JP2006317470A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor which yields high reliability and accurate measurement values by using a trace amount of sample. <P>SOLUTION: The biosensor comprises a working electrode substrate 11, a counter electrode substrate 14, and a reagent layer including at least an enzyme and an electron carrier. In the biosensor, a working electrode 12 disposed on the working electrode substrate and a counter electrode 15 disposed on the counter electrode substrate are arranged facing each other, and terminals of a measuring instrument can be made to come into contact with terminals 13, 16 of both the electrodes from through-holes 25, 24. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biosensor for quickly and accurately quantifying a substrate contained in a sample.

As a quantitative analysis method for saccharides such as sucrose and glucose, a photometric method, a colorimetric method, a reduction titration method, a method using various chromatographies, and the like have been developed. However, none of these methods have low accuracy because the specificity for saccharides is not so high. Among these methods, the photometer method is easy to operate, but is greatly affected by the temperature during operation. Therefore, the photometry method is not appropriate as a method for ordinary people to quantify saccharides easily at home.
By the way, in recent years, various types of biosensors utilizing specific catalytic action of enzymes have been developed.

Below, the quantitative method of glucose is demonstrated as an example of the quantitative method of the substrate in a sample solution. As an electrochemical glucose determination method, a method using glucose oxidase (EC 1.1.3.4: hereinafter abbreviated as GOD) and an oxygen electrode or a hydrogen peroxide electrode is generally known (for example, Non-Patent Document 1).
GOD selectively oxidizes β-D-glucose, which is a substrate, to D-glucono-δ-lactone using oxygen as an electron carrier. In the presence of oxygen, oxygen is reduced to hydrogen peroxide during the oxidation reaction by GOD. The amount of decrease in oxygen is measured with an oxygen electrode, or the amount of increase in hydrogen peroxide is measured with a hydrogen peroxide electrode. Since the decrease amount of oxygen and the increase amount of hydrogen peroxide are proportional to the glucose content in the sample solution, glucose is quantified from the decrease amount of oxygen or the increase amount of hydrogen peroxide.
In the above method, as can be inferred from the reaction process, the measurement result is greatly affected by the concentration of oxygen contained in the sample liquid, and measurement is impossible when oxygen is not present in the sample liquid.

Therefore, a new type of glucose sensor has been developed that does not use oxygen as an electron carrier but uses an organic compound or metal complex such as potassium ferricyanide, a ferrocene derivative, or a quinone derivative as an electron carrier. In this type of sensor, the concentration of glucose contained in the sample solution is obtained from the amount of oxidation current by oxidizing the reduced form of the electron carrier generated as a result of the enzyme reaction on the electrode. By using such an organic compound or metal complex as an electron carrier instead of oxygen, a known amount of GOD and those electron carriers are supported on the electrode accurately in a stable state to form a reaction layer. Is possible. In this case, since the reaction layer can be integrated with the electrode system in a state close to a dry state, a disposable glucose sensor based on this technique has attracted much attention in recent years (for example, see Patent Document 1). . In the disposable glucose sensor, the glucose concentration can be easily measured with the measuring instrument simply by introducing the sample solution into the sensor detachably connected to the measuring instrument. Such a method is applicable not only to the determination of glucose but also to the determination of other substrates contained in the sample solution.
Shuichi Suzuki volume "Biosensor" Kodansha Japanese Patent No. 2517153

In the measurement using the glucose sensor as described above, the substrate concentration in the sample can be easily obtained with a sample amount of the order of several μl. However, in recent years, development of a high-performance biosensor that can be measured with a smaller amount of sample, particularly 1 μl or less, is easy to handle, and is highly desired in various fields.
However, in most conventional electrochemical glucose sensors, the electrode system is arranged on one plane. When the electrode system is on one plane and the sample is extremely small, the resistance to charge transfer between the electrodes, mainly ion transfer, increases. As a result, variations in measurement results may occur.

In order to solve the above problems, a biosensor of the present invention comprises a working electrode substrate, a counter electrode substrate, and a reagent layer including at least an enzyme and an electron carrier, and the working electrode and the counter electrode arranged on the working electrode substrate. The counter electrode arranged on the substrate is arranged at a position facing each other across the space.
The present invention comprises a working electrode substrate, a counter electrode substrate, a spacer member interposed between the two substrates, and a reagent layer including at least an enzyme and an electron carrier, and the working electrode and the counter electrode substrate disposed on the working electrode substrate. Provided is a biosensor in which a counter electrode arranged on the top is arranged at a position facing each other through a spacer member.
Here, it is preferable that at least one of the working electrode substrate and the counter electrode substrate has a through-hole that allows the electrode terminal of the other substrate to face the outside.
One of the working electrode substrate and the counter electrode substrate has a notch that exposes the electrode terminal of the other substrate to the outside, and the lead connected to the electrode of the one substrate passes through the side surface of the substrate. It is preferable to be led out to the back side of the surface on which the electrode is disposed.
Alternatively, one of the working electrode substrate and the counter electrode substrate has a cutout portion that exposes the electrode terminal of the other substrate to the outside, and the lead connected to the electrode of the one substrate fills the through hole of the substrate It is preferable to be led out to the back side of the surface on which the electrode is disposed via the conductive material formed.
The present invention also provides an insulating substrate having a groove on the surface, a cover member which is bonded to the insulating substrate and forms a space for accommodating a sample in the groove, and is disposed opposite to the groove. Provided is a biosensor comprising an electrode and a counter electrode, and a reagent layer including at least an enzyme and an electron carrier disposed in the groove.

  According to the present invention, a biosensor that provides a highly reliable and accurate measurement value with a small amount of sample can be obtained.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
FIG. 1 is an exploded perspective view of the glucose sensor excluding the reagent layer in this embodiment, and shows an example of an electrode / lead arrangement.
The working electrode substrate 11 was produced by the following procedure. First, a silver paste was printed on an insulating substrate made of polyethylene terephthalate by screen printing to form a lead 13. Next, a conductive carbon paste containing a resin binder was printed on the substrate to form the working electrode 12. The working electrode 12 is in contact with the lead 13. Furthermore, an insulating paste 17 was formed on the substrate 11 by printing an insulating paste. The insulating layer 17 covers the outer periphery of the working electrode 12, thereby keeping the area of the exposed portion of the working electrode 12 constant.

A counter electrode substrate 14 was produced in the same procedure. That is, the silver paste was printed on the back surface of the insulating substrate to form the lead 16, the conductive carbon paste was then printed to form the counter electrode 15, and the insulating paste was further printed to form the insulating layer 18. Air holes 19 were formed in the counter electrode substrate.
The spacer 21 sandwiched between the working electrode substrate 11 and the counter electrode substrate 14 has a slit 22, and this slit 22 forms a sample supply path between the working electrode substrate and the counter electrode substrate.

An aqueous solution containing GOD and potassium ferricyanide as an electron carrier was dropped onto the working electrode 12 of the working electrode substrate 11 and dried to prepare a reagent layer. Thereafter, the working electrode substrate 11, the counter electrode substrate 14, and the spacer 21 were bonded with a positional relationship as indicated by a one-dot chain line in FIG. 1 to produce a biosensor. The working electrode having the counter electrode and the reagent layer faces each other in the sample supply path formed in the slit 22 portion of the spacer 21. Since the air hole 19 of the counter electrode substrate communicates with the sample supply path, if the sample solution is brought into contact with the sample supply port 23 formed at the open end of the slit, the sample solution is easily supplied by capillary action. Reach the reagent layer in the road.
Next, a solution containing a certain amount of glucose was supplied as a sample to the sample supply path of the sensor, and after a predetermined time had elapsed, glucose was measured by applying a voltage of 500 mV to the working electrode 12 with reference to the counter electrode 15. .
By interposing a spacer between the two substrates, the strength of the sensor increases with respect to the physical pressure acting on the substrate. Therefore, the volume of the sample supply path is easily kept constant, and the influence of physical pressure or the like on the sensor response is reduced.
As a result of the measurement, a current response proportional to the glucose concentration in the liquid was observed, and the variation in response was reduced.

Example 2
FIG. 2 is an exploded perspective view of the glucose sensor excluding the reagent layer according to the present embodiment.
The configuration is the same as that of the first embodiment, except that the working electrode substrate 11 and the counter electrode substrate 14 are provided with through holes 24 and 25 for facing terminals to the outside.
By providing the through holes in both substrates, a part of the lead / terminal 16 of the counter electrode substrate 14 is exposed from the through hole 24 of the working electrode substrate 11. On the other hand, a part of the lead / terminal 13 of the working electrode substrate 11 is exposed from the through hole 25 of the counter electrode substrate 14. When the spacer 21 extends to the terminal portion, a corresponding through hole may be provided in the spacer.
Thereby, the attachment of the bonded sensor chip to the measuring instrument, that is, the electrical connection between the sensor chip and the measuring instrument becomes more reliable, and the measurement accuracy is improved.

Example 3
FIG. 3 is an exploded perspective view of the glucose sensor excluding the reagent layer in the present embodiment.
The working electrode substrate 11 and the spacer 21 have the same configuration as in the first embodiment.
On the other hand, the counter electrode substrate 34 was produced by sputtering palladium on the entire surface including the side surface on an insulating substrate having a cutout portion 36 in which a portion corresponding to the terminal portion 13 of the working electrode substrate 11 was cut out. Therefore, the palladium layer 35 formed on the lower surface of the counter electrode substrate 34 functions as a counter electrode, and this counter electrode is electrically connected to the terminal portion of the palladium layer formed on the upper surface from the side surface of the substrate.
An aqueous solution containing GOD and potassium ferricyanide as an electron carrier was dropped onto the working electrode 12 of the working electrode substrate 11 and dried to prepare a reagent layer. Thereafter, the working electrode substrate 11, the counter electrode substrate 34 provided with the air holes 39, and the spacers 21 were bonded together in a positional relationship as indicated by a one-dot chain line in FIG. 3 to produce a biosensor.
By providing the notch 36 in the counter electrode substrate 34, a part of the lead / terminal of the working electrode substrate 11 is exposed from the notch 36. If the spacer 21 extends to the terminal portion, a corresponding notch may be provided in the spacer 21 as well. On the other hand, the lead electrically connected to the counter electrode 35 is led to the upper surface via the side surface of the counter electrode substrate 34.
As a result, both terminal portions are exposed only on one side. Therefore, for the sensor having such a structure, the connection terminal on the measuring instrument side that has been generally used in the past can be applied as it is, which is effective in reducing the manufacturing cost.
The lead portion disposed on the side surface portion of the sheet-like substrate may have a problem in physical strength as compared with the upper surface portion and the lower surface portion. In such a case, as shown in FIG. 4, a through hole 37 may be provided in the counter electrode substrate 34, and a conductive material such as silver paste or carbon paste may be filled therein. In that case, the lead of the electrode arranged on the lower surface of the substrate is connected to the terminal portion on the upper surface of the substrate via this conductive material.

  In this embodiment, the example in which the notched portion 36 or the through hole 37 is provided in the counter electrode substrate 34 has been described. can get. However, in that case, it is necessary to define the working electrode area using an insulating layer or the like.

Example 4
FIG. 5 is an exploded perspective view of the glucose sensor excluding the reagent layer of the present embodiment.
The insulating substrate 40 has a groove 41 with a free end surface and an upper surface. Sputtering palladium across the upper surface from the opposite side walls of the groove 41, and then trimming with a laser, the working electrode 42 and the counter electrode 45 and lead / terminal portions 43 and 46 corresponding to the respective electrodes were formed. Further, an insulating layer 47 was formed so as to partially cover the lead portion. Next, an aqueous solution containing GOD and potassium ferricyanide was dropped into the groove 41 and dried to form a reagent layer. And the cover 48 which has the air hole 49 in the position corresponding to the back | inner side of the groove | channel 41 was adhere | attached on the board | substrate 40 with the positional relationship as shown by the dashed-dotted line in FIG. 5, and the biosensor was produced.
In this biosensor, the portion of the groove 41 of the substrate is the sample accommodating portion, and the sample solution easily moves to the air hole side by capillary action if the sample solution is brought into contact with the open portion of the groove 41 on the end face of the substrate. In contact with both electrodes.
In a sensor assembled by bonding substrates having electrodes as in the above-described embodiment, there may be a positional shift between the electrodes in the bonding process. In the sensor of the present embodiment, since the electrode system is formed on the inner wall surface of the groove 41, there is no positional deviation between the electrodes in the attaching process, and therefore the measurement accuracy is not lowered due to the positional deviation between the electrodes. .

In the above embodiment, the voltage applied to the electrode system is 500 mV, but the present invention is not limited to this. Any voltage may be used as long as the electron carrier reduced with the enzyme reaction is oxidized.
As the oxidoreductase contained in the reaction layer, one corresponding to the substrate to be measured contained in the sample solution is used. Examples of the oxidoreductase include fructose dehydrogenase, glucose oxidase, alcohol oxidase, lactate oxidase, cholesterol oxidase, xanthine oxidase, and amino acid oxidase.
Examples of the electron carrier include potassium ferricyanide, p-benzoquinone, phenazine methosulfate, methylene blue, and ferrocene induction. A current response can also be obtained when oxygen is used as an electron carrier. One or more of these electron carriers are used.
The reagent layer may be immobilized on the working electrode to insolubilize the enzyme or electron carrier. In the case of immobilization, a cross-linking immobilization method or an adsorption method is preferable. Moreover, you may mix in electrode material.

In the above embodiment, the through holes and notches provided in the substrate are shown in order to bring the specific electrode system, lead / terminal, and measuring instrument side connection terminal into contact with the terminal. The present invention is not limited to the examples.
Moreover, in the said Example, although carbon or palladium was described as an electrode material, it is not limited to this. As the working electrode material, any conductive material that does not oxidize itself when the electron carrier is oxidized can be used. As the counter electrode material, any commonly used conductive material such as silver or platinum can be used.

  The biosensor of the present invention is useful as a sensor for easily quantifying saccharides at home and the like.

It is a disassembled perspective view of the glucose sensor in one Example of this invention. It is a disassembled perspective view of the glucose sensor of the other Example of this invention. It is a disassembled perspective view of the glucose sensor of other Example of this invention. It is a disassembled perspective view of the glucose sensor of other Example of this invention. It is a disassembled perspective view of the glucose sensor of other Example of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Working electrode board 12 Working electrode 13, 16 Terminal 14, 34 Counter board 15, 35 Counter electrode 17, 18 Insulating layer 19, 39 Air hole 21 Spacer 22 Slit 23 Sample supply port 24, 25, 37 Through-hole 36 Notch

Claims (2)

A working electrode substrate, a counter electrode substrate, and a reagent layer including at least an enzyme and an electron carrier,
The working electrode disposed on the working electrode substrate and the counter electrode disposed on the counter electrode substrate are disposed at positions facing each other across the space portion,
One of the working electrode substrate and the counter electrode substrate has a notch for facing the electrode terminal of the other substrate to the outside,
In addition, the lead connected to the electrode of the one substrate is led out to the back side of the surface on which the electrode is disposed via the side surface of the substrate or the conductive material filled in the through hole of the substrate. The biosensor characterized by being made. A working electrode substrate, a counter electrode substrate, a spacer member interposed between the two substrates, and a reagent layer including at least an enzyme and an electron carrier,
The working electrode arranged on the working electrode substrate and the counter electrode arranged on the counter electrode substrate are arranged at positions facing each other through the spacer member,
One of the working electrode substrate and the counter electrode substrate has a notch for facing the electrode terminal of the other substrate to the outside,
In addition, the lead connected to the electrode of the one substrate is led out to the back side of the surface on which the electrode is disposed via the side surface of the substrate or the conductive material filled in the through hole of the substrate. The biosensor characterized by being made.

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