JPH01265150A - Biosensor - Google Patents

Abstract

PURPOSE:To maintain the specified pH in an immobilizing film holding microorganisms, etc., by forming both side faces of the immobilizing film as a flow passage for a buffer soln. and a flow passage for a sample and bringing a platinum cathode into contact with the side face where the buffer soln. flows via a gas permeable membrane. CONSTITUTION:A flow cell 40 is constituted by sandwiching the immobilizing film 34 which holds the microorganisms or enzyme reacting by coming into contact with the sample and providing the flow passage 36 for guiding the buffer soln. in contact with one side face of the immobilizing film 34 and the flow passage 38 for guiding the same in contact with the other side face to said film. A detector 42 is provided to this flow cell 40. The buffer soln. flows in the circumference of the immobilizing film 34 an a platinum cathode electrode 4 which comes into contact with said film via the gas permeable membrane 60. The ammonia oxidizing bacteria held in the immobilizing film 34 in this constitution are held in the state of nearly specified pH by the buffer soln. penetrating in the immobilizing film 34 and, therefore, the sample does not require a pH adjustment and is not diluted by the buffer soln. The working conditions of the sample and the immobilizing film 34 are thus maintained constant.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a biosensor that measures the components of a sample by immobilizing microorganisms, enzymes, cells, etc. as molecular identification elements (receptors) on a porous membrane. Regarding.

[Conventional technology]

This type of biosensor immobilizes microorganisms, enzymes, etc. on an immobilization membrane as a molecular recognition element (receptor).

This is attached to a flow cell, combined with an electrochemical detector using electrodes such as a dissolved oxygen detector, and the sample to be measured (hereinafter referred to simply as the sample) is brought into contact with the immobilized membrane, and the This is a sensor that detects changes caused by biochemical reactions as the output current of the electrode of an electrochemical detector, and processes these measured values in the arithmetic and control circuit to measure the components of the sample. It has characteristics.

(b) It has excellent selectivity (1) Receptors for microorganisms, enzymes, etc. can be used repeatedly (3) Analysis can be performed with simple operations and in a short time) (5) A small amount of sample can be used for analysis. Measurement results can be taken directly as electrical signals, making it suitable for automatic measurement.Therefore, it is expected to have a wide range of applications in medical fields such as blood testing, food industry fields such as food quality control, and environmental measurement fields such as wastewater treatment. is being actively promoted.

[Problem to be solved by the invention]

However, since biosensors use living organisms and biological materials, they have the following characteristics.

(b) The metabolic functions and enzymatic reactions of microorganisms are affected by the surrounding pH conditions, and there is generally a region called optimum p) for the reaction, and when using microorganisms and enzymes as biosenna, stable sensors are It is necessary to keep the pH constant in order to obtain output.

(b) Living organisms and biological substances are generally unstable, and in order to maintain their action and activity, it is necessary to supply trace amounts of metal elements, etc. In some cases, these are added to a buffer solution and supplied. The buffer solution used in this process is important and indispensable for its practical application.

(c) Generally, the measurement range of biosenna is narrow.

Dilution of the sample is often required, and a buffer solution is also required for sample dilution.

Because of these characteristics, in the case of continuous measurement devices using biosensors.

(a) Buffer solution consumption is high (C) Reagent costs are high (d) In the case of a sample with a low concentration, adding a buffer solution to adjust the pH is necessary. There were problems such as dilution of the liquid below the measurement limit.

The present invention was made to solve the above-mentioned problems, and it reduces the amount of buffer solution consumed by circulating and reusing the buffer solution without requiring pH and Jl adjustment of the sample. The purpose of the present invention is to provide a biosenna with improved equipment maintenance.

[Means to solve the problem]

In order to solve the above-mentioned problems, the present invention comprises an immobilized membrane holding microorganisms or enzymes, one side of the immobilized membrane is a flow path for a buffer solution, and the other side is a flow path for a sample. A flow cell configured as a channel and a detector provided on one side of the immobilized membrane to detect the amount of oxygen consumed or the amount of products generated in the reaction between the sample and the immobilized membrane as a current value (+7) above. 71:I-7k17 immobilized biosensor.

(B) A tubular body fixed to the flow cell (B) An anode fixed to one end of the body opposite to the part fixed to the flow cell and provided inside the body (C) An immobilized membrane in contact with the buffer mti mentioned above. A platinum cathode is in contact with one side of the main body via a gas permeable membrane provided at the other end of the main body, and is provided at the seven nodes via an insulating layer). The sensor shall be equipped with a detector consisting of an electrolyte.

[Effect]

In the present invention, a flow cell is constructed by sandwiching an immobilized membrane holding microorganisms or enzymes, and using one side of the immobilized membrane as a flow path for a buffer solution and the other side as a flow path for a sample. This biosenner has a detector equipped with a platinum cathode that is in contact with one side of the immobilized membrane through which the buffer solution flows through a gas permeable membrane. The components of the sample are measured by detecting the amount of oxygen consumed or the amount of products produced in the reaction between the immobilized membrane and the sample as a current value with a detector. Furthermore, the pH inside the immobilized membrane is kept constant by the action of the buffer solution that circulates in contact with the immobilized membrane, so the pH of the sample
Stable continuous measurement is possible without fA adjustment.

〔Example〕

1 to 3 show embodiments of the present invention, in which FIG. 1 is a flow diagram showing the configuration of a measuring device, FIG. 2 is a sectional view showing the configuration of a biosensor 16 of the present invention, and FIG. The figure is the second
It is a partially enlarged view of the figure. In this example, sewage wastewater was used as a sample, and ammonia nitrogen (N
Although the measurement of the components of I(;-N) will be explained as an example, it is also possible to similarly measure the components of other samples using a suitable biosenna.

In FIG. 1, a sample 2 passes through a switching valve 4 and is sent to a measuring section 8 by a liquid sending tube 6.

At this time, air may be mixed into the sample 2 using the air pump 10 to saturate the sample 2 with dissolved oxygen. In the measuring section 8, the sample 2 is passed through the heat exchanger 14 in the constant temperature bath 12.
is heated to a constant temperature of 300CK.

After passing through the biosenna 16 and being measured, it is discharged outside the system. Buffer solution 20 in container 18 is pumped through circulation pump 22
By passing through the heat exchanger 23, the liquid is sent to the biosensor 16, and after contacting the biosensor 16, it is returned to the container 18, and this circulation is repeated. The first standard solution 24 and the second standard solution 26 are stored in respective containers, and are sent to the measuring section 8 by the liquid sending pump 6 by switching the switching valves 28 and 4 at desired times. The biosensor 16 is connected to the calculation/control circuit section 3 by the signal line 3°.
2, and the measurement results are calculated and displayed.

FIG. 2 is a sectional view showing the structure of the biosensor 16 of the present invention, and FIG. 3 is a partially enlarged view of FIG. 2.

The configuration of the biosensor 16 will be explained below with reference to FIGS. 2 and 3. Biosensor 16 was created by the procedure described below. A flow path 36 that sandwiches the immobilized membrane 34 holding microorganisms or enzymes that react with the sample 2 and guides the buffer solution 20 that comes into contact with one side (the right side in the figure) of the immobilized membrane 34. and a flow path 38 that guides the sample 2 that comes into contact with the other side (the left side in the figure), forming a flow cell 40.
2. An inflow portion 44 into the flow path 36 .
The outflow section 46 is connected to the flow path 38 and the inflow section 48 . An outflow portion 50 is provided. The detector 42 is located on one side of the immobilized membrane 34 that contacts the buffer solution 20 (the right side 1i in the figure).
lll), and the amount of oxygen consumed by the reaction between the sample 2 and the immobilized membrane 34 or the product f generated by the reaction is set to 1! It is detected as a flow value.

(a) Tubular main body 52 fixed to the flow cell 40 (b) Main body 52 on the opposite side from the part 54 fixed to the flow cell 40
An anode fixed to one end and provided inside the main body 52 ←→
One side (right side in the figure) of the immobilization membrane 34 that comes into contact with the buffer solution 20 comes into contact with the other end of the main body 52 through a gas permeable membrane 60 that is sealed with an O-ring 58 and An electrolytic solution 6 filled between the anode 56 and the platinum cathode 64 inside the main body 52
The biosensor 16 is equipped with a detector 42 consisting of the following: 6, and is characterized in that a platinum cathode 64 is in contact with the immobilized M34 through a gas permeable membrane 6°.

Next, the immobilized membrane 34 of the biosenna 16 shown in FIGS. 2 and 3 will be described by taking as an example a membrane for measuring the component of ammonia nitrogen (anatomy-N). Ammonia oxidizing bacteria, such as Nitrosomonas eurobaea, were added to 50 m4 of a liquid medium having the composition shown in Table 1.
s europaea ATCC25978) about 10
1Vm! Inoculate 5m/ of culture solution containing this into Loom/
3000 in Erlenmeyer flask. Rotary shaking culture was performed for 6 days at 120 revolutions per minute.

1. Next, this culture solution was used as a liquid medium 500ml having the composition shown in Table 1.
The whole amount was added to M1, and cultured with aeration and stirring at 30'C for 6 days. 100m4 of this culture solution at 20°C, 7 min.
．． After centrifuging at 000 rpm to concentrate the bacterial cells, the bacterial cell suspension was transferred to the skin layer side of the porous asymmetric membrane (
A porous asymmetric membrane is layered with a pore diameter of 0.2 μm), and a porous asymmetric membrane is layered in the same way on the skin layer side, and the skin layers are in contact with each other, and the membranes are bonded to form an immobilized membrane that retains microorganisms. I got 34. This immobilized film 34 is configured as a flow cell 40 having the structure described above, and a detector 42t-
The biosensor 16 is configured to measure the components of ammonia nitrogen (NH-N), and the components are measured as described in FIG. 1 above.

Note that the buffer solution 20 flows around the platinum cathode electrode 64 that contacts the immobilization membrane 34 through the gas permeable membrane 60 in FIGS. 2 and 3, and the sample 2 is on the left side of the immobilization membrane 34. flows. With this configuration, the pH of the ammonia-oxidizing bacteria held on the immobilization membrane 34 is kept almost constant by the buffer solution that permeates the immobilization membrane 341C, so sample 2 does not require pH adjustment.

It is not diluted with the buffer solution 20, and the working conditions of the sample 2 and the immobilized membrane 34 are maintained constant.

FIG. 4 is a diagram showing the characteristics of the biosensor 16 according to the embodiment of the present invention, in which the horizontal axis is time (time) and the vertical axis is senna output
It is a characteristic diagram shown by taking A). Measurement examples of the present invention will be explained below with reference to the drawings of FIGS. 1 to 4.

First, the flow path is switched by the switching valve 4.28, and the first standard solution 24 (for example, ion-exchanged water) containing no ammonia nitrogen (4-N) (Omf/υ) is sent by the pump 6.
Mix air with the air pump 10 to saturate dissolved oxygen,
At the same time, the buffer solution 2
0 is sent by the circulation pump 22 and passed through the flow path 36 of the biosensor 16 to be circulated. The circulation pump 22 is constantly operated during the measurement, and continuously circulates the buffer solution 20 at a constant flow rate of 3 to 4 ml per minute.

At this time, the detector 42 of the biosenna 16 detects the anode 5
A current flows between the first standard solution 24 and the platinum cathode 64 via the electrolytic solution 66, which is connected to the calculation/control circuit section 32 by the signal line 30, and the measurement results are calculated and displayed.
The output current of the biosenna 16 at this time using
Set to 1.

Next, the switching valve 28 is switched to ammonia nitrogen (NH4-
A second standard solution 26 with a known concentration of N) is sent by the pump 6, air is mixed in with the air pump 10 to saturate the dissolved oxygen, and the solution is flowed into the flow path 38 of the biosensor 16 and measured in the same manner as above. Then, the ammonia-oxidizing bacteria retained within the immobilized membrane 34 of the biosensor 16 are exposed to this second standard solution 2.
In order to act on ammonia nitrogen (NH-N) of 6 and oxidize it, the amount of dissolved oxygen near the immobilization membrane 34 decreases, and the output current of the biosenna 16 gradually decreases.
After 0 to 15 minutes, the output becomes constant I2. The difference from the above 11* "1-" 2 is obtained as a value corresponding to the concentration of ammonia nitrogen (+; -N) in the second standard solution 26, and in this way, the biosensor shown in FIG. 16 calibration is completed.

Next, switch the selector valve 4, and ammonia nitrogen (NH; -N
) The sample 2 whose concentration is to be measured is fed by the pump 6, air is mixed in with the air pump 10 to saturate the dissolved oxygen, and the sample 2 is fed into the flow path 38 of the biosensor 16 in the same manner as described above. Measurement results corresponding to the concentration of ammonia nitrogen (NH-N) are obtained. Note that during the above measurements, the buffer solution 20 was cyclically stirred.

Since the liquid level in the container 18 is in contact with air, the dissolved oxygen in the buffer solution 20 is always saturated even without mixing air with an air pump.

In the above measurements. The change in the output current of the biosensor 16 depends on the immobilization membrane 34 and the platinum cathode 64.

If contact is not made through the gas permeable membrane 60 (in this embodiment, an oxygen permeable membrane such as a Teflon membrane) as in the present invention, dissolved oxygen is supplied to the immobilization membrane 34 by the buffer solution 20. This rate is higher than the oxygen consumption rate of the microorganisms retained on the immobilized membrane 34, and therefore, the decrease in dissolved oxygen in sample 2 due to annular oxidation cannot be detected. In the present invention, as explained above,
A platinum cathode 6 is attached to the immobilization membrane 34 via a gas permeable membrane 60.
4 is in contact with each other, the dissolved oxygen in sample 2 is immobilized [3
4, the oxygen is consumed by microorganisms and decreased.

Because it passes through the gas permeable membrane 60 and reaches the platinum cathode 64, the structure of the detector 42 of the present invention allows the ammonia nitrogen (+N ) has become possible to measure the concentration of

Figure 5 shows the concentration of ammonia nitrogen (mN) (m#/Z) on the horizontal axis and the senna output current difference (μA) on the vertical axis. This is the test Ji line between the concentration of ammonia nitrogen (Peng-N) obtained for the standard solution and the sensor output current difference.
The concentration of N) can be determined.

Furthermore, it has been found that by using the buffer solution 20 used in the measurement having the composition shown in Table 2 below, it is possible to perform long-life measurements over a long period of one month or more.

Table 2 A trace amount of nutritional components for ammonia-oxidizing bacteria was added to the buffer solution with the composition shown in Table 2, and chloramphenicol, a substance that inhibits the growth of other heterotrophic bacteria that causes measurement errors, was added. [Effects of the Invention] According to the present invention, a gas such as a Teflon membrane is attached to an immobilized membrane holding microorganisms such as ammonia-oxidizing bacteria or enzymes. Through the permeable membrane, the platinum cathode of a detector that detects the amount of oxygen consumed in the reaction between the immobilized membrane and the sample or the amount of products produced by the five reactions as a current value is brought into contact with the surrounding area. A buffer solution is made available by flowing and circulating, and the pH can now be maintained at a nearly constant state by the buffer solution permeating the immobilized membrane. Therefore, a large amount of buffer solution, which was conventionally consumed for adjusting the optimum measurement conditions (pH condition, concentration condition) of the sample, can be reused and can be significantly reduced.

In addition to cost savings, we also had the effect of reducing maintenance and management costs for the measuring equipment used. In addition, the amount of buffer solution retained in the equipment used can be reduced, making the equipment more compact, and by constantly circulating the buffer solution, the components necessary for the catalytic activity of microorganisms or enzymes and the growth of heterotrophic bacteria can be prevented. Since inhibiting substances can be continuously supplied, stable measurements can be performed continuously over a long period of time, and the biosensor of the present invention can be used as an environmental measurement (water quality monitor) or a monitor of fermented food industrial processes. It becomes applicable.

[Brief explanation of the drawing]

1 to 3 show an embodiment of the present invention, and FIG. 1 is a flow diagram showing the configuration of a measuring device. Figure WJ2 is a sectional view showing the configuration of the biosensor of the present invention,
Figure 3 is a partially enlarged view of Figure 2, and Figure 4 shows time on the horizontal axis)
Sensor output 1 on the vertical axis! The characteristic diagram of the biosensor shown in Figure 5 shows the current (μA), and the horizontal axis shows ammonia nitrogen (
FIG. 3 is a diagram showing a calibration curve in which the sensor output current difference (μA) is plotted against the concentration (mF/Z) of oyster-N) on the vertical axis. 2... Sample to be measured (sample), 4.28... Switching valve,
6...Liquid pump & 8...Measuring unit, 10...Air pump, 16...Biosensor, 20...Buffer solution, 22...Circulation pump. 32... Arithmetic/control circuit section, 34... Immobilization membrane, 3
6.38...Flow path, 40...Flow cell, 42...
・Detector, 52...Main body. 56... Anode, 60... Gas permeable membrane, 64...
・Shirokane Kang-8/! ! So fixed fηp Figure 1 Darkness m) Figure 4 VU Figure 5

Claims (1)

[Scope of Claims] 1) A channel that sandwiches an immobilized membrane holding a microorganism or enzyme that reacts with a sample to be measured, and that leads a buffer solution that contacts one side of the immobilized membrane, and the other side. a flow cell configured with a flow channel that guides the sample to be measured that contacts the side surface of the buffer solution, and a flow channel that is provided on one side surface of the immobilized membrane that contacts the buffer solution to prevent the reaction between the sample to be measured and the immobilized membrane. A biosensor equipped with a detector that detects the amount of oxygen consumed by or the amount of products produced by a reaction as a current value, (a) a tubular body fixed to the flow cell, and (b) opposite to the part fixed to the flow cell. (c) an anode fixed to one end of the main body and provided inside the main body;
A biosensor comprising: (d) a platinum cathode provided on the anode via an insulating layer; and an electrolytic solution filled between the anode and the platinum cathode inside the main body.