JPS6029657A - Glucose sensor - Google Patents

Abstract

PURPOSE:To permit easy miniaturization and manufacture of multisensors with a simple process and to extend the life of the sensors by forming directly a glucose oxidase-immobilized film on a pH-ISFET by using a photosensitive resin. CONSTITUTION:A pH-ISFET element 1 which is an underlying electrode is provided with sources 2 and 4, drains 3 and 5, pseudo-reference electrode 6 and lead wires 7. This pH-ISFET element is the composite type pH-ISFET element constituted of one pH-ISFET(A) consisting of the source 2 and the drain 3 and another pH-ISFET(B) consisting of the source 4 and the drain 5 as well as the (pseudo) reference electrode 6. The glucose sensor is manufactured by a method for mounting a glucose oxidase-immobilized film on the part 8 of the pH- ISFET(A) and not mounting said film on the another pH-ISFET(B). If there is glucose in the sample soln., the glucose is decomposed and the pH of the glucose oxidase-immobilized film generates a difference in the pH of the sample soln. itself monitored by the pH-ISFET(B) having no glucose oxidase-immobilized film.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel glucose measuring enzyme in which a glucose oxidase-immobilized membrane is formed on the ion-sensitive surface of a hydrogen ion-sensitive field effect transistor (pH-xgFET) using a photosensitive resin. It is related to sensors.

Measurement of glucose concentration in body fluids such as blood and urine is very important for clinical diagnosis, and various methods for quantifying glucose have been developed and improved. Among these, various so-called enzyme cells have been proposed which are capable of rapid and continuous measurement by combining glucose oxidase-immobilized membranes and various electrodes such as hydrogen peroxide electrodes.

The operation of a sensor that combines a glucose immobilization membrane and a hydrogen peroxide electrode, which has been proposed as an enzyme sensor for measuring glucose, will be explained. This enzyme sensor can be manufactured by attaching a membrane in which glucose oxidase is entrappingly immobilized or a membrane in which glucose oxidase is cross-linked and immobilized to the sensitive part of a hydrogen peroxide electrode. Glucose in the sample solution is decomposed into gluconol δ-lactone and hydrogen peroxide within the glucose immobilized membrane according to the following formula (1), and the former is further converted into glucose gluco'-δ-lactone↑↓ OOH HCOH OCH C0II ■ COH CH , 20H gluconic acid Hydrolyzed to gluconic acid. When glucose is contained in the sample solution, hydrogen peroxide is generated according to reaction formula (1), so the glucose concentration can be measured by quantifying the hydrogen peroxide using a hydrogen peroxide electrode.

Various attempts have been made to miniaturize this type of glucose sensor, but it is inherently difficult to miniaturize the glucose sensor because it is difficult to miniaturize the base electrode. Furthermore, it is extremely difficult to create a multi-sensor that can simultaneously measure glucose and various substrates with one sensor. In addition, the manufacturing method is complicated, as it is necessary to prepare the glucose oxidase-immobilized membrane separately and then attach it to the base electrode, and the structure is also complicated.

The present invention eliminates the above-mentioned drawbacks of the conventional ones and provides a glucose sensor that can be easily miniaturized and made into a multi-sensor, and that can be manufactured using a simple manufacturing method by forming the sensor directly on the sensitive surface. I'm self-centered.

The present invention comprises a hydrogen ion-sensitive field effect transistor in which a glucose oxidase-immobilized membrane is bonded to an ion-sensitive surface using a photosensitive resin, a hydrogen ion-sensitive field effect transistor, and a reference electrode. This is a characteristic glucose sensor.

In the present invention, the glucose oxidase-immobilized membrane is prepared by using a photosensitive resin containing glucose oxidase at pH-15F.
It can be applied directly to the ion-sensitive surface of ET and cured, and pH-rsp'g'
It can be patterned onto the ion-sensitive surface of r.

In the present invention, pH-ISF is very small (about several millimeters).
Since the glucose oxidase-immobilized film is formed using ET only on the ion-sensitive surface, it is necessary to limit the light irradiation area using a photosensitive resin, and to apply glucose oxidase y---L only to the necessary areas. ``It is possible to form an immobilized membrane.

Next, the glucose sensor according to the present invention will be explained with reference to the drawings.

FIG. 1 is a perspective view of a pH-15IT device used as a base electrode of a glucose sensor according to the present invention. The pH-15FET element/ which is the base electrode is the source and ψ, and the drain 3
, a similar reference electrode B, and a lead bond 7. This pH-15FET element is one pH-15FBT (A) consisting of a source and a drain 3.

Consisting of source and drain S, the same pH-l as 5/f
This is a composite pH-Ispg'r element composed of 5FET (B) and a K (similar) reference electrode 6. This element can be manufactured in accordance with the manufacturing method of ordinary metal oxide field effect transistors. Here, the reference electrode 6 is a deposited gold film. pH-I5FE'1% ~ and pH-I5FE
Each T (B) is individually sensitive to hydrogen ions, and is necessary to measure the current flowing between the source and drain by applying a constant voltage between them, or to flow a constant current between the source and drain. By measuring the source voltage, it is possible to measure the TIH (hydrogen ion concentration) in the solution.

Next, a glucose oxidase-immobilized membrane was attached to the part of the pH-15FET (A) shown in FIG. 2g, and the other membrane was attached.

By not attaching it to the pH-l5FET (B), the pH inside the oxidase-immobilized membrane is different from the pH of the sample solution itself monitored by the T3H-rsFET (B) without the glucose oxidase-immobilized membrane. What happens is pressure.

Therefore, this glucose sensor has a pH of 1.2
By measuring the source voltage required to flow a constant current between the source and drain of FETs (A) and (B), and amplifying the differential output of the source voltages of both pH-1FF':T, The glucose concentration in the solution can be measured.

The present invention will be explained below based on Examples.

Example l N-methyl-p-formylstyrylpyridinium methosulfate was added to the hydroxyl group of polyvinyl alcohol (the addition rate was og
A 5% by weight aqueous solution of photosensitive resin (Yamamura, described in JP-A-Sho SA-Left 7 Otsu No. 1! (?) was prepared. 3 ml of glucose oxidase was dissolved in 02 ml of this aqueous solution to make a homogeneous Fe solution. This enzyme/photosensitive resin mixed aqueous solution is applied to the source λ and drain 3 as shown in part g in Figure 2.
The channel portion of the pH-15FFT, that is, the ion-sensitive surface of the pH-15FFT, which is made up of the following coatings, was coated widely, and a spinner was used to form a uniform film, and the mixture was dried. after that.

,? 3! Cuts light with a liquid length of less than 1 nm! rOW
Using water anchor lights,! The enzyme/photosensitive resin mixture was irradiated with light for r minutes to form glucose oxidase immobilization (t[).

The response characteristics of the glucose oxidase sensor prepared as described above were measured using -00.2M phosphate buffer (pI-I) at a glucose concentration of 1.
The study was carried out within the range of /l. Figure 3 shows the glucose concentration. O
Fl+? 2 illustrates a response curve of the present example glucose sensor at /t VCts. Curve A in FIG. 5 shows the calibration curve of this glucose sensor. As can be seen from Fig. 3, the response of this glucose sensor is quick, and the response of Fig. q.
As shown in A, there is a linear response to the urea concentration in the range from left to t, oovg/l. Also, the lower limit of detection is r mg
/t. We also evaluated the lifespan of this sensor and found that it was 2! under the condition of 7870 measurements! The output decrease after r days had passed was 3% or less, and the lifespan was sufficiently long.

Example A photosensitive resin aqueous solution containing 0 or 2% by weight of S as described in Example
20 ml of urease and 2 pg of bovine serum albumin were added to make a homogeneous solution. Example 1
Similarly, as shown in FIG. 2, a glucose oxidase-immobilized film G was formed on the ion E and end surface of a pH-15 FET consisting of a source and a drain 3. Next, an operation was performed to increase the mechanical strength of this glucose oxidase-immobilized membrane. That is, the urease-immobilized membrane was immersed in a 1.2S% glutaraldehyde aqueous solution for 10 minutes to mutually crosslink protein molecules by covalent bonds.

This glucose oxidase-immobilized membrane was thoroughly washed with water, and in order to remove remaining glutaraldehyde, 0.1 mol%
The sample was immersed in an aqueous glycine solution for 1 minute. This glucose sensor was then washed with water.

The response characteristics of the glucose sensor manufactured in this way are 00
Evaluation was performed using λM phosphate buffer (pH!rl).

The curve B in FIG. 9 is the calibration curve of this sensor. The linear response range and detection lower limit were almost the same as in Example 1, but the response amount was about %. Note that the life span was also the same as that of Example 1.

Example 3 The photosensitive resin aqueous solution containing 0% by weight of S described in Example 3, . 2
ml K 5 pg of glucose oxidase was processed into a homogeneous solution. Similar to Example 1, this solution was mixed with a pH-l solution consisting of source J and drain 3 as shown in FIG.
Apply it to cover the ion-sensitive surface of the 5FET, and
The floor/photosensitive resin mixture was made into a uniform film using the following method and dried. Next, a glucose oxidase-immobilized membrane was formed only in the ion-sensitive area and its surroundings using a mask that irradiated light only to the area indicated by 9 in FIG. Light irradiation was performed using the same equipment and conditions as described in Example 7.

The response characteristics and lifespan of the glucose sensor in which the glucose oxidase-immobilized membrane was patterned only on the ion-sensitive surface in this manner were the same as those described in Example 1.

In the above examples, N-methyl-p-
Although polyvinyl alcohol having pendant formylstyrylpyridinium methosulfate was used, any photosensitive material that can immobilize urease without deactivating it will have the same effect. For example, polyethylene glycol dimethacrylate!・(For example, benzoin ethyl ether added as a sensitizer), Moon? Rivinyl alcohol (mixed with a diazide compound as a crosslinking agent) can be used. Furthermore, although a noble metal was used to provide the gate voltage, a stable reference electrode such as a silver or silver chloride electrode may also be used.

As described above, according to the present invention, pH is adjusted using a photosensitive resin.
Since the glucose oxidase-immobilized membrane was directly formed on the -l5FET, a glucose sensor that can be easily miniaturized and made into a multi-sensor, and has a simple manufacturing method, and has a sufficient lifespan can be obtained.

[Brief explanation of drawings]

FIG. 7 is a perspective view of a pH-15FET element which is a base electrode of a glucose sensor according to the present invention, FIG. 2 is a perspective view of a glucose sensor according to an embodiment of the present invention, and FIG.
The figure on the left is a perspective view of the glucose sensor in Example 3. In the figure, l...pH-l5FIIT element, 2,9...source. 3. S...Drain, 6... (similar) reference electrode, 7...
Lead wire? ...Glucose oxidase immobilized membrane. Note that the same reference numerals in each figure indicate the same or corresponding parts. Agent Masuo Oiwa Procedural Amendment “Voluntary” 1. Description of the case Japanese Patent Application No. 1314111 2, Name of the invention: Curcose sensor 3, Representative Hitoshi Katayama, Part 6, Contents of the amendment (1) The scope of the claims is amended as shown in the attached sheet. (2) The specification shall be amended as follows. (Attachment) Claims (11) A hydrogen ion-sensitive field effect transistor in which a glucose oxidase-immobilized membrane is bonded to an ion-sensitive surface using a photosensitive resin, a hydrogen ion-sensitive field effect transistor, and a reference electrode. (2) The glucose oxidase-immobilized membrane is obtained by directly spraying and curing a photosensitive resin containing glucose oxidase on the ion-sensitive surface of a hydrogen ion-sensitive field effect transistor. A glucose sensor according to claim 1. (3) A glucose sensor according to claim 1, wherein a glucose oxidase-immobilized membrane is patterned on the ion-sensitive surface of a hydrogen ion-sensitive field effect transistor using photolithography technology. The glucose sensor according to range 2.

Claims (1)

[Claims] (1) A hydrogen ion-sensitive field effect transistor in which a glucose oxidase-immobilized membrane is bonded to an ion-sensitive surface using a photosensitive resin, a hydrogen ion-sensitive field effect transistor, and a reference electrode. glucose sensor. (,l) The glucose sensor according to claim 1, wherein the glucose oxidase-immobilized membrane is obtained by directly coating and curing a photosensitive resin containing glucose oxidase on the ion-sensitive surface of a hydrogen ion-sensitive field effect transistor. . (-?) The glucose sensor according to claim 1, wherein the glucose oxidase-immobilized membrane is patterned on the ion-sensitive surface of a hydrogen ion-sensitive field effect type Tora/Jinuta using photolithography technology.