehyde, formaldehyde, acetaldehyde, etc.
The aldehyde group of the aldehyde compound, preferably represented by glutaraldehyde, is bonded to the amino group of the enzyme, with 1,8-diamino-
4-Aminomethyloctane acts as a spacer to weaken the above bond. The active ingredient that binds to the enzyme is fixed on an inorganic carrier as a mixture film, but as shown in the table below, among various polymer film materials, only one using polyvinyl butyral is used. shows excellent peeling resistance against various liquids. That is, a dope solution consisting of a 1% organic solvent solution of various polymer film-forming materials was cast onto a silicon wafer whose surface had been treated with silicon nitride to form a film at room temperature, and the film was formed in an open environment at 60°C for 20 minutes. The dried pieces were immersed in various liquids at 40°C, taken out after 2 hours, and visually evaluated for the degree of adhesion of the membrane to the silicon nitride surface.

【table】

〔Effect of the invention〕

The field effect transistor-urea sensor provided with the membrane for enzyme immobilization according to the present invention can immobilize a large amount of enzyme in terms of thickness, and
An enzyme immobilization membrane using polyvinyl butyral provided on the inorganic support surface of the ISFET shows good adhesion, and its responsiveness as a urea sensor is also good. [Example] Next, the present invention will be explained with reference to an example. Example Using a P-type wafer as a substrate, silicon island creation - field oxidation - phosphorus diffusion (source/drain formation) - gate oxidation. The ISFET was fabricated through a series of steps: chemical vapor deposition of silicon nitride, contact hole drilling using plasma etching, electrode formation using aluminum vapor deposition, hydrogen annealing, and wire bonding. This ISFET is
It was small with a width of 300 μm and a length of 4.0 mm, and the silicon nitride film deposited on it by chemical vapor deposition was approximately 1500 Å thick. Next, a mixture film was formed on the silicon nitride surface of this ISFET. That is,
1% polyvinyl butyral solution in methylene chloride 10
ml of 50% glutaraldehyde aqueous solution and 1 ml of 1,8-diamino-4-aminomethyloctane.
The ISFET was immersed and then pulled out to dry at room temperature. One of the two ISFETs with a mixture film formed on the silicon nitride surface in this way,
The membrane was immersed in an aqueous solution of urease (EC3.5, 1.5 Sigma product) with a concentration of 5 mg/ml and left at 4°C for 24 hours to immobilize the urease enzyme on the surface of the membrane. These two ISFETs, urease enzyme-immobilized ISFET 1 and reference ISFET 2 without enzyme immobilization, are placed in a test tube 4 along with a grounded silver/silver chloride reference electrode 3, as shown in FIG. They are each immersed in the test liquid 5. The responsiveness to urea using a differential measurement system having each of these elements, a differential amplifier 6, and a recorder 7 was investigated under the conditions of 37°C and PH7.0 as follows. Buffer solution (PH7.0, 5mM Tris-
Adjust the final concentration after injection of urea aqueous solutions of various concentrations to 0.08, 0.1, 0.25, 0.5, 0.75 or 1.0 mg/ml to 3.9 ml of HCl), and add 100μ of each.
was injected with a micro cylinder. The response potential change for each sample reached a steady value in about 2 minutes. When the initial rate of response potential change (ΔVg/Δt) and the logarithm of the urea concentration were plotted, a glass as shown in FIG. 2 was obtained. From this result, urease-immobilized ISFET is
At least within the urea concentration range of 0.08 to 1.0 mg/ml,
It is shown that urea can be detected quantitatively.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of a differential measurement system using a field effect transistor-urea sensor according to the present invention. Further, FIG. 2 is a graph showing the relationship between the initial rate of response potential change and urea concentration by this system. Explanation of symbols, 1... Enzyme-immobilized ISFET, 2...
...Reference ISFET, 3...Silver/silver chloride reference electrode, 5
...Test liquid.

Claims (1)

[Claims] 1. On an inorganic carrier, polyvinyl butyral,
An enzyme immobilization membrane comprising a mixture membrane of 1,8-diamino-4-aminomethyloctane and an aldehyde compound. 2. The membrane for enzyme immobilization according to claim 1, wherein a mixture membrane is formed on the surface of an inorganic carrier of a field effect transistor.