JP2003014764A - Bio-device, quantitative measuring device using the same and quantitative measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bio-device in which a testing substance in a sample solution can be quantitative-measured promptly and with high accuracy and superior reproducibility, a quantitative measuring device using the same and a quantitative measuring method. SOLUTION: In a bio-device for measuring a testing substance included in a test solution, the bio-device comprises a sample introduction part, a detected substance holding part and a determination part. The sample introduction part, the detected substance holding part and the determination part are arranged so that the sample solution introduced into the sample introduction part moves to the determination part through the detected substance holding part, and at least the detected substance holding part and the determination part are constituted by the same member. A first substance group containing a substance which reacts particularly on the test substance is included in the detected substance holding part in a state of being able to elute with the sample solution, and after the first substance group has been eluted from the sample introduced into the sample introduction part, the first substance group is spread as an agglomeration having a start end and a terminal end in elution and spread processes.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biodevice provided for a test method by dry chemistry and for measuring a test substance contained in a sample solution, a quantitative measuring apparatus and a quantitative measuring method using the biodevice.

[0002]

2. Description of the Related Art In recent years, various test methods have been used in clinical tests, and one of the test methods is a dry chemistry test method. The dry chemistry is a method in which a liquid sample is spotted on a reagent stored in a dry state in a solid layer matrix such as a film or a test paper, and a test substance in the sample is measured. As a device, there is a multi-layer type in which a developing layer, a reaction layer, a reagent layer and the like are laminated on a layer, as opposed to a single-layer type in which a reagent is supported on a filter paper. The characteristics are that the reagent is already supported on the solid layer matrix, so that the reagent does not need to be adjusted, the reagent can be stored in a small space, and the amount of the sample to be tested can be small. An immunochromatographic method is a typical test method using dry chemistry. The immunochromatographic method is a test method that utilizes an antigen-antibody reaction and a capillary phenomenon, and the device has an immobilized antibody (antigen) and a detection reagent on a carrier made of a porous material typified by a membrane filter. The antibody (antigen) labeled with is carried in a dry state. During the test, an analyte sample containing an antigen (antibody) is added onto the device, developed by capillary action, and the reaction site is colored by a sandwich-type antigen-antibody reaction at the reaction site. The (antibody) is identified, the presence or absence thereof, or the amount of antigen (antibody) is measured. As a form of the antigen-antibody reaction, there is an immunochromatography method using a competitive type reaction in addition to the sandwich type reaction, but the device structure and the inspection method are the same.

FIG. 1 shows the structure of a conventional immunochromatographic device. FIG. 1A is a top view and FIG. 1B is a side view. A determination unit 16 is formed on a substrate 11 made of a porous material, and a sample introduction unit 13, a detection substance holding unit 12 and a water absorption unit 14 each made of a separate member are provided on the substrate 11, The sample introducing part 13, the detection substance holding part 12, and the water absorbing part 14 are laminated on the backing substrate 15.

In the detection substance holding section 12, a first substance labeled with a labeling substance which reacts specifically with the test substance
The antibody of FIG.
A second antibody, which specifically reacts with the test substance, is immobilized on. The sample solution introduced into the sample introduction unit 13 develops while eluting the first antibody carried by the detection substance holding unit 12, and reaches the determination unit 16. When the test substance is contained in the sample solution, the determination unit 16 forms a complex consisting of the first antibody, the test substance, and the second antibody through the test substance. Detection substance holding unit 12 and determination unit 16
Since the first antibody carried by the detection substance holding unit 12 diffuses from the detection substance holding unit 12 toward the determination unit 16 so as to gradually exude, During the measurement, the flow of the first antibody is not interrupted from the detection substance holding unit 12.

The advantages of the test method using the immunochromatographic method include, in addition to the above-mentioned advantages of dry chemistry, convenience of operation, rapid determination, and low price, and are not limited to clinical tests. Point of Care Testing (hereinafter POCT)
Abbreviated as “)” can be said to be an applicable inspection method.
POCT refers to a clinical test at a medical site, and is a general term for tests in which the time from the collection of a sample to the production of a result is most important.

[0006]

The conventional POCT-compatible biodevice requires 3 to 5 minutes until the determination result is obtained, but further rapidity is required. In addition to qualitative determination, many test substances require quantitative measurement, but conventional quantitative measurement of POCT-compatible biodevices is not sufficient from the viewpoint of reproducibility. It was the current situation.

In view of the above-mentioned conventional problems, the present invention provides a biodevice capable of qualitatively and quantitatively measuring a test substance in a sample solution rapidly, with high accuracy and good reproducibility, and a quantification using the biodevice. An object is to provide a measuring device and a quantitative measuring method.

[0008]

In order to solve the above-mentioned problems, a biodevice of the present invention is a biodevice for measuring a test substance contained in a sample liquid, which comprises a sample introduction part, a detection substance. A holding unit and a determination unit, the sample introduction unit, the detection substance holding unit and the determination unit so that the sample liquid introduced into the sample introduction unit moves to the determination unit via the detection substance holding unit. A first member that is disposed and at least the detection substance holding unit and the determination unit are configured by the same member, and the detection substance holding unit includes a substance that specifically reacts with the test substance. A substance group is contained in a state capable of being eluted by the sample liquid, and after the first substance group is eluted by the sample introduced into the sample introduction part, the first substance group is subjected to an elution and development process. At the beginning Characterized in that it deployed in mass with termination.

According to another aspect of the present invention, the present invention is a biodevice for measuring a test substance contained in a sample liquid, which comprises a sample introduction part, a detection substance holding part and a determination part. The sample introducing unit, the detection substance holding unit and the determination unit are arranged so that the sample liquid introduced into the sample introduction unit moves to the determination unit via the detection substance holding unit, and at least the detection The substance holding unit and the determination unit are configured by the same member, and the detection substance holding unit includes a first substance group containing a substance that specifically reacts with the test substance by the sample solution. It is contained in a state capable of being eluted, and the determination part contains a second substance group containing a substance that specifically reacts with the test substance in a fixed state, The first substance group included in the substance holding unit is In the process of being eluted by the sample solution introduced into the sample introducing section and reaching the determination section together with the sample solution while diffusing in the moving direction of the sample solution, the first sample generated in the moving direction of the sample solution When the end of the diffusion width of the substance group reaches the determination unit, the width in the moving direction of the sample liquid in the portion containing the first substance group in the detection substance holding unit before the introduction of the sample liquid. The ratio A: B between A and the diffusion width B of the first substance group is 1: 0.25.
It is characterized in that it becomes ˜1: 1.

In one embodiment of the present invention, the area of the region between the detection substance holding portion and the determination portion is 3 mm ² or more and 150 mm ² or less.

One embodiment of the present invention is characterized in that the sample introducing part, the detection substance holding part and the judging part are in a dry state before the sample liquid is introduced.

In one embodiment of the present invention, the sample liquid is a body fluid.

In one embodiment of the present invention, the substance that specifically reacts with the test substance contained in the first substance group is a coloring substance, a fluorescent substance, a phosphorescent substance, a luminescent substance, a redox substance, It is characterized by being labeled with an enzyme, a nucleic acid or an endoplasmic reticulum.

One embodiment of the present invention is characterized in that the coloring substance is colloidal gold particles.

One embodiment of the present invention is characterized in that the first substance group contains a first antibody against the test substance and the second substance group contains a second antibody against the test substance.

In one embodiment of the present invention, the first substance group contains a first antibody and a second antibody against a test substance,
And the second antibody is labeled with biotin,
The second substance group includes avidin that specifically reacts with the biotin.

In one embodiment of the present invention, the first substance group includes a first antibody and a second antibody against a test substance,
Moreover, the second antibody is labeled with a magnetic substance, and the second substance group includes a substance for magnetically capturing the magnetic substance.

One embodiment of the present invention is characterized in that the detection substance holding portion and the determination portion are made of a porous material.

One embodiment of the present invention is characterized in that the porous material is a nitrocellulose membrane.

One embodiment of the present invention is characterized in that a sample introducing section is laminated on a porous material which constitutes a detection substance holding section and a determination section.

In one embodiment of the present invention, a sample solution is provided on a porous material in which a detection substance holding part and a determination part are formed, and on the side opposite to the detection substance holding part with respect to the determination part. It is characterized in that a water absorbing portion for absorbing water is laminated.

In one embodiment of the present invention, at least a part of the sample introduction part, the detection substance holding part and the determination part is
It is characterized in that an impermeable material is provided in close contact with the sample liquid.

Further, the quantitative measuring device of the present invention is a quantitative measuring device for measuring a test substance contained in a sample liquid, and is a physical or chemical substance in the biodevice and a determination part of the biodevice. And a measuring device for quantitatively measuring a specific signal.

Further, the quantitative measurement method of the present invention is a quantitative measurement method for measuring a test substance contained in a sample solution using the quantitative measurement device, wherein a fixed amount of the sample solution is introduced into the sample. And a step of quantitatively measuring a physical or chemical signal in the determination part with a measuring device.

Therefore, the invention described herein is a rapid,
Provided are a biodevice that realizes qualitative and quantitative measurement of a test substance in a sample solution with high accuracy and high reproducibility, and a quantitative measurement device and a quantitative measurement method using such a biodevice. The above and other advantages of the present invention will be apparent to those skilled in the art from the following detailed description with reference to the accompanying drawings.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION A biodevice for measuring a test substance contained in a sample solution according to an example of the present invention,
Description will be given using the reference numerals shown in FIGS. 5A and 5B.

The biodevice 50 according to the present invention comprises a sample introduction section 52, a detected substance holding section 51, and a determination section 54. The sample introduction unit 52, the detection substance holding unit 51, and the determination unit 54 are arranged so that the sample liquid introduced into the sample introduction unit 52 moves to the determination unit 54 via the detection substance holding unit 51. At least the detection substance holding unit 51 and the determination unit 54 are configured by the same member.

The detection substance holding section 51 contains a first substance group containing a substance which specifically reacts with the test substance in a state capable of being eluted by the sample solution. After the substance group is eluted by the sample introduced into the sample introduction part 52, the first substance group is developed in a mass having a start end and an end in the elution and development process.

More specifically, the first substance group contained in the detection substance holding section 51 is eluted by the action of the sample solution applied to the sample introducing section 52, and the moving direction of the sample solution (Fig. 5A, while reaching the determination unit 54 together with the sample liquid while diffusing in the direction indicated by arrow M).

The determination section 54 includes a second substance group containing substances that specifically react with the test substance. The second substance group is immobilized on the determination unit 54.

As used herein, the term "holding width A"
Is defined as the width in the sample movement direction of the detection substance holding unit formed by including the first substance group in the same member before applying the sample liquid.

As used herein, the term "diffusion width B"
With respect to the retention width A, the first substance group moves together with the sample liquid after the sample liquid is applied, and the first substance group moves in the moving direction of the sample liquid when the end reaches the determination unit. It is defined as the width of one substance group. According to the present invention, the holding width A: diffusion width B is 1: 0.25 to 1: 1.

The term "moving direction" used in this specification is defined as the moving direction of the sample solution indicated by the arrow A in FIG. 5A.

In the biodevice of the present embodiment, the detected substance holding section 51 and the determination section 54 are made of the same member, so that when the sample solution is introduced into the sample introduction section 52, it is eluted by the sample solution. The first substance group starts moving in the state of a lump, moves while diffusing in the moving direction, and passes through the determination unit 54 in a short time. On the other hand, in the conventional biodevice, the first antibody corresponding to the first substance group carried on the detection substance holding unit 12 is the detection substance holding unit 1
Since the flow diffuses from 2 to the determination unit 16 such that the flow gradually exudes from the detection substance holding unit 12 without interruption, a sufficient amount of the first device is provided as compared with the biodevice of the present embodiment. It took a long time for the antibody to reach the determination unit 16. As described above, in the biodevice according to the present embodiment, the reaction in the determination unit takes place in a shorter time than in the conventional device, so that the determination time is shortened. For example, most of pregnancy diagnostic agents known as immunochromatography devices require 3 to 5 minutes to obtain a sufficient determination result, but the biodevice of the present invention requires less than 3 minutes. The determination result can be surely obtained.

Here, FIG. 13 schematically shows the movement of the first substance group in the biodevice of the present invention in comparison with the conventional device.

FIG. 13 (a) shows the biodevice of the present invention, and FIG. 13 (b) shows the first of the conventional device.
The states of the movement of the substance groups are schematically shown. As shown in (a) of FIG. 13, in the biodevice of the present invention, when the sample liquid is applied to the detection substance holding unit 51, the first substance group in the dry state is detected substance holding unit 51 (deployment direction). It has a predetermined holding width A in the (moving direction); it is eluted as a lump from (shown in the left diagram of FIG. 13 (a)), and moves with the sample liquid with a predetermined diffusion width. Go (as shown in the diagram on the right of FIG. 13 (a)). In contrast,
As shown in FIG. 13B, in the conventional device,
The first antibody corresponding to the first substance group is continuously eluted from the detection substance holding unit toward the determination unit without interruption from the detection substance holding unit, and diffuses so that it gradually exudes. Therefore, it cannot be said that the first antibody migrates in a lump state to the determination part (as shown in the diagram on the right side of FIG. 13B).

The state of elution and migration of the first substance group from the detected substance holding portion in the biodevice of the present invention will be described in more detail.

When the sample liquid is applied to the detection substance holding unit 51, the first substance group in a dry state is eluted from the detection substance holding unit 51 as a lump, and starts to move together with the sample liquid. The width in the moving direction of the mass of the first substance group at the start of movement is usually smaller than 25% of the holding width A. Then, as the first substance group moves toward the determination unit 54, the width of the first substance group increases.

The reason why the width of the first substance group becomes large is as follows.

The developing speed of the sample liquid which is moving the first substance group becomes maximum immediately after the sample liquid is applied to the detection substance holding section 52, and becomes smaller as time passes thereafter. Since the diffusion of the first substance group becomes easier as the developing speed becomes slower, the width of the first substance group increases as time elapses immediately after the introduction of the sample solution.

When the biodevice of the present invention is used to quantitatively measure a test substance contained in a sample solution, it is necessary that the reaction in the judging section 54 occurs uniformly in order to improve the accuracy of the quantitative measurement. Becomes As the width of the first substance group increases, the uniformity of the diffusion state of the first substance group decreases due to the difference in velocity at each point of the moving first substance group, so the accuracy of quantitative measurement is , Depends on the size of the diffusion width B when the first substance group reaches the determination unit 54.

Coefficient of variation is one of the indicators of accuracy in quantitative measurement.
device (hereinafter, abbreviated as CV value), and a device having a smaller CV value is a device with higher accuracy. The CV value is the standard deviation of the measured values / average value * 10.
It is calculated as 0.

In the biodevice of this embodiment, A:
Since the determination unit 54 is provided at a position such that B becomes 1: 0.25 to 1: 1, that is, the ratio B / A × 100 becomes 25 to 100%, it takes a short time and Measurements can be performed with higher accuracy than before, and therefore reproducibility among devices is also improved.

When the determination unit is provided at a position where the ratio B / A × 100 is less than 25%, the time from the introduction of the sample to the passage of the analyte-first substance group complex from the determination unit 54. Is short, the test substance-first substance group complex is the determination unit 5
Since the speed of passing 4 increases and the reaction passes before the reaction sufficiently occurs, a sufficient judgment result cannot be obtained.

On the other hand, when the determination unit 54 is provided at the position where the ratio B / A × 100 is larger than 100%,
On the contrary, when the test substance-first substance group complex passes through the determination unit 54, the time has passed since the sample was introduced, and the development speed decreases, so that the test substance-first substance group complex Although the reaction with the second substance group sufficiently occurs, it takes a long time to pass through the determination unit 54, so that the determination cannot be performed quickly.
Furthermore, since the diffusion width B is large and the uniformity is reduced,
The accuracy in quantitative measurement becomes low.

When the sample liquid has a high viscosity like a blood sample, the developing speed on the biodevice becomes relatively slow, so that the ratio A: B of A and B should be set so that the sample solution passes through the judging section 54 quickly. , 1: 0.25 to 1: 0.50 is preferable. On the other hand, when the sample liquid has a low viscosity such as a urine sample, the developing speed on the biodevice becomes relatively fast, so that the ratio A:
B is preferably 1: 0.50 to 1: 1.

Here, the holding width A and the diffusion width B are, for example,
It can be determined by optical methods. First, for the retention width A, a biodevice is installed on a scanning stage of a reflection absorption spectrometer, the stage is scanned, and the reflection absorbance due to the first substance group is measured. As shown in FIG. A reflection absorbance measurement diagram for is obtained. From FIG. 2, the scanning width in the range where the absorbance was 0.01 or more was defined as the retention width A.

Next, the diffusion width B (with respect to the holding width A, the first substance group moves together with the sample liquid after the sample liquid is applied, and the end of the first substance group reaches the determination part. Regarding the width in the moving direction), the state of the development of the first substance group on the biodevice when the sample solution is introduced into the biodevice is examined by time-dependent reflection absorbance measurement. It is set in advance so that the position where the determination unit 54 is to be provided is irradiated with the incident light on the biodevice. Next, the sample liquid is spotted on the biodevice to develop the first substance group. 1st at the position where the incident light is set
When the group of substances arrives, the absorption starts to be measured and the absorbance value increases rapidly. When the first substance group further moves and the incident light has finished passing through the set position, the absorbance value decreases. The positions of the start end and the end of the first substance group can be confirmed from the change in the absorbance value, and the width of the first substance group when the end of the first substance group reaches the determination unit 54 can be obtained. Specifically, when a reference was made to a portion not containing the first substance group on the biodevice, the once increased absorbance value began to decrease, and was set to a point at which it became 0.3 or less. At the same time as confirming the position of the terminal end, the scanning stage on which the biodevice is mounted is caused to scan in the direction opposite to the developing direction of the sample solution (the direction opposite to the direction indicated by arrow M in (a) of FIG. 5). Then, as shown in FIG. 3, a measurement diagram of the reflection absorbance with respect to the scanning distance is obtained. From FIG. 3, the point at which the once-increased absorbance value decreased and became substantially stable when scanning in the reverse direction from the end of the first substance group was taken as the start of the first substance group. The width of the first substance group in the moving direction is the first
The distance between the starting end and the ending end of the substance group and the scanning distance from the starting end to the ending end was defined as the diffusion width B.

The form of diffusion of the first substance group is not uniform depending on the spread of the sample solution and the surface condition of the biodevice,
In particular, the tail end may be tailed. The reason why the absorbance value at the time of obtaining the end point is set to 0.3 is to remove the tail portion of the tailing from the diffusion width when tailing occurs.

In addition to the optical method, for example, when the detection substance contained in the first substance group has an electrochemical physical property, the electrochemical method is the same as the above optical method. The holding width and diffusion width can be determined by

A biodevice according to another embodiment of the present invention is a biodevice for measuring a test substance contained in a sample liquid, and comprises a sample introducing section 52, a detected substance holding section 51 and a judging section 54. And the sample introduction part 52
The sample introducing section 5 is so arranged that the sample solution introduced into the sample solution moves to the determining section 54 via the detection substance holding section 51.
2, the detection substance holding unit 51 and the determination unit 54 are arranged, and at least the detection substance holding unit 51 and the determination unit 54 are configured by the same member, the detection substance holding unit 51, A first substance group containing a substance that specifically reacts with the test substance is included in a state that can be eluted by the sample solution, and the determination unit 54 is specific for the test substance. The second substance group including the substance that reacts with is contained in a fixed state, and the area of the region between the detection substance holding unit 51 and the determination unit 54 is 3 m.
It is characterized in that it is not less than m ^{2 and not} more than 150 mm ² . By doing so, the time from the addition of the sample liquid to the passage of the first substance group through the determination unit 54 can be controlled, and the determination unit 54 can be controlled.
Since the diffusion width B of the first substance group when passing through is uniform, the same effect as that of the biosensor in the one embodiment of the present invention can be obtained.

When the sample liquid has a high viscosity such as a blood sample, the developing speed on the biodevice becomes relatively slow, so that the detection substance holding section 51 and the determination section 54 should pass through the determination section 54 quickly. Area of the area between 3mm ² or more 2
It is preferably 5 mm ² or less.

On the other hand, when the sample solution has a low viscosity like a urine sample, the developing speed on the biodevice becomes relatively high, so that the sample substance holding unit 51 and the detection substance holding unit 51 should pass slowly through the determination unit 54. The area of the area between the determination unit 54 and the determination unit 54 is 2
It is preferred that the 5 mm ² or more 150 mm ² or less.

In the biodevice of the present invention, it is preferable that the sample introduction part 52, the detected substance holding part 51 and the determination part 54 are in a dry state before the sample liquid is introduced. In this way, the form before the biodevice is used is provided as a dry device, which is excellent in operability,
It can support POCT as a simple device.

The applications of the biodevice of the present invention include:
For example, there are urine test, pregnancy test, water quality test, stool test, soil analysis, and food analysis. The sample liquid may be either an aqueous solution or an organic solution, and examples thereof include body fluid, river water, seawater, groundwater, and an aqueous solution in which soil or food is dissolved. Among these, examples of the body fluid include blood, plasma, serum, urine, saliva, sweat and tears.

As the test substance in the present invention, bacteria,
Examples thereof include proteins and viruses. Examples of bacteria include erythrocytes, tubercle bacilli, and Escherichia coli, and examples of proteins include hemoglobin, hemoglobin A1c, high-density lipoprotein (HDL), and low-density lipoprotein (L
DL), C-reactive protein (CRP), albumin,
Alternatively, examples of the virus such as α-fetoprotein (AFP) include AIDS virus, hepatitis C virus and the like.

Here, the substance which specifically reacts with the test substance contained in the first substance group is a coloring substance, a fluorescent substance, a phosphorescent substance, a luminescent substance, a redox substance, an enzyme, a nucleic acid or an endoplasmic reticulum. Is preferably labeled with. As the coloring substance, gold colloid, silver colloid, selenium colloid, colored latex, cyanine and azo, etc., as the fluorescent substance, aromatic compounds such as pyrene, those having a functional group substituted with an aromatic compound such as dansyl, Fluorescein, rhodamine, coumarin and the like, phosphorescent substances such as benzophenone, and luminescent substances such as luciferin and AT
A substance that emits light by a form such as a luminescence reaction with P, a redox substance that generates an electric current by a form such as a redox reaction between glucose and glucose oxidase, and an endoplasmic reticulum include micelles and liposomes. To be Among these, gold colloid particles are more preferable.

In the present invention, the substance that specifically reacts with the test substance is, for example, an antigen, an antibody, a nucleic acid,
Enzymes and receptors. Here, it is preferable that the first substance group contains a first antibody against the test substance, and the second substance group contains a second antibody against the test substance. First
The antibody and the second antibody may be any antibody that specifically reacts with the test substance, and examples thereof include an anti-cell antibody, an anti-protein antibody, an anti-glycoprotein antibody, an anti-enzyme antibody, an anti-polysaccharide antibody, and an anti-polysaccharide antibody. Bacterial antibodies, and antiviral antibodies are included.
The antibody may be a monoclonal antibody or a polyclonal antibody. The first antibody and the second antibody that specifically react with the same type of test substance may be those that recognize different antigenic determinants (epitope) of the test substance.

In the biodevice of the present invention, the first substance group contains the first antibody and the second antibody against the test substance, the second antibody is labeled with biotin, and the second substance group is It may contain avidin that specifically reacts with the biotin. By doing so, the first antibody and the second antibody labeled with biotin are developed while forming a complex through the test substance in the sample solution, and the complex of the complex is determined in the determination unit 54. By specifically reacting biotin and avidin, the test substance can be captured by the determination unit 54.

In the biodevice of the present invention, the first substance group contains a first antibody and a second antibody against a test substance, and the second antibody is labeled with a magnetic substance,
The second substance group may include a substance for magnetically capturing the magnetic substance. By doing so, the first antibody and the second antibody labeled with the magnetic substance are developed while forming a complex via the test substance in the sample solution, and the complex is determined in the determination unit 54. Since the magnetic substance in the body is magnetically captured, the test substance can be captured by the determination unit 54. Here, examples of the magnetic substance include magnetic particles such as iron oxide and aluminum oxide, and examples of the method of magnetically capturing include a method of providing a magnet or the like in the determination unit.

In the biodevice of the present invention, the material for the sample introduction part 52, the detected substance holding part 51 and the determination part 54 may be any as long as the sample solution is developed at an appropriate speed by the capillary phenomenon. Examples include porous materials such as nitrocellulose membranes, cellulose acetate membranes, and glass filter papers. Of these, nitrocellulose membranes are preferred.

Here, the detection substance holding unit 51 and the determination unit 5
The sample introduction part 52 may be laminated on the porous material of which No. 4 is configured. By doing so, a sufficient amount of the sample liquid can be supplied to the porous material forming the detection substance holding unit 51 and the determination unit 54. Here, as the material of the sample introducing portion 52, for example, a water-absorbing porous material such as a non-woven fabric can be used.

Further, the detected substance holding unit 51 and the determination unit 54
On the porous material having the structure
A water absorbing part 56 for absorbing the sample liquid may be laminated on the side opposite to the detection substance holding part 51 with respect to 4.
By doing so, the detection substance holding unit 51 and the determination unit 54
It is possible to absorb the excess sample liquid on the porous material which is composed of. Here, examples of the material of the water absorbing portion 56 include a porous material such as glass fiber filter paper.

The biodevice of the present invention is preferably housed in a hollow case. Here, the hollow case may be made of, for example, plastic and at least the determination unit and the sample introduction unit are open,
The effect of preventing the sample liquid from leaking to the outside can be obtained.

Further, the sample introduction section 52 and the detected substance holding section 5
It is preferable that a material impermeable to the sample liquid is provided in close contact with at least a part of the first and determination sections 54. For example, an adhesive tape made of a material impermeable to the sample solution may be attached to prevent the sample solution from leaking to the outside, and also to control the flow rate of the sample solution on the biodevice and the flow of the sample solution. The effect of making uniform is obtained.

The quantitative measuring device of the present invention is a quantitative measuring device for measuring a test substance contained in a sample liquid,
The present invention is characterized by including the above biodevice and a measuring device for quantitatively measuring a physical or chemical signal in the determination unit 54 of the biodevice. Here, as the measuring device for quantitatively measuring the physical or chemical signal in the determination unit 54, a change amount of the physical or chemical signal in the determination unit 54 can be digitized. It suffices to use, and for example, in the case of quantifying the change in the coloration intensity of the determination unit 54, one that can measure the reflection spectral absorbance can be mentioned.

The quantitative measuring method of the present invention is a quantitative measuring method for measuring a test substance contained in a sample solution using the above quantitative measuring apparatus, wherein a fixed amount of the sample solution is sampled. The method is characterized by including a step of introducing into the introducing section 52 and a step of quantitatively measuring a physical or chemical signal in the judging section 54 with a measuring device. Here, in the step of introducing a fixed amount of the sample solution into the sample introducing section 52, for example, the sample solution may be accurately weighed using an instrument such as a pipette and spotted on the sample introducing section 52.

In the biodevice of the present embodiment, the detected substance holding section 51 and the determination section 54 are made of the same member, so that when the sample solution is introduced into the sample introduction section 52, it is eluted by the sample solution. The first substance group starts moving in the state of a lump, moves while diffusing in the moving direction, and passes through the determination unit 54 in a short time. On the other hand, in the conventional biodevice, the first antibody corresponding to the first substance group carried on the detection substance holding unit 12 is the detection substance holding unit 1
Since the flow diffuses from 2 to the determination unit 16 such that the flow gradually exudes from the detection substance holding unit 12 without interruption, a sufficient amount of the first device is provided as compared with the biodevice of the present embodiment. It took a long time for the antibody to reach the determination unit 16. As described above, in the biodevice according to the present embodiment, the reaction in the determination unit takes place in a shorter time than in the conventional device, so that the determination time is shortened. For example, most of pregnancy diagnostic agents known as immunochromatography devices require 3 to 5 minutes to obtain a sufficient determination result, but the biodevice of the present invention requires less than 3 minutes. The determination result can be surely obtained.

In the biodevice of this embodiment, A:
Since the determination unit 54 is provided at a position such that B becomes 1: 0.25 to 1: 1, that is, the ratio B / A × 100 becomes 25 to 100%, it takes a short time and Measurements can be performed with higher accuracy than before, and therefore reproducibility among devices is also improved.

[0070]

EXAMPLES The biodevice according to the present invention, a quantitative measurement apparatus and a quantitative measurement method using the same will be described in more detail with reference to the following examples. The present invention is not limited to the examples below.

Example 1 A biodevice for measuring hCG in urine will be described as an example.

(A) Preparation of Judgment Section Position Setting Device First, the judgment section position setting device 4 is subjected to the following procedure.
0 was prepared. FIG. 4 shows the structure of the determination unit position setting device. The position setting device 40 includes a nitrocellulose film 43 having a sample introduction part 42 and a detection substance holding part 41 thereon.

Colloidal gold particles, which are coloring substances, are 0.0
Prepared by adding 1% citric acid solution to a 100 ° C. solution of 1% chloroauric acid at reflux. After refluxing was continued for 30 minutes, the mixture was left standing at room temperature for cooling. After the anti-hCG-α antibody was added to the gold colloid solution adjusted to pH 9 with 0.2 M potassium carbonate solution and stirred for several minutes, p
H9 10% BSA (bovine serum albumin) solution final 1
%, And the mixture was stirred to prepare an antibody-gold colloid complex (labeled antibody) as a detection substance. After the labeled antibody was isolated by centrifuging the labeled antibody solution at 20000 G for 50 minutes at 4 ° C. and suspending it in a washing buffer (1% BSA / phosphate buffer),
The centrifugation was performed to wash and isolate the labeled antibody. The labeled antibody was suspended in a washing buffer, filtered through a 0.8 μm filter, adjusted to 1/10 of the original amount of gold colloid solution, and stored at 4 ° C.

The labeled antibody solution was set in a solution discharge device, applied onto the nitrocellulose film 43, and then the film was dried. As a result, the detection substance holding unit 41 containing the labeled antibody that is the first substance group is attached to the nitrocellulose membrane 4
A biodevice reaction layer carrier having the above 3 was obtained.

The reaction layer carrier 40 containing the detection substance holding portion 41 thus prepared was cut into 0.5 cm wide strips to prepare a biodevice.

(B) Setting of Judgment Section Position 40 μl of a urine sample was spotted on the sample introduction section 42 of the prepared biodevice, the biodevice was installed on the scanning stage of the reflection absorption spectrometer, and the sample solution was introduced. The state of development of the labeled antibody on the biodevice at that time was examined by measuring the reflection absorbance with time. The incident light was set at the position where the judgment part was provided, and the retention width A and the diffusion width B when the end of the diffusion width of the labeled antibody reached the judgment part position were obtained using the above method. The relationship between the determination portion position and the diffusion width B was obtained by changing the setting position of the incident light and performing the measurement.

(C) Preparation of biodevice Next, a biodevice was prepared. The structure of the prepared biodevice is shown in FIG. 5A is a plan view thereof, and FIG. 5B is a sectional view thereof.
The biodevice 50 includes a support body 55 and a support body 5.
5, the detection substance holding unit 51 and the determination unit 5
The nitrocellulose membrane 53 having the number 4 and the water absorbing portion 56 arranged on the nitrocellulose membrane 53.
The sample introduction part 52 is arranged on the nitrocellulose membrane part not containing the detection substance in the vicinity of the detection substance holding part 51, and the water absorption part 56 is provided on the nitrocellulose membrane 53 and with respect to the determination part 54. It is provided on the opposite side. This biodevice 50 was prepared as follows.

First, an anti-hCG-β antibody solution having a concentration adjusted by diluting it with a phosphate buffer solution was prepared. This antibody solution was applied onto the nitrocellulose film 53 using a solution discharge device. As a result, the antibody immobilization line, which is the determination unit 54, was obtained on the nitrocellulose membrane 53. After drying this nitrocellulose membrane, it was immersed in a Tris-HCl buffer solution containing 1% skim milk and gently shaken for 30 minutes. After 30 minutes, the membrane was transferred to a Tris-HCl buffer solution tank and shaken gently for 10 minutes, and then another Tri
Gently shake for another 10 minutes in the s-HCl buffer solution tank,
The membrane was washed. After washing twice, the membrane was taken out of the liquid bath and dried at room temperature.

Next, the detection substance holding part 51 was prepared as follows.

Gold colloids were prepared by adding a 1% citric acid solution to a 100 ° C. solution of 0.01% chloroauric acid at reflux. After refluxing was continued for 30 minutes, the mixture was left standing at room temperature for cooling. With a 0.2 M potassium carbonate solution, p
After the anti-hCG-α antibody was added to the gold colloid solution prepared in H9 and stirred for several minutes, 10% BSA at pH 9 was added.
An antibody-gold colloid complex (labeled antibody), which is a detection substance, was prepared by adding (bovine serum albumin) solution in an amount of 1% at the final and stirring. Add the labeled antibody solution to 4
The labeled antibody is isolated by centrifuging at 20000 G for 50 minutes at 0 ° C and washed with a washing buffer (1% BS).
A. Phosphate buffer) and then the centrifugation was performed to wash and isolate the labeled antibody. The labeled antibody was suspended in a washing buffer, filtered through a 0.8 μm filter, adjusted to 1/10 of the original amount of gold colloid solution, and stored at 4 ° C.

After the labeled antibody solution was set in the solution discharge device and applied to the nitrocellulose membrane 53 on which the anti-hCG-β antibody had been immobilized, at a position away from the determination part 54,
The membrane was dried. As a result, a biodevice reaction layer carrier having the detection substance holding unit 51 on the biodevice was obtained.

The reaction layer carrier containing the detection substance holding section 51 thus prepared was attached on a support 55, a non-woven fabric was added as a sample introducing section 52, and a glass fiber filter paper was added as a water absorbing section 56. 0.5c after covering the area excluding parts with transparent tape (Nitto Denko, not shown)
Biodevices were made by cutting into m-wide strips. Using the result of the examination by the determination unit position setting device 40, the ratio A: B of the retention width A and the diffusion width B when the end of the labeled antibody reaches the determination unit is 1: 0.05,1. : 0.2
5, 1: 0.5, 1: 0.75, 1: 1, 1: 1.0
5, 1: 1.1 and 1: 1.2, ie the ratio B / A × 1
00 is 5, 25, 50, 75, 100, 105, 11
Eight types of biodevices were prepared, five each, so that the ratio was 0 and 120%.

The performance of the biodevice thus manufactured was evaluated.

(D) Evaluation of biodevice 1000 U / in the sample introduction part 52 on each biodevice
About 40 μl of urine containing 1 hCG was added, and the determination unit 54
The state of color development after 5 minutes based on the antigen-antibody reaction was evaluated by reflection absorption measurement. Reflective spectrophotometer (CS9
300: Shimadzu Corporation) was used to measure the absorbance at 520 nm. FIG. 6 shows the absorbance value and the measurement result of the CV value obtained from the measurement results of the five devices. The CV value is calculated by standard deviation of measured values / average of measured values × 100. As can be seen from FIG. 6, a good CV value and a good absorbance value were obtained when the diffusion width B / holding width A was 25% to 100% when the end of the labeled antibody reached the determination unit 54. .

Next, the same measurement was carried out for sample solutions having hCG concentrations of 0, 100, 1000 and 10000 U / l using three types of devices having a ratio B / A × 100 of 5, 50 and 120%. It was

HC is added to the sample introduction unit 52 on the biodevice.
Urine containing G was added in an amount of about 40 μl, and after 5 minutes from the addition of the sample, the color development was measured using the reflection spectrophotometer, and then the coloration calculation processing was performed.

The absorbance at 520 nm was measured and substituted into a calibration curve showing the relationship between the hCG concentration and the absorbance prepared in advance. 7 shows a biodevice having a ratio B / A × 100 of 5% when the end of the diffusion width B of the labeled antibody reaches the determination unit 54, and FIG. 8 shows a biodevice having a ratio B / A × 100 of 50%. And the measurement results in the case of the biodevice having a ratio B / A × 100 of 120% are shown in FIG.

Ideally, for example, 1000 U / l hC
When the absorbance of urine containing G is measured and the absorbance is substituted into the calibration curve, the hCG concentration should be 1000 U / l, but a slight deviation occurs in the measurement using an actual device. The accuracy of the measurement can be known from the magnitude of the deviation.

As can be seen from FIG. 7, the ratio B / A × 100
In the case of a 5% biodevice, it was not possible to measure 100 U / l sample solution. It is considered that this is because the sensitivity in the low-concentration region is low because the light passes through the determination unit 54 before a sufficient reaction occurs. Further, as can be seen from FIG. 9, in the case of the biodevice having a ratio B / A × 100 of 120%, the measurement accuracy was particularly poor in the high concentration range. It is considered that this is because it takes time to pass through the determination unit, so that the flux is disturbed and the reaction is varied. On the other hand, as can be seen from FIG. 8, the ratio B / A × 100
In the case of a biodevice with 50%, accurate and highly accurate quantitative results were obtained.

Example 2 A biodevice was prepared in the same manner as in Example 1. However, three types of biodevices were manufactured so that the areas of the regions between the detection substance holding unit and the determination unit were 175, 50, and 5 mm ² . Sample solutions with hCG concentrations of 0, 100, 1000 and 100
The same measurement as in Example 1 was performed using 00 U / l of urine.

When the area between the detected substance holding portion and the determination portion is 175 mm ² in FIG. 10, and when the area between the detected substance holding portion and the determination portion is 50 mm ² in FIG.
Further, FIG. 12 shows the measurement results in the case where the area between the detection substance holding unit and the determination unit is 5 mm ² . The horizontal axis represents the hCG concentration of the sample added to the biodevice. The vertical axis represents the converted value of hCG concentration obtained by substituting the signal in the determination unit into the calibration curve.

From FIG. 10, when the area of the region between the detection substance holding portion and the determination portion is 175 mm ² , the linearity in the hCG concentration cannot be obtained in the high concentration region of 10000 U / l, and the CV is further increased. The value shows a large variation of 10 to 100%, indicating that the quantitative performance is poor. On the other hand, FIG.
From No. 12 and 12, when the area of the region between the detection substance holding unit and the determination unit is 50 mm ² or 5 mm ² , linearity is obtained up to the high concentration region, and the CV value of each is 3 to 28%.
It can be seen that the quantitative performance is good.

[0093]

EFFECTS OF THE INVENTION By optimizing the positional relationship between the detection substance holding part and the determination part, a biodevice capable of qualitative and quantitative measurement of a test substance in a sample solution quickly, with high accuracy and good reproducibility. A quantitative measurement device and a quantitative measurement method using the same can be provided. The biodevice of the present invention is suitable as a POCT-compatible device.

[Brief description of drawings]

FIG. 1 is a diagram showing the structure of a conventional immunochromatographic device.

FIG. 2 is a diagram showing an example of measuring a retention width by an optical method in the biodevice of the present invention.

FIG. 3 is a diagram showing an example of measurement of a diffusion width by an optical method in the biodevice of the present invention.

FIG. 4 is a diagram showing a structure of a determination unit position setting device used in one embodiment of the present invention.

FIG. 5 is a view showing a structure of a biodevice used in one example of the present invention.

FIG. 6 is a diffusion width B / a retention width A when hCG in urine is measured using a biodevice in one example of the present invention.
And a graph showing the relationship between the CV value and the absorbance value.

FIG. 7 is a result of measuring hCG concentration in urine using a biodevice having a diffusion width B / holding width A of 5% when the end of the diffusion width B reaches the determination part in one example of the present invention. The graph showing.

FIG. 8 is a result of measuring hCG concentration in urine using a biodevice having a diffusion width B / holding width A of 50% when the end of the diffusion width B reaches the determination part in one example of the present invention. The graph showing.

FIG. 9 is a result of measuring the hCG concentration in urine using a biodevice having a diffusion width B / holding width A of 120% when the end of the diffusion width B reaches the determination part in one example of the present invention. The graph showing.

FIG. 10 is a graph showing the results of measuring hCG concentration in urine using a biodevice having an area of a region between the detection substance holding unit and the determination unit of 175 mm ² in another example of the present invention.

FIG. 11 is a graph showing the results of measuring hCG concentration in urine using a biodevice in which the area between the detection substance holding unit and the determination unit is 50 mm ² in another example of the present invention.

FIG. 12 is a graph showing the results of measuring hCG concentration in urine using a biodevice in which the area between the detection substance holding unit and the determination unit is 5 mm ² in another example of the present invention.

FIG. 13 is a diagram schematically showing the state of elution and transfer of the first substance group in the biodevice of the present invention in comparison with conventional Dehys.

[Explanation of symbols]

11 board 12, 41, 51 Detection substance holding part 13, 42, 52 Sample introduction part 14,56 Water absorption part 15 Backing substrate 16, 54 Judgment unit 40 Judgment unit position setting device 43,53 Nitrocellulose membrane 50 bio devices 55 Support

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Tatsuro Kawamura             1006 Kadoma, Kadoma-shi, Osaka Matsushita Electric             Sangyo Co., Ltd. (72) Inventor Masayoshi Nadaoka             1 Juden Matsushita, 1-8 Koshinmachi, Takamatsu City, Kagawa Prefecture             Child Industry Co., Ltd. (72) Inventor Hirohashi Tanaka             1 Juden Matsushita, 1-8 Koshinmachi, Takamatsu City, Kagawa Prefecture             Child Industry Co., Ltd. (72) Inventor Mie Takahashi             1 Juden Matsushita, 1-8 Koshinmachi, Takamatsu City, Kagawa Prefecture             Child Industry Co., Ltd.

Claims (17)

[Claims] 1. A biodevice for measuring a test substance contained in a sample solution, comprising a sample introducing part, a detection substance holding part, and a judging part, wherein the sample solution introduced into the sample introducing part. However, the sample introduction unit, the detection substance holding unit and the determination unit are arranged so as to move to the determination unit via the detection substance holding unit,
And at least the detection substance holding unit and the determination unit is configured by the same member, the detection substance holding unit, a first substance group containing a substance that specifically reacts with the test substance The first substance group is contained in a state capable of being eluted by the sample solution, and after the first substance group is eluted by the sample introduced into the sample introducing part, the first substance group is a starting point in an elution and development process. A biodevice characterized by being developed in a lump having an end. 2. A biodevice for measuring a test substance contained in a sample solution, comprising a sample introducing section, a detected substance holding section and a judging section, wherein the sample solution introduced into the sample introducing section. Is arranged so that the sample introduction unit, the detection substance holding unit and the determination unit are moved to the determination unit via the detection substance holding unit, and at least the detection substance holding unit and the determination unit are the same member. The detection substance holding unit includes a first substance group containing a substance that specifically reacts with the test substance in a state capable of being eluted by the sample solution, The part includes a second substance group containing a substance that specifically reacts with the test substance in a fixed state, and the first substance group included in the detection substance holding unit. Is the sample introduced into the sample introduction part. In the process of being eluted by the raw material liquid and reaching the determination part together with the sample liquid while diffusing in the moving direction of the sample liquid, the end of the diffusion width of the first substance group generated in the moving direction of the sample liquid is Upon reaching the determination unit, the width A in the moving direction of the sample liquid and the diffusion of the first substance group in the portion containing the first substance group in the detection substance holding unit before the introduction of the sample liquid. Width B
A biodevice having a ratio A: B of 1: 0.25 to 1: 1. 3. The biodevice according to claim 1, wherein the area between the detection substance holding unit and the determination unit has an area of 3 mm ² or more and 150 mm ² or less. 4. The sample introduction part, the detection substance holding part, and the determination part are in a dry state before the introduction of the sample liquid.
The biodevice according to claim 1. 5. The sample liquid is a body fluid,
The biodevice according to claim 1. 6. The substance which specifically reacts with the test substance contained in the first substance group is a coloring substance, a fluorescent substance, a phosphorescent substance, a luminescent substance, a redox substance, an enzyme, a nucleic acid or an endoplasmic reticulum. The biodevice according to claim 1, which is labeled. 7. The biodevice according to claim 6, wherein the coloring substance is colloidal gold particles. 8. The biodevice according to claim 2, wherein the first substance group contains a first antibody against the test substance, and the second substance group contains a second antibody against the test substance. . 9. The first substance group includes a first antibody and a second antibody against a test substance, the second antibody is labeled with biotin, and the second substance group is specific to the biotin. Characterized in that it contains avidin that reacts with
The biodevice according to claim 2. 10. The first substance group includes a first antibody and a second antibody against a test substance, the second antibody is labeled with a magnetic substance, and the second substance group is the magnetic substance. The biodevice according to claim 2, comprising a substance for magnetically capturing the substance. 11. The biodevice according to claim 1, wherein the detection substance holding portion and the determination portion are made of a porous material. 12. Biodevice according to claim 11, characterized in that the porous material is a nitrocellulose membrane. 13. The biodevice according to claim 11, wherein the sample introduction section is laminated on the porous material that constitutes the detection substance holding section and the determination section. 14. A water absorbing part for absorbing a sample liquid, which is on a porous material in which a detection substance holding part and a determination part are formed, and which is on the side opposite to the detection substance holding part with respect to the determination part. The biodevice according to claim 11, wherein the biodevice is laminated. 15. A material impermeable to a sample liquid is provided in close contact with at least a part of the sample introduction part, the detected substance holding part and the determination part. The biodevice according to. 16. A quantitative measurement device for measuring a test substance contained in a sample solution, comprising: the biodevice according to claim 1; and a physical or chemical signal in a determination unit of the biodevice. And a measuring device for quantitatively measuring the. 17. A quantitative measurement method for measuring a test substance contained in a sample solution using the quantitative measurement device according to claim 16, wherein a fixed amount of the sample solution is introduced into a sample introduction part. And a step of quantitatively measuring a physical or chemical signal in the determination unit with a measuring device.