JP5813481B2 - Biosensor and test liquid measurement method - Google Patents

Description

  The present invention relates to a biosensor for quantifying a measurement target component in a test liquid and a test liquid measurement method.

Analytical methods for quantifying components to be measured in test fluids using the specificity of immune responses have been put into practical use for a long time. In particular, lateral flow (immunochromatography) biosensors (immunochromatography sensors) that flow from one end to the other along the surface of a porous membrane such as a membrane are simple to operate and require a short analysis time. It is popular as a general inspection tool.
As a method for detecting a measurement target component (for example, an antigen) in a test solution using an immunochromatographic sensor, the analysis target component is composed of two types of specific binding substances (ligands, for example, antibodies) having different specific binding sites for the measurement target component. A sandwich method is generally used in which a complex is formed with a sandwich.

For example, a labeled antibody in which a label such as gold colloid is bound to a strip-shaped development layer, one end of which is a test liquid addition part for adding a test liquid, is held in a state where it can be eluted with the development of the test liquid At the same time, an immunochromatographic sensor in which an immobilized antibody without a label is immobilized on a detection line on the downstream side in an insoluble state is used.
According to this immunochromatographic sensor, first, a complex (antigen-labeled antibody) in which an antigen in a test solution and a labeled antibody are bound to each other is generated. This complex reaches the detection line with the development of the test solution, and sandwiches. It is captured on the detection line as a complex (immobilized antibody-antigen-labeled antibody). Since the amount of the sandwich complex to be captured corresponds to the antigen concentration in the test solution, the antigen concentration in the test solution can be determined by visually or otherwise detecting the label of the complex.

However, the region where the amount of sandwich complex to be captured corresponds to the concentration of the measurement target component in the test solution is narrow, and the detection concentration range of the measurement target component is limited to one to two digits. When the concentration of the measurement target component in the test solution exceeds the detection concentration range, the antigen-antibody reaction is saturated, and no further antigen-antibody reaction occurs.
Furthermore, if the concentration of the measurement target component in the test solution greatly exceeds the detection concentration range, a prozone phenomenon (false negative due to excessive measurement target component) occurs, and the measurement target component in the actual test solution is high in concentration. Regardless, a result that is apparently worthy of low concentration may be obtained.

Therefore, when the concentration of the measurement target component is high, it is necessary to dilute the test solution in advance, and naturally, dilution accuracy is required in order to perform dilution and perform highly accurate quantification. Dilution operations, particularly high-magnification dilution operations, are usually very complicated for unskilled persons with little experience in chemical experiments, and it is difficult to dilute accurately. Therefore, the users of the immunochromatography sensor are limited to those who have a certain experience. Moreover, in order to obtain dilution accuracy, a dedicated device or the like is required.
In addition, if the approximate concentration of the component to be measured is unknown, it is difficult to determine an appropriate dilution factor, and trial and error must be repeated many times to obtain an appropriate concentration that falls within the detection concentration range. There was also a problem.

  In addition, when the prozone phenomenon has occurred and it is not an appropriate dilution factor, and it is not noticed, the measurement target component in the actual test solution has a high concentration, It is possible to misunderstand that the apparently low concentration results are correct. For example, in the case of measurement in a clinical test, the prescription for the patient is selected according to the test result, and in extreme cases, it may be related to the survival of life, and false negatives due to the prozone phenomenon can be misidentified as true negatives Sex is the most deadly issue.

In order to detect the prozone phenomenon and make a wider detection concentration range, for example, an immunochromatographic sensor (Patent Document 1) provided with a plurality of detection lines to which an immobilized antibody is immobilized has been proposed.
Specifically, the labeled antibody is held in a strippable development layer, one end of which is a test solution addition part to which the test solution is added, in a state where it can be eluted with the development of the test solution, and on the downstream side thereof There has been proposed a biosensor in which first and second detection lines are sequentially provided, and immobilized antibodies having different affinities with respect to an antigen to be measured are respectively fixed to the detection lines.
According to this biosensor, when a prozone phenomenon occurs in the detection in the first detection line arranged on the upstream side, it can be detected by the second detection line arranged on the downstream side. Yes.
In addition, by making the affinity of the immobilized antibody of each detection line different from that of the antigen, each detection line has a different detection concentration range, and as a result, the detection concentration of each detection line. It is said that it is possible to deal with a wide range of concentration ranges.

International Publication No. 03/014740

However, in the biosensor of Patent Document 1, since the antigen concentration changes to some extent when it passes through the first detection line, it is difficult to obtain an accurate antigen concentration in the second detection line. . In Patent Document 1, it is said that three or more detection lines may be provided. However, when a large number of detection lines are provided, the length of the entire biosensor becomes long, and the test solution can be developed only by capillary action. It becomes difficult. Therefore, the number of detection lines is limited, and the effect of expanding the detection density range is limited.
This invention is made in view of the said situation, Comprising: It aims at providing the measuring method of the biosensor and test liquid which can perform an accurate concentration measurement simply over a wide detection concentration range. .

In order to achieve the above object, the biosensor and the test liquid measurement method of the present invention are as follows.
[1] A sheet-like water absorbing body having a plurality of measurement areas divided by two or more line segments extending from an arbitrary point to the periphery is provided, and the arbitrary point and its vicinity on one surface of the water absorbing body are a test solution In the plurality of measurement areas, a marker part and a result display part are arranged in order in the direction away from the test liquid addition part, and the test liquid added to the test liquid addition part develops the water absorbent. The labeling unit and the result display unit are configured to be sequentially wetted, and in each labeling unit, a labeled ligand that specifically binds to the measurement target component in the test solution can be eluted by wetting the test solution. The fixed ligand that is carried on the water absorbent and specifically binds to the measurement target component in the test liquid is fixed to the water absorbent in a state in which it cannot be eluted even when the test liquid is wet. , Labeled ligands and analytes Min and reactive, and at least one reactant with the fixed ligand and the measurement target component is a biosensor characterized in that it is adjusted to be different from each reactivity in one or more other measurement area.
[2] The part other than the test liquid addition part on one side of the water-absorbing body, and at least a part from the label part to the result display part is covered with a water-impermeable transparent sheet. Biosensor.
[3] The biosensor as set forth in [1] or [2], wherein the other surface of the water-absorbing body is covered with a water-impermeable base sheet.
[4] A marker part is further arranged in each of the plurality of measurement areas, and the marker part is provided with a display indicating the detection concentration range of the measurement target component that can be measured by the measurement area. [3] The biosensor according to any one of [3].
[5] The water absorber is a regular polygon having n vertices (where n is an integer of 3 or more), and has n measurement areas, according to any one of [1] to [4]. Biosensor.
[6] The biosensor according to [5], wherein each of the two or more line segments that divide the n measurement areas is a line segment that extends from the center point of the water absorber to the midpoint of two adjacent vertices.
[7] The biosensor according to any one of claims 1 to 6 is folded so that the test solution adding section is positioned at the tip along the line segment dividing a plurality of measurement areas, and the test solution A method for measuring a test liquid, wherein the adding portion is immersed in the test liquid.

According to the biosensor and the test liquid measurement method of the present invention, it is possible to provide a biosensor capable of simply performing accurate concentration measurement over a wide detection concentration range. Therefore, the test solution can be measured by the biosensor without diluting the test solution and also when diluted.
In addition, even if the approximate concentration of the component to be measured is unknown, the possibility of entering one of the multiple detection concentration ranges is increased. There is no need for trial and error.

It is a top view of the biosensor which concerns on one Embodiment of this invention. It is II-II sectional drawing of FIG. It is explanatory drawing of one usage pattern of the biosensor which concerns on one Embodiment of this invention. It is a schematic diagram of the antigen antibody reaction in the biosensor which concerns on one Embodiment of this invention.

A biosensor 10 according to an embodiment of the present invention will be described with reference to FIGS. 1 and 2. As shown in FIG. 1, the biosensor 10 has a square shape with four apexes T1 to T4 and four sides P1 to P4 as a periphery, and as shown in FIG. A water absorbent 2 and a transparent sheet 3 are sequentially laminated on the base sheet 1.
The biosensor 10 has a center point C viewed from the top surface and the vicinity thereof as the test liquid addition unit 4. In this test liquid addition part 4, the transparent sheet 3 is provided with a circular hole 3a so that the water absorbing body 2 is exposed.

The water-absorbing body 2 may be any material that can be wetted with the test liquid, and for example, a porous film made of filter paper, non-woven fabric, cloth, glass fiber or the like can be used.
The base sheet 1 is not particularly limited as long as it can reinforce the water-absorbing body 2, but it is preferable to use a water-impermeable material. By being non-permeable, it is possible to prevent the person in charge of the test from coming into contact with the test solution, and it can be handled appropriately even when a test solution is used that may cause infection, such as blood, saliva, or urine. . Further, unintended contamination of the water absorbent body 2 can be avoided.
Moreover, it is preferable that the base material sheet 1 is opaque. If it is opaque, the light from the outside of the base sheet 1 is blocked, and the labeled ligand in the water absorbent 2 can be easily observed. As the base sheet 1, for example, an opaque resin film such as a milky white polyester sheet, a laminate film in which at least one layer of opaque resin is laminated, a laminated paper in which paper is laminated with a resin film, and the like can be used.
The transparent sheet 3 is not particularly limited as long as it is a transparent sheet. Here, the term “transparent” means that a signal from a labeling substance of a labeling ligand described later can be detected visually or mechanically from the outside of the transparent sheet 3. The transparent sheet 3 is preferably made of a water-impermeable material for the same reason as the base sheet 1. As the transparent sheet 3, for example, a transparent resin film such as a transparent polyester sheet or a transparent polypropylene film, a laminate film obtained by laminating two or more transparent resins, and the like can be used.

The biosensor 10 is divided into four measurement areas A1 to A4 by line segments L1 to L4 extending from the center point C to the midpoints of the sides P1 to P4 (midpoints of two adjacent vertices). The center point C is preferably a true geometric center point of the biosensor 10. However, it may be slightly deviated from the true geometric center point as long as the effect of the present invention is not impaired.
The line segments L1 to L4 may be printed with a solid line, a broken line, or the like, or may be subjected to processing such as a line push or a perforation. By performing printing and processing, the person in charge of the inspection can easily recognize the boundary between the measurement areas. Further, the bending along the line segments L1 to L4 is also easy. When printing is performed, the water absorbent body 2 may be directly printed, or the substrate sheet 1 may be printed on the water absorbent body 2 side or the transparent sheet 3. In the case of performing processing such as line pushing and perforation, it is preferable to apply to the entire laminate of the base sheet 1, the water absorbent body 2, and the transparent sheet 3 for the convenience of processing.
Note that it is not essential to print or process the line segments L1 to L4. That is, the line segments L1 to L4 may be virtual lines that are assumed only for dividing the measurement areas A1 to A4.

In each of the measurement areas A1 to A4, label portions R1 to R4 are provided, respectively. Each of the label portions R1 to R4 has an arc shape, and has a ring shape centered on the center point C as a whole. In addition, each label | marker part R1-R4 may mutually be continuous and may be spaced apart.
Further, in each of the measurement areas A1 to A4, result display portions D1 to D4 are provided on the apexes T1 to T4 side from the label portions R1 to R4, respectively. The result display portions D1 to D4 each have an arc shape, and have a ring shape centered on the center point C as a whole. In addition, each result display part D1-D4 may mutually be continuous, and may be spaced apart.
Further, in each of the measurement areas A1 to A4, marker portions M1 to M4 are provided on the vertexes T1 to T4 side of the result display portions D1 to D4, respectively.

In the labeling portions R1 to R4, a labeled ligand that specifically binds to the component to be measured in the test solution is carried on the water absorber 2 in a state where it can be eluted by wetting the test solution.
On the other hand, in the result display units D1 to D4, a fixed ligand that specifically binds to the measurement target component in the test solution is fixed to the water absorber 2 in a state where it cannot be eluted even when the test solution is wet.
The ligand in the present invention is a substance that specifically binds to a substance having a specific structure (in the case of the present invention, a component to be measured), and includes an antigen, an antibody, a nucleic acid sequence fragment, an effector molecule, a receptor molecule, an enzyme and its Inhibitors, avidin, biotin, sugar chain compounds, lectins, aptamers and the like can be mentioned.
The bond between the measurement target component and the ligand includes ionic bond, covalent bond, coordinate bond and the like in physical adsorption and chemical bond.

The labeled ligand carried on the labeling portions R1 to R4 is a ligand to which a labeling substance that itself emits a signal is bound. Here, the signal is a signal that can be detected visually or mechanically. The type of signal is preferably absorption or emission at a specific wavelength (color that is confirmed in the visible range when visually observed). Moreover, radioactivity, light emission, phosphorescence, fluorescence, etc. may be sufficient.
Examples of labeling substances include gold colloids, metal colloids such as selenium colloids, colored latex particles colored with dye dyes, fluorescent latex particles colored with fluorescent dyes, semiconductor nanoparticles, and other colored lipid vesicles (liposomes) and vesicles Etc.
On the other hand, the fixed ligand fixed to the result display units D1 to D4 is a different type of ligand from the ligand of the labeled ligand, and the ligand of the labeled ligand is a ligand that binds to a different part of the measurement target component.

At least one of the reactivity between the labeled ligand and the measurement target component and the reactivity between the fixed ligand and the measurement target component in each measurement area is adjusted to be different from each reactivity in the other measurement areas. Thereby, the detection concentration ranges of the measurement target components in the result display portions D1 to D4 in the respective measurement areas are different from each other.
The reactivity between the ligand and the measurement target component can be adjusted, for example, by selecting an appropriate epitope involved in binding, or introducing a group that causes steric hindrance to the binding of the measurement target component to the ligand.
The reactivity between the labeled ligand and the component to be measured can be adjusted by adjusting the amount of binding of the ligand per labeling substance.

The marker portions M1 to M4 are provided with a display indicating the detected concentration range of the measurement target component that can be measured by the measurement area where the marker portion is present.
The display is not particularly limited as long as the detection density range can be grasped. For example, a mark corresponding to the detection density range as shown in FIG. 1 may be added, and a color corresponding to the detection density range may be added. . Further, a numerical value directly indicating the detected concentration range may be described.
The display may be applied directly to the water absorbent body 2 by printing, or may be applied to the water absorbent body 2 side of the base sheet 1 or the transparent sheet 3 by printing. Further, the display position is not particularly limited as long as the detection of signals from the result display units D1 to D4 is not hindered. You may change the color of the whole water absorbing body 2 or the base material sheet 1 for every measurement area.

In order to measure the component to be measured in the test liquid using the biosensor 10, the test liquid may be dropped into the test liquid adding section 4 in a state where the whole is expanded as shown in FIG. As shown, the transparent sheet 3 is folded outside in the line segments L1 to L4 so that the test solution addition part 4 is located at the tip, and the exposed test solution addition part 4 is directly placed in a beaker 20 or the like. You may immerse in the liquid S.
In any of the methods, the test solution is wetted (developed) by capillary action from the test solution addition unit 4 toward the vertices T1 to T4 and the surroundings P1 to P4.

FIG. 4 shows the reaction state in each measurement area, taking as an example the case where the component to be measured in the test solution is antigen A, and the labeled ligand and fixed ligand are labeled antibody R and fixed antibody D, respectively.
When the test liquid is dropped into the test liquid addition part 4 of the biosensor 10 or the test liquid addition part 4 is immersed in the test liquid, the test liquid absorbed in the test liquid addition part 4 instantly infiltrates toward the periphery. Then, the labeled antibody R carried on each of the labeling portions R1 to R4 is dissolved or dispersed in the test solution and the movement is continued. If antigen A is present in the test solution, labeled antibody R binds to antigen A while moving to form complex X (labeled antibody R-antigen A).
When this complex X reaches the fixed antibody D fixed to each of the result display portions D1 to D4, the antigen A of the complex (labeled antibody R-antigen A) binds to the fixed antibody D, and there is a sandwich complex. Y (fixed antibody D-labeled antibody R-antigen A) is captured.
The concentration of the antigen A in the test solution can be determined by visually or mechanically detecting the amount of the sandwich complex Y captured.
The labeled antibody R that has not been captured as the sandwich complex Y continues to move and reaches the vicinity of the biosensor 10. By confirming the labeled antibody R reaching the vicinity, it can be confirmed that the test solution has been normally developed.

In the detection density ranges of the measurement areas A1 to A4, the measurement area A1 has the lowest density, the measurement area A2 has a slightly higher density, the measurement area A3 has a higher density, and the measurement area A4 has the highest density. In this case, the concentration of antigen A in the test solution is determined as follows.
When the sandwich complex Y is detected only in the result display portion D1 in the measurement area A1, the concentration of the antigen A is obtained based on the amount of the sandwich complex Y detected in the result display portion D1.
When the sandwich complex Y is detected in the result display part D2 of the measurement area A2, and not detected in the result display part D3 of the measurement area A3 and the result display part D4 of the measurement area A4, it is detected in the result display part D2. Based on the amount of sandwich complex Y, the concentration of antigen A is determined.
When the sandwich complex Y is detected in the result display part D3 of the measurement area A3 and not detected in the result display part D4 of the measurement area A4, the amount is determined based on the amount of the sandwich complex Y detected in the result display part D3. The concentration of antigen A is determined.
When the sandwich complex Y is detected in the result display part D4 of the measurement area A4, it is possible to determine the concentration of the antigen A based on the amount of the sandwich complex Y detected in the result display part D4. Since the prozone phenomenon may occur, it is preferable to perform the test again using another biosensor 10 after diluting the test solution or further increasing the dilution factor.
Note that even when the sandwich complex Y is not detected in the result display section of any measurement area, the prozone phenomenon may occur, so care should be taken.

In the above description, the case where the detection density ranges of the measurement areas A1 to A4 are all different from each other has been described as an example. However, the detection density ranges of all the measurement areas may not be the same. For example, the detection density ranges of the measurement area A1 and the measurement area A2 are the same, the detection density ranges of the measurement area A3 and the measurement area A4 are the same, the detection density ranges of the measurement area A1 and the measurement area A2, the measurement area A3, and the measurement The detection density range of area A4 may be different.
That is, in any one set of measurement areas, at least one of the reactivity between the labeled ligand and the measurement target component and the reactivity between the fixed ligand and the measurement target component is equal to each reactivity in the other one or more measurement areas. It only needs to be adjusted differently.

Since the biosensor 10 has a plurality of measurement areas having different detection concentration ranges, the biosensor 10 can cope with a wide detection concentration range as a whole. Therefore, even if the test solution is not diluted, the detection concentration range in any area is likely to cover the concentration of antigen A in the test solution.
In addition, even if the concentration of antigen A in the test solution is too high, it can be measured by diluting the test solution at a low dilution rate, so that higher dilution accuracy can be obtained than in the case of a high dilution rate, and as a result, measurement can be performed with high accuracy. Can do.

In addition, even if the approximate concentration of the measurement target component is unknown, multiple detection concentration ranges can be confirmed at a time, so trial and error can be repeated many times by changing the dilution factor in order to adjust the test solution to an appropriate concentration. There is no need to do it.
In the present embodiment, since the line segment that divides each measurement area is a line segment that extends from the center point C to the midpoint of two adjacent vertices, it reaches from the center point C to one vertex in each measurement area. The development distance can be taken. Therefore, a sufficient development distance can be obtained without increasing the entire area. Moreover, the area of each measurement area can be made equal.
Moreover, in this embodiment, since it was set as the square which has four vertices, it can cut and manufacture material from a large sheet material etc. without waste. Therefore, it is easy to suppress the manufacturing cost.

The biosensor 10 of the present embodiment has a configuration in which the outer shape is a square having four vertices and four measurement areas are provided. However, the outer shape is n (however, n is 3 or more). Another regular polygon having an (integer) vertex may be used, and n measurement areas may be provided. Also in this case, in order to provide each measurement area equally and to obtain a sufficient development distance, it is preferable that the line segment dividing each measurement area is a line segment from the center point to the midpoint of two adjacent vertices. .
The outer shape may be a regular polygon having m vertices (where m is an even number of 4 or more), and m / 2 measurement areas may be provided. In this case, when dividing by m / 2 line segments from the center point C to one vertex (however, every other line), the development distance from the center point C to one vertex is taken in each measurement area. Is preferable.

The outer shape and the number of measurement areas can be set separately. For example, the outer shape may be a polygon having more than 20 vertices, and the number of measurement areas may be 10 or less.
In the case where the outer shape of the absorber is a regular polygon, the number of vertices is preferably an even number. Among regular polygons, squares and regular hexagons are preferred because they can be produced efficiently using materials, and squares are particularly preferred because they are easy to produce.
Further, the outer shape does not necessarily need to be a regular polygon, and can be, for example, a circle.
In addition, when the test solution addition part is folded along the line segment that divides the plurality of measurement areas so that the test solution addition part is positioned at the tip, and the test solution addition part is immersed in the test solution for measurement, bending is easy. Therefore, the number of measurement areas is preferably an even number.
If the number of measurement areas is too large, the area of each area becomes too small and is impractical, so it is preferably 20 or less, and more preferably 10 or less.

Further, in the biosensor 10 of the present embodiment, each of the label portions R1 to R4 and the result display portions D1 to D4 are each arc-shaped so as to form a ring shape as a whole. For example, it may be provided along a straight line connecting line segments that divide each measurement area.
Moreover, in the biosensor 10 of the present embodiment, the transparent sheet 3 is configured to cover the water absorbing body 2 over the entire surface other than the test solution addition portion. However, as long as at least a portion from the label portion to the result display portion is covered. Good. For example, the water absorbing body 2 may be exposed in the vicinity of the periphery.
Furthermore, the base sheet 1 and the transparent sheet 3 are not necessarily essential, and either one or both may be omitted.
Moreover, the water absorbing body 2 does not need to be a single-leaf sheet, and may be one in which one or more ring-shaped sheets are stacked on a circular sheet, for example.

  Moreover, in the biosensor 10 of the present embodiment, the common starting point of a plurality of line segments that divide a plurality of measurement areas is the center point C. However, the common starting point is as long as the line segment can be drawn with the periphery as the end point. Any point is acceptable. In other words, the plurality of measurement areas only need to be divided by two or more line segments starting from an arbitrary point inside the periphery and starting from the periphery.

DESCRIPTION OF SYMBOLS 1 ... Base material sheet, 2 ... Water absorption body, 3 ... Transparent sheet, 4 ... Test liquid addition part,
10 ... Biosensor, 20 ... Beaker,
T1-T4 ... vertex, P1-P4 ... side, C ... center point, L1-L4 ... line segment,
A1 to A4 ... measurement area, R1 to R4 ... label part, D1 to D4 ... result display part,
M1 to M4 ... marker part, A ... antigen, R ... labeled antibody, D ... fixed antibody,
X ... complex, Y ... sandwich composite

Claims (6)

A method for measuring a test liquid, in which the following biosensor is folded along a line segment that divides a plurality of measurement areas so that the test liquid addition part is positioned at the tip, and the test liquid addition part is immersed in the test liquid.
Biosensor: A sheet-like water absorbent body having a plurality of measurement areas divided by two or more line segments from an arbitrary point to the periphery,
The arbitrary point on the one surface of the water-absorbent body and its vicinity are the test solution addition part,
In each of the plurality of measurement areas, a label unit and a result display unit are sequentially arranged in a direction away from the test solution addition unit, and the test solution added to the test solution addition unit develops the water absorbent and It is configured to wet the result display part sequentially,
In each labeling part, a labeled ligand that specifically binds to the component to be measured in the test solution is carried on the water absorbent in a state where it can be eluted by wetting the test solution,
In each result display section, a fixed ligand that specifically binds to the measurement target component in the test solution is fixed to the water absorbent in a state where it cannot be eluted even when the test solution is wet,
At least one of the reactivity between the labeled ligand and the measurement target component and the reactivity between the fixed ligand and the measurement target component in each measurement area is adjusted to be different from each reactivity in one or more other measurement areas. The The part other than the test liquid addition part on one surface of the water absorbent body in the biosensor , and at least the part from the label part to the result display part is covered with a water-impermeable transparent sheet. Method for measuring the test fluid . The method for measuring a test liquid according to claim 1 or 2, wherein the other surface of the water-absorbing body in the biosensor is covered with a non-permeable base sheet. The marker part is further arrange | positioned at each of the several measurement area in the said biosensor, The display which shows the detection density | concentration range of the measuring object component measurable by the said measurement area is attached | subjected to this marker part. The measuring method of the test liquid as described in any one of -3 . The test according to any one of claims 1 to 4, wherein the water absorbent body in the biosensor is a regular polygon having n vertices (where n is an integer of 3 or more) and has n measurement areas. Liquid measurement method . The two or more line segments that divide the n measurement areas in the biosensor are each a line segment from the center point of the water-absorbing body to the midpoint of two adjacent vertices . Measuring method .

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