JP2010032396A - Biosensor - Google Patents

Abstract

A biosensor that can measure a substance to be inspected in a liquid to be inspected with high accuracy in a wide concentration range and can be easily manufactured at low cost.
A developing layer for developing a liquid to be inspected includes a labeling reagent part holding a labeling reagent for labeling a substance to be inspected in the liquid to be inspected, and an immobilizing reagent for immobilizing the substance to be inspected. In the biosensor provided with at least a holding immobilization reagent part, the labeling reagent includes an insoluble granular marker, and the insoluble granular marker is separately prepared and mixed with at least two particle groups having different particle size distributions. It is characterized by.
[Selection] Figure 8

Description

  The present invention relates to a biosensor that qualitatively or quantitatively detects a test substance in a test liquid.

  In recent years, with the improvement of home medical care and community medical care, the demand for early diagnosis, and the increase in urgent clinical tests, there is a demand for equipment that can perform high-precision measurements easily and quickly even if you are not a clinical laboratory specialist. ing. A small analyzer for POCT (POINT OF CARE TESTING), which can perform highly reliable measurement in a short time without requiring complicated operation, has been in the spotlight.

  POCT is a general term for tests performed in the “patient's vicinity” such as a doctor's office or ward. Doctors immediately determine the test results, take prompt actions, and monitor the treatment process and prognosis. It is attracting attention as something that greatly helps improve the quality of medical care. Compared with laboratory testing, testing with a small analyzer in POCT is said to reduce the cost of sample transport and equipment and reduce total testing costs. The POCT market is streamlining hospital management It is expanding rapidly in the United States, and is expected to become a growing market globally.

  With the widespread use of POCT, immunochromatographic test strips that can be quantitatively measured have been distributed in the market as products. A dry analytical element typified by an immunochromatographic test piece does not require reagent adjustment, and a test substance contained therein can be obtained simply by dropping a test liquid such as blood or urine onto the analytical element. Since it is very useful for analyzing a substance to be inspected easily and quickly, many of them have been put to practical use today for POCT.

  In general, immunochromatographic test strips have antibodies immobilized on a part of the development layer and are held in a dry state in which the labeled antibody can be eluted by the test solution at a position closer to the spotted part than the antibody immobilized part. It is a configuration. When the required amount of test liquid is added to the spotted part, it penetrates the spreading layer, and when the test target substance is contained in the test target liquid, the test target substance binds to the labeled antibody and further becomes the immobilized antibody. Since it binds, it is possible to detect the substance to be examined by detecting the binding of the labeled antibody to the antibody-immobilized portion. An example of a labeled substance constituting a labeled antibody is gold colloid particles, which can be visually observed by a color reaction by the gold colloid particles, so the presence and concentration of the test target substance in the test liquid is detected from the coloration degree. (Measurement).

  The above is the case where the sandwich principle of the antigen-antibody reaction using the labeled antibody is used as the measurement principle, but the subject to be examined depends on the coloration state due to the binding of the labeling reagent to the antibody-immobilized portion. The presence and concentration of the substance can be detected. As the labeling substance, in addition to the above-mentioned gold colloid, latex is generally used, but magnetic particles, enzymes, metal colloids other than gold colloid, and the like are used in various ways. When the label is an enzyme, the user may need to add an enzyme substrate or a reaction stopping reagent during measurement. An immunochromatographic test piece in which measurement is performed only by an operation in which a user drops a test liquid is called a one-step immunochromatographic test piece.

The POCT market demands higher analysis accuracy in addition to being able to measure anyone anytime and anywhere. In other words, it is required to be able to measure a wider concentration range with higher sensitivity, more easily and quickly. However, in an analysis using the binding between a ligand and a receptor, particularly an analysis using an immunochromatographic test strip, a prozone phenomenon occurs when an inspection target substance is excessively present in a test solution. The prozone phenomenon in the immunochromatography method is that the labeled antibody is originally captured by the immobilized antibody via the test substance, but if there is a large amount of the test substance, the free test that could not react to the labeled antibody. This means that the target substance competes with the immobilized antibody, and as a result, the binding of the test target substance labeled with the labeled antibody to the immobilized antibody is prevented. In order to cope with this, immunochromatography that can quantitate a wide range of concentrations with high accuracy by arranging immobilized reagents with different affinity to the substance to be tested in the test solution in multiple stages on the development layer. A test piece has been developed (see Patent Document 1).
WO03 / 014740

  In the immunochromatographic test piece of Patent Document 1, it is necessary to prepare a plurality of immobilization reagents having different affinities with respect to the measurement object (test substance) in the test liquid. The reaction in the immunochromatographic test strip is originally an immune reaction from the viewpoint of the word source, and in particular, an antigen-antibody reaction is often used. In many cases, since an antibody having affinity for a test substance is used as an immobilization reagent, it is necessary to prepare a plurality of antibodies having different affinity.

  However, it is difficult to control the affinity for an antigen during antibody production. A plurality of antibodies capable of covering the measurement range necessary for detecting the target test substance as an antigen, that is, a plurality of antibodies having a combination of affinity capable of covering the measurement range must be prepared. Therefore, a large number of antibodies having different affinities must be prepared and selected from them. Producing a large number of antibodies with different affinities requires a great amount of labor, time, and cost, and it is not easy to find an antibody with an affinity that can realize a desired combination. The same applies when considering the use of an antigen instead of an antibody as an immobilization reagent.

  An object of the present invention is to provide a biosensor that can measure a substance to be inspected in a liquid to be inspected with high accuracy in a wide concentration range and can be easily manufactured at low cost.

  In order to solve the above-described problem, the biosensor of the present invention includes a labeling reagent unit that holds a labeling reagent for labeling a substance to be inspected in a development layer for developing the test liquid, and the test object In a biosensor provided with at least an immobilization reagent part holding an immobilization reagent for immobilizing a substance, the labeling reagent includes an insoluble granular marker, and the insoluble granular marker is separately prepared and has a particle size. It is characterized in that at least two particle groups having different distributions are mixed.

The main peak of the particle size distribution in each particle group of the insoluble granular marker is in the range of an average particle size of 1 to 100 nm.
In the present invention, the insoluble granular marker refers to a granular marker that is insoluble in the liquid to be inspected. The particle group refers to a group of particles contained in a particle solution manufactured at a time, and the particle size distribution refers to the particle size of the particle group by observation with a microscope, a counting method such as a light scattering method, or a specific gravity balance. This refers to the frequency distribution or cumulative distribution of particle diameters obtained when measured by any analysis method such as the method, light transmission method, sedimentation method, and sieving method. The main peak of the particle size distribution in each particle group refers to the maximum peak of the frequency distribution or cumulative distribution of the particle size in the particle group. In each particle group, there is no problem even if the average particle diameter of the second peak, the third peak, or a peak below it is in any range. The particle size distribution may be 1 or less or 100 nm or more.

  The insoluble granular marker is characterized in that a complex is formed by labeling a specific protein having a specific affinity for a substance to be examined. Specific proteins are proteins that bind to any small molecule, such as proteins with affinity for proteins, enzymes for any substrate, receptors for any hormone, and proteins with affinity for any vitamin or any toxin Any protein that has a specific affinity for an arbitrary substance such as a protein that binds to a nucleic acid such as a DNA-binding protein or an RNA-binding protein may be used.

  The specific protein is a substance involved in an immune response. The substance involved in the immune reaction is not limited as long as it is a substance involved in the immune reaction, such as complement and its capture substance, T cell receptor and its specific substance, cytokine and its receptor.

  The immune reaction is an antigen-antibody reaction. The antigen involved in the antigen-antibody reaction is a target test substance in the present invention. For example, proteins and protein derivatives such as immunoglobulins, hormones, enzymes, peptides; bacteria, viruses, fungi, mycoplasmas, parasites, And infectious substances such as products and components thereof; drugs such as therapeutic drugs and drugs of abuse; tumor markers; low molecular substances such as vitamins and toxins. Antibodies have specificity for antigens such as single chain antibodies, modified antibodies such as Fab and F (ab ′) 2, and recombinant antibodies such as scFv, dsFv, diabody, mSEA-diabody and minibody As long as it has, it may have any shape.

  For example, a metal colloid can be used as the insoluble granular marker. A metal colloid refers to any particle composed of a metal or an alloy that is colloidally dispersed in a solution. In addition to metal colloids, colored polymer beads such as latex particles, polymer dye particles, and magnetic particles can be used as the insoluble granular marker without any limitation. A preferred example of the metal colloid is a gold colloid. In addition, colloids such as silver, copper, and platinum can be used.

  The spreading layer is composed of a single layer or a plurality of layers of porous carriers. The spreading layer only needs to be wettable by the liquid to be inspected, and a carrier made of any porous material such as filter paper, non-woven fabric, membrane, cloth, glass fiber can be used.

  The biosensor of the present invention can be configured as an immunochromatographic test piece.

  The biosensor of the present invention has a simple configuration in which at least two particle groups having different particle size distributions are mixed and used as an insoluble granular marker constituting a labeling reagent, so that a substance to be inspected in a test liquid has a wide concentration range. Therefore, it is possible to measure with high accuracy, and the manufacture thereof can be performed easily and inexpensively.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(Embodiment 1)
FIGS. 1A and 1B are an exploded view and a perspective view, respectively, of an immunochromatographic test piece which is a biosensor according to Embodiment 1 of the present invention. In (b), it is shown in a partially transparent state.

  In FIG. 1, reference numeral 2 denotes a development layer, which is composed of a membrane filter (synthetic polymer film) mainly composed of nitrocellulose. Any porous material such as other membranes, filter paper, non-woven fabric, cloth, glass fiber, and the like can be used as long as the material is not limited to nitrocellulose membranes and can be wetted by the liquid to be inspected. It may be a single layer or a multilayer.

  Reference numeral 4 denotes a labeling reagent portion. A labeling reagent capable of labeling a substance to be inspected in a test solution, here a labeled antibody labeled with colloidal gold (hereinafter also referred to as a gold colloid labeled antibody) is in a dry state, It is held in a state where it can be eluted by the liquid.

  The colloidal gold labeled antibody is composed of a group of several types of colloidal gold particles having different particle size distributions. For example, a colloidal gold labeled antibody having a main peak average particle diameter of 20 nm, a colloidal gold labeled antibody of 30 nm, and a colloidal gold labeled antibody of 40 nm are mixed at 1: 1: 1. However, the blending ratio is arbitrary, and it is possible to correspond to the measurement range according to each substance to be inspected by the blending ratio. Not only particles of 20 nm, 30 nm, and 40 nm, but particles in the range of 1 to 100 nm can be used.

  Reference numeral 5 denotes a reagent immobilization unit, which is in a dry state so that an immobilization reagent capable of capturing a test target substance in a test liquid, here an antibody to the test target substance can form a complex with the test target substance and the labeling reagent. It is fixed with. The antibody to be used is only required to form a ternary complex with the labeling reagent and the test substance, and the epitope for the test substance may be the same as or different from the antibody of the labeling reagent.

  In the drawing, an example in which one reagent immobilization unit 5 is provided is shown, but it is not always necessary to have one, and if it is one or more, it may be freely selected according to the purpose. Moreover, although the example which made the reagent immobilization part 5 the line shape (band | belt shape) is shown, it does not necessarily need to be a line shape and can select freely, such as a spot shape, a character shape, and a key shape.

  The labeling reagent of the labeling reagent unit 4 described above is selected to detect the fixation of the substance to be inspected in the reagent immobilization unit 5, and the gold colloid as a marker is merely an example, and other metals Colored polymer beads such as colloids, alloys and latex particles, insoluble particulate markers such as polymer dye particles and magnetic particles can be arbitrarily selected according to the needs of the user.

  Reference numeral 6 denotes a liquid impermeable sheet, which is made of PET. The liquid-impermeable sheet 6 covers the surface of the development layer 2 in close contact except for the start end portion and the end portion along the development direction of the test liquid, thereby blocking the coating portion from contact with the test liquid. Protect and prevent external contamination. Contamination from outside refers to inadvertent contact of the liquid to be inspected or direct contact of a subject with a hand or the like. When more accurate measurement is required, the liquid impermeable sheet 6 tightly seals the upper surface of the development layer 2, particularly the upper surface extending from the labeling reagent unit 4 to the reagent immobilization unit 5, and both side surfaces are sealed. What is necessary is just to take the structure sealed tightly. The material of the liquid-impermeable sheet 6 is not limited to the above-described PET, and can be arbitrarily selected as long as it can cover the spreading layer 2 and is liquid-impermeable. It is preferable to use a transparent material for the part covering the reagent immobilization part 5 so that at least the state of the reagent immobilization part 5 can be seen through. Reference numeral 3 denotes an open portion in the development layer 2, that is, a terminal portion that is not covered with the liquid impermeable sheet 6.

  Reference numeral 7 denotes a substrate that supports the development layer 2. The substrate 7 has a role of reinforcing the spreading layer 2 and also has a role of blocking the liquid to be inspected, such as blood, saliva, urine, and the like, and is called a sheet or a film. It may be a thickness. In the case where the spreading layer 2 is light-transmitting when wetted, the substrate 7 can have an effect of blocking light. It is preferable to use white PET as a material, but there is no particular limitation as long as it has the above effects.

  Reference numeral 8 denotes a fine space forming material, and the role of forming the fine space 1 into which the liquid to be inspected flows by capillary action on the starting end portion of the development layer 2 not covered with the liquid impermeable sheet 6, and the object to be inspected It has the role of protecting the exterior from contamination during handling after the liquid is spotted. The protection here refers to inadvertent scattering and adhesion of the liquid to be inspected. The fine space forming material 8 can be composed of any liquid-impermeable material such as ABS, polystyrene, polyvinyl chloride or other synthetic resin material, metal, glass or the like. Transparent or translucent is preferable, but it may be colored or opaque.

  9 is a cell contraction reagent (cell component contraction agent), which is disposed in the fine space 1. The cell contraction reagent 9 only needs to have an effect of contracting cells, such as salts such as potassium chloride, sodium chloride and sodium phosphate, amino acids such as glycine and glutamic acid, imino acids such as proline, glucose, sucrose, trehalose and the like. Sugar alcohols such as saccharides and glucitol can be used. Such a system containing the cell contraction reagent 9 is particularly effective when whole blood is used as a test solution, and is not particularly necessary when a test solution containing no cell components is intended.

The inspection method using the biosensor will be described.
When the liquid to be inspected is brought into contact with the end of the fine space 1 (hereinafter referred to as the introduction part), it naturally flows into the fine space 1 by capillary action, and the cell components in the liquid to be examined are contracted by the cell contraction reagent 9. While spreading through the development layer 2. Whether the inflow amount of the liquid to be inspected is sufficient can be confirmed through the transparent fine space forming material 8. When the addition amount of the liquid to be inspected is limited, for example, when a certain amount is required, the addition amount can be accurately limited by setting the volume of the fine space 1 to a certain volume in advance. What is necessary is just to adjust the volume of the fine space 1 according to the required quantity of to-be-inspected liquid.

  When the liquid to be inspected reaches the labeling reagent section 4, elution of the labeling reagent is started, and when a substance to be inspected is present in the liquid to be inspected, it reacts with the eluted labeling reagent and combines the “labeling reagent-substance to be inspected” When the penetration proceeds while forming a body and reaches the reagent immobilization section 5, a gold colloid contained in the labeling reagent is captured by forming a "immobilized reagent-substance to be examined-labeling reagent" complex according to the amount. The color reaction due to is shown.

  On the other hand, when the substance to be inspected does not exist in the liquid to be inspected or the amount is below the detection sensitivity, none of the above-mentioned complex is formed, and the labeling reagent passes through without being bound. In both cases where the inspection target substance is not present and present, the liquid to be inspected that has permeated the development layer 2 and reached the opening 3 gradually evaporates or evaporates. In some cases, the liquid to be inspected may ooze out into the open part 3, but only on the development layer 2 in the open part 3, and the same height as the test liquid on the development layer 2 in the fine space 1 or the same Up to the height.

  Thus, by providing the open part 3, the permeation and permeation direction of the liquid to be inspected in the development layer 2 are controlled in a constant direction during the measurement. In general, the spreading layer 2 is often provided with a water absorbing portion in place of the open portion 3, and a porous material having a higher wetting effect and water absorbing effect than the material used for the reaction portion of the spreading layer 2 is used for the water absorbing portion. In addition, it has a function of absorbing and sucking the liquid to be inspected to pass over the development layer and a function of shortening the measurement time. The opening part 3 here has the same effect as this water absorption part. Providing the open portion 3 and the fine space 1 is particularly suitable when the amount of liquid to be inspected is extremely small, such as blood by puncturing a fingertip.

  The inspection result is obtained by confirming the coloration state due to the binding of the labeling reagent in the reagent immobilization unit 5. Visual determination is also possible for the qualitative purpose of examining the presence or absence of a substance to be examined. If high-precision measurement is required, quantitative results can be obtained by measuring the degree of coloration and the amount of label attached by optical means using reflected or transmitted light of diffuse electromagnetic radiation or images. it can. This will be described later.

  The electromagnetic radiation here is preferably in the visible region or near-visible region, and the electromagnetic radiation source may be an LED (Light Emitting Diode), LD (Laser Diode), or the like according to the needs of the user. Can be selected. The optical means by image means any analysis means that captures an image with a CCD image sensor or a CMOS image sensor.

  The sandwich reaction in the antigen-antibody reaction has been described above. However, a competitive reaction can be performed using a reagent that reacts competitively with the substance to be inspected. In addition to the antigen-antibody reaction, a reaction system that forms a specific bond can be used.

(Embodiment 2)
2 (a) and 2 (b) are an exploded view and a perspective view, respectively, of an immunochromatographic test piece that is a biosensor according to Embodiment 2 of the present invention. In (b), it is shown in a partially transparent state.

  In this immunochromatographic test piece, in addition to the reagent immobilization section 5, a reagent immobilization section 10 and a reagent immobilization section 11 are provided, each of which has an antibody against a substance to be inspected in a test liquid, It is immobilized in a dry state so as to form a complex with the test substance and the labeling reagent. Others are the same as those of the first embodiment described above.

  The antibody used for the reagent immobilization unit 5, the antibody used for the reagent immobilization unit 10, and the antibody used for the reagent immobilization unit 11 are selected so as to have different affinities for the test substance. Since the closer to the spot of the liquid to be inspected (upstream side), the liquid to be inspected and the substance to be inspected come into contact sooner.

  The antibodies used in the reagent immobilization units 5, 10, and 11 need only be able to form a ternary complex with the labeling reagent and the substance to be inspected, and the epitope for the substance to be inspected is the same as the antibody of the labeling reagent. It can be either different.

  In the figure, there are provided three reagent immobilization parts, reagent immobilization parts 5, 10, and 11. However, it is not always necessary that there are three reagent immobilization parts. Can be placed. When arranging in three or more places, you may use the immobilization reagent from which affinity differs in all the places as mentioned above, or may combine the place using two types of immobilization reagents from which affinity differs. The combination in that case can be freely selected according to the purpose of the user.

  In addition, the reagent immobilization units 5, 10, and 11 do not need to have a line shape as illustrated, and can be freely selected from a spot shape, a character shape, a key shape, and the like. As shown in the drawing, it is not always necessary to be separated from each other, and it is possible to make contact so that it looks like a single line. The colloidal gold is merely an example of an insoluble granular marker, and other metals, alloys, colored polymer beads, polymer dye particles, magnetic particles, and other insoluble granular markers can be arbitrarily selected according to the needs of the user.

The inspection method using the biosensor will be described.
When the liquid to be inspected is brought into contact with the end of the fine space 1 (hereinafter referred to as the introduction part), the spreading layer 2 penetrates in the same manner as described in the first embodiment, and the object to be inspected is in the liquid to be inspected. When a substance is present, penetration proceeds while reacting with the labeling reagent eluted by the labeling reagent unit 4 to form a “labeling reagent-substance to be examined” complex.

  When the liquid to be inspected reaches the reagent immobilization unit 5, a "immobilized reagent-test object-labeling reagent" complex is formed according to the amount of the test object substance, and is presented by the gold colloid constituting the labeling reagent. A color response is indicated.

  When the liquid to be inspected reaches the reagent immobilization unit 10, a "immobilized reagent-substance to be inspected-labeled reagent" complex is formed according to the amount of the substance to be inspected (if it remains) to form the labeling reagent The color reaction due to the colloidal gold is shown.

  When the liquid to be inspected reaches the reagent immobilization unit 11, a "immobilized reagent-substance to be inspected-labeled reagent" complex is formed according to the amount of the substance to be inspected (if it remains), thereby constituting the labeling reagent. The color reaction due to the colloidal gold is shown.

  If the substance to be inspected does not exist in the liquid to be inspected or the amount is lower than the detection sensitivity, none of the above-mentioned complex is formed, and the labeling reagent passes through without binding, and the development layer The liquid to be inspected that has penetrated 2 and reaches the opening 3 gradually evaporates or evaporates, or oozes into the opening 3 and accumulates only on the spread layer 2 in the opening 3.

  The test result is obtained by confirming the coloration state due to the binding of the labeling reagent in the reagent immobilization units 5, 10, and 11. Visual judgment is also possible for qualitative purposes. If high-precision measurement is required, quantitative results can be obtained by measuring the degree of coloration and the amount of label binding by means of reflected or transmitted light of diffuse electromagnetic radiation and optical means using images (see above). Obtainable. This will be described later. It is also possible to use the reagent immobilization unit 5 for detecting the concentration of the substance to be inspected and the reagent immobilization units 10 and 11 (using antibodies with low affinity) for detecting the prozone.

  The sandwich reaction in the antigen-antibody reaction has been described above. However, a competitive reaction may be performed using a reagent that reacts competitively with the test substance. In addition to the antigen-antibody reaction, a reaction system that forms a specific bond can be used.

The present invention will be further described below with reference to specific examples. These examples are not intended to limit the invention.
Example 1
The immunochromatographic test piece of FIG. 1 described above was manufactured as follows. A membrane mainly composed of nitrocellulose is used for the development layer 2, an anti-CRP antibody A is used for the reagent immobilization part 5, and a complex of anti-CRP antibody B and gold colloid is used for the labeling reagent part 4.

a) Preparation of antibody-immobilized nitrocellulose membrane An anti-CRP antibody A solution diluted with a phosphate buffer solution to adjust its concentration was prepared, and this antibody solution was applied onto the nitrocellulose membrane with a solution discharge device to prepare an antibody. An immobilization line (reagent immobilization part) was provided. The nitrocellulose membrane was dried, immersed in a Tris-HCl buffer solution containing 1% skim milk, and gently shaken for 30 minutes. After 30 minutes, the membrane was washed by transferring into Tris-HCl buffer solution, gently shaking for 10 minutes, and gently shaking for another 10 minutes in another Tris-HCl buffer solution. This membrane was treated with a surfactant by gently shaking in Tris-HCl buffer containing 0.05% sucrose monolaurate for 10 minutes. The membrane after the surfactant treatment was taken out and dried at room temperature.

b) Preparation of 20 nm colloidal gold labeled antibody Gold colloid was prepared by adding 1% citric acid solution to a final concentration of 0.03% to a 100 ° C. solution under reflux of 0.01% chloroauric acid. Refluxing was continued for 25 minutes, followed by cooling at room temperature. About the gold colloid obtained in this way, when the particle size distribution which is a particle size distribution was measured with the dynamic light scatterometer (Zeta Nano ZS, Sysmex Corporation), the peak of the particle size distribution was 19.1 nm. A group of colloidal gold particles having a peak with an average particle diameter of 19.1 nm is called a 20 nm colloidal gold.

  By adding anti-CRP antibody B to the 20 nm gold colloid prepared to pH 9 and stirring for 5 minutes, and further adding 10% BSA (bovine serum albumin) solution adjusted to pH 9 to a final 1%, and stirring, A 20 nm gold colloid-antibody complex (labeled antibody) was prepared. This labeled antibody solution is centrifuged at 4 ° C. and 20100 G for 50 minutes, the separated product is suspended in 1% BSA / 5% sucrose (pH 8.9) solution, and the above-mentioned centrifugation is performed again to wash the labeled antibody. Released. This labeled antibody was filtered through a 0.45 μm filter, and diluted with 1% BSA / 5% sucrose (pH 8.9) solution so that the absorbance at 520 nm was 150, and this was used as a 20 nm gold colloid-labeled antibody. Stored at ° C.

c) Preparation of 30 nm gold colloid-labeled antibody Gold colloid was prepared by adding 1% citric acid solution to a final concentration of 0.023% to a 100 ° C. solution under reflux of 0.01% chloroauric acid. Refluxing was continued for 25 minutes, followed by cooling at room temperature. About the gold colloid obtained in this way, when the particle size distribution which is a particle size distribution was measured with the dynamic light scatterometer (Zeta Nano ZS, Sysmex Corporation), the peak of the particle size distribution was 29.4 nm. A group of colloidal gold particles having a peak with an average particle diameter of 29.4 nm is referred to as a 30 nm colloidal gold. 30 nm gold colloid-antibody complex (labeled antibody) was prepared using the 30 nm gold colloid prepared at pH 9 in the same manner as the 20 nm gold colloid described above, and washed and isolated (centrifuged at 15000 G for 50 minutes at 4 ° C.) After filtration and dilution, it was stored at 4 ° C. as a 30 nm colloidal gold labeled antibody.

d) Preparation of 40 nm gold colloid-labeled antibody Gold colloid was prepared by adding a 1% citric acid solution to a final concentration of 0.015% to a 100 ° C. solution in 0.01% chloroauric acid under reflux. Refluxing was continued for 25 minutes, followed by cooling at room temperature. About the gold colloid obtained in this way, when the particle size distribution which is a particle size distribution was measured with the dynamic light scatterometer (Zeter Nano ZS, Sysmex Corporation), there were two peaks of scattered light intensity, and the main peak was 42. 0.5 nm. A group of gold colloid particles having a main peak in the vicinity of the average particle diameter of 40 nm is called 40 nm gold colloid. Using the 40 nm gold colloid prepared at pH 9 in the same manner as the 20 nm gold colloid described above, a 40 nm gold colloid-antibody complex (labeled antibody) was prepared and washed and isolated (centrifuged at 4 ° C., 8000 G for 50 minutes) After filtration and dilution, it was stored at 4 ° C. as a 40 nm colloidal gold labeled antibody.

e) Preparation of mixed gold colloid-labeled antibody I The above-mentioned 30 nm gold colloid-labeled antibody and 40 nm gold colloid-labeled antibody are mixed so that the liquid amount becomes 1: 1 and mixed well, and then mixed gold colloid-labeled antibody. I was stored at 4 ° C as I.

f) Preparation of mixed gold colloid-labeled antibody II The above-mentioned 20 nm gold colloid-labeled antibody, 30 nm gold colloid-labeled antibody and 40 nm gold colloid-labeled antibody were mixed at a volume ratio of 1: 1: 1 and stirred well. In addition, it was stored at 4 ° C. as mixed gold colloid-labeled antibody II.

g) Application of colloidal gold-labeled antibody The above five types of colloidal gold-labeled antibody solutions are each set in a solution discharge device so that the test solution introduction part is closer to the nitrocellulose membrane than the reagent-immobilized part. The nitrocellulose film after coating was lyophilized under vacuum. As a result, five types of spreading layers having a reagent immobilization part and a labeling reagent part were obtained.

h) Assembly The above-mentioned five kinds of development layers are each pasted on a substrate made of white PET having a thickness of 0.5 mm, cut to a width of 2.0 mm, and each piece after cutting is started from the labeling reagent portion. A fine space forming material that covers a predetermined range from a position closer to the end to a position closer to the end than the reagent immobilization section by wrapping a transparent tape with a thickness of 100 μm and forming a fine space in the start end not covered with the transparent tape. By pasting, a plurality of five types of immunochromatographic test pieces were obtained. In addition, the fine space forming material is retained with a shrinkage agent in which potassium chloride is kept in a dry state by spotting a potassium chloride aqueous solution adjusted to 1.5M in advance and then immediately freezing with liquid nitrogen and freeze-drying. I gave a part. Each immunochromatographic test piece is referred to as a 20 nm gold colloid labeled antibody test piece, a 30 nm gold colloid labeled antibody test piece, a 40 nm gold colloid labeled antibody test piece, a mixed gold colloid labeled antibody test piece 1A, and a mixed gold colloid labeled antibody test piece 2A. .

FIG. 3 shows the results of examining the particle size distribution (particle size distribution) of a 20 nm gold colloid, a 30 nm gold colloid, and a 40 nm gold colloid using a dynamic light scatterometer (Zeter Nano ZS, Sysmex Corporation).
[Test Example 1]
a) Preparation of sample Human blood added with EDTA · 2K as an anticoagulant was adjusted to a hematocrit value of 40%, a CRP solution was added to the adjusted blood (plasma), and the CRP concentration was 0.03 mg / dl, 0.06 mg / dl, 0.1 mg / dl, 0.3 mg / dl, 0.6 mg / dl, 1.0 mg / dl, 3.0 mg / dl, 6.0 mg / dl, 10.0 mg / dl A blood sample was prepared. The CRP concentration of each blood sample was confirmed using a commercially available apparatus (7020 type biochemical automatic analyzer, manufactured by Hitachi, Ltd.) using a latex immunoagglutination method and a reagent.

b) Measurement The above five types of immunochromatographic test pieces, that is, 20 nm gold colloid labeled antibody test piece, 30 nm gold colloid labeled antibody test piece, 40 nm gold colloid labeled antibody test piece, and mixed gold colloid labeled antibody test piece according to the present invention. 5 μL of each concentration of blood sample was added to each sample introduction part of 1A and the mixed gold colloid-labeled antibody test piece 2A, and the coloration state of the reagent immobilization part was measured 5 minutes after the addition, using a reflection absorbance meter (CS9300 Shimadzu Corporation). Was used to measure the reflection absorbance at 635 nm. The measurement was performed 5 times for each concentration, and an average value was taken.

c) Results FIG. 4 shows the measurement results. The horizontal axis shows the CRP concentration in logarithm, and the vertical axis shows the absorbance in logarithm. The diamond plot is the result obtained with a 20 nm gold colloid labeled antibody test piece, the square plot is the result obtained with a 30 nm gold colloid labeled antibody test piece, the triangle plot is the result obtained with a 40 nm gold colloid labeled antibody test piece, the white circle plot Is the result obtained with the mixed gold colloid labeled antibody test piece 1A, and the black circle plot is the result obtained with the mixed gold colloid labeled antibody test piece 2A. The response to CRP is clearly different for each specimen.

The measurement results of FIG. 4 are schematically shown in FIG. As is clear from FIG. 5, each test piece has a different measurement range as a result of a different response to CRP.
In the concentration A in the figure, a signal is obtained only from the mixed gold colloid-labeled antibody test piece 1A, and this concentration A can be measured only by the mixed gold colloid-labeled antibody test piece 1A. At concentration B, a signal was obtained with the mixed gold colloid-labeled antibody test piece 1A, and a signal near the measurement limit of the measuring device was obtained from the 40 nm gold colloid-labeled antibody test piece. Measurement is possible.

  Concentration C can be similarly measured with the mixed gold colloid-labeled antibody test piece 1A, the 40 nm gold colloid-labeled antibody test piece, and the mixed gold colloid-labeled antibody test piece 2A. Concentration D can be similarly measured using mixed gold colloid-labeled antibody test piece 1A, 40 nm gold colloid-labeled antibody test piece, mixed gold colloid-labeled antibody test piece 2A, and 30 nm gold colloid-labeled antibody test piece. For concentration E, similarly, mixed gold colloid labeled antibody test piece 1A, 40 nm gold colloid labeled antibody test piece, mixed gold colloid labeled antibody test piece 2A, 30 nm gold colloid labeled antibody test piece, and 20 nm gold colloid labeled antibody test piece. That is, measurement can be performed on all test pieces.

  At concentration F, signals were obtained for all test pieces, but the signals for the mixed gold colloid-labeled antibody test piece 1A and the 40 nm gold colloid-labeled antibody test piece are not in the region where linear regression is possible, and are already It is considered to be in the zone area. Therefore, for this concentration E, the mixed gold colloid-labeled antibody test piece 1A, the test piece excluding the 40 nm gold colloid-labeled antibody test piece, that is, the mixed gold colloid-labeled antibody test piece 2A, the 30 nm gold colloid-labeled antibody test piece, and the 20 nm gold Measurement is possible only with colloid-labeled antibody test strips.

  Similarly, the concentration G can be measured only with a 30 nm gold colloid-labeled antibody test piece and a 20 nm gold colloid-labeled antibody test piece. Similarly, the concentration H can be measured only for the 20 nm gold colloid-labeled antibody test piece.

  In other words, concentrations E to H (and higher) for 20 nm colloidally labeled antibody test strips, concentrations D to H for 30 nm gold colloid labeled antibody test strips, concentrations B to F for 40 nm gold colloid labeled antibody test strips, mixed gold colloid labels The antibody test piece 1A can measure concentrations A to F, and the mixed gold colloid-labeled antibody test 2A can measure concentrations C to G.

  The measurable concentration range varies depending on the type of labeling reagent carried on the test piece, that is, depending on the particle size of the gold colloid constituting each labeling reagent. As the particle size increases, the measurable range shifts to the low concentration side.

  On the other hand, the relationship between the colloidal gold particle size and mobility is not constant. First, 20 nm gold colloid-labeled antibody test piece (hereinafter, gold colloid-labeled antibody test piece is simply referred to as gold colloid test piece), 30 nm gold colloid test piece, and 40 nm gold colloid test piece. , E, the measurement limit concentration D of the 30 nm colloidal gold specimen is expected to be an intermediate point between the concentration B and the concentration E, but is actually closer to the concentration E.

  Secondly, for the mixed gold colloid test piece 1A, a 1: 1 mixed solution of 30 nm gold colloid labeled antibody and 40 nm gold colloid labeled antibody is immobilized, so the measurement limit concentration A is 30 nm gold colloid test. It is expected to be an intermediate point between the measurement limit concentrations D and E of the strip and the 40 nm colloidal gold test strip. However, the actual measurement limit concentration A is significantly lower than the measurement limit concentration B of the 40 nm gold colloid test piece, and the measurement range is expanded toward the low concentration side.

  Thirdly, for the mixed gold colloid test piece 2A, a 1: 1 mixed solution of 20 nm colloid-labeled antibody, 30 nm colloid-labeled antibody, and 40 nm colloid-labeled antibody is immobilized. It is expected to be in the vicinity of the measurement limit concentration D. However, the actual measurement limit concentration C is not near the concentration D, but closer to the lower concentration side, that is, the measurement limit concentration B of the 40 nm gold colloid test piece.

  As a gold colloid preparation method, the above-described method using chloroauric acid and citric acid has been widely used in the past because it is easy to control the particle size, but it is easy to control the particle size distribution. is not. The first result is assumed to be due to this.

  According to the mixed gold colloid test pieces 1A and 2A according to the present invention, the second and third results show that the CRP concentration-absorbance curve is theoretically higher than when using a colloidal gold labeled antibody having the same average particle diameter. This shows that it is possible to expand the measurement range to the low concentration side by shifting to the low concentration side.

(Example 2)
The immunochromatographic test piece of FIG. 2 described above was manufactured as follows. Using a membrane mainly composed of nitrocellulose for the developing layer 2, a complex of anti-CRP antibody B and gold colloid is used for the labeling reagent part 4, and an anti-CRP antibody A is used for the reagent immobilizing part 5, thereby immobilizing the reagent. Anti-CRP antibody C having a lower affinity than anti-CRP antibody A is used for part 10, and anti-CRP antibody D having a lower affinity than anti-CRP antibody C is used for reagent immobilization part 11.

a) Preparation of antibody-immobilized nitrocellulose membrane An anti-CRP antibody A solution diluted with a phosphate buffer solution to adjust its concentration is prepared. An immobilization line for CRP antibody A was provided. Similarly, an anti-CRP antibody C immobilization line was provided 2 mm downstream of the anti-CRP antibody C, and an anti-CRP antibody D immobilization line was further provided 2 mm downstream of the anti-CRP antibody C.

  The nitrocellulose membrane was dried, immersed in a Tris-HCl buffer solution containing 1% skim milk, and gently shaken for 30 minutes. After 30 minutes, the membrane was washed by transferring into Tris-HCl buffer solution, gently shaking for 10 minutes, and gently shaking for another 10 minutes in another Tris-HCl buffer solution. This membrane was treated with a surfactant by gently shaking in Tris-HCl buffer containing 0.05% sucrose monolaurate for 10 minutes. The membrane after the surfactant treatment was taken out and dried at room temperature.

b) Application of colloidal gold labeled antibody 20 nm colloidal labeled antibody prepared in Example 1, colloidal gold labeled antibody 30 nm, colloidal gold labeled antibody 40 nm, colloidal gold labeled antibody I, mixed colloidal gold labeled antibody II, respectively. It set to the discharge device, it apply | coated to the above-mentioned nitrocellulose film so that it might be closer to the test liquid introduction | transduction part rather than the reagent immobilization part, and the nitrocellulose film | membrane after application | coating was vacuum-freeze-dried. As a result, five types of spreading layers having a reagent immobilization part and a labeling reagent part were obtained.

c) Assembling Using the above-described five types of spreading layers, in the same manner as in Example 1, a 20 nm gold colloid labeled antibody test piece, a 30 nm gold colloid labeled antibody test piece, a 40 nm gold colloid labeled antibody test piece, and the present invention are applied. The mixed gold colloid labeled antibody test piece 1A and the mixed gold colloid labeled antibody test piece 2A were assembled.
[Test Example 2]
a) Sample and Measurement The CRP concentrations prepared in Example 1 were 0.03 mg / dl, 0.06 mg / dl, 0.1 mg / dl, 0.3 mg / dl, 0.6 mg / dl, 1.0 mg / dl 3.0 mg / dl, 6.0 mg / dl, 10.0 mg / dl blood samples were used. The CRP concentration of each blood sample was confirmed using a commercially available apparatus (7020 type biochemical automatic analyzer, manufactured by Hitachi, Ltd.) using a latex immunoagglutination method and a reagent.

  In the same manner as in Example 1, the above five types of immunochromatographic test pieces, namely, 20 nm gold colloid labeled antibody test piece, 30 nm gold colloid labeled antibody test piece, 40 nm gold colloid labeled antibody test piece, mixed gold colloid labeled antibody test. 5 μL of each concentration of blood sample was added to each sample introduction part of the piece 1A and the mixed gold colloid-labeled antibody test piece 2A, and the coloration state of the reagent-immobilized part was measured 5 minutes after the addition by a reflection absorbance measuring device (CS9300). Shimadzu Corporation) was used to measure the reflection absorbance at 635 nm. The measurement was performed 5 times for each concentration, and an average value was taken.

b) Results FIG. 6 shows the measurement results. The horizontal axis shows the CRP concentration in logarithm, and the vertical axis shows the absorbance in logarithm. FIG. 6A shows the results obtained with a 20 nm colloidal labeled antibody test piece, FIG. 6B shows the results obtained with a 30 nm colloidal labeled antibody test piece, and FIG. 6C shows the 40 nm colloidal labeled antibody test. FIG. 6 (d) shows the result obtained with the mixed gold colloid-labeled antibody test piece 1A, and FIG. 6 (e) shows the result obtained with the mixed gold colloid-labeled antibody test piece 2A. . 6 (a) to (e), the black circle plot is obtained from the reagent immobilization unit I (5 in FIG. 2), and the black triangle plot is the reagent immobilization unit II (10 in FIG. 2), The black square plot shows the result obtained from the reagent immobilization part III (11 in FIG. 2). In each test piece, the measurement range is significantly wider than the test piece of Example 1, that is, the test piece having one reagent immobilization part.

  The measurement results of FIGS. 6A to 6E are schematically shown in FIGS. As is clear from FIG. 7, each test piece shows different CRP responses in the reagent immobilization unit I, the reagent immobilization unit II, and the reagent immobilization unit III. 7A to 7E, the measurement limit concentration on the low concentration side (minimum measurement limit concentration in the three reagent-immobilized parts) is represented as A (A1, A2, A3, A4, and A5). The measurement limit concentration on the high concentration side (the maximum value of the limit concentration allowing linear regression for the CRP concentration-absorbance curves of the three reagent-immobilized parts) was expressed as B (B1, B2, B3, B4 and B5). In each test piece, the range of concentrations A to B is the measurement range.

  As is clear from FIGS. 7A to 7E, the measurement range is different for each test piece. As described in Example 1, the difference in the measurement range is largely due to the difference in the measurement range on the low concentration side. The signal of the reagent fixing part III of the 40 nm gold colloid-labeled antibody test piece and the mixed gold colloid-labeled antibody test piece 1A does not show a curve shape in which the CRP concentration-absorbance is correlated, and the high concentration side is the reagent fixing part II. As a result, the measurement range 3 and the measurement range 4 were narrower than the other measurement ranges.

  FIG. 8 shows the measurement ranges 1 to 5 obtained in FIGS. 7A to 7E side by side as black bars. The horizontal axis indicates the CRP concentration. A1 to A5 and B1 to B5 described on the horizontal axis have the same meaning as in FIG. The measurement range 3 and the measurement range 4 are narrowed for the reasons described above. In measurement range 1, measurement range 2, and measurement range 5, the measurement limit concentration on the high concentration side is approximately similar, but the measurement limit concentration on the low concentration side is different.

  The mixed gold colloid-labeled antibody test piece 1A was able to greatly expand the measurement range on the low concentration side compared to the 20 nm gold colloid-labeled antibody test piece and the 30 nm gold colloid-labeled antibody test piece. As a result. In contrast, the mixed gold colloid-labeled antibody test piece 2A was able to widen the measurement limit on the low concentration side without sacrificing the measurement limit on the high concentration side.

  According to the results of Example 1 and Example 2, according to the mixed gold colloid-labeled antibody test piece 1A of the present invention in which 30 nm gold colloid-labeled antibody and 40 nm gold colloid-labeled antibody were mixed at a ratio of 1: 1, The measurement range is expanded; according to the mixed gold colloid-labeled antibody test piece 2A in which 20 nm gold colloid-labeled antibody, 30 nm gold colloid-labeled antibody and 40 nm gold colloid-labeled antibody are mixed at 1: 1: 1, even on the low concentration side or the high concentration side However, the measurement range is widened; any test piece 1A, 2A can obtain a measurement range that is equal to or greater than the measurement range obtained from colloidal gold-labeled antibodies of 20 nm, 30 nm, and 40 nm. In addition, the measurement range with test strips 1A and 2A using any mixed gold colloid-labeled antibody does not expand on average or proportionally from the measurement range obtained with gold colloid-labeled antibodies of 20 nm, 30 nm, and 40 nm. Uniformly spread. These results indicate that the measurement range can be set more freely by mixing gold colloid-labeled antibodies composed of gold colloid particles having different particle diameters, such as the mixed gold colloid-labeled antibody test pieces 1A and 2A of the present invention. It shows that it becomes possible.

  The above Examples 1 and 2 are exemplifications, and the mixing ratio of 20 nm colloid-labeled antibody, 30 nm colloidal gold-labeled antibody, and 40 nm colloidal gold-labeled antibody is arbitrary, and by appropriately changing the mixing ratio, The measurement range can be set according to various substances to be inspected. In addition, the measurement range can be set using particles having a particle size of 1 to 100 nm, not limited to particles having a particle size of 20 nm, 30 nm, and 40 nm.

  The particle size distribution of colloidal gold was measured using the dynamic light scattering method, but is not limited to this. Light scattering methods such as laser diffraction and static light scattering, measurement with a microscope, and screening There is no problem even if other measuring methods such as sedimentation methods such as a method, a specific gravity balance method and a light transmission method are employed. The labeling substance constituting the labeling reagent is not limited to gold colloid, and any insoluble particulate marker may be used such as other metal colloids, alloys, colored polymer beads such as latex particles, and polymerized dye particles.

  It goes without saying that a larger number of reagent immobilization units can be provided depending on the purpose of measurement, and the relationship of each reagent immobilization unit can be changed depending on the affinity of the antibody used. The signal used for the measurement is a signal from the bound labeling reagent in the reagent immobilization section, and can be determined visually depending on the necessity, but in order to measure more accurately, the above-mentioned is used. It is preferable to use an arbitrary detector such as an absorbance meter.

  The labeling reagent part does not necessarily have to be one place, and can be configured by a combination of a plurality of reagent immobilization parts and a plurality of labeling reagent parts. For example, it is possible to adopt a configuration in which a labeling reagent unit is arranged upstream of each of a plurality of reagent immobilization units. Although the manufacturing method is complicated, it can be installed in any number and at any position.

  The biosensor (immunochromatographic test piece) is not limited to the above-described configuration in which the labeling reagent part and the reagent immobilization part are provided on the nitrocellulose membrane, but a material different from nitrocellulose, for example, a porous carrier such as a nonwoven fabric There is no problem even if a sample carrying a labeling reagent is placed on the support.

  Examples of liquids to be inspected include water, body fluids such as urine, blood, plasma, serum, saliva, (water) solutions in which solids and powders and gases are dissolved, urine tests, blood tests, fecal tests, The biosensor of the present invention can be used for environmental analysis such as water quality inspection and soil analysis, and food analysis.

  Although C-reactive protein (CRP) was exemplified as the test substance, in addition to this, proteins and protein derivatives such as antibodies, immunoglobulins, hormones, enzymes and peptides, bacteria, viruses, fungi, mycoplasmas, parasites and those Drugs and tumor markers such as infectious substances such as products and components, therapeutic drugs and drugs of abuse. Specific examples are chorionic gonadotropin (hCG), luteinizing hormone (LH), thyroid stimulating hormone, follicle forming hormone, parathyroid stimulating hormone, adrenal lipid stimulating hormone, estradiol, prostate specific antigen, hepatitis B surface antigen, myoglobin , CRP, cardiac troponin, HbA1c, albumin and the like.

  In any case, with the above-described configuration of the biosensor, the measurement range of the concentration of the substance to be inspected is sufficiently wide with high sensitivity and high performance, and the measurement can be performed easily and quickly. Is also easier.

  The biosensor according to the present invention is not limited to medical diagnosis sites such as clinical fields, but can be used as simple, accurate, and quick measurements in various fields such as food hygiene related fields and environmental measurement fields. .

The exploded view and perspective view which show the biosensor in Embodiment 1 of this invention Exploded view and perspective view showing a biosensor in Embodiment 2 of the present invention Particle size distribution of colloidal gold used in the biosensors of FIGS. Measurement results with the biosensor of FIG. Schematic diagram of FIG. Measurement result with the biosensor of Fig. 2 Schematic diagram of FIG. Schematic diagram of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fine space 2 Expanding layer 3 Opening part 4 Labeling reagent 5 Reagent immobilization part 6 Liquid impervious sheet material 7 Substrate 8 Fine space forming material 9 Cell contraction reagent 10 Reagent immobilization part 11 Reagent immobilization part

Claims (9)

A labeling reagent part that holds a labeling reagent for labeling the substance to be tested in the test liquid and an immobilization reagent that holds an immobilizing reagent for immobilizing the substance to be inspected on a development layer for developing the test liquid In a biosensor provided with at least a reagent part,
The biomarker is characterized in that the labeling reagent includes an insoluble granular marker, and the insoluble granular marker is prepared separately and at least two particle groups having different particle size distributions are mixed.   The biosensor according to claim 1, wherein the main peak of the particle size distribution in each particle group of the insoluble granular marker is in the range of an average particle size of 1 to 100 nm.   2. The biosensor according to claim 1, wherein the insoluble granular marker forms a complex by labeling a specific protein having a specific affinity for the substance to be examined.   The biosensor according to claim 3, wherein the specific protein is a substance involved in an immune reaction.   The biosensor according to claim 4, wherein the immune reaction is an antigen-antibody reaction.   The biosensor according to claim 1, wherein the insoluble granular marker is a metal colloid.   The biosensor according to claim 6, wherein the metal colloid is a gold colloid.   The biosensor according to claim 1, wherein the development layer is composed of a single layer or a plurality of layers of porous carriers.   The biosensor according to claim 1, which is an immunochromatographic test piece.

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