JPH0696A - Preservation of microorganism immobilized membrane - Google Patents

Abstract

PURPOSE:To ensure starting assays immediately after installing a microorganism immobilized membrane on a biosensor. CONSTITUTION:A microorganism immobilized membrane is held, together with a buffer solution containing substrate nutrient components at specified concentrations, in a polyvinyl chloride pack followed by immersion in a buffer solution and sealing the pack which is then wet-preserved at low temperatures. When the membrane is to be used, the porous membrane is in a swollen state and the microorganisms exist in the substrate nutrient components; therefore, the microorganisms sustain activity similar to that during use immediately after installing the membrane on a biosensor. Thus, there becomes unnecessary for microorganisms activation prior to the above installation and the aging operation until sensor output gets stabilized, leading to ensuring assays to be started immediately after installing the membrane on the biosensor.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an immobilized microorganism used in a biosensor for analyzing components of a sample liquid by an immobilization membrane in which an enzyme, a microorganism or the like is used as a molecular identification element (receptor) and immobilized on a porous membrane. A method for storing a membrane.

[0002]

2. Description of the Related Art This biosensor uses an immobilization membrane immobilized on a porous membrane by utilizing a biofunctional substance such as an enzyme or a microorganism as a molecular identification element for recognizing a substance to be measured in a sample solution. In combination with an electrochemical detector using electrodes, the component analysis of the sample liquid is performed. In principle, the sample solution is brought into contact with the immobilization membrane, the change caused by the biochemical reaction caused by this is detected as the output current of the electrode, and the measured value is the value obtained by signal processing in the calculation / control section. Since it has excellent selectivity in measurement, such that various substances to be measured can be selected by changing microorganisms and enzymes, it is used in the medical field such as blood tests and in the fermentation / food industry such as food quality control. It is widely used in environmental measurement fields such as measurement and wastewater treatment.

Immobilized microbial membranes used in biosensors are
It is prepared and stored as follows. FIG. 3 is a schematic diagram for explaining this. In FIG. 3, for example, a culture solution 4 of a microorganism in a container such as an Erlenmeyer flask and a porous material such as acetyl cellulose to which an annular spacer 5 is attached. An appropriate amount is collected on the membrane 6, and at this time, suction is performed from below the porous membrane 6 using an aspirator or the like (not shown). In FIG. 3, the collection of the culture solution 4 is shown by a dotted line, and the spacer 5 and the porous membrane 6 are drawn separately in order to make it easy to understand. Next, another porous film 7 which is the same as the porous film 6 is attached onto the spacer 5 so that the spacer 5 is sandwiched between these two porous films 6 and 7, and the microorganisms Among them, the immobilized microbial membrane 2 can be obtained by being immobilized on the porous membranes 6 and 7. The obtained immobilized microbial membrane 2 is then naturally dried and stored in a dry state in a dry state.

[0004]

However, the dry storage of the immobilized microbial membrane 2 has the following problems.
Before the immobilized microbial membrane 2 is attached to a biosensor and used, the material of the porous membranes 6 and 7 is swollen and dried to activate microorganisms in a dormant state, for example, in a phosphate buffer solution. It is necessary to soak it all day and night. Since the activation of the microorganisms gradually increases with the swelling of the porous membranes 6 and 7, stable measurement cannot be performed for 2 to 3 days even after the biosensors are attached. Aging operation to provide nutrients such as glutamate BOD standard solution must be performed. From these things, immobilized microbial membrane 2 attached to biosensor
Immediately after the replacement, it takes 3 to 4 days before stable measurement can be resumed, which causes a time loss.

The present invention has been made in view of the above-mentioned points, and an object of the present invention is to store an immobilized microbial membrane capable of promptly starting measurement after attaching the immobilized microbial membrane to a biosensor. To provide a method.

[0006]

In order to solve the above problems, the method for preserving an immobilized microbial membrane according to the present invention comprises a fixed microbial membrane in a vinyl pack having a high gas permeability, and a predetermined amount of a substrate nutrient component. Then, the vinyl pack is sealed by immersing it in a buffer solution containing the above-mentioned concentration, immersing it in the buffer solution, and storing it in a wet condition at a low temperature of about 5 ° C.

[0007]

Since the method for storing the immobilized microbial membrane of the present invention is wet preservation as described above, the porous membrane which is a constituent material of the immobilized microbial membrane is in a swollen state from the beginning when used. Moreover, since the microorganisms are present in the nutrient component of the substrate, immediately after the immobilized microbial membrane is attached to the biosensor, the same microbial activity as in use is maintained. Therefore, as in the case of conventional dry storage, activation of microorganisms before attachment to the biosensor and aging operation until the sensor output stabilizes are no longer necessary, and after attaching the immobilized microbial membrane to the biosensor. Therefore, the sample liquid can be promptly measured.

[0008]

EXAMPLES The present invention will be described below based on examples. Figure 1
FIG. 3 is a schematic diagram for explaining the method for storing the immobilized microbial membrane of the present invention. As shown in FIG. 1, the present invention includes a substrate micronutrient component (for example, glucose / glutamic acid BOD) in a non-plastic flexible vinyl pack 1 having high gas permeability.
A phosphate buffer solution (for example, pH 7.0, added with a standard solution) so as to have a predetermined concentration (for example, 220 mg / l)
0.1 mg / l) 3 was added, and the immobilized microbial membrane 2 was placed in this
Is immersed, the open end of the vinyl pack 1 is sealed, the immobilized microbial membrane 2 and the buffer solution 3 are sealed, and this is stored in, for example, a refrigerator at a low temperature of 5 ° C. or lower. At this time, in order to prevent the propagation of miscellaneous bacteria during storage and the spoilage of the buffer solution 3, before sealing the immobilized microbial membrane 2 , all of the vinyl pack 1 and the buffer solution 3 are autoclaved to 120
It is desirable to sterilize at 20 ° C for 20 minutes.

Immobilized microbial membrane 2 stored in this way
FIG. 2 shows the initial fluctuation of the sensor output immediately after being attached to the biosensor. FIG. 2 is a diagram in which the vertical axis represents relative output with the sensor output in the first measurement being 1, and the horizontal axis represents elapsed time (hr). For comparison, immobilized microorganisms by a conventional storage method are shown. The case where the film 2 is used is also shown. As can be seen from FIG. 2, in the immobilized microbial membrane 2 dry-stored by the conventional method, before the sensor output appears,
It takes about 1 day, about 1 day for a total of 2 days for the output to stabilize. On the other hand, the biosensor using the immobilized microbial membrane 2 according to the wet preservation method of the present invention is The sensor output is stable in about 3 hours, and the sample liquid can be quickly measured. As described above, this is because the preservation method of the present invention preserves the microbial membrane 2 in the substrate nutrient component of the microorganism in the same swelling state as when used after attaching the biosensor to the biosensor. This is because the activity continues.

The biosensor is generally an immobilized microbial membrane 2
It has a limited lifespan and must be replaced on a regular basis. Therefore, after exchanging the immobilized microbial membrane 2 , it is impossible to measure the sample solution until the sensor output becomes stable, and the shorter the time until the output becomes stable, the more efficient and excellent the practicality. It can be seen that the method of the present invention is effective.

[0011]

EFFECTS OF THE INVENTION In a biosensor, it is necessary to periodically replace the immobilized microbial membrane, but since the conventional method for storing the immobilized microbial membrane is the dry method, the output is stable after being attached to the biosensor. Although it took about 2 days until the preparation, in the present invention, the method for preserving the immobilized microbial membrane is a wet method, and the swelled immobilized microbial membrane is preserved in the nutrient component to maintain the microbial activity. Therefore, after attaching this to the biosensor, the sensor output becomes stable within about 3 hours, the sample solution can be measured quickly, and a highly practical biosensor that operates extremely efficiently can be obtained. it can.

[Brief description of drawings]

FIG. 1 is a schematic diagram for explaining a method for storing an immobilized microbial membrane of the present invention.

FIG. 2 is a biosensor output diagram showing a comparison between the method for preserving the immobilized microbial membrane of the present invention and the conventional method.

FIG. 3 is a schematic diagram for explaining a conventional method for producing and storing an immobilized microbial membrane.

[Explanation of symbols]

1 Vinyl pack 2 Immobilized microbial membrane 3 Buffer solution 4 Culture solution 5 Spacer 6 Porous membrane 7 Porous membrane

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigeru Hatsumata 1-1, Tanabe Nitta, Kawasaki-ku, Kawasaki-shi, Kanagawa Fuji Electric Co., Ltd.

Claims (4)

[Claims] 1. A method of preserving the immobilized microbial membrane of a biosensor for analyzing the components of a sample solution by combining an immobilized microbial membrane comprising a microorganism immobilized on a porous membrane and an electrochemical detector. Then, the method for preserving an immobilized microbial membrane is characterized by immersing the membrane in a liquid in a sealed container and preserving it at low temperature in a wet manner. 2. The method according to claim 1, wherein the sealed container is a vinyl pack. 3. The method for storing an immobilized microbial membrane according to claim 1 or 2, wherein the immobilized microbial membrane is immersed and stored in a buffer solution containing a substrate nutrient component at a predetermined concentration. 4. The method for preserving an immobilized microbial membrane according to claim 1, wherein the immobilized microbial membrane is preserved at 5 ° C. or lower.