JPS6186644A - Manufacture of biosensor - Google Patents

Abstract

PURPOSE:To obtain economical manufacturing method of a biosensor characterized by a small amount of consumption of enzyme, by forming a plurality of ion sensitive parts on a chip, coating a part other than the sensitive parts by a hydrophobic resin beforehand, holding a liquid including the enzyme, and forming an immobilized enzyme film. CONSTITUTION:A part on a substrate 1 other than ion sensitive parts 2 is coated by a hydrophobic resin 3. A solution including enzyme 4 having albumin and enzyme is formed on one gate by a syringe. The solution including enzyme 4 loses water and a film including enzyme 5 is formed in several minutes. Then glutaraldehyde is supplied thereon as a bringing agent 6. It is repelled by a resin layer (fluoride) and held on the previously formed protein film. This is made to remain for several minutes. Then the protein bridge is favorably expanded, and a fixed enzyme film 7 is formed. Then the product is made to remain intact for a specified time period. After washing with water and glycine treatment, the product is stored in a buffer. Thus a small amount of the enzyme can be well fixed on a minute sensor.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Application of the Invention) The present invention relates to a method for producing a biosensor that has at least two types of minute ion-sensitive parts on one chip and partially has enzymatic activity.

(Prior Art) The present inventor has previously proposed the following as a chip having two types of ion-sensing regions and only one having enzymatic activity. Here, in order to make only one of a pair of electrodes have enzymatic activity on an enzyme-immobilized film previously formed on the entire surface of an ion-sensitive field effect transistor (I8FET) wafer, etc., the enzyme was covered with a quartz photomask. The entire differential electrode was created by shielding only the active part from light and then irradiating it with light to deactivate the remaining part, especially the other ion-sensing part.

In this method, by simply irradiating the enzyme membrane with light, one of the pair of ion-sensitive sections can be used for enzyme activity measurement and the other for comparison, making it possible to easily provide a differential biosensor. On the other hand, most of the enzyme drugs used are inactivated, so the amount of drug used is large, and the disadvantage is that it is basically impossible to create a multi-biosensor that measures two or more types of substrates at the same time. Ta.

Furthermore, to date, there has been no report on an example in which two or more types of enzymes are immobilized on one chip.

(Objective of the Invention) The present invention provides an effective and economical way to develop a multi-biosensor using an element having two or more ion-sensing parts on one chip.
This provides a method for sending r and d.

(Structure of the Invention) The present invention involves forming a plurality of ion-sensing regions on a chip and completely immobilizing at least one enzyme (in an iosensor production method, parts other than the ion-sensing regions are preliminarily coated with a hydrophobic resin). A method for producing a biosensor, which comprises forming an immobilized enzyme membrane by causing the ion-sensitive portion to retain an enzyme-containing liquid after coating.

(Detailed description of the structure) According to the present invention, it is possible to immobilize an enzyme in a very fine ion-sensitive region where it is normally difficult to form an immobilized enzyme. A well-known method for immobilizing enzymes on electrodes is to mix enzymes with soluble proteins and crosslink them with glutaraldehyde or the like. An example of using albumin as a soluble protein is M, Matsushiji and G.
G, Guilbeault ('M, Mascini and
Analytical Chemistry Volume 49, No. 6, May 1977 (An.
analytic chemistry, vol.
49.

No. 6). Here, urease is immobilized to obtain an electrode for urea measurement. In the literature, it is instructed to mix a mixture of enzyme and albumin with gel tar aldehyde just before applying it to the electrode, and to quickly apply it to the electrode surface. According to water droplets, crosslinking reacts quickly in the atmosphere, so the viscosity increases quickly. Urea is quantified using ammonia produced as a result of an enzyme reaction using a Matsushi Niraha ammonia gas sensor.

In this case, since the coating is applied over a relatively wide area, the operation is simple.

The gate section of the smallest φ one that the inventors are using as an ion-sensing section is 50 μm x 400 mm.
．． Two am-sized ones are lined up at 250 μm intervals.
It is a Puer type 15 FET with a total width of 0.6 strips. This structure is shown in FIG. In Fig. 2, 11 is a sapphire substrate, 12 is a lead wire extension, 13 is a drain region, 1
4 is a gate portion and 15 is a source region.

It was previously considered difficult to retain the immobilized enzyme in only one of these. For example, in the method of Massi et al. shown above, the viscosity of the mixture for immobilized enzymes makes it extremely difficult to apply the enzyme to only one side of the thin needle-like sensor.

In order to solve the impractical enzyme immobilization method, the present inventors improved Massini et al.'s method. First, a mixture of enzyme and albumin was held on one gate, and then geltaraldehyde was placed on top of it. We have found a way to create cross-links by attaching them.

However, in this case, the wettability of the albumin-enzyme mixture to the semiconductor surface is very poor, so it was found that although it was easy to adhere, the enzyme film flowed over the entire sensor area.

The present inventors investigated a method for selectively immobilizing an enzyme in such a micro region, and found that by pre-coating the area other than the area where the enzyme is immobilized with a hydrophobic polymer, the enzyme-albumin mixture can be transferred to a mascrosyringe. Therefore, we discovered a method of hepivectoring on gates. That is, a wafer having the above-mentioned 15FET whose parts other than the gate part are covered with a negative resist material usually used for patterning integrated circuits, and whose parts other than the gate part are covered with a fluororesin is prepared, and one of the pair of electrodes is prepared. A solution containing enzyme and albumin was held in a microsyringe. FIG. 1 (al) is a cross-sectional view of the wafer gate portion, showing that portions of the substrate 1 other than the ion-sensitive portion 2 are covered with the hydrophobic resin 3.

Figure 1(b) shows a cross section when an enzyme-containing solution 4 containing albumin and NF element is formed on one gate using a syringe. Due to the hydrophobic nature of the resin layer, it is repelled,
Holds well on the gate. Two types of resin were compared, and it was found that fluorinated resin was superior. This is thought to be because the fluororesin has greater hydrophobicity than the cyclized rubber that is the material of the negative resist material.

The enzyme and albumin mixture shown in FIG. 1(b) does not lose water, and an enzyme-containing film 5 as shown in FIG. 1(c) is formed in a few minutes. If, for example, geltaraldehyde is subsequently supplied thereon as a crosslinking agent 6, the resin F'f4K is also formed.
It is retained on the protein membrane at the tip of the tip (Fig. 1(d)
). When this is left for several minutes, the crosslinking of the proteins progresses smoothly, and an identified enzyme membrane 7 is formed (FIG. 1(e)). Afterwards, as described in Massini's paper, it was left to stand for a certain period of time, washed with water, treated with glycine, and then stored in a buffer.

In this way, by fully adopting the present invention, it has become possible to successfully immobilize an enzyme on a microsensor using only a small amount of enzyme.

Here we have described a differential biosensor that immobilizes a single enzyme, but by repeating the same operation with different types of enzymes, it is easy to create a multi-biosensor that measures multiple substrates simultaneously with a single chip. I can do it.

(Example) Using ISF'ET with three gates '2 on one chip, urease and glucose oxidase are immobilized on each of the two gates, one 'ip)! By using it as an electrode, we have integrated a differential multi-biosensor into one chip. The reference electrode was developed by the present inventors at the 1984 International Conference on Element Materials.
nce on 5olid 5tate Dev
A gold electrode is provided on the back surface as a pseudo reference electrode, as described in the publication titled "Unintegrated SOS/FET Biosensor" in ICES and M; Berials) J.

A plan view of the device and a cross section taken along line A-A' are shown in Figure 3 (
a+, shown in (b). The parts other than the gate part of the element are coated with fluororesin in advance. FIG. 4 shows a cross section of the enzyme immobilized.

Enzyme immobilization basically followed the method of Massey et al.

51n9 nourease (Boehringer Mannheim Lot.

No. 1513. (39,111In915000U
) Add 450 μl of distilled water to the dissolved solution! urease stock solution containing the same ('15% albumin solution 5
2 μl of glucose oxidase (Boehringer Mannheim Lot, No. 1273334.
90'7/25,0OOIJ) dissolved in distilled water 450
Glucose oxidase stock containing 2.5 μl
An aqueous gel taraldehyde solution was prepared for immobilized enzyme. Fluororesin coating on the element surface was easily achieved by the following method. That is, only the three gate portions 22 of the device were previously coated with a positive resist material, and then the entire surface was coated with fluororesin. The thickness of the fluororesin was approximately 5 μm.

Subsequently, the resist material made of novolac resin was dissolved by abrasion with acetone, and at the same time, the fluororesin on the gate was removed. In this way, only the gate part can be easily closed to expose the surface Si3N4 (ferrous silicon) layer, and the remaining parts can be covered with fluororesin by excluding the lead wire extension part to obtain an element covered with fluororesin as shown in fc0 Fig. 3. Of the three gates 22 shown, about 0.05 μl of the above-mentioned urease stock was placed in the leftmost gate using a microsyringe. The results were well preserved as shown in Figure 1 (bl).

Subsequently, the kiglufus oxidase stock was introduced to the rightmost gate surface, and as a result, the culm was successfully maintained as shown in FIG. 1(b). The time required for the above two operations was about 1 minute. After holding both stock solutions on the gate,
After a few minutes, as shown in Figure 1(c), a layer of enzyme and albumin was formed on both gates.Subsequently, a 2.5 tiglutaraldehyde aqueous solution was injected with the same microsyringe as shown in Figure 1(c).
It was guided onto a membrane made of enzyme and albumin as shown in d).

This was left as it was for 15 minutes to obtain the enzyme-immobilized membrane shown in FIG. 1(e)K. The results measured using this element are shown in Figure 5 and Figure 6.
As shown in the figure. The measured values are differential; the difference between the gate voltages at the left end and the center in FIG. 3 is the urea sensor output, and the difference between the gate voltages at the right end and the center is the glucose sensor output. The measurement results were good, demonstrating the superiority of this multi-sensor. The responses of the glucose sensor to urea and the urea sensor to glucose were close to zero.

(Effects of the Invention) As described above, the present invention is an epoch-making invention that can reduce the consumption of enzymes and easily obtain a multi-biosensor. In order to impart hydrophobicity to areas other than the gate part, the most effective method is to form a highly hydrophobic resin such as fluororesin on the surface as shown here, and it is also useful for protecting the sensor.

However, it is clear that the same effect can be brought about by applying wax or the like to areas other than the gate area, for example.

[Brief explanation of drawings]

Figure 1 (al~(elid) is a schematic diagram explaining the details of the present invention, Figure 2 is a schematic diagram of ion-sensitive PET. Figure 3 (a), (b) H1 has three ion-sensitive parts and a common A schematic diagram of an ion-sensitive FFT with a drain. Figure 4 is a cross-sectional view of the gate part of the multi-biosensor. Figure 5 is a correlation diagram between the differential output of the gate voltage of ViFBT and the urea concentration. Figure 6 is the diagram of the gate of PET. A correlation diagram between voltage difference output and glucose concentration is shown. In the figure, 1 is the substrate, 2 is the ion sensing part, 3 is the hydrophobic resin,
4 is an enzyme-containing solution, 5 is an enzyme-containing membrane, 6 is a crosslinking agent, 7 is an immobilized enzyme membrane, 12 is a lead wire extension part, 13.23
14.22 is a gate region, 15.21 is a source region, 30 is a urease-immobilized film, and 31 is a glucose oxidase-immobilized film. E 1 Figure 2 Figure 3 A-A' cross section

Claims (6)

[Claims] (1) In a method for manufacturing a biosensor in which a plurality of ion-sensitive parts are formed on one chip and at least one type of enzyme is immobilized, the parts other than the ion-sensitive parts are coated with a hydrophobic resin in advance, and then the A method for producing a biosensor, comprising forming an immobilized enzyme membrane by holding an enzyme-containing liquid in an ion-sensing part. (2) A claim in which the enzyme-containing liquid comprises a first liquid containing at least an enzyme, and a second liquid that crosslinks the enzyme contained in the first liquid or this enzyme and other substances. 2. A method for producing a biosensor according to item 1. (3) The method for manufacturing a biosensor according to claim 2, wherein the first liquid is first applied to the ion-sensitive part, and then the second liquid is impregnated. (4) Claim 2 or 3, wherein the first solution is a solution in which an enzyme and a soluble protein are dissolved in a buffer solution, and the second solution is a solution containing a compound having reactive groups on both sides of the molecule. The method for manufacturing the biosensor described in Section 1. (5) The method for producing a biosensor according to claim 4, wherein the soluble protein is albumin. (6) The method for producing a biosensor according to claim 4, wherein the compound having reactive groups on both sides of the molecule is glutaraldehyde.