Biosensor

Abstract

There is provided a cholesterol sensor with high-accuracy and excellent response, whose object to be measured is whole blood, where plasma with which is obtained by filtering out hemocytes therein filtered in blood can rapidly reach an electrode system. In a biosensor where plasma with which is obtained by filtering out hemocytes therein filtered with by a filter is sucked into a sample solution supply pathway due to capillarity, there are formed: a first pressing part for holding a primary side portion of the filter from the bottom; a second pressing part for holding a secondary side portion of the filter from the top and the bottom; a third pressing part for holding the central portion of the filter from the top; and a void for surrounding the filter between the second pressing part and third pressing part.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Section 371 of International Patent Application No. PCT/JP02/04826, filed May 17, 2002, the disclosure of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a biosensor, specifically a cholesterol sensor, capable of carrying out speedy, highly-sensitive, simple determination of a specific component in a sample.

BACKGROUND ART

A description will be given to an example of a conventional biosensor in terms of a glucose sensor.

A typical glucose sensor is obtained by forming an electrode system including at least a measurement electrode and a counter electrode on an insulating base plate by a method such as screen printing and then forming an enzyme reaction layer including a hydrophilic polymer, oxidoreductase and an electron mediator on the electrode system. As oxidoreductase used is glucose oxidase; as the electron mediator used is a small complex, an organic compound or the like, such as potassium ferricyanide, ferrocene derivative or quinone derivative. A buffer is added to the enzyme reaction layer as required.

When a sample solution containing a substrate is added dropwise onto the enzyme reaction layer in this biosensor, the enzyme reaction layer dissolves to cause a reaction of the enzyme with the substrate, which accompanies reduction of the electron mediator. After completion of the enzyme reaction, the substrate concentration in the sample solution can be determined from a value of oxidation current, which is obtained when the reduced electron mediator is electrochemically oxidized.

In this type of glucose sensor, a reductant of the electron mediator generated as a result of the enzyme reaction is oxidized at the electrode, to determine the glucose concentration from the oxidation current value.

Such a biosensor is theoretically capable of measuring diverse substances by using an enzyme whose substrate is an object to be measured. For example, when cholesterol oxidase or cholesterol dehydrogenase is used as oxidoreductase, it is possible to measure a cholesterol value in a serum to be used as a diagnostic indicator in various medical institutions.

Because the enzyme reaction of cholesterol esterase proceeds very slowly, with an appropriate surfactant added thereto, activity of cholesterol esterase can be improved to reduce the time required for the overall reaction.

However, the surfactant, as being included in the reaction system, has an adverse effect on hemocytes, making it impossible to measure whole blood itself, as done in the glucose sensor.

Thereat, a proposal has been made to provide a filter (hemocyte-filtering which out from into the upper surface of the primary side portion of the filter 5 thereinto . In the filter 5, plasma exudes from the termination of the secondary side portion of the filter 5 because the infiltrating rate of hemocytes is slower than that of the plasma which is a liquid component. The exuded plasma then fills the entire sample solution supply pathway 9′ constituted by the slit 9 extended to the vicinity of the electrode system and further to the part of the air aperture 12, while dissolving a reaction layer carried on the position covering the electrode system and/or the reverse face of the cover. Once the entire sample solution supply pathway 9′ is filled with the liquid, the flow of the liquid in the filter 5 also stops and hence the hemocytes are held in the filter 5 at that time, without arriving at the termination of the secondary side portion of the filter 5. It is therefore necessary to design the filter 5 so as to have a difference in flow resistance between the plasma and the hemocytes to the extent that, when the plasma of enough an amount to fill the entire sample solution supply pathway 9′ passes through the filter, the hemocytes do not reach the secondary side portion of the filter 5. A depth filter with a pore size of about 1 to 7 μm is favorably applied to the filter of the present invention. In the case of the example of the present invention, the filter favorably has a thickness of 300 to 400 μm.

After undergoing such a process of filtering out the hemocytes, a chemical reaction of the reaction layer dissolved by the plasma with a component to be measured (cholesterol in the case of a cholesterol sensor) in the plasma occurs, and a current value in the electrode reaction is measured after a lapse of a certain period of time to determine a component in the plasma.

FIG. 4 shows an example of disposition of the reaction layer in the vicinity of the electrode system of the sample solution supply pathway 9′. On the electrode system of the base plate 1 formed are the hydrophilic polymer layer 21 such as sodium carboxymethyl cellulose (hereinafter simply referred to as “CMC”) as well as the reaction layer 22a including a reaction reagent e.g. the electron mediator. The reaction layer 22b including oxidoreductase is formed on the surface exposed to the sample solution supply pathway 9′ on the reverse face of the cover member, which is given by combining the cover 10 and the spacer 6.

As shown in FIGS. 1 to 4, the cross sectional area, vertical to the direction of the flowing liquid, of the sample solution supply pathway 9′ constituted by the slit 9 is made smaller than the cross sectional area of the primary side portion of the filter 5, however, the part at a distance of 1 mm from the secondary side portion of the filter 5 is compressed and disposed in the vicinity of the opening 8 of the sample solution supply pathway 9′. In the case of suction power of a sensor having the size described in the example of the present invention, the part of the filter 5 to be compressed was favorably at a distance of no longer than about 1 mm from the termination of the secondary side portion. Further, with respect to the degree of compression of the secondary side portion of the filter 5, it was preferably that the secondary side portion was compressed into about one fourth to one third of the primary side portion. While it is difficult to represent the suction power to the sensor by a numeric value in such a compressing condition, in the case of a spacer with a thickness of 100 μm, a filter with a thickness of 370 μm exhibited a favorable measurement result (flow-in rate). It should be noted that, in the case of the filter with a thickness of 310 μm or less, the flow-in rate was slower.

As thus described, making the cross sectional area of the sample solution supply pathway 9′ smaller than the cross sectional area of the primary side portion of the filter 5 allows rapid suction of plasma, with which is obtained by filtering out hemocytes therein filtered with in blood by the filter 5, into the sample solution supply pathway 9′ due to capillarity.

The reaction layer generally comprises an easy-to-dissolve part and a hard-to-dissolve part. A portion of the reaction layer at the edge of the sample solution supply pathway 9′, i.e. the part along the wall face of the slit 9 in the spacer 6 is easy to dissolve, whereas the central portion of the reaction layer in the flowering direction of the liquid is hard to dissolve. Since the sample solution having passed through the filter 5 flows along the spacer 6 by priority, there may be cases when the sample solution fills in the air aperture before complete dissolution of the central portion of the reaction layer. Protrusion of the central portion of the secondary side portion of the filter 5 into the sample solution supply pathway 9′ more than the both the right and left terminations thereof enables the priority flow of the sample solution through the central portion of the sample solution supply pathway 9′, whereby the plasma can be rapidly flown into the senor without leaving bubbles at the central portion of the sample solution supply pathway 9′.

In-measurement, when blood as the sample solution is supplied from the sample solution supply part constituted by the aperture 17 of the auxiliary upper cover 16 to the filter 5, the blood infiltrates from the upper surface of the primary side portion of the filter 5 thereinto. In the presence of the third pressing part “c” to serve as a partition at that time, dropwise addition of the sample solution onto the surface of the filter 5 will not be followed by priority flowing of the sample solution along the surface of the filter 5 directly into the sample solution supply pathway 9. Further, in the projection thereof drawing to the plane face which is the same as the base plate 1, the position of the third pressing part “c” does not correspond to that of a first pressing part “a”, whereby neither the expansion of the filter 5 is obstructed nor there is the fear of destroying the hemocytes.

It is preferable that the electrode system comprises a noble metal electrode. With the width of the sample solution supply pathway being preferably not more than 1.5 mm, accuracy in determination of an electrode area is poor in a printing electrode processed by screen printing. As opposed to this, the noble metal electrode exhibits a high accuracy in determination of the electrode area as being able to be subjected to laser trimming by a width of 0.1 mm.

Below, an example of the present invention will be described; however, the present invention is not limited to this.

EXAMPLE

A cholesterol sensor having the configurations of FIGS. 1 to 4, where the reaction layer 22a included the electron mediator and the reaction layer 22b included cholesterol oxidase, cholesterol esterase and a surfactant, was produced.

First, 5 μl of an aqueous solution containing 0.5 wt % of CMC was dropped onto the electrode system of the base plate 1, and dried in a drying apparatus with warm blast at 50° C. for 10 minutes to form the CMC layer 21.

Next, 4 μl of potassium ferricyanide aqueous solution (corresponding to 70 mM of potassium ferricyanide) was dropped onto the CMC layer 21, and dried in the drying apparatus with warm blast at 50° C. for 10 minutes to form the layer 18a including potassium ferricyanide.

Polyoxyethylene (10) octyl phenyl ether (Triton X100) as the surfactant was added to an aqueous solution with cholesterol oxidase originating from Nocardia (EC1.1.3.6) and cholesterol esterase originating from Pseudomonas (EC3.1.1.13) dissolved therein. 0.64 μl of this mixed solution was dropped onto the part (sample supply pathway 9′) of the slit 9 formed by integrating the cover 10 with the spacer 6, prefrozen with liquid nitrogen at −196° C., and dried in a freeze-drying apparatus for two hours, to form the reaction layer 22b including 570 U/ml of cholesterol oxidase, 1,425 U/ml of cholesterol esterase, and 2 wt % of the surfactant.

The slit 9 had a width of 0.08 mm and a length (the length between the opening of the sample solution supply pathway 9′ and the air aperture) of 4.5 mm. The spacer 6 had a thickness (the distance between the base plate 1 and the cover 10) of 100 μm.

As for the filter 5 used is one made of a glass fiber filter having a thickness of about 370 μm in an isosceles triangle shape with a bottom of 3 mm and a height of 5 mm. The tip of the secondary side portion (the part in contact with the opening 8 of the sample solution supply pathway 9′) was roundly processed and then placed between the joint base plate A comprising the base plate 1 and the auxiliary lower cover 20 and the joint base plate B comprising the cover 10 and the spacer 6.

Subsequently, the member obtained by placing the filter 5 between the joint base plate A and the joint base plate B was bonded to the member obtained by integrating the auxiliary plate 13 with the auxiliary upper cover 16, to produce a cholesterol sensor having the structures shown in FIGS. 1, 2 and 4.

10 μl of whole blood as the sample solution was introduced into the sample solution supply part of this sensor; three minutes later, a pulse voltage of +0.2 V was applied to the measuring electrode toward the anode relative to the counter electrode, and five second later, a value of a current flowing between the working electrode and the counter electrode was measured. The results were shown in FIG. 5. FIG. 5 is a graph showing relations between the total cholesterol concentration and the response current.

As is evident from FIG. 5, according to the sensor of the present invention, a favorable linearity between the cholesterol concentration and the response current value was obtained. In FIG. 5, “x” indicates the result of plasma with a ratio of red cell volume of 0%, “O” indicates the result of whole blood with a ratio of red cell volume of 35%, and “□” indicates the result of whole blood with a ratio of red cell volume of 60%.

Industrial Applicability

According to the present invention, hemocytes as interfering substances can be removed without the destruction thereof by a filter, and plasma with which is obtained by filtering out hemocytes therein removed in blood can be supplied with rapidity to an electrode system even with the thickness of the filter being thin. Accordingly, there can be provided a chemical biosensor excellent in response characteristic.

Claims

1. A biosensor comprising:

An an insulating base plate;

an electrode system having a working electrode and a counter electrode which are provided on said base plate;

a reagent including at least oxidoreductase and an electron mediator;

a sample solution supply pathway which includes said electrode system and said reagent and has an air aperture on the termination side thereof;

a sample supply part; and

a filter which is disposed between said sample solution supply pathway and said sample supply part and which filters out hemocytes, where plasma with hemocytes therein filtered with said filter is sucked into said sample solution supply pathway due to capillarity ,

2. The biosensor in accordance with claim 1, characterized in that said primary side portion of said filter is exposed outside at the upper face of the biosensor.

3. The biosensor in accordance with claim 1, characterized in that said secondary side portion of said filter and said working electrode are not in contact with each other.

4. The biosensor in accordance with claim 2, characterized in that said secondary side portion of said filter and said working electrode are not in contact with each other.