JPH0810207B2 - Biosensor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a biosensor used in the fields of chemical / food industry, medical care, environmental measurement, and the like, and particularly to an enzyme sensor which is required to have high responsiveness.

[Prior Art and Problems] In recent years, enzyme sensors have been attracting attention. For example, an enzyme immobilization membrane formed by immobilizing an enzyme on a hydrophobic porous inorganic carrier or a polymer carrier and a transducer such as an electrode are combined to immobilize the enzyme. An enzyme sensor has been developed in which changes in chemical substances and the like caused by an enzymatic reaction when passing through a sample solution in a chemical film are converted into electric signals by a transducer to measure a specific substrate concentration.

However, the enzyme-immobilized membrane is an obstacle to the diffusion of the sample solution,
In particular, when the enzyme used is low in activity and the membrane is thickened to increase the amount of enzyme immobilized, a remarkable delay in responsiveness occurs.

Therefore, making the enzyme-immobilized membrane as thin as possible has been studied.

However, if the enzyme immobilization membrane is made thin, the amount of enzyme immobilization decreases and the output signal becomes small, so the sensitivity to low-concentration samples deteriorates and the miniaturization of the sensor is hindered.
In addition, the strength of the enzyme-immobilized membrane is reduced.

[Means for Solving the Problem and Action] Therefore, the present invention has solved the problem by the following means.

(1) To have electrodes provided on both sides of the enzyme-immobilized membrane for electropermeating a sample solution through the enzyme-immobilized membrane, and an electrode for converting a substance change of an enzyme reaction in the enzyme-immobilized membrane into an electric signal. Characteristic biosensor.

(2) Electrodes that are provided on both sides of the non-immobilized membrane located on the front side of the enzyme-immobilized membrane and allow the sample solution to electropermeate through the non-immobilized membrane, and an electrical signal indicating the substance change of the enzyme reaction in the enzyme-immobilized membrane. A biosensor, comprising: an electrode that converts into.

(3) Electrodes that are provided on both sides of the enzyme-immobilized membrane and that allow the sample solution to electropermeate through the enzyme-immobilized membrane and also convert the substance change of the enzyme reaction in the enzyme-immobilized membrane into an electric signal,
A biosensor comprising:

Generally, the enzyme-immobilized membrane or non-immobilized membrane (solid phase) is charged positively or negatively with respect to the sample solution (liquid phase) passing through it, and this charge is especially the electrokinetic potential (ζ potential). Is measured as Therefore, taking the electrode type enzyme sensor as shown in FIG. 1 as an example, this ζ potential is-(minus).
In this case, when a DC voltage is applied by the electrode 5 for electroosmosis generation as shown in FIG.
The sample liquid diffuses in the enzyme-immobilized membrane 4 and is quickly transferred to the measurement electrode 2 (in the direction of the broken arrow).

In particular, the present invention can be preferably applied to an electrode-type enzyme sensor that employs a measurement electrode as a transducer, has excellent responsiveness, and enables highly accurate analysis.

As the porous carrier constituting the enzyme-immobilized membrane or the non-immobilized membrane (also referred to as “enzyme-immobilized membrane or the like”), it is preferable to use a porous carrier that is easily charged due to the relationship with the sample solution. This chargeability is
It depends on the adsorption of ions from the sample solution and the ionization of the carrier itself. Therefore, depending on the sample liquid to be measured from that point of view,
It is preferable to select and use the most suitable material from various inorganic and organic materials. As inorganic carriers, oxide ceramics such as alumina, magnesia, titania, zirconia; phosphates (apatite, calcium phosphate, etc.), perovskites, titanates (barium titanate), aluminates (mullite, spinel), silicic acid Double oxide ceramics such as salts (zircon); glass such as silica and borosilicate; and crystallized glass such as β-spodumene and cordierite. On the other hand, nitride ceramics and carbide ceramics are not preferable because the adsorption of sample liquid ions and the ionization of the ceramics themselves are insufficient. Examples of the organic carrier include polymeric materials such as polyethylene, polystyrene and polyurethane. In particular, when the porous carrier made of these materials is actually used as an enzyme-immobilized membrane, the absolute value of ζ potential generated between the sample solution and the sample solution is at least 5 mV, especially 20 mV.
A material of mV or more is preferable. The higher the ζ potential is, the greater electroosmotic force is obtained, which is advantageous for improving the responsiveness. Also, regarding the porous carrier, the porosity is 60% and the average pore size is
A thickness of about 0.3 and a thickness of about 0.5 mm is preferable.

The electrode for electroosmosis generation may be directly attached to both sides of the enzyme-immobilized membrane or the like. As the material, there can be widely used ordinary electrode materials such as precious metals, refractory metals, metal oxides, etc., especially precious metals (Au, Pt) with excellent chemical stability and corrosion resistance.
Etc.) are preferred. Regarding the porosity, it is preferable that the electrode material is present in an amount of 40 to 70% with respect to the total area in the cross section in the transfer direction. This electrode may be provided by applying a conductive material to the enzyme immobilization film or the like by printing, plating, sputtering, vapor deposition or the like, or by sandwiching the enzyme immobilization film with a separate mesh-shaped electrode. .

On one or both sides of the enzyme-immobilized membrane, harmful substances that cause measurement errors (eg, ascorbic acid and uric acid in blood glucose measurement; other alcohols, acids, and aldehydes in ethanol measurement) adhere to the measurement electrodes. In order to prevent this, it is preferable to have a permselective membrane. The permselective membrane is cellulose acetate,
Made of Teflon, Alumina gel, etc., thickness 0.1 ~ 10μm,
Porosity should be around 30-70%.

In addition, it is preferable that each element (enzyme-immobilized membrane, selective membrane, electrode, etc.) is in close contact. However, there may be some gaps between the elements.

[Example] An ethanol sensor will be described below as an example.

(1) Immobilization of enzyme In advance, alumina purity 99.9%, porosity 36%, average pore diameter
0.27 [mu] m, a bulk density of 2.6 g / cm ^3, to prepare a porous alumina (carrier) in size 3 φ × 0.5 ^{t mm.}

The alumina porous body is subjected to immobilization treatment by the following steps (a) to (e).

(A) The porous body is immersed in a solution containing 10 g of γ-aminopropyltriethoxysilane and 90 g of toluene at room temperature for 15 hours.

(B) 5 minutes with 100 ml of toluene, 5 minutes with 100 ml of ethanol
And wash with 100 ml of pure water for 10 minutes.

(C) Glutaraldehyde after air-drying for 1 hour 2.
Immerse in a solution consisting of 5 g and 97.5 g of phosphate buffer for 2 hours at room temperature.

(D) Immerse in a solution consisting of 0.5 g of alcohol oxidase (commercially available product) and 100 ml of phosphate buffer at room temperature for 1 hour.

(E) Rinse with phosphate buffer for 2 hours at room temperature.

 Thus, the enzyme-immobilized membrane was obtained.

(2) Fabrication of sensor element As shown in Fig. 2 and Fig. 2, alumina porcelain substrate (size 10 x
A platinum electrode 2 as a measuring electrode is vapor-deposited on a 10 × 0.6 ^t mm) 1. Cellulose acetate membrane (thickness 2 μm) 3 as selective permeation membrane 3, gold mesh (existing portion: non-existing portion ≒ 3: 7) 5 as electrode for electrode permeation generation, enzyme immobilized membrane
4, Gold mesh 5 is overlaid, and finally an adhesive is used to cover and seal the cover 6 having the liquid discharge port 6a. The sensor element is now assembled (Figs. 3 and 4).

(3) Measurement An electrometer was connected to the terminals a and b of the platinum electrode 2, a DC voltage was applied to the terminals c and d of the gold mesh electrode 5, and 0.1 ml of test solutions of various ethanol concentrations were dropped. , Response time and current value (amperometry signal conversion method) were measured.

Test solution: Phosphate buffer solution (pH 7.0) (reagent) Ethyl alcohol (reagent) The results are shown in Table 1 and FIG.

As is clear from Table 1 and FIG. 5, it is possible to apply a slight DC voltage (about 2 V) to the electrode for electroosmosis generation.
The response time was greatly shortened and the measured current value was large, so high sensitivity could be maintained even for low-concentration samples. In particular, when the enzyme-immobilized membrane was made thick (0.5 mm), it was confirmed that the response was remarkably improved as compared with the case where no voltage was applied (applied voltage 0 V). In addition, even when a selective permeation membrane is interposed between the enzyme-immobilized membrane (electrode for electroosmosis generation) and the measurement electrode as in the present embodiment, sufficient responsiveness can be maintained. It could be confirmed. Although it depends on the measurement conditions, in the case of an ordinary sensor, if the measured current value is less than 1 μA, accurate measurement becomes difficult.

FIG. 6 shows that a non-immobilized siliceous membrane (a porous carrier on which no enzyme is immobilized) 7 is separately provided on the front side of the enzyme-immobilized membrane 4, and the electroosmotic electrodes 5 are provided on both sides of the non-immobilized membrane 7. This is an example. In addition, 8 is a liquid discharge hole.

In the above example, the electrode active material (hydrogen peroxide) generated by the enzyme reaction in the enzyme-immobilized membrane 4 is converted into a current value by the electrode reaction (oxidative decomposition) by the electrode 5 for electroosmosis generation, which causes a measurement error. May result in However, in this embodiment, although there is a slight delay in response as compared with the above-mentioned example, the electrode 5 for electroosmosis generation is used.
The sample liquid can pass through the enzyme-immobilized membrane 4 (and thus can be reliably transferred to the measurement electrode 2) by utilizing the electroosmotic force of the electrode. Moreover, the electrode active material generated by the enzyme reaction can be accurately measured by the measurement electrode 2. It can be converted into a current value, and more accurate measurement is possible.

FIG. 7 shows an embodiment in which one electrode (pair) 5 serves both as an electroosmosis generating electrode and a measuring electrode. Further, a selective permeation membrane 3 is interposed between the pair of electrodes 5 together with the enzyme immobilization membrane 4.

In the present embodiment, one electrode 5 is used to efficiently utilize the electroosmotic force to cause the electrode reaction of the electrode active substance generated by the enzymatic reaction, so that it is the most responsive and has a simplified structure. it can.

The sensor element of each embodiment is inserted into a sample solution for measurement, a flow system for measuring by injecting the sample solution into a through-type cell, and further a batch flow system. It is applicable to any of.

The present invention is not limited to the above embodiment. For example, the electrode for electroosmosis generation, as long as it can exert its action,
The shape, thickness, size, etc. are not critical. Further, the biosensor of the present invention can be applied not only to the ethanol sensor but also to other alcohol sensors, further sugar sensors (measurement of glucose, maltose, etc.), lipid sensors (measurement of cholesterol, etc.). Also, it goes without saying that various methods such as a covalent bond method, a cross-linking method, and an encapsulation method can be used as the method for immobilizing the enzyme on the porous carrier.

In addition, various other biosensors that use immobilized membranes,
It will be obvious that the present invention can be applied to, for example, an immunosensor, a microorganism sensor, an odor sensor, a freshness sensor and the like.

[Brief description of drawings]

FIG. 1 is a sectional view showing an example of a biosensor element for explaining the operation of the present invention, and FIGS. 2 to 4 are views showing an embodiment of the biosensor element according to the present invention (claim 1). 2 is a plan view showing the sensor element for each constituent element (enzyme-immobilized membrane etc.), FIG. 3 is a perspective view of the sensor element, and FIG. 4 is IV- of FIG.
IV cross section, Fig. 5 shows the applied voltage is 0 with the enzyme immobilization membrane of 0.5mm thickness.
FIG. 6 is a test result at V and 10 V, showing the relationship between ethanol concentration and measured current. FIG. 6 is a cross-sectional view showing an embodiment of the biosensor element according to the present invention (claim 2), FIG. 7 is a sectional view showing an embodiment of the biosensor element according to the present invention (claim 3), respectively. A ... Biosensor element 2 ... Pt (for measurement) electrode 3 ... Cellulose acetate (selective permeation membrane) 4 ... Alcohol oxidase (enzyme) immobilization membrane 5 ... Au mesh (for electroosmosis generation) electrode 7 ... … Non-immobilized membrane

Front page continuation (72) Inventor Junichi Tokumoto 14-18 Takatsuji-cho, Mizuho-ku, Nagoya, Aichi Nihon Special Ceramics Co., Ltd. (72) Hideho Aoki 14-18 Takatsuji-cho, Mizuho-ku, Aichi Inside the Special Ceramics Co., Ltd. (72) Inventor Okura Tsuneto 14-18 Takatsuji-cho, Mizuho-ku, Nagoya, Aichi Japan Nihon Special Ceramics Co., Ltd. (72) Inventor Kurokawa 14-14 Takatsuji-cho, Mizuho-ku, Nagoya, Aichi No. 18 Nihon Special Ceramics Co., Ltd.

Claims (3)

[Claims] 1. An electrode, which is provided on both sides of an enzyme-immobilized membrane, for electropermeabilizing a sample solution through the enzyme-immobilized membrane, and an electrode for converting a substance change of an enzyme reaction in the enzyme-immobilized membrane into an electric signal. A biosensor characterized in that 2. An electrode provided on both sides of a non-immobilized membrane located on the front side of the enzyme-immobilized membrane for electropermeabilizing a sample solution through the non-immobilized membrane, and a substance change of an enzyme reaction in the enzyme-immobilized membrane. A biosensor, comprising: an electrode for converting into an electric signal; 3. An electrode, which is provided on both sides of an enzyme-immobilized membrane, for electropermeating a sample solution through the enzyme-immobilized membrane and converting a substance change of an enzyme reaction in the enzyme-immobilized membrane into an electric signal. Characteristic biosensor.