JPH0481739B2 - - Google Patents

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for forming an enzyme-immobilized membrane in an integrated semiconductor biosensor in which an enzyme-immobilized membrane is provided on the surface of a semiconductor field-effect ion sensor. It is related to.

(Prior technology) Conventionally, a membrane with an enzyme immobilized on the surface of a semiconductor field-effect ion sensor (hereinafter referred to as "ISFET") is a type of semiconductor biosensor that measures the concentration of a specific organic substance in a solution. something is known. This biosensor detects changes in hydrogen ion concentration that occur when a specific organic substance in a solution undergoes a chemical reaction due to the catalytic action of an enzyme on an enzyme-immobilized membrane.
It measures the concentration of specific organic substances by detecting them with ISFET. Examples of enzyme-immobilized membranes with this selectivity include urease-immobilized membranes for urea detection and glucose oxidase membranes for glucose detection (Sensors and Actuators).
Actu-ators, Vol. 7, pp. 1-10 (1985)). In addition, an ISFET with an enzyme-immobilized film and an ISFET with a deactivated enzyme-immobilized film were formed on the same chip using island-shaped silicon provided on a sapphire substrate, and the back side was used as a reference electrode. A one-chip biosensor has also been developed by providing a gold electrode (16th, 1984 International Conference on Solid State Devices and Materials, Late News Abstracts). on Solid State
Devices and Materials，Late News
Abstracts) pp. 66-67 (1984)). In manufacturing such a biosensor, it is necessary to form an enzyme-immobilized membrane on a predetermined ISFET. Several methods are known for forming an oxygen-immobilized film, but the method described in the cited document above is one that allows the formation of an enzyme-immobilized film at the wafer stage and enables mass production of biosensors. After forming an enzyme-immobilized film using cellulose triacetate as a carrier on the entire surface of the wafer, irradiation with ultraviolet rays through a photomask was performed to form a predetermined film.
There is a method of deactivating the enzyme in the enzyme-immobilized membrane other than on the ISFET.

(Problems to be Solved by the Invention) However, the above method requires more than one day to form the enzyme immobilization, and the properties of the enzyme immobilization membrane, such as thickness, enzyme content, etc. The problem was that it was difficult to change factors that had a significant impact on On the other hand, a lift-off method has been proposed that allows an enzyme-immobilized membrane to be obtained in a short time and allows for easy changes in the enzyme content in the enzyme-immobilized membrane (Japanese Patent Application No. 59-209165). However, in this lift-off method, the enzyme-immobilized membrane comes into contact with the organic solvent, so
The lift-off method could not be used to form a semiconductor biosensor enzyme-immobilized membrane using enzymes that are easily inactivated by organic solvents.

(Means for Solving the Problems) The present invention provides a method for forming an enzyme-immobilized film for a semiconductor biosensor, in which an enzyme-immobilized film is provided on the surface of a semiconductor field-effect ion sensor. a step of applying a photoresist on a semiconductor wafer on which an ion sensor is formed, and then removing the photoresist on the surface of a predetermined semiconductor field-effect ion sensor on which an enzyme immobilization film is to be provided by a photoringography method; b) spin-coating a hydrophilic primer solution on the surface of the semiconductor wafer to treat the surface of the predetermined semiconductor field effect ion sensor with a hydrophilic primer; (d) Further, after forming an enzyme-immobilized film on the semiconductor wafer surface by spin-coating a protein solution containing an enzyme and a crosslinking agent on the semiconductor wafer surface that has undergone the above steps, By immersing a semiconductor wafer in an aqueous solution with a specified pH range and performing ultrasonic treatment,
A semiconductor biosensor characterized in that an enzyme-immobilized film existing on a surface other than the surface of the predetermined semiconductor field-effect ion sensor is removed, and an enzyme-immobilized film is formed on the surface of the predetermined semiconductor field-effect ion sensor. This is a method for forming an enzyme-immobilized membrane.

(Function) According to the method of the present invention, after coating a photoresist on a semiconductor wafer, a predetermined area on which an enzyme-immobilized film is to be provided is formed using a photorinography method.
The photoresist on the surface of the ISFET is removed, and then the surface of the ISFET from which the photoresist has been removed is treated with a hydrophilic primer by spin coating. Through this step, a reactive group of a hydrophilic primer is introduced onto the surface of a predetermined ISFET, and is subjected to reaction with a crosslinking agent contained in the protein solution. The photoresist remaining on the semiconductor wafer after the hydrophilic primer treatment process is removed using an organic solvent or the like. The chemicals used to strip the photoresist must not damage the reactive groups of the hydrophilic primer. A protein solution containing an enzyme and a crosslinking agent is spin-coated onto the semiconductor wafer that has undergone the above steps. After the crosslinking reaction by the crosslinking agent is completed, the semiconductor wafer is immersed in an aqueous solution having a pH higher than a predetermined value. In an aqueous solution with a predetermined pH or higher, both the enzyme-immobilized membrane and the semiconductor wafer are negatively charged, and due to electrical repulsion, the surface of the predetermined ISFET is held by chemical bonds with the reactive groups of the hydrophilic primer. The enzyme-immobilized film other than the above is lifted from the semiconductor wafer surface, and when ultrasonic waves are applied in this state, the lifted enzyme-immobilized film is mechanically removed, leaving the enzyme-immobilized film only on the designated ISFET surface. is formed. Although it depends on the material of the enzyme-immobilized membrane and other conditions, an aqueous solution with a pH of 8 or higher is preferable.

(Example) Examples of the present invention will be described below with reference to the drawings.

FIG. 1 is a process explanatory diagram of an embodiment of the method for forming a semiconductor biosensor enzyme-immobilized membrane according to the present invention.
A pair of pairs formed on a sapphire substrate.
A case is shown in which an enzyme-immobilized membrane is formed only on one side of the ISFET. In Figures 1a-e,
1 is a sapphire substrate, 2 is a high impurity concentration n-type silicon region, 3 is a p-type silicon region, 4 is a silicon oxide film, 5 is a nitrogen silicon film, 6 is a photoresist film, and 7 is a gold electrode. Next, the formation process will be explained step by step. An ISFET is formed using the island-like silicon layer on the front surface of the sapphire substrate 1, and an ISFET is formed using the island-like silicon layer on the back surface of the sapphire substrate 1.
A photoresist film 6, such as Microposit 1300-27 manufactured by Shipley Co., Ltd., is spin-coated on the surface of the wafer with gold 7 deposited on the back surface of the sapphire (FIG. 1a). Next, the photoresist film on the surface 8 of the ISFET on which the enzyme-immobilized film is provided is removed by exposure and development using a photomask (FIG. 1b).
Thereafter, a hydrophilic primer, such as a 1% aqueous solution of γ-aminopropyltriethoxysilane, is spin-coated onto the wafer and heat treated at 110°C for 5 minutes.
γ-
Attach aminopropyltriethoxysilane. This wafer is immersed in a 5% glutaraldehyde aqueous solution for 15 minutes to cause the amino groups of γ-aminopropyltriethoxysilane bonded on the surface of the ISFET to react with the aldehyde groups of glutaraldehyde, thereby forming an enzyme-immobilized film. Modify the surface of ISFET with aldehyde groups. Next, the semiconductor wafer is immersed in acetone to remove the photoresist (FIG. 1c). After washing with water and drying, add a protein solution containing an enzyme and a crosslinking agent, such as 0.1 M piperazine-N,N'-bis(2) containing 300 mg bovine serum albumin.
- ethanesulfonic acid) - sodium hydroxide (PH
6.8) Add 1 volume part of a 50 mg/ml urease (manufactured by Miles Laboratories) aqueous solution to 2 volume parts, and then add 1 volume part of a 2% by weight glutaraldehyde aqueous solution, and then spin-coat 900 μ of the well-mixed solution. The mixture was left at room temperature for 1 hour to complete the crosslinking reaction with glutaraldehyde, forming an enzyme-immobilized membrane (FIG. 1d). The semiconductor wafer on which the enzyme-immobilized film 9 has been formed in this manner is immersed in an aqueous solution with a pH of 8 or higher, for example, 0.1 M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, and the enzymes other than those on the ISFET treated with a hydrophilic primer are After the immobilized film is lifted from the surface of the semiconductor wafer, ultrasonic treatment is performed to remove the lifted enzyme immobilized film (FIG. 1e).

Through the above steps, it was possible to form an enzyme-immobilized membrane only on the surface of a predetermined ISFET.

(Effects of the Invention) According to the method of the present invention, since the enzyme-processed film is formed by spin-coating a protein solution, the thickness of the enzyme-immobilized film is uniform on each ISFET on the wafer, and the thickness of the enzyme-immobilized film is uniform on each ISFET on the wafer. does not contain any factors that affect enzyme activity, making it possible to mass produce biosensors using ISFET.

[Brief explanation of the drawing]

FIGS. 1a to 1e are process explanatory diagrams when the method of the present invention is applied to the manufacturing process of a semiconductor biosensor. In the figure, 1 is a sapphire substrate, 2 is a high impurity concentration n-type silicon region, 3 is a p-type silicon region, 4 is a silicon oxide film, 5 is a nitrogen silicon film,
6 is a photoresist film, 7 is a gold electrode, 8 is an ISFET surface provided with an enzyme-immobilized film, and 9 is an enzyme-immobilized film.

Claims (1)

[Claims] 1. A method for forming an enzyme-immobilized film of a semiconductor biosensor, in which an enzyme-immobilized film is provided on the surface of a semiconductor field-effect ion sensor, comprising: (a) a semiconductor field-effect ion sensor is formed; (b) removing the photoresist from the surface of a predetermined semiconductor field effect ion sensor on which an enzyme immobilization film is to be provided by photolithography after applying a photoresist on the semiconductor wafer; , a step of spin-coating a hydrophilic primer solution to treat the surface of the predetermined semiconductor field-effect ion sensor with a hydrophilic primer, and (c) a step of peeling off the photoresist remaining on the semiconductor wafer after the above step. (d) Furthermore, after forming an enzyme-immobilized film on the surface of the semiconductor wafer by spin-coating a protein solution containing an enzyme and a cross-linking agent on the surface of the semiconductor wafer that has undergone the above steps, the semiconductor wafer is heated to a predetermined pH range. By immersing it in an aqueous solution and performing ultrasonic treatment,
A semiconductor biosensor characterized in that an enzyme-immobilized film existing on a surface other than the surface of the predetermined semiconductor field-effect ion sensor is removed, and an enzyme-immobilized film is formed on the surface of the predetermined semiconductor field-effect ion sensor. Method for forming an enzyme-immobilized membrane.