JPS62144062A - Enzyme sensor - Google Patents

Abstract

PURPOSE:To suppress the permeation of cation other than ammonia without use of a reference electrode, by providing an ammonia selectively permeating film between an si3N4 film covered with an ion sensitive FET and an enzyme immobilized membrane. CONSTITUTION:A hydrophobic Si3N4 protective film 8 is formed on an SiO2 film 7 covering a gate section 6 of an ion sensitive FET10. A sample aqueous solution immersed portion of the film 8 is provided with an ammonia selectively permeating film 20 comprising a polymer such as poly-gamma-trichloro ethyl glutamate and an non-actin having ammonia selective permeating property. An enzyme immmobilized membrane 30 is applied covering the film 20. With such an arrangement, H<+>, Na<+>, K<+> and the like in the sample aqueous solution is suppressed in the permeation with the membrane 20 and ammonia which was generated from an enzyme reaction with the membrane 30 premeates selectively to change the gate potential. This can eliminate any reference electrode thereby simplyfying the equipment.

Description

[Detailed description of the invention] [Technical part L'F to which the invention pertains]

The present invention relates to an enzyme sensor using an ion-sensitive field effect transistor (hereinafter abbreviated as 15FET), and particularly to an enzyme sensor used for quantifying urea in a sample aqueous solution.

[Prior art and its problems]

Biosensors using 15FET as a signal conversion section include a penicillin sensor for measuring penicillin published by J. Janata et al. in 1980, a urea sensor, a glucose sensor, and the like. All of these are formed on the surface of the 15FET, and the p++ change that occurs when organic matter is decomposed by the enzyme-immobilized membrane, which is the molecular identification part, is converted into an electrical signal by the signal conversion part [5FET]. This has the advantage that the sensor can be made smaller and have a higher intensity. However, in this type of sensor, H and Na are present in the sample aqueous solution due to causes other than enzyme reactions.
If there is a 4 degree change or a change in pl+ of cations such as ', K', etc., there is a problem in that measurement errors will occur.In order to eliminate this shadow, a reference electrode made of a JSFET etc. It is necessary to take measures such as eliminating the above error factors by installing a reference electrode inside the reference electrode to detect potential changes caused by causes other than the enzyme reaction and subtracting the output signal of this reference electrode from the output signal of the l5FET. This method requires a signal processing circuit for determining the difference signal between the two signals, which has the disadvantage of complicating the device and causing economic disadvantage.

[Purpose of the invention]

The present invention was made in view of the above-mentioned situation, and it is possible to eliminate the influence of canon other than ammonia without using a reference electrode, and therefore, the measurement accuracy of urea in a sample aqueous solution is high, and it is simplified. The aim is to provide an inexpensive enzyme sensor.

Summary of the Invention The present invention is characterized in that the surface of the FET, including the gate portion, is made of silicon oxide (S).
iO In addition, the ammonia-selective membrane was double coated with an enzyme-immobilized membrane on which an enzyme having a property of involving ammonia in the substrate or product of the enzymatic reaction formed on the outside of the ammonia-selective membrane was immobilized. Nonactin in the ammonia selectively permeable membrane is an ionophore that binds to biological membranes in living organisms and selectively permeates monovalent cations, and its affinity for cations is N11a'> K ''>> Na, especially for ammonia. Transmits well. Therefore, the permeation of cations such as H', Na'', K'' in the test aqueous solution is blocked by the ammonia permselective membrane, and changes in the gate potential of <15FET based on these cations can be prevented, and the enzyme By selectively permeating ammonia produced by the reaction, changes in gate potential due to ammonia groups NHa' in the sample water solution can be measured with high sensitivity as changes in drain current.

[Embodiments of the invention]

The present invention will be explained below based on examples. FIG. 1 is a side sectional view of an enzyme sensor showing an embodiment of the present invention. In the figure, IO is an ion-sensitive field effect transistor (ISFET), and l is a P-9i group)
On the other hand, 2 is a source electrode, 3 is a drain electrode, 4 is an n diffusion region, 5 is a p channel) 7 bar, 6 is a gate part from which the metal electrode is removed, and 7 is a metal electrode so as to cover the FET surface including the gate part 6. Formed SiJ Zetsu! ! The film 8 is a silicon nitride (Si3N) protective film formed on the outside of the 5iOz insulating film, and by covering the part that will be immersed in the test aqueous solution with the silicon nitride protective film 8, the water resistance of the 15FET'4fA8! It is possible to maintain one's sexuality. 20 is an ammonia selective i3 permeable membrane, and the polymer forming the membrane and the ammonia selective i! It consists of the antibiotic nonactin with 1IAta. Nonactin is produced in large quantities by actinomycetes, and can be extracted and purified using known methods from the same country, but a commercially available product manufactured by Streptomyces griseus may also be used. The polymers that form the ammonia selective membrane are:
It is necessary that the membrane formed from the polymer does not allow protons to pass through and is easily soluble in tetrahydrofuran (THF), which is a good medium for nonactin. Polymethyl glucmate derivative 1 polyvinylidene chloride is an example of a polymer that satisfies these conditions. Examples include acetylcellulose and its g-11 form. These polymers and nonactin are dissolved in an organic solvent to obtain a polymer dope, and a thin film is formed from this polymer dope. A dip coating method is used to form a thin film on the surface of the 15FET. 15FET may be used after being subjected to appropriate surface treatment to improve adhesion to the polymer. Regardless of the treatment, the 15FET is immersed in a polymer dope of an appropriate concentration, pulled up, and dried by an appropriate method to form a thin film. At this time, ensure that not only the gate part of the 15FET but also the part that is inserted into the test aqueous solution during use is completely removed. Ammonia permselectivity If! to be 1N! Form 20. 30 is an enzyme immobilization membrane formed on the ammonia permselective membrane 20, and the enzyme to be immobilized is effective as a sensor if ammonia is involved in the substrate or product of the enzyme reaction. . Examples of such enzymes include urease, creatinine diminase, and glutamate oxidase. The enzyme may be immobilized by any known method as long as it can form a thin film. For example, the enzyme can be immobilized by an entrapping immobilization method using polyvinyl alcohol, di-cauginan, agarose gel, etc., a carrier cross-linking method using albumin, chitosan, etc. as a carrier, or a combination method thereof. An enzyme sensor can be obtained by partially or completely covering the tip portion of the 15FETIO including the gate portion 6 with the enzyme-immobilized membrane 30. Example 1 15FE with silicon nitride protective film 8 formed by CVD method
The surface of TIO was subjected to surface treatment by vapor depositing T-aminopropyltriethoxylane (Cr-APTES) at 80°C and 0.5mH[+] for 2 hours, followed by heat treatment at 100°C for 2 minutes. - An ammonia permselective membrane 20 was formed using poly-γ-trichloroethylglutamate, which is a derivative of methyl-L-glutamate, as a polymer. Poly-T-trichloroethylglutamate is
Polyγ-methyl- and monoglutamate methyl group ββ
Transesterification with β-trichloroethanol, exchange rate 7
0% was used. The polymer toomg and nonactin 2m manufactured by Streptomyces griseus
A polymer dope was obtained by dissolving 1-g of the polymer in tetrahydrofuran (THF). T-APTES using the above method
The treated 15FET was immersed in this polymer dope, then slowly pulled up and dipped, and dried at room temperature under a pressure of 10 mHg to prepare an ammonia permselective membrane 20. Further, as the enzyme-immobilized membrane 30, 15 mg of bovine serum albumin and 10 mg of urease were mixed with O, OIM, p
Dissolve in 1 ml of H7.0 phosphate buffer, add glutaraldehyde (GA) to a concentration of 1%, stir, and immediately dip the 15FET on which the ammonia permselective membrane 20 has been formed into this solution. Coat and place in desiccator. It is formed by drying at 5°C, and further contains 0, O
By washing thoroughly in IM, Tris-HCI, pH 7.0 buffer, an enzyme sensor for urea measurement as shown in FIG. 1 was obtained. FIG. 2 is a response characteristic diagram for urea of the enzyme sensor according to the above-mentioned example, and the horizontal axis in the figure is urea (Co(Nl!)).
2) The vertical axis shows the time before and after injecting the sample solution containing 2 into the enzyme sensor container kept at 30°C. The vertical axis shows the change in drain current of 15FET converted to gate potential of gate section 6. . In the figure, the time of sample liquid injection (1=0)
The potential Eo in the previous example is an interfacial potential based on the electric double layer at the interface between the silicon nitride protective film 8 and the ammonia permselective film 20 of 15FETIO, and is the interfacial potential based on the electric double layer at the interface between the silicon nitride protective film 8 and the ammonia permselective film 20. Urea is decomposed by an enzymatic reaction and ammonia (1ficNHa') is generated. The generated ammonia stores pass through the ammonia permselective filter 20 and reach the interface of the silicon nitride protective film 8, and by changing the interface potential E0, the gate potential rises to about 2 After a few minutes, a steady state corresponding to the concentration of urea in the sample solution is reached. Therefore, if the correlation with gate potential or drain current is calibrated in advance using sample solutions with different urea concentrations,
The urea content in the sample solution can be quantitatively measured. Furthermore, if cations such as H', Na., Ni', etc. are present in the sample solution, these cations are prevented from approaching the interface of the silicon nitride protective film 8 of the 15FETIO by the ammonia permselective membrane 20. It is possible to obtain an enzyme sensor for measuring urea that does not change the gate potential and can therefore eliminate the influence of cations other than ammonia without using a reference bridge. Example 2 An ammonia selectively permeable membrane was formed by dip coating the surface of an 15FET that had been surface-treated with γ-APTES twice using a polymer dope having the same composition as in the previous example, and coated with ethylenediamine. 0.1
, J were vapor-deposited at 80°C, and glutaraldehyde (
GA) 0.1- was deposited at 80°C. Next, 20 mg of urease and 20 mg of bovine serum albumin were added at a pH of 1-7.
After dissolving in 0,0,01M phosphate buffer, GA was
．． It was added to a concentration of 5% and stirred. The above-treated 15FET was dipped in this solution and dried at 5°C for one day to form an enzyme-immobilized film, which was then further coated with O, OIMTr.
The enzyme sensor for urea measurement was obtained by washing for 8 hours with is-Hcl, pt 17.0 mild Si solution. When urea was quantified using this sensor, it was found that 1O~200m
It was possible to quantify urea in the range of g//j.

【Effect of the invention】

As described above, the present invention constitutes an enzyme sensor for measuring urea by depositing an ammonia selective perms membrane and an enzyme immobilization membrane on the surface including the gate part of a +5FET on which a silicon nitride retention 8i membrane is formed. . As a result, the ammonia permselective membrane eliminates the problem with conventional technology, where ions other than ammonia change the gate potential, reducing measurement accuracy, making it possible to accurately quantify urea in the sample solution. An enzyme sensor for measuring urea can be provided. In addition, since the influence of ions other than ammonia is eliminated by the ammonia selective permselective membrane, it becomes possible to omit the reference electrode that was used in the conventional technology to cancel the influence of the ions.
In addition, a signal processing circuit for subtracting the output signal of the reference electrode from the output signal of the 15FET becomes unnecessary, so that
The advantage is that the urea measuring device can be simplified, the measurement operation and calibration work are easy, and the urea measuring device with high urea measurement accuracy can be economically advantageously provided.

[Brief explanation of drawings]

FIG. 1 is a side sectional view of an enzyme sensor showing an embodiment of the present invention, and FIG. 2 is a response characteristic diagram for urea of the sensor in the embodiment. lO: Ion-interceptive field-effect transistor (ISF)
ET), 6: gate section, 7: SiO□ insulating film, 8: silicon nitride protective film, 20: ammonia permselective film, 30: enzyme immobilization film.・1. T9・)

Claims (1)

[Claims] 1) An ion-sensitive field effect transistor in which the surface portion of the device including the gate portion is covered with a hydrophobic silicon nitride protective film, and a silicon nitride protective film of this ion-sensitive field effect transistor. An enzyme sensor comprising: an ammonia selectively permeable membrane attached to cover a portion immersed in a test aqueous solution; and an enzyme immobilization membrane attached to cover the ammonia selectively permeable membrane. 2) The enzyme sensor according to claim 1, wherein the ammonia selectively permeable membrane is composed of a membrane-forming polymer and nonactin having ammonia selectively permeable ability. 3) The enzyme sensor according to claim 1, wherein the polymer is poly-γ-trichloroethylglutamate.