JPH02108954A - Formation of immobilized enzyme film of semiconductor enzyme sensor - Google Patents

Abstract

PURPOSE:To obviate the contamination of the immobilized enzyme by other enzymes and to make simultaneous and exact measurement of samples of multiple components by applying a water soluble photosensitive resin contg. the enzyme to be immobilized on a water soluble photosensitive resin. CONSTITUTION:The water soluble photosensitive resin is applied by spin coating on a hydrogen ion-sensitive film 6 of a substrate 1 integrated with 3 pieces of hydrogen ion-sensitive transistor elements to form a thin bovine serum albumin film 13. The photosensitive resin is then so exposed that the immobilized bovine serum albumin film 11 remains only on the part of a transistor 12 for reference. The water soluble photosensitive resin is then applied thereon to form a thin glucose oxidase film 10. The unexposed part of the thin film 13 is then partly developed. Further, only the sensitive film 6 of the transistor desired to be formed with the immobilized glucose oxidase film 8 is exposed and developed. After the substrate 1 is dried, the thin film 13 is applied and coated thereon. A thin urease film 9 is then formed. The thin film 9 is photocrosslinked to form an immobilized urease film 7 and the thin film 9 is removed, by which the immobilized enzyme film is formed.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for forming an immobilized enzyme membrane for a semiconductor enzyme sensor that can simultaneously measure a plurality of chemical components.

[Prior art] Figures 4A to 4F are, for example, disclosed in Japanese Patent Application Laid-Open No. 60-39547.
1 is a side cross-sectional view showing the manufacturing process of a semiconductor enzyme sensor by the conventional immobilized enzyme film forming method disclosed in the above publication; in these figures, (1) is a substrate; (2) is this substrate (1);
), (3) and (4) are respectively a source electrode and a drain xfI! arranged in each element isolation region (2). , (5) is the substrate (
1) Insulating film formed on top, (6) this insulating film (5)
(7) is the immobilized urease membrane formed on the hydrogen ion sensitive membrane (6) on one element separation region (2), and (8) is the other hydrogen separation region. (
2) An immobilized glucose oxidase membrane formed on the hydrogen ion sensitive membrane (6) above.

Next, a method for producing an immobilized enzyme membrane using the above-mentioned method for forming an immobilized enzyme membrane will be explained.

First, it has three element isolation regions (2), a source electrode (3) and a drain electrode (4) are formed in each element isolation region (2), and an insulating film (5) and a hydrogen electrode are formed on the substrate (1). Integrated hydrogen ion-sensitive field effect transistors each having an ion-sensitive film (6) formed thereon are prepared. A water-soluble photosensitive resin containing urease is spin-coated on the hydrogen ion-sensitive wA (6) on this transistor to form a urease thin film (9) with a uniform thickness (Figure 4A). First, immobilization. Expose only the area where you want to form the urease film (7).
The water-soluble photosensitive resin is made insoluble in water by photo-crosslinking it by irradiating it with light using a photomask (not shown, the same applies hereafter) as indicated by the arrow in the figure (Figure 4B). . Next, by developing with water, the unexposed portion is removed to obtain the immobilized urease film (7) (4th C).
figure).

Next, a water-soluble photosensitive resin containing glucose oxidase is spin-coated onto this transistor to form a glucose oxidase thin film (10) with a uniform thickness (Fig. 4D), and an immobilized glucose oxidase film (8) is formed. ) The water-soluble photosensitive resin is photo-crosslinked by irradiating light using a photomask that exposes only the desired area to make it insoluble in water (Figure 4E) 1. Next, by developing with water. An immobilized glucose oxidase membrane (8) is formed (Figure 4F) to obtain the semiconductor enzyme senna. In addition,
The order of forming the immobilized urease membrane (7) and the immobilized glucose oxidase membrane (8) may be reversed.

[Issues to be Solved by the Invention] In the method for forming an immobilized enzyme film as described above, there is an opportunity for a water-soluble photosensitive resin solution containing different types of enzymes to come into direct contact with a certain immobilized enzyme film. enzymes may contaminate certain immobilized enzyme membranes. Furthermore, when measuring other components at the same time, there is a problem in that reactions caused by foreign enzymes that cause this contamination may interfere with the intended response. Furthermore, there was a problem in that the surface of the ion-sensitive field effect transistor used as a reference was contaminated with the enzyme.

FIG. 5 is a response curve of a semiconductor enzyme sensor specifically showing such a state. One constant is 10mM PIPES (
Piperazine-N,N'-bis(2-ethanesulfonic acid)
-NaOHNFfi solution (pH 7,0) was used at 25°C with a glucose concentration of 3mM and a urea concentration of 5mM. Showing greenhouse results when using a mixed solution of urea 5mM and glucose 5mM,
In the case of Figure 5, the glucose response outputs a positive voltage, and the urea response outputs a negative voltage. A glucose response occurs (Fig. 5(a)), and in a mixed solution of urea and glucose,
The response of glucose alone due to the influence of urea (Fig. 5 (b)
), the glucose response is smaller than that of the fifth
Figure (C)) shows that accurate glucose concentration cannot be measured.

This invention was made to solve these problems, and it eliminates the contamination of the immobilized enzyme membrane with other enzymes and the surface of the reference transistor with the enzyme, and allows simultaneous and accurate measurement of multi-component samples. The purpose of this study is to obtain a method for forming an immobilized enzyme membrane for a semiconductor enzyme sensor that can be used for measurement.

[Means for Solving the Problems] The method for forming an immobilized enzyme film according to the present invention is such that when forming different types of immobilized enzyme films by photolithography using a water-soluble photosensitive resin, it is possible to directly form a film with other enzymes. In order to prevent the enzyme from contacting or adhering to the surface of the reference transistor, use a water-soluble photosensitive resin containing other enzymes or proteins that does not affect the response of the enzyme used, or anything else. A water-soluble photosensitive resin containing no enzyme is coated, and a water-soluble photosensitive resin containing an enzyme to be immobilized is coated on top of the coated water-soluble photosensitive resin, followed by exposure and development.

[Function] In the present invention, there is a gap between a certain type of immobilized enzyme film that has already been formed and a water-soluble photosensitive resin solution for forming the immobilized enzyme film that is then formed thereon, as well as between a reference transistor. By interposing a thin film of water-soluble photosensitive resin solution that serves as a spacer between the hydrogen ion-sensitive membrane and the water-soluble photosensitive resin solution for forming the immobilized enzyme membrane, the immobilized enzyme membrane that has already been formed or the reference This prevents contamination by enzymes on the surface of the hydrogen ion sensitive membrane of commercial transistors.

[Example] Figures 1A to 1G are side sectional views showing the manufacturing process of a semiconductor enzyme sensor according to an example of the present invention, in which (1) represents a substrate, and (2) represents this substrate. <
1), three element isolation regions formed in the upper part of the element isolation region (3), a source electrode formed in this element isolation region (2), and (3) a source electrode formed in the element isolation region (2);
4) is a drain electrode formed in the element isolation region (2), (5) is an insulating film formed on the substrate (1), and (5) is an insulating film formed on the substrate (1).
6) is a hydrogen ion sensitive film formed on this insulating film (5), (7) is an immobilized urease film formed on the hydrogen ion sensitive film (6) on one element isolation region (2), (8
) is hydrogen ion sensitive 11 on one element isolation region (2)
K (6) is an immobilized glucose oxidase membrane formed on the reference transistor (12), and (11) is an immobilized bovine serum albumin membrane formed on the hydrogen ion sensitive membrane (6) on the reference transistor (12). Note that the water-soluble photosensitive resin has a molecular weight of 3
60,000 polyvinylpyrrolidone aqueous solution (10% by weight),
2.5-bis(4'-azido-2'-sulfobenzal)
Cyclopentanone sodium salt (1% by weight) (manufactured by Tokyo Ohka Kogyo Co., Ltd.) was used.

Example 1 A water-soluble photosensitive resin containing a protein called bovine serum albumin was spin coated on the hydrogen ion sensitive film (6) of the substrate (1) on which three hydrogen ion sensitive transistor elements were integrated, and the thickness was Of the three hydrogen ion-sensitive transistors on which a uniform bovine serum albumin thin film (13) was formed (Fig. 1A), the middle one was used as a reference transistor (12), and the bovine serum albumin immobilized only on the upper part was used as the reference transistor (12). The serum albumin membrane (11) was exposed using a photomask (not shown, the same applies hereafter) (indicated by an arrow in the figure, the same applies hereafter) (Fig. 1B).Next, glucose oxidase was applied on top of this. A water-soluble photosensitive resin containing Glucose Oxidase III (10) with a uniform thickness is formed by spin coating.

At this time, simultaneously apply the bovine serum albumin thin film (1
3) A part of the unexposed area is developed (Figure 1C)
Further, only the hydrogen ion sensitive membrane (6) of the transistor on which the immobilized glucose oxidase membrane (8) is to be formed was exposed using a photomask and developed with water (Figure 1D). After drying the substrate (1), Then, a water-soluble photosensitive resin containing bovine serum albumin was applied again to form a bovine serum albumin thin film (13) of uniform thickness (Fig. 1E). Next, a water-soluble photosensitive resin containing urease was spin-coated to form a urease thin film (9) with a uniform thickness. At this time, most of the previously applied bovine serum albumin thin film (13) is dissolved, but not all of it is removed, and the bovine serum albumin thin film forms a thin film between the urease thin film (9) applied above and the base. (13) is formed (Fig. 1F).

Then, using a photomask that can expose only the part where the immobilized urease film (7) is desired to be formed, the urease thin film (
9) was photocrosslinked to form an immobilized urease film (7).Next, the urease thin 11K (9) that was not photocrosslinked was removed by developing with water to obtain a semiconductor enzyme sensor. Figure) 6 Example 2 Since glucose oxidase does not easily contaminate the reference transistor, Example 1 can be simplified as follows. Side sectional views showing the manufacturing process of the semiconductor enzyme sensor according to Example 2 are shown in FIGS. 28 to 2E.

A water-soluble photosensitive resin containing glucose oxidase is spin coated on the hydrogen ion sensitive membrane (6) of the substrate (1) on which three hydrogen ion sensitive transistor elements are integrated, and glucose oxidase 411 with a uniform thickness is formed! l (10>
was formed (Figure 2A), exposed using a photomask that exposed light only to the part where the immobilized glucose oxidase membrane (8) was desired to be formed, and then developed with water (Figure 2B).
Next, a water-soluble photosensitive resin containing bovine serum albumin was spin-coated to form a bovine serum albumin thin film (13) of uniform thickness. Then, a photomask was used to expose only the hydrogen ion sensitive membrane (6) of the reference transistor (12) (Fig. 2C)6.Next, the water-soluble photosensitive resin solution containing urease was rotated. A thin urease film 9) of uniform thickness was formed. At this time, the previously applied bovine serum albumin thin film (13) is almost dissolved except for the exposed part, but not all of it is removed, leaving a thin film between the urease thin film (9) applied above and the base. It remains as (Figure 2D).

Finally, a semiconductor enzyme sensor was obtained by exposing only the portion where the immobilized urease film (7) was desired to be formed using a photomask and developing with water (FIG. 2E).

The response of the semiconductor enzyme sensor thus formed to glucose and urea is shown in FIG.
7.0), the urea concentration was 5mM (curve (a) in the figure), and the glucose concentration was 3mM (curve (a) in the figure).
b)), the mixed solution is 3mM glucose and 5mM urea.
Curve (C) in the figure for each concentration of M was used. In addition, in the case of Fig. 3, the glucose response outputs a positive voltage,
Urea response outputs a negative voltage.

As is clear from the figure, there is no difference in response when each component is mixed alone, indicating that each component can be measured simultaneously and accurately.

Note 1. In Example E, a water-soluble photosensitive resin consisting of polyvinylpyrrolidone and 2,5-bis(4'-azido-2'sulfobenzal)cyclopentanone sodium salt was used, but it could be easily oxidized without deactivating the enzyme. Other water-soluble photosensitive resins that can be made insoluble in water by crosslinking can also be used, and the same effects as described above can be achieved.

In addition, in Example 1 above, an immobilized bovine serum albumin film was first formed on the ion-sensitive membrane of the reference transistor, but this film could be formed even after the immobilized glucose oxidase film was formed or after the immobilized urease film was formed. The same effect can be achieved even if

In addition, in Example E, a water-soluble photosensitive resin containing bovine serum albumin was used, but in addition to this, it may contain an enzyme or protein that does not affect the reaction of the enzyme to be immobilized, such as urease or glucose oxidase. Similar effects can be obtained by using a water-soluble photosensitive resin or a water-soluble photosensitive resin that does not contain such enzymes or proteins.

[Effects of the Invention] As explained above, the present invention applies a water-soluble photosensitive resin containing an enzyme or protein that does not affect the response reaction of the enzyme to be immobilized on a hydrogen ion-sensitive film of a substrate, and then A water-soluble photosensitive resin containing the enzyme to be immobilized is applied onto the photoresin, and then this water-soluble photosensitive resin is exposed to light.
Since it is developed, it is possible to obtain a semiconductor enzyme sensor for simultaneous measurement of multiple components, which is free from contamination by enzymes and capable of highly accurate measurement.

[Brief explanation of the drawing]

1A to 1G are side sectional views showing the manufacturing process of a semiconductor enzyme sensor according to one embodiment of the present invention, and FIGS. 2A to 2E are side sectional views showing the manufacturing process of a semiconductor enzyme sensor according to another embodiment of the invention. 3 is a diagram showing the response curve of a semiconductor enzyme sensor formed according to an embodiment of the present invention, and FIGS. 4A to 4F show the manufacturing process of a semiconductor enzyme senna using a conventional forming method. FIG. 5, a side cross-sectional view, is a diagram showing a response curve of a semiconductor enzyme sensor formed by a conventional fabrication method. In each figure, the same reference numerals indicate the same or corresponding parts. Figure 1A Figure 1C Figure 1D Figure Year 10 Hydrogen ion sensitive/immobilized urease N (Immobilized glucose oxy production enzyme) Albumin odor p-table transisph beef m-clear fulbumin turmoil Fig. 2C Fig. 4A Procedural amendment January 11, 1990

Claims (1)

[Claims] A method in which different types of immobilized enzyme films are formed using a water-soluble photosensitive resin on the hydrogen ion-sensitive film of an integrated hydrogen ion-sensitive field effect transistor in which multiple hydrogen ion-sensitive field effect transistors are stacked. After coating a water-soluble photosensitive resin containing an enzyme or protein that does not affect the response reaction of the enzyme to be immobilized on the hydrogen ion-sensitive membrane, the water-soluble photosensitive resin containing the enzyme to be immobilized is coated on the hydrogen ion-sensitive membrane. A method for forming an immobilized enzyme film for a semiconductor enzyme sensor, which comprises applying a water-soluble photosensitive resin, then exposing and developing the water-soluble photosensitive resin.