JPH02168153A - Method for forming immobilized enzyme film - Google Patents

Abstract

PURPOSE:To form an immobilized enzyme film with uniform thickness by developing a wafer and a solution of protein while cooling the both to equal to or lower than 15 deg.C when said wafer onto which said solution of protein is developed is rotated to form a thin film. CONSTITUTION:A photoresist solution is developed on a substrate 4 of sapphire having an ion sensor 5 of semiconductor field effect type thereon, and the substrate 4 is rotated at 3,000rpm for 30 seconds. Then, the substrate is pre-baked, exposed and developed with the use of a photomask, so that a part of a photoresist film 6 where an immobilized enzyme film 7 is to be formed is removed. Thereafter, 0.6ml PIPES buffer solution containing, e.g., 60mg/ml glucose oxidase, 15% bovine serum albumin and 1% glutaraldehyde is developed on the wafer. At this time, the buffer solution is 0 deg.C and the wafer is 4 deg.C. The wafer is immediately rotated at 2,000rpm for 30 seconds so that the solution is applied all over the surface of the wafer. After the enzyme film is completely crosslinked through one-hour reaction at 20 deg.C, the wafer is soaked in acetone, whereby the film 6 and the film 7 applied on the film 6 are removed. Thus, the film 7 having uniform thickness can be formed.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for manufacturing a biosensor, and particularly to a method for manufacturing a biosensor in which a large number of immobilized enzyme films are formed on a wafer during the manufacturing process.

(Prior Art) In recent years, there have been many attempts to miniaturize biosensors that measure the concentration of organic matter in a solution. Among these, we utilize semiconductor manufacturing technology to form a large number of potentiometric ion sensors such as field effect devices and amperometric electrochemical electrodes such as noble metal electrodes on wafers made of semiconductors or insulators. Is this method of manufacturing a biosensor by forming an immobilized enzyme membrane on the sensitive part low in cost due to mass production? It is attracting attention because it is possible to obtain a sensor with a large number of characteristics at the same time (for example, Japanese Patent Application No. 59-1561).
23, Jitsugan Sho 59-134995).

Conventionally, in semiconductor manufacturing processes, spin coating has often been used to form organic thin films such as photoresist films. Spin coating is a method in which a solution developed on a wafer is spread over the entire upper surface of the wafer by rotating the wafer, thereby forming a thin film with a uniform thickness.

The inventors applied this to the formation of an immobilized enzyme membrane, and by spreading a protein solution containing an enzyme and a crosslinking agent on a wafer and rotating it, they were able to obtain an immobilized enzyme membrane of uniform thickness. (Patent application No. 59-209165).

That is, after coating a photoresist on a wafer on which semiconductor field-effect transistors are formed, the photoresist is removed from the surface of a given semiconductor field-effect ion sensor on which an enzyme-immobilized film is provided using a photolithography method. The enzyme film is formed by spin-coating a protein solution containing a crosslinking agent, and when the photoresist is dissolved and removed, the immobilized enzyme film existing on areas other than the surface of the specified semiconductor field effect ion sensor is removed to form an enzyme film. Ta.

(Problem to be Solved by the Invention) However, with this method, when the concentration of the solution to be applied is low, an enzyme film with a fairly uniform thickness can be obtained, but when the concentration is high, the enzyme film can only be formed on a part of the wafer. A film cannot be formed. Alternatively, even if it can be formed, the enzyme film has the disadvantage that it is thick at the center and periphery of the wafer, and thinner in the middle, making it impossible to obtain a uniform film. This was thought to be due to the fact that the solution to be developed is reactive, so that the crosslinking reaction proceeds during solution preparation and development, resulting in changes in the viscosity, etc. of the solution.

An object of the present invention is to provide a method for obtaining an immobilized enzyme membrane with a uniform thickness.

(Means for Solving the Problem) The present invention forms an immobilized enzyme membrane by applying a protein solution containing an enzyme and a crosslinking agent to a specific chemically sensitive site on Ueno 1, which is provided with a large number of microelectrodes. The forming method is characterized in that when the protein solution is spread on the sea urchin/% and the wafer is rotated to form a thin film, both the wafer and the protein solution are cooled to 15°C or less before being spread. This is a method for forming an immobilized enzyme membrane.

(Function) When preparing a solution in which a crosslinking agent is added to a protein solution, the crosslinking reaction proceeds rapidly at the same time as the solution preparation, and the solution rapidly increases its viscosity. This reaction rate is largely related to temperature, and it is possible to reduce the reaction rate by lowering the temperature.

Therefore, if the temperature of the solution itself and the temperature of the wafer being developed is kept low, it becomes easier to obtain a uniform enzyme film. The lower the temperature of the solution, the lower the reaction rate; however, if the temperature is too low, problems such as increased viscosity and freezing may occur. In order to prevent a decrease in enzyme activity, the temperature of the enzyme solution should be kept cool enough to prevent freezing. Cooling the wafer itself has a similar effect. A certain degree of effect can be obtained if either the solution or the wafer is cooled, but it is more effective if both are cooled, preferably to 10° C. or lower.

(Example) The present invention will be explained with reference to the drawings.

3(a) to 3(d) are cross-sectional views showing the process of forming an immobilized enzyme membrane. First, a photoresist solution (Microposit MP1 manufactured by Silafray Co., Ltd.
300-37) Expand and rotate at 3.00 rpm for 30 seconds (Figure 3(a)). This was prebaked at 90°C for 30 minutes, exposed and developed using a photomask to provide the immobilized enzyme membrane (150pm x 500μm).
The photoresist film was removed (FIG. 3(b)). The thickness of the photoresist film was 3.14±0.09 pm. After that, PIFE containing 60 mg/ml of glucose oxidase, 15% bovine serum albumin, and 1% glutaraldehyde was added as an example of a protein solution containing an enzyme and a crosslinking agent.
0.6 ml of S buffer (pH 6, 8, 50 mM) was spread on the wafer (Fig. 3 (C)). At this time, the temperature of the solution was set to 0°C, and the temperature of the wafer was set to various temperatures from 4°C to 25°C. It was immediately rotated at 200 rpm for 30 seconds to coat the entire wafer surface. In order to improve the adhesion of the film to the wafer, it is also possible to perform a primer treatment before spin coating the enzyme film.

The reaction was carried out at 20° C. for 1 hour to complete the crosslinking reaction of the enzyme membrane. Thereafter, the wafer was immersed in acetone to dissolve the photoresist, and at the same time, the immobilized enzyme film coated on the photoresist was removed (FIG. 3(d)).

As a result of measuring the film thickness of the enzyme film at each point on the diameter of the wafer, a film thickness distribution as shown in FIG. 1 was observed. Figure 1 1) to 3) show the film thickness distribution when each enzyme film was formed at a wafer temperature of 4°C, 115°C, and 125°C. Measured using It was confirmed that the film thickness was uniform when the wafer temperature was low. Also, the wafer temperature is 25℃
The shape of the enzyme film formed at each temperature of 115°C and 14°C is shown in Figures 2 (a) to (C) in order.As can be seen from the figure, when the wafer temperature is high, the enzyme film has a shape with a lower center. However, when the temperature was low, a good film with uniform thickness within one film was obtained. When an enzyme film was formed in the same way by changing the composition of the enzyme solution, the glutaraldehyde concentration was 0.4%.
An enzyme film with a uniform thickness was obtained at 1.2%. Regarding the protein concentration, the BSA concentration was 18
Although it is possible to form an enzyme film up to 10%, the effect of cooling is particularly significant in a concentration range higher than 10%.

A good immobilized membrane was obtained when the enzyme concentration was 30% or less of the protein concentration. When the type of enzyme was changed, for example, urease and catalase, the cooling effect during enzyme film formation was similar. From the above experiments, when an enzyme film was formed by cooling both the wafer and the protein to 15° C. or lower, a uniform film thickness was obtained in a practical sense. however,
It has been found that cooling to 10° C. or less is preferable from the viewpoint of reproducibility.

(Effects of the Invention) As explained above, the present invention has the advantage that when forming an enzyme film by spin coating on the wafer surface, an enzyme film with a uniform thickness can be obtained by cooling the protein solution and the wafer. There is. Furthermore, if individual enzyme membranes are also formed by this method, there is an effect that good membranes with no change in thickness can be obtained.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the film thickness distribution of an enzyme film on a wafer according to the present invention. FIGS. 2(a) to 2(c) are diagrams showing the shape of the enzyme membrane under each condition. FIGS. 3(a) to 3(d) are diagrams showing the enzyme membrane forming process.

Claims (1)

[Claims] In a method of forming an immobilized enzyme film by applying a protein solution containing an enzyme and a crosslinking agent to a specific chemically sensitive site on a wafer provided with a large number of microelectrodes, the protein solution is spread on the wafer. A method for forming an immobilized enzyme film, characterized in that when the wafer is rotated to form an enzyme film on the entire upper surface of the wafer, both the wafer and the protein solution are cooled to 15° C. or lower and then developed.