JP2008237334A - Method of measuring glucose level in living fish and biosensor used for the method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of measuring a glucose level in living fish which imposes a lesser load on the fish, restrains enzymes from getting deactivated and can measure a glucose level for a long time, and to provide a biosensor used for the method. <P>SOLUTION: The measuring method sticks a biosensor A having a short line working electrode 1 covering the periphery with an insulator 2 except for the tip portion, a counter electrode 5 made up of a silver-silver chloride layer applied to the periphery of the insulator 2, a spherical body 6 mounted on the tip of the working electrode 1 and having a diameter larger than the diameter of the working electrode 1, a measuring portion 8 measuring electrical current flowing through a circuit connecting the working electrode 1 and the counter electrode 5, and an enzyme fixing portion 11 provided on the periphery of the tip portion of the working electrode 1 between the spherical body 6 and the insulator 2 into an interstitial liquid in the interior of a mucous membrane present in the vicinity of an eyeball of fish and measures a glucose level in the living fish. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for measuring a glucose concentration in a fish body using a stromal fluid in the vicinity of fish eyeballs as a specimen, and a biosensor used therefor.

Due to the global decline in fish stocks, the growing fishery of various fish species is thriving year after year. Not only are sick fish hated by consumers, but high fish mortality causes economic losses. Because it grows large, it is necessary to cultivate fish in a stable state without stress, and to keep the amount of antibiotics used to a minimum.
By the way, there is a close relationship between the stress of fish and the blood glucose concentration, and it is said that when the blood glucose concentration is high, the fish feels strong stress and the mortality rate tends to increase.

Conventionally, as a method for examining the health condition of fish, there is a method for examining abnormalities in fish by comparing a test value obtained by examining one or more individuals selected from a school of fish by a hematological test and a blood biochemical test with a test standard range. It is known (see Patent Document 1).
However, in this method, blood must be collected from the arteries and heart of the fish, and the burden on the fish is large. In some cases, laparotomy is required, so the fish to be examined may be killed. Further, since the collected blood is used as a specimen, it was impossible to continue the examination for a long time.

Furthermore, a biosensor has been developed in which a sensor detection unit is formed in a needle shape having a diameter of about 1 mm, and is directly inserted into a living body and measures a body fluid component in an embedded state (see Patent Document 2).
However, a normal bisensor has a diameter of about 0.5 mm to 1.5 mm, and the burden on the fish is not only large, but it is difficult to attach it to live fish for a long time.

JP 2001-37368 A JP-A-2005-230521

  The problem to be solved by the present invention is to measure the glucose concentration in the fish body that can be measured continuously over a long period of time, suppressing the inactivation of the enzyme that is the receptor of the biosensor, with less burden on the fish And a biosensor used for the method.

In the measurement method of the present invention, the glucose concentration in the fish body is measured by a biosensor inserted into the interstitial fluid in the mucous membrane existing near the fish eyeball.
To attach the biosensor, an outer cylinder is fitted to the outer periphery of the injection needle, the injection needle and the outer cylinder are inserted into the mucous membrane near the eyeball of a living fish through the skull, and the injection needle is removed and removed. It is preferable to embed only the cylinder, then insert the biosensor into the outer cylinder, and then bond the biosensor to the skull.

A measurement value such as current measured by a biosensor may be converted into a glucose concentration in the interstitial fluid.
In this case, the measurement value of the biosensor is wirelessly transmitted by the transmission unit, the signal from the transmission unit is received by the reception unit arranged in the vicinity of the living fish, and the conversion unit connected to the reception unit is used. The measurement value of the biosensor may be converted into the glucose concentration in the interstitial fluid.

The biosensor of the present invention comprises a short-line working electrode whose outer periphery excluding the tip is coated with an insulator, a counter electrode composed of a silver / silver chloride layer applied to the outer periphery of the insulator, and a tip of the working electrode. A sphere having a diameter larger than the diameter of the working electrode, a measuring unit for measuring a current flowing in a circuit connecting the working electrode and the counter electrode, and the working electrode between the sphere and the insulator. And an enzyme immobilization part provided on the outer periphery of the tip part.
Alternatively, the short electrode working electrode whose outer periphery excluding the tip is coated with an insulator, a counter electrode composed of a silver / silver chloride layer coated on the outer periphery of the insulator, and the tip of the working electrode are mounted, A sphere having a diameter larger than the diameter of the pole, a measurement unit that measures a current flowing in a circuit that connects the working electrode and the counter electrode, a transmission unit that wirelessly transmits a measurement value of the measurement unit, and the sphere and the insulator And an enzyme immobilization portion provided on the outer periphery of the tip end portion of the working electrode.

According to the inventions according to claims 1 to 4, since the biosensor is inserted into the mucous membrane in the vicinity of the eyeball and the measurement is performed, the burden on the fish can be reduced as compared with the case where the biosensor is inserted into the blood vessel. It is possible to measure the glucose concentration as it is.
In addition, since blood is not used as a measurement target, it is possible to suppress the inactivation of the enzyme (glucose oxidase) due to adhesion of coagulated blood.

In particular, according to the invention of claim 2, since the biosensor is supported by a hard skull, it can be stably attached to a living fish that moves around, and is prevented from coming off due to the movement of the fish.
Further, according to the invention according to claim 4, since it is not necessary to connect the biosensor and the conversion means with a conducting wire, the fish equipped with the biosensor can move freely and the biosensor can be tried on. It can be measured over a long period of time, and there is no fear that the glucose concentration will fluctuate due to stress.

According to the inventions according to claims 5 and 6, the counter electrode is obtained by winding a silver wire subjected to silver / silver chloride treatment electrochemically many times while being extremely fine with a diameter of 0.3 mm to 0.5 mm. It is possible to increase the surface area of the counter electrode as in the case of forming the electrode, and as a result, the polarization can be prevented and the potential can be stabilized.
Also, unlike the conventional ones, the silver / silver chloride treatment on the surface of the silver wire is peeled off, and there is no concern that the exposed silver wire and the two metals of silver / silver chloride will play the role of counter electrodes. High stability is obtained.

In addition, since a sphere with a larger diameter than this is attached to the tip of the working electrode, when immersed in an enzyme solution, an appropriate surface tension is created, so that the enzyme can be securely fixed to a fine enzyme fixing part, and a biosensor can be inserted. It is possible to prevent the enzyme fixed to the enzyme fixing part from peeling off at times.
In addition, since the sphere is provided at the tip, even when it is inserted into the eyeball mucous membrane of a living fish for a long period of time, the stimulation can be reduced and the burden on the fish can be reduced.

1 to 7 show Embodiment 1 of the present invention.
The biosensor A of the present invention is inserted into the interstitial fluid in the mucous membrane existing near the eyeball of a living fish, and as shown in FIG. Attached to the tip of the working electrode 1, an insulator 2 coated on the outer periphery, a silver wire 3 provided on the outer periphery of the insulator 2, a counter electrode 5 composed of a silver / silver chloride layer applied to the outer periphery of the working electrode 1 A spherical body 6, a constant voltage device 7 connected to the working electrode 1 and the counter electrode 5, a measuring unit 8 connected to the constant voltage device 7 and measuring a current flowing through a circuit connecting the working electrode 1 and the counter electrode 5, Is provided.
The constant voltage device 7 and the measurement unit 8 are incorporated in a potentiostat 17. The measurement unit 8 is connected to a conversion means (computer) 15 for converting the measurement value into a glucose concentration via the transmission line 16, and the measurement value of the measurement unit 8 is transmitted to the conversion means 15 via the transmission line 16. It has become so.

The working electrode 1 is obtained by cutting a platinum iridium wire having a diameter of 0.178 mm to a length of 2 cm, and the outer periphery thereof is made of an insulator 2 made of Teflon (registered trademark) or the like except for a length of about 1 mm from the tip. It is covered.
Further, in order to solder the conducting wire 10, a silver wire 3 having a diameter of 0.1 mm is wound about 10 times near the end of the outer periphery of the insulator 2.
The silver / silver chloride layer as the counter electrode 5 is formed by applying a silver paste containing 0.2 to 40% by weight of fine silver chloride powder to the outer periphery of the insulator 2 so as to cover the silver wire 3. The
A constant voltage device 7 is connected to the working electrode 1 and the counter electrode 5 via a soldered lead 10 so that a constant voltage of +0.650 mV can be applied from the constant voltage device 7 to the working electrode 1. Yes.

The sphere 6 is made of an epoxy resin or the like and has a diameter slightly larger than the diameter of the working electrode 1. A recessed portion on the outer periphery of the tip end of the working electrode 1 between the sphere 6 and the insulator 2 serves as an enzyme fixing portion 11.
Before the actual measurement, the biosensor A is immersed in a glucose oxidase solution, and glucose oxidase is fixed to the enzyme fixing unit 11. Glucose oxidase is reliably fixed to the enzyme fixing part 11 by surface tension.

When this biosensor A is inserted into the mucous membrane C, glucose in the interstitial fluid comes into contact with glucose oxidase fixed to the enzyme fixing part 11, and glucose is oxidized by the catalytic reaction of glucose oxidase to generate hydrogen peroxide. .
Since a constant voltage of +0.650 mV is applied to the working electrode 1, the potential of +0.650 mV is always maintained at the working electrode 1 when no glucose oxidation reaction occurs. When this occurs, electrons are received and the potential rises, and the current flowing through the circuit connecting the working electrode 1 and the counter electrode 5 increases.

Since the concentration of generated hydrogen peroxide is proportional to the glucose concentration in the interstitial fluid, the higher the glucose concentration, the larger the current that flows through the circuit connecting the working electrode 1 and the counter electrode 5.
In addition, since the current measured by the biosensor A is proportional to the glucose concentration, it can be easily converted into the glucose concentration by multiplying the current value by a proportionality constant.
Therefore, when the current measured by the measuring unit 8 is transmitted to the conversion unit 15, the conversion unit 15 multiplies the received measurement value by this proportionality constant to convert it into a glucose concentration.

Using the biosensor A of this example, the glucose concentration in the living body of fish (in this example, tilapia) was measured.
Pull tilapia out of the water and anesthetize with 2-phenoxy ethanol.
Next, as shown in FIG. 2, by using a surf flow in which an outer cylinder 13 made of Teflon (registered trademark) is fitted to the outer periphery of the injection needle 12, the injection needle 12 and the outside are placed inside the mucous membrane C near the eyeball B of the tilapia through the skull. After inserting the tube 13, as shown in FIG. 3, the injection needle 12 is pulled out and only the outer tube 13 is embedded.

Next, the outer cylinder 13 protruding from the skin is cut off, the biosensor A is inserted into the outer cylinder 13 from the sphere 6 side, and the biosensor A is inserted into the mucous membrane C near the eyeball B. The outer cylinder 13 may be pulled out after that, or may be left.
Next, the biosensor A is adhered to the skin with an adhesive for soft tissue bonding (for example, trade name: Aron Alpha A, cyanoacrylate adhesive such as “Sankyo Co., Ltd.”).

In addition, 1 ppm of anesthetic (2-phenoxy ethanol) is added to a water tank with a width of 40 cm, a depth of 24 cm, and a height of 15 cm, and two separators are placed inside the water tank. Opened and installed.
Then, the tilapia with the biosensor A attached in the vicinity of the eyeball was housed between two separators, and the current was measured by the measuring unit 8.
After throwing tilapia into the aquarium, wait for a while until the potential stabilizes, then remove it from the aquarium 1.07 hours later (time point a), and measure the glucose concentration in the interstitial fluid near the eyeballs (manufactured by ARKRAY, Inc.) DIAmeter αGLUCOCARD).
Thereafter, tilapia was returned to the water tank, and tilapia was taken out after 1.85 hours (time point b) and 3.44 hours (time point c), respectively, and the glucose concentration in the interstitial fluid was measured with a blood glucose meter.
Table 1 and FIG. 4 show measured current values and blood glucose levels by the biosensor A.

The measured value of biosensor A at time a is 4.37 nA, the glucose concentration by a blood glucose meter is 194 mg / dL, the measured value of biosensor A at time b is 15.11 nA, the glucose concentration by a blood glucose meter is 272 mg / dL, Since the measured value of biosensor A at time point c is 22.46 nA, the blood glucose level G (t) at time point c is obtained from these values by a two-point calibration method.
Since the current and the glucose concentration are proportional to each other, the current value at the time point c is expressed by an equation:
22.46 = S × G (t) + I ₀

When the proportionality constant S and the intercept I ₀ are obtained from the measurement results at time points a and b,
S = (15.11-4.37) / (272-194) = 0.14
I ₀ = 4.37− (0.14 × 194) = − 22.35984.
Therefore,
G (t) = (22.46−I ₀ ) / S = {22.46 − (− 22.36)} / 0.14 = 325 mg / dL.
Since the glucose concentration measured with the blood glucose meter at time c was 317 mg / dL, it was found that the glucose concentration calculated from the current measured by the biosensor A was almost correct.

In addition, FIG. 5 shows the results of measuring the glucose concentration in the interstitial fluid collected from the inside of the mucous membrane near the eyeball of tilapia and the blood glucose concentration at regular intervals using a disposable blood glucose meter.
From FIG. 5, the glucose concentration in the interstitial fluid tends to be slightly lower than the blood glucose concentration, and the glucose concentration in the interstitial fluid changes at a constant rate with the change in the blood glucose concentration. Is highly correlated.
Therefore, blood glucose concentration can be monitored by calculating the glucose concentration in the interstitial fluid from the output of the biosensor A.

Furthermore, the durability and stability of the biosensor A electrode of the present invention and the conventional biosensor electrode which is a counter electrode obtained by winding a silver wire coated with silver / silver chloride on the surface many times were compared.
The biosensor electrode before immobilizing the enzyme to the biosensor A of the present invention and the conventional biosensor electrode are immersed in PBS of pH 6.5, a constant voltage of 0.650 mV is applied, and the current value is Changes were monitored for 1 week. The monitoring results are shown in FIGS.
As can be seen from FIGS. 6 and 7, the biosensor electrode of the present invention showed very good stability, whereas the conventional biosensor electrode showed a large potential shift with time. This difference occurs because conventional biosensor electrodes have a very thin coating because the surface of an ultrafine silver wire is wrapped with an electrochemical coating of silver and silver chloride. If the silver and silver chloride are peeled off easily, the two metals, the inner silver wire and the surface silver chloride, will play the role of counter electrodes, and the potential may be very unstable.

8 and 9 show a second embodiment of the present invention.
As shown in FIG. 8, a transmission unit 9 that wirelessly transmits the measurement value is connected to the measurement unit 8 of the biosensor A, and reception is performed to receive a signal from the transmission unit 9 in the vicinity of a living fish that is a measurement target. The means 14 is installed, and the receiving means 14 is connected to a conversion means 15 for converting the measured current into a glucose concentration.
The constant voltage device 7, the measurement unit 8, and the transmission unit 9 are unitized as a wireless potentiostat 17 ′, and the constant voltage device 7 is connected to the conductor 10 of the biosensor A.

In this embodiment, a commercially available wireless potentiostat base (3102BP, manufactured by PINNACK TECHNOLOGY Co., Ltd.) provided with a transmission means 9 that converts the output signal of the measuring unit 8 into an FM wave and transmits it as the wireless potentiostat 17 ′. Was used after waterproofing. Since this wireless potentiostat 17 'has a small diameter of about 3 cm and a weight of 3 g, it can be attached to the skin of fish using a belt or an adhesive for soft tissue.
Further, since the FM wave can be transmitted in fresh water, it can be received by the receiving means 14 that receives the FM wave.

FIG. 9 shows the result of receiving the signal transmitted from the biosensor A by the receiving means (FM receiver, 3100RX) 14 installed on land, by submerging the biosensor A of the present embodiment in fresh water.
According to the present embodiment, since it is not necessary to directly connect the biosensor A and the conversion means 15 with the transmission line 16, the fish equipped with the biosensor A can move freely, and the biosensor A was tried on. It is possible to measure over a long period of time, and there is no concern that the glucose concentration fluctuates due to stress.

1 is a diagram illustrating a biosensor according to Example 1. FIG. FIG. The figure which shows the state which embed | buried the outer cylinder in the living fish. The figure which shows the measured electric current value. The figure which shows the relationship between the glucose level in interstitial fluid, and the blood glucose level. The figure which shows the change of the electric current value of the biosensor of this invention and the conventional biosensor within a narrow electric current range. The figure which shows the change of the electric current value of the biosensor of this invention and the conventional biosensor in a wide electric current range. The figure which shows the biosensor which concerns on Example 2, and its peripheral device. The figure which shows the result of having monitored the output signal from the biosensor of Example 2. FIG.

Explanation of symbols

A Biosensor B Eyeball C Mucosa 1 Working electrode 2 Insulator 3 Silver wire 5 Counter electrode 6 Sphere 7 Constant voltage device 8 Measuring unit 9 Transmitting means 10 Conducting wire 11 Enzyme fixing unit 12 Injection needle 13 Outer tube 14 Receiving means 15 Converting means 16 Transmitting Line 17 Potentiostat 17 'Wireless Potentiostat

Claims (6)

  A method for measuring the glucose concentration in a fish by using a biosensor inserted into the interstitial fluid in the mucous membrane existing near the eyeball of the fish.   The outer cylinder is fitted to the outer periphery of the injection needle, and the injection needle and the outer cylinder are inserted into the mucous membrane in the vicinity of the eyeball of the living fish through the skull, and then the injection needle is removed to embed only the outer cylinder, The method for measuring a glucose concentration in a fish body according to claim 1, wherein the biosensor is inserted into an outer cylinder, and then the biosensor is adhered to a skull.   The method for measuring a glucose concentration in a fish body according to claim 1 or 2, wherein the measurement value of the biosensor is converted into a glucose concentration in an interstitial fluid.   The measurement value of the biosensor is wirelessly transmitted by a transmission means, a signal from the transmission means is received by a reception means arranged in the vicinity of the living fish, and the biosensor is converted by a conversion means connected to the reception means The method for measuring the glucose concentration in a fish body according to claim 3, wherein the measured value is converted into a glucose concentration in the interstitial fluid.   A biosensor used in the method for measuring a glucose concentration in a fish living body according to any one of claims 1 to 3, wherein a short wire-like working electrode whose outer periphery excluding the tip is covered with an insulator; The working electrode and the counter electrode are connected to a counter electrode made of a silver / silver chloride layer applied to the outer periphery of the insulator, a sphere attached to the tip of the working electrode and having a diameter larger than the diameter of the working electrode. A biosensor comprising: a measurement unit that measures a current flowing through a circuit; and an enzyme immobilization unit provided on an outer periphery of a distal end portion of the working electrode between the sphere and the insulator.   A biosensor used in the method for measuring a glucose concentration in a fish body according to claim 4, wherein a short wire-like working electrode whose outer periphery excluding a tip is covered with an insulator, A counter electrode composed of a silver / silver chloride layer applied to the outer periphery, a sphere attached to the tip of the working electrode and having a diameter larger than the diameter of the working electrode, and a current flowing through a circuit connecting the working electrode and the counter electrode A biosensor comprising: a measuring unit for measuring; a transmitting unit for wirelessly transmitting a measurement value of the measuring unit; and an enzyme fixing unit provided on the outer periphery of the tip of the working electrode between the sphere and the insulator .

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