JP2011220705A - Complex particle for immunochromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide complex particles for immunochromatography with which not only absorbed light but also fluorescence can be detected by immunochromatography, no drop of the intensity of the absorbed light occurs and the detection of target substances can be sufficiently performed even by visual inspection, visual inspection is possible when a certain amount of target substances is included in specimen while target substances can be detected using a more sensitive device when determination based on light absorbing properties of target particles is impossible because the amount of the target substances is minute.SOLUTION: Complex particles for immunochromatography include a structure in which the outside of fine particles made from metal is covered with at least one layer of silica containing at least one fluorescence substance, and fine particles whose surface is modified with indicator substances enabling specific identification of target substances.

Description

  The present invention relates to composite particles for immunochromatography.

With immunochromatography (immunochromatography), the detection target (target substance) moves through the porous support by capillary action, and is captured by the labeled particles by a labeling substance that specifically recognizes the target substance, such as an antigen-antibody reaction. Further, the labeled particles bound to the detection target are concentrated by efficiently contacting with the capture substance that captures the detection target locally (for example, in a line) immobilized on the porous support, and the capture target is concentrated. This is an immunoassay method in which the presence or absence of a detection target is determined by color development of a line on which is immobilized.
The following three points can be mentioned as features of the immunochromatography method. (1) The time required for determination is 20 minutes or less, and rapid inspection is possible. (2) Since the measurement can be performed simply by dropping the specimen and the operation is simple, a plurality of specimens can be processed. (3) Since a determination is easy without requiring a special detection device, a general consumer can inspect himself / herself.
Utilizing these features, immunochromatography is used for pregnancy tests and influenza tests, and has attracted attention as a new POCT (Point Of Care Testing) technique (see, for example, Patent Document 1).

  Currently, gold nanoparticles and colored latex particles are used as labeling particles in the immunochromatography method, and a method of visually confirming the color development of the line is common (for example, see Patent Document 2). However, although the method of visually determining the color development of the line is simple, there is a problem that the sensitivity is not sufficiently high.

On the other hand, in the immunochromatography method, a method is known in which fluorescent particles are used as labeling particles and the fluorescent color development of the line is confirmed with a fluorescence detection device (see, for example, Patent Document 3). Although the fluorescence type immunochromatography test using a fluorescence detection device has improved sensitivity as compared with the conventional type visually confirmed, it has the following problems.
<1> In the immunochromatography reagent manufacturing process, labeled particles sensitized with an antibody are contained in a pad and dried to prepare a conjugate pad. The colored particles, such as gold particles and colored latex particles, whose label particles visually determine color development, have a high absorbance, so it is easy to determine whether the pad contains particles or whether they are contained uniformly. it can. However, when the labeled particles are fluorescent particles, the absorbance of the particles is low, so when the fluorescent particles are included in the pad, it is not easy to determine how much fluorescent particles are contained, and they must be contained in a certain amount. Is difficult. Whether it is contained in a certain amount needs to be determined by a fluorescence detection device, and the production cost increases.
<2> The fluorescence detection type immunochromatography reagent cannot be used in an environment where the fluorescence detector cannot be used, such as when the fluorescence detector fails or when the fluorescence detection device is not present.
<3> Since the fluorescence intensity of fluorescent particles decreases due to fading when the storage period is long, correct determination may not be possible using a fluorescent immunochromatographic reagent stored for a long period of time.
<4> When the immunochromatography reagent after the inspection is stored as the inspection result, the line disappears due to fading of the fluorescent particles in the fluorescent immunochromatography reagent, and the determination result after the inspection of the immunochromatography reagent cannot be stored for a long time.

  In order to solve the above problem, in immunochromatography, a method is known in which a target substance is detected by antigen-antibody reaction or the like using a mixture of colored particles and colored particles as labeled particles (for example, see Patent Document 4). However, since the antigen is separated and captured in both colored particles and colored particles, the detection sensitivity is lowered due to a decrease in colored luminance.

JP 2006-667979 A JP 2003-262638 A JP 2009-115822 A JP 2010-014631 A

In view of the above problems, the present invention can detect both absorption and fluorescence in immunochromatography, and can detect a target substance sufficiently even with visual observation without a decrease in absorption intensity. Immunochromatographic complex that allows visual determination when the amount is more than the amount, but can detect target substance by using a more sensitive instrument when the target substance is too small to be judged by the absorbance of the labeled particles It is an object to provide particles.
It is another object of the present invention to provide an immunochromatographic test strip that enables visual determination without detection sensitivity being lowered even after a long period of time has passed since preparation.
It is another object of the present invention to provide an immunochromatographic test strip that can be stored for a long period of time as a result of the test after the test strip for immunochromatography.

The above problem is solved by the following means.
(1) The outer side of the fine particle made of metal has a structure covered with at least one layer of silica containing at least one kind of fluorescent substance, and consists of fine particles whose surface is modified with a labeling substance that specifically recognizes the target substance. , Composite particles for immunochromatography.
(2) The immunochromatographic composite particle according to (1), wherein the metal substance is a substance that causes plasmon absorption.
(3) The immunochromatography as described in (1) or (2) above, wherein the metal substance is at least one selected from the group consisting of gold, silver, copper, platinum and a mixture thereof. Composite particles.
(4) Any of the above (1) to (3), wherein the fluorescent substance is an organic substance, and the difference between the maximum absorption wavelength of the fluorescent substance and the maximum plasmon absorption wavelength of the metal is 100 nm or more The composite particle for immunochromatography according to claim 1.
(5) The target substance is specifically captured using the immunochromatographic composite particles according to any one of (1) to (4), and the color of the metal of the composite particles capturing the target substance is visually observed. The target substance detection is determined, and the target substance detection determination and / or target substance quantification is performed by observing the fluorescence of the fluorescent substance of the composite particles capturing the target substance with a fluorescence detection device. A method for detecting a target substance.
(6) A test strip for immunochromatography comprising the composite particles for immunochromatography according to any one of (1) to (4).

  In the present specification, the composite particle for immunochromatography of the present invention is also simply referred to as “composite particle”.

In the composite particle for immunochromatography of the present invention, the labeled particle contains both absorptive and fluorescent functions, and the target substance can be detected even by visual observation without a decrease in the light absorption intensity. Therefore, when the target substance contains a certain amount or more, it is possible to detect the target substance visually without using a fluorescence detection device. On the other hand, if the target substance is in a small amount and cannot be judged by the absorbance of the labeled particles, it is higher. The target substance can be detected by using a sensitive instrument.
Since the test strip for immunochromatography of the present invention can detect a target substance by visual observation through absorption and emission of metal fine particles such as plasmon absorption, the detection sensitivity of the target substance is high even after the test strip has been stored for a long period of time. It does not decline.
Furthermore, the test strip for immunochromatography of the present invention can be reconfirmed by the visual determination result by absorption emission of metal fine particles such as plasmon absorption, even after the test strip after inspection is stored. Long-term storage of results is possible.

FIG. 1A is a plan view of an embodiment of an immunochromatographic test strip of the present invention, and FIG. 1B is a longitudinal sectional view of the immunochromatographic test strip shown in FIG. is there.

First, the composite particles of the present invention will be described.
The composite particle of the present invention is a composite particle whose surface is modified with a labeling substance that specifically recognizes a target substance (for example, a biomolecule such as an antibody, an antigen, DNA, or RNA).
In this invention, it is preferable that the average particle diameter of the said composite particle is 20-1000 nm, It is more preferable that it is 20-600 nm, It is further more preferable that it is 60-300 nm. If the particle size is too small, the detection sensitivity decreases, and if the particle size is too large, the porous support (membrane) used in the immunochromatography method becomes clogged.

In the present invention, the average particle size is determined from the total projected area of 50 labeled particles randomly selected from an image of a transmission electron microscope (TEM), a scanning electron microscope (SEM), etc. Is obtained by an image processing apparatus, and an average value (average circle equivalent diameter) of the diameters of circles corresponding to a value obtained by dividing the total occupied area by the number of selected composite particles (50 particles) is obtained.
The composite particles of the present invention are preferably monodispersed as a particulate material, and the so-called CV value of the variation coefficient of the particle size distribution is not particularly limited, but is preferably 15% or less, more preferably 10% or less.

The composite particles of the present invention have both coloring and fluorescent functions. The composite particle of the present invention has a plasmon absorption having a molar extinction coefficient ε of 5 × 10 ⁵ M ⁻¹ cm ⁻¹ or more (more preferably 1 × 10 ⁷ M ⁻¹ cm ⁻¹ or more) in the core part (plasmon). It preferably contains a metal that absorbs light.
Here, the molar extinction coefficient ε can be calculated from the following Lambert-Bale equation.

A = Log ₁₀ (I ₀ / I) = εbc

[A: absorbance, I: intensity of transmitted light, I ₀ : intensity of incident light, ε: molar extinction coefficient (M ⁻¹ cm ⁻¹ ), b: optical path length (cm), c: concentration of composite particles (M (Mol / L)), a _s : specific absorbance, c ′: concentration of composite particles (g / L)]

  The concentration c (mol / L) of the composite particle is obtained by determining the size of the labeled particle from the TEM photograph, determining the volume of one particle, and determining the density of the particle (for example, the volume ratio of silica as the shell to the metal as the core). The mass of one particle is determined from a weighted average), and only the composite particles are recovered from a fixed amount (for example, 1 mL) of the composite particle dispersion and dried to determine the number of moles from the mass of the composite particles. This is the value obtained.

In this specification, “molar extinction coefficient ε of the composite particles” means the absorbance of the composite particles in the dispersion obtained by measuring the absorbance of the composite particle dispersion and applying it to the Lambert-Beer equation. It refers to the molar extinction coefficient ε.
The absorbance, absorption spectrum, and ε of the composite particles should be measured using a dispersion such as an aqueous dispersion, ethanol dispersion, N, N-dimethylformamide dispersion, etc., using an arbitrary absorptiometer or plate reader. Can do.

  The metal constituting the core part of the composite particle of the present invention is preferably a substance that causes plasmon absorption peculiar to metal fine particles (absorbs light by plasmon). Examples of the metal include gold, silver, copper, platinum, and a mixture thereof.

Although there is no restriction | limiting in particular in the magnitude | size of the core part (fine particle which consists of metals) of the composite particle of this invention, 10 nm-100 nm are preferable from a plasmon absorptive viewpoint, and 30 nm-60 nm are more preferable.
Since the plasmon wavelength depends on the particle size (curvature) of the metal, the maximum plasmon absorption wavelength can be appropriately adjusted by adjusting the particle size.

The composite particles of the present invention have a structure in which the outside of fine particles made of metal is covered with at least one layer of silica containing at least one kind of fluorescent substance.
The fluorescent substance is not particularly limited, and examples thereof include organic fluorescent molecules (for example, fluorescein, rhodamine, Texas red, Alexa (trade name, manufactured by Invitrogen), Cy (trade name, manufactured by Applied Biosystems), etc.), semiconductors Examples thereof include nanoparticles (for example, CdSe, InGaP, ZnSSe, etc.).

In the composite particles of the present invention, the thickness of the shell portion is not particularly limited, but is preferably 100 to 400 nm, and more preferably 200 to 300 nm.
The molar extinction coefficient of the fluorescent substance contained in the shell part is preferably 1 × 10 ⁵ to 1 × 10 ⁹ M ⁻¹ cm ^−1, more preferably 1 × 10 ⁷ to 1 × 10 ⁸ M ⁻¹ cm ^−1. .

  In the composite particles of the present invention, the difference between the maximum absorption wavelength of the fluorescent material and the maximum plasmon absorption wavelength of the metal is preferably 50 nm or more, and more preferably 100 nm or more. In this way, by adjusting the maximum absorption wavelength of the fluorescent material and the maximum plasmon absorption wavelength of the metal, the amount of light absorption necessary for excitation of the fluorescent material can be increased, and the fluorescence intensity from the fluorescent material can be increased.

In the composite particles of the present invention, one or more silica layers containing metal particles as nuclei and containing at least one kind of fluorescent substance are formed on the outside by a core-shell method. Here, for example, it is preferable from the viewpoint of silica layer formation to prepare composite particles using a metal dispersion in which metal particles are dispersed in a commercially available citric acid aqueous solution.
The composite particles of the present invention may be composite particles obtained by any arbitrary preparation method. Examples thereof include composite particles prepared by the sol-gel method described in Journal of Colloid and Interface Science, 159, 150-157 (1993).
In the present invention, it is particularly preferable to use composite particles obtained according to the method for preparing fluorescent dye compound-containing colloidal silica particles described in International Publication No. 2007 / 074722A1.
For example, APS which is silane coupling is added to colloidal gold, and APS is adsorbed on the surface of colloidal gold particles. Furthermore, Na ₂ O · nSiO ₂ (water glass) is added to the solution, and silica coating is performed on the APS to obtain composite particles. Alternatively, the surface of the silica coating with Na ₂ O · nSiO _2, may form a shell further contains a fluorescent material.

Specifically, the formation of a shell containing a fluorescent substance is performed by using one or two kinds of products obtained by reacting a fluorescent substance with a silane compound and covalently bonding, ionic bonding, or other chemical bonding or adsorption. It can be carried out by polymerizing the above silane compounds.
As an embodiment of a shell forming method containing a fluorescent material, N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group, epoxy group, A fluorescent substance having or added an active group such as a cyano group is reacted with a silane coupling agent having a substituent (for example, an amino group, a hydroxyl group, or a thiol group) that reacts correspondingly with the active group, and is covalently bonded. It can be prepared by polymerizing one or two or more kinds of silane compounds to the product obtained.

  Specific examples of the fluorescent substance include a fluorescent substance having an NHS ester group such as 5- (and -6) -carboxytetramethylrhodamine succinimidyl ester (trade name, manufactured by emp Biotech GmbH) or added thereto. Can do.

  Specific examples of the silane coupling agent having a substituent include γ-aminopropyltriethoxysilane (APS), 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, N-2 ( Examples thereof include silane coupling agents having an amino group such as (aminoethyl) 3-aminopropylmethyldimethoxysilane and 3-aminopropyltrimethoxysilane. Of these, APS is preferable.

  The silane compound to be polymerized is not particularly limited, but includes tetraethoxysilane (TEOS), γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS). ), 3-thiocyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxy Mention may be made of silane. Among these, TEOS, MPS, or APS is preferable.

  In the present invention, the fluorescent material is in a state of being immobilized in silica particles in the silica layer.

When prepared as described above, spherical or nearly spherical composite particles can be produced using metal particles such as gold as the core. More specifically, the composite particles having a nearly spherical shape have a shape in which the ratio of the major axis to the minor axis is 2 or less.
In order to obtain composite particles having a desired average particle size, ultrafiltration is performed using an ultrafiltration membrane such as YM-10, YM-100 (both trade names, manufactured by Millipore), and the particle size is large. It is possible to remove particles that are too small or too small, or to centrifuge at an appropriate gravitational acceleration and collect only the supernatant or precipitate.

The composite particles are surface-modified with a labeling substance that specifically recognizes a target substance (analyte) (for example, a biomolecule such as an antibody, an antigen, DNA, or RNA).
There is no particular limitation on the method of surface-modifying the composite particles with a labeling substance that specifically recognizes the target substance, and the target substance is specifically identified by electrostatic attraction, van der Waals force, hydrophobic interaction, or the like. The labeling substance to be recognized may be adsorbed on the composite particle, or may be bound by a chemical bond with a crosslinking agent or a condensing agent. Further, when the composite particle surface has a thiol group, it may be bonded to the thiol group of a substance that specifically recognizes the target substance by an SS bond.
Further, when the composite particles are aggregated when the labeling substance is bonded to the surface of the composite particles, the surface of the composite particles may be previously subjected to a surface treatment by an alternate adsorption method. The alternating adsorption method is a method of forming a polymer thin film on the surface of a substrate or particle by adsorbing a charged polymer to the surface of the substrate or particle having a charge by electrostatic attraction. Since charge can be imparted to the surface of the composite particles by performing alternate adsorption treatment on the surface of the composite particles, an electrostatic repulsive force is generated between the particles, and dispersibility is improved. In addition, the polymers bonded to the composite particles overlap each other as the distance between the particles approaches, thereby generating entropy repulsion and improving dispersibility.

For the purpose of improving the dispersibility of the composite particle of the present invention and modifying the antibody, it is preferable to adsorb or bind a polymer to the surface of the composite particle. The polymer has a functional group in the side chain that can bind to the labeling substance such as an antibody by an amide bond, disulfide bond, etc., and the functional group is ionized in a buffer solution or the like to act as a hydrophilic group. Molecules are preferred, and specific examples include alginic acid, polyallylamine hydrochloride, sodium polyacrylate, and chitosan.
Here, “antibody modification” means that a side chain of a polymer (for example, a carboxyl group of the side chain) and an antibody (for example, an amino group of an antibody) are chemically bound (peptide bond or the like). Say. However, in the present invention, it is not always necessary to use a polymer as a method for binding the antibody to the particle. As described above, the surface of the particle directly using electrostatic attraction, van der Waals force, hydrophobic interaction, etc. May be modified with an antibody or the like.
In the present invention, the surface of the composite particle may be modified with a surfactant, a biological substance or the like in addition to a labeling substance that recognizes a target substance.

The immunochromatographic test strip of the present invention comprises:
(1) A sample addition member (sample pad) and a member (conjugate pad) containing the composite particles of the present invention,
(2) The conjugate pad and a membrane having an antibody immobilization part (antibody immobilization membrane), and (3) the antibody immobilization membrane and the absorption pad are connected in series so that a capillary phenomenon occurs between them. Preferably it is.

A preferred embodiment of the immunochromatographic test strip of the present invention will be described with reference to FIGS. 1 (a) and 1 (b), but the present invention is not limited thereto.
FIG. 1 (a) is a plan view of a preferred embodiment of the immunochromatographic test strip of the present invention, and FIG. 1 (b) is a longitudinal sectional view of the immunochromatographic test strip shown in FIG. 1 (a). FIG.
The immunochromatographic test strip 1 of the present invention preferably comprises a sample pad 2, a conjugate pad 3, an antibody-immobilized membrane 4, and an absorption pad 5. Each of the above constituent members is preferably lined with a backing sheet 6 with an adhesive.

  The antibody immobilization section of the antibody immobilization membrane 4 is provided with a test line 41 in which a target substance capturing antibody for determining the presence or absence of a target substance, that is, positive / negative is determined. The antibody-immobilized membrane 4 preferably includes a control line 42 on which an antibody that recognizes the antibody labeled on the composite particle is immobilized.

In the present invention, examples of target substances to be detected and quantified include antigens, antibodies, DNA, RNA, sugars, sugar chains, ligands, receptors, peptides, chemical substances, and the like.
In the present invention, the sample containing the target substance is not particularly limited, and examples thereof include liquid samples such as urine and blood.

Next, each member will be described.
1) Sample pad 2
The sample pad 2 is a component that drops a sample containing a target substance.

2) Conjugate pad 3
The conjugate pad 3 is a structural member containing the composite particles of the present invention, and the target substance contained in the sample moved from the sample pad 2 by capillary action is a specific molecular recognition reaction such as an antigen-antibody reaction, and the composite The part that is captured and labeled by the particle.

3) Antibody-immobilized membrane 4
The membrane 4 is a structural member in which the target substance labeled with the composite particles moves by capillary action, and an antibody immobilization part (determination) in which a sandwich-type immune complex forming reaction consisting of immobilized antibody-target substance-composite particles is performed. Part).
The shape of the antibody immobilization part (determination part) in the membrane is not particularly limited as long as the capture antibody is locally immobilized, and includes a line shape, a circular shape, a belt shape, etc. It is preferable that there is a line shape having a width of 0.5 to 1.5 mm.

  The target substance labeled with the composite particles is captured by the antibody immobilization part (determination part) by the sandwich-type immune complex formation reaction consisting of the immobilized antibody-target substance-composite particles, and the complex in the formed complex The presence or absence of the target substance can be determined based on the degree of color development or fluorescence derived from the composite particles, that is, positive / negative can be determined. That is, the composite particles are concentrated in the antibody immobilization section (determination section), and can be detected and determined visually or using a detection device as a colored or fluorescent signal.

  In order to sufficiently complete the sandwich-type immune complex formation reaction, or to avoid the influence on the measurement by the colored or fluorescent substance in the liquid sample or the measurement by the composite particles not bound to the target substance, the antibody The determination part in the immobilized membrane is preferably provided at a position (for example, the middle of the membrane) at a certain distance from the connection end with the conjugate pad and the connection end with the absorption pad.

The amount of antibody immobilization in the antibody immobilization part (determination part) is not particularly limited, but when the shape is a line, it is preferably 0.1 μg to 5 μg per unit length (cm). Examples of the immobilization method include a method in which an antibody solution is applied, dropped or sprayed and then dried and immobilized by physical adsorption.
After the above-described antibody immobilization, the whole antibody-immobilized membrane is preferably subjected to a so-called blocking treatment in order to prevent the influence of nonspecific adsorption on the measurement. For example, a method of immersing in a buffer solution containing a blocking agent such as albumin, casein, polyvinyl alcohol or the like for an appropriate period of time and then drying may be mentioned. Examples of the commercially available blocking agent include skim milk (manufactured by DIFCO), 4% block ace (manufactured by Meiji Dairies), and the like.

4) Absorption pad 5
The absorption pad 5 is a constituent member that absorbs the sample and composite particles that have moved through the membrane by capillary action and always generates a constant flow.

There are no particular limitations on the material of each of these constituent members, and members used for immunochromatographic test strips can be used, but glass fibers such as Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE) are used as sample pads and conjugate pads. A fiber pad is preferable, a nitrocellulose membrane such as Hi-Flow Plus 120 membrane (trade name, manufactured by MILLIPORE) is preferable as the membrane, and a cellulose membrane such as Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) is preferable as the absorption pad. Is preferred.
Examples of the backing sheet with an adhesive include AR9020 (trade name, manufactured by Adhesives Research).

  The test strip is produced by adjoining members at both ends of each member in order of the sample pad, conjugate pad, antibody-immobilized membrane, and absorption pad in order to facilitate capillary action between the members. And 1 to 5 mm in a stack (preferably on a backing sheet).

Next, a method for detecting a target substance using the composite particles of the present invention will be described.
In the method for detecting a target substance of the present invention, first, a sample containing the target substance is dropped onto a sample pad of a test strip for immunochromatography.
If the target substance contained in the sample is more than a certain amount (amount that can be detected visually), the color of the test line provided on the antibody-immobilized membrane of the immunochromatographic test strip can be confirmed visually and is positive or negative. Judgment is possible. On the other hand, color development on the test line is not visually confirmed for samples with a target substance of a certain amount or less, but positive or negative determination is performed with high sensitivity by observing the strip using a fluorescence detection device for immunochromatography. Is possible. As described above, the test method using the immunochromatographic composite particles of the present invention can easily and quickly make a positive determination visually when the target substance is a certain amount or more, and the amount of the target substance is less than a certain amount. In this case, it is possible to determine with high sensitivity using a fluorescence detection apparatus for immunochromatography. Thus, in the method for detecting a target substance of the present invention, the composite particle of the present invention uses particles that complement the functions of the fluorescent particles and the colored particles and do not decrease the detection sensitivity of the target substance. An appropriate means for detecting the target substance can be appropriately selected according to the amount.

The target substance detection method of the present invention uses the composite particle of the present invention to capture the target substance by a specific molecular recognition reaction such as an antigen-antibody reaction, and visually observe the color of the metal of the composite particle that has captured the target substance. Thus, the target substance detection is determined, and the fluorescence of the fluorescent substance of the composite particle that has captured the target substance is observed with a fluorescence detection device, so that the target substance detection is determined and / or the target substance is quantified.
In this specification, “quantitative” means, for example, the relative amount of the target substance based on the difference in fluorescence intensity from the composite particles obtained by irradiating the test line of the test strip with predetermined light. To express. Specifically, the fluorescence intensity is defined by the output value of a light receiving element (CCD detector or the like) used for fluorescence detection, and the amount of the target substance can be measured based on the magnitude.

Next, the fluorescence detection apparatus used for the target substance detection method of the present invention will be described.
The fluorescence detection apparatus used for the target substance detection method of the present invention comprises an excitation light source and a filter.
Examples of the excitation light source include a mercury lamp, a halogen lamp, a xenon lamp, a laser diode, and a light emitting diode. The filter is a filter for transmitting only light of a specific wavelength from an excitation light source, and further a filter that removes the excitation light and transmits only fluorescence from the viewpoint of detecting only fluorescence. It selects suitably from a fluorescence wavelength and a fluorescence wavelength.
The fluorescence detection apparatus may include a photomultiplier tube or a CCD detector that receives the fluorescence. As a result, fluorescence having an intensity or wavelength that cannot be visually confirmed can be detected. Further, since the fluorescence intensity can be measured, the target substance can be quantified, and highly sensitive detection and quantification are possible.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these Examples at all.

Production Example 1 (Preparation of composite particles)
5.8 mg of a fluorescent dye (trade name: HiLyte Fluor 647 succinimide ester, manufactured by AnaSpec) was dissolved in 1 mL of dimethylformamide (DMF). 2.6 μL of APS was added thereto and reacted at room temperature (23 ° C.) for 1 hour.
A mixture of 400 μL of the resulting reaction solution and 1 mL of Au particles (trade name, Au citric acid colloid solution) having a particle diameter of about 40 nm dispersed in an aqueous citric acid solution was mixed with 128 mL ethanol, 400 μL TEOS, 28.8 mL distilled water, 28 mass. % Ammonia water 400 μL was added, and the reaction was performed at room temperature for 24 hours.
The reaction solution was centrifuged for 30 minutes at a gravitational acceleration of 18000 × g, and the supernatant was removed. 4 mL of distilled water was added to the precipitated composite particles to disperse the composite particles, and the mixture was again centrifuged at 18000 × g for 30 minutes. This washing operation was further repeated twice to remove unreacted TEOS, ammonia and the like contained in the composite particle dispersion, thereby obtaining 94.2 mg of composite particles having an average particle diameter of about 200 nm. It was confirmed by an electron micrograph that the core of the obtained composite particles was gold fine particles and the shell was silica. Further, in the obtained composite particles, the maximum absorption wavelength of the fluorescent material was 647 nm, and the maximum plasmon absorption wavelength of gold was 530 nm.

When the absorbance of the composite particles of the labeled silica nanoparticles 1 g / L was measured (measurement wavelength: 600 nm to 700 nm) and the silica density was 2.3 mg / cm ³ , the molar extinction coefficient corresponding to silica of the composite particles was determined. 7.4 × 10 ⁷ M ⁻¹ cm ⁻¹ . Further, the fluorescence intensity of 1 g / L of the composite particles was measured, and the fluorescence intensity per particle was determined by setting the density of silica to 2.3 mg / cm ³ , which corresponds to the fluorescence intensity of 44,000 fluorescent dyes. .

Production Example 2 (Production of Silica Particle / Alginic Acid Composite Particle)
Colloid 500 μL (dispersion medium: distillation) of silica particles (average particle size 183 nm) containing a fluorescent dye (trade name: HiLyte Fluor 647 succinimide ester, manufactured by AnaSpec) having a concentration of 40 mg / mL obtained by the same method as in Production Example 1. To the water), 2.9 mL of distilled water and 400 μL of a sodium alginate aqueous solution (weight average molecular weight 70000) having a concentration of 10 mg / mL were added and stirred well with a stir bar. Subsequently, 200 μL of 28 mass% ammonia water was added, and the colloid was stirred at room temperature (23 ° C.) for 1 hour. The resulting colloid was centrifuged for 30 minutes at a gravitational acceleration of 15,000 × G, and the supernatant was removed. 3.4 mL of distilled water was added to the precipitated particles to redisperse the particles. Subsequently, 400 μL of 10 mg / mL sodium alginate aqueous solution was added and stirred well with a stirrer, and then 200 μL of 28 mass% aqueous ammonia was added and stirred for 1 hour. The colloid was centrifuged at a gravitational acceleration of 15,000 × G for 30 minutes, and after removing the supernatant, 1 mL of distilled water was added to disperse the particles. Similarly, centrifugal separation twice and dispersion in distilled water were repeated to wash the particles, and the particles were dispersed in 1 mL of distilled water to obtain a colloid of fluorescent dye-containing silica particles / alginic acid composite particles (yield 20 mg / mL × 1 mL). It was confirmed by an electron micrograph that the core of the obtained composite particles was gold fine particles and the shell was silica. Further, in the obtained composite particles, the maximum absorption wavelength of the fluorescent material was 647 nm, and the maximum plasmon absorption wavelength of gold was 530 nm.

Production Example 3 (Modification of composite particles with anti-hCG antibody)
100 μL, 50 mg / mL of 0.5 M MES (2-Morpholinoethanesulfonic acid) buffer solution (pH 6.0) is added to 500 μL of the fluorescent dye-containing silica particle / alginate composite particle having a concentration of 20 mg / mL prepared in Production Example 2. 461 μL of NHS, 19.2 mg / mL EDC (1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide) 150 μL, and distilled water 1.089 mL were added and mixed for 30 minutes.
The reaction solution was centrifuged at a gravitational acceleration of 15,000 × G for 30 minutes, and after removing the supernatant, 1 mL of distilled water was added to disperse the particles. Similarly, centrifugation was repeated twice and dispersion in distilled water was repeated to wash the particles. Finally, the particles were dispersed in 1 mL of 50 mM KH ₂ PO ₄ buffer (pH 8.0). Here, 1 mL of 1 mg / mL anti-hCG antibody (Anti-hCG mouse IgG1, manufactured by Medix Biochemica) dissolved in 50 mM KH ₂ PO ₄ buffer (pH 8.0) was added and mixed for 2 hours. Subsequently, 100 μL of 100 mg / mL BSA solution was added and further mixed for 1 hour.
The reaction solution was centrifuged at a gravity acceleration of 15,000 × G for 30 minutes, and after removing the supernatant, 1 mL of 50 mM KH ₂ PO ₄ buffer (pH 8.0) was added to disperse the particles. Similarly twice more, by repeating the dispersion into KH ₂ PO ₄ buffer centrifugation and 50 mM (pH 8.0) to wash the particles, finally KH ₂ PO ₄ buffer 50 mM (pH 8.0) 1 mL To obtain a colloid of rhodamine-containing silica particles modified with an anti-hCG antibody (yield 10 mg / mL × 1 mL).

Reference Example 1 (Preparation of conjugate pad containing mixed particles)
For 1 mL of colored latex particles (DC02B / 5641, manufactured by Bangs Laboratories) having a particle size of 0.16 μm and a concentration of 10 mg / mL, the anti-hCG antibody was modified in the same manner as in Production Example 3, and a 50 mM KH ₂ PO ₄ buffer solution was used. 1 mL (10 mg / mL) of colored latex particle colloid modified with anti-hCG antibody dispersed in (pH 8.0) was obtained.
Subsequently, with respect to 1 ml of fluorescent latex particles (FS02F / 8234, manufactured by Bangs Laboratories) having a particle size of 0.19 μm and a concentration of 10 mg / mL, the anti-hCG antibody was modified in the same manner as in Production Example 3, and 50 mM KH ₂ PO 1 mL (10 mg / mL) of fluorescent latex particle colloid modified with anti-hCG antibody dispersed in ₄ buffer solution (pH 8.0) was obtained.

0.05 mL of colored latex particle colloid modified with the anti-hCG antibody (10 mg / mL), 0.25 mL of fluorescent latex particle colloid modified with the anti-hCG antibody (10 mg / mL), 3 mL of 50 mg / ml L sucrose, and distillation 1.7 mL of water was mixed. 0.8 mL of the obtained mixed particle colloid was contained in one conjugate pad (trade name: Glass Fiber Conjugate Pad, manufactured by MILLIPORE, 8 × 150 mm).
The conjugate pad containing the mixed particles of colored latex particles and fluorescent latex particles was light blue, and it was visually confirmed that the pads contained particles.

Reference Example 2 (Preparation of conjugate pad containing fluorescent particles)
Fluorescent latex particles obtained by mixing 0.3 mL of fluorescent latex particle colloid (10 mg / mL) modified with anti-hCG antibody obtained in Reference Example 1, 3 mL of 50 mg / mL sucrose, and 1.7 mL of distilled water 0.8 mL of colloid was contained per one conjugate pad (trade name: Glass Fiber Conjugate Pad, manufactured by MILLIPORE, 8 × 150 mm).
The conjugate pad containing only fluorescent latex particles was colorless, and it could not be visually confirmed that the pad contained particles.

Production Example 4 (Preparation of conjugate pad containing composite particles)
Colored fluorescent silica particles (10 mg / mL) modified with an anti-hCG antibody prepared in Production Example 3 (0.3 mL), 50 mg / mL sucrose (3 mL), and distilled water (1.7 mL) were mixed. 0.8 mL of the resulting colored fluorescent silica particle (composite particle) colloid was added to one conjugate pad (trade name: Glass Fiber Conjugate Pad, manufactured by MILLIPORE, 8 × 150 mm).
The conjugate pad containing the composite particles was light red, and it was visually confirmed that the composite particles were contained in the pad.

Example 1 (Detection of recombinant hCG using immunochromatographic test strip prepared using a conjugate pad containing colored fluorescent silica particles)
As shown below, a test strip for immunochromatography was prepared using the conjugate pad prepared in Production Example 4, and recombinant hCG was detected.

(1) Preparation of antibody-immobilized membrane An anti-hCG antibody (approximately 12 mm from the end) near the center of the membrane (length 25 mm, trade name: Hi-Flow Plus120 membrane, manufactured by MILLIPORE) is used as an anti-hCG antibody ( Alpha subunit of FSH (LH), clone code / 6601, manufactured by Medix Biochemica) 1 mg / mL solution ((50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) 0.75 μL / cm It was applied with.
Subsequently, as a control line having a width of about 1 mm, 0.75 μL of a solution ((50 mM KH ₂ PO ₄ , pH 7.0) sugar free) containing 1 mg / mL of an anti-IgG antibody (Anti Mouse IgG, manufactured by Dako) was used. It was applied at a coating amount of / cm and dried at 50 ° C. for 30 minutes. The distance between the test line and the control line was 1 cm.
Next, the entire membrane was immersed in a blocking buffer (0.5% casein-containing 50 mM borate buffer (pH 8.5)) at room temperature for 30 minutes as a blocking treatment.
The membrane was transferred to a membrane washing / stabilization buffer (0.5 mM sucrose, 0.05 mM sodium cholate-containing 50 mM trishydroxymethylaminomethane hydrochloride buffer (pH 7.5)) and allowed to stand at room temperature for 30 minutes. The membrane was pulled up, placed on a paper towel and dried overnight at room temperature to prepare an antibody-immobilized membrane.

(2) Preparation of test strip Sample pad (trade name: Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), conjugate pad prepared in Reference Example 1, the antibody-immobilized membrane, and absorption pad (trade name: Cellulose) Fiber Sample Pad (CFSP), manufactured by MILLIPORE, is assembled in this order on a backing sheet (trade name AR9020, manufactured by Adhesives Research), and cut into strips of 5 mm width and length 60 mm, and FIG. A test strip having the structure shown in 1 (b) was produced.
As shown in FIGS. 1 (a) and 1 (b), each component member was pasted with its both ends overlapped with an adjacent member by about 2 mm (the same applies hereinafter).

(3) Detection of recombinant hCG 100 μL of 100, 50, 20, 10, 5, 2, or 0.2 IU / L of recombinant hCG (manufactured by Rohto Pharmaceutical) was dropped on the sample pad portion of the test strip and left for 5 minutes. Then, the color development of the test line coated with the anti-hCG antibody and the control line coated with the anti-IgG antibody was visually confirmed. As a result, when the recombinant hCG was 100, 50 and 20 IU / L, the color development of the test line and the control line could be visually confirmed. When the recombinant hCG was 10, 5, 2, 0.2 IU / L, no color development on the test line was visually confirmed.

  A test strip onto which a sample containing 10, 5, 2 or 0.2 IU / L of the recombinant hCG was dropped was irradiated with a mercury lamp (103 W), and a CCD detector (trade name: C2741-35A, manufactured by Hamamatsu Photonics) was used as a detector. ) Was used to image fluorescence. As a result, the fluorescent color development of the test line and the control line was confirmed for any sample having the recombinant hCG of 10, 5, 2, 0.2 IU / L.

  From the above results, the test strip using the colored fluorescent silica particles can be easily confirmed visually when the recombinant hCG exceeds a certain amount, and even with a small amount of the recombinant hCG, it can be determined by the fluorescence detection apparatus. It was.

Comparative Example 1
A test strip was prepared in the same manner as in Example 1 except that the conjugate pad prepared in Reference Example 1 was used as the conjugate pad. Recombinant hCG was detected by the same method as in Example 1 (3) using the obtained test strip.
As a result, when the recombinant hCG was 100, 50, or 20 IU / L, the color development of the test line and the control line could be confirmed visually, but when the recombinant hCG was 10, 5, 2, or 0.2 IU / L, it was visually The color of the test line could not be confirmed. In the evaluation using the fluorescence detection apparatus, the fluorescent color development of the test line and the control line was confirmed for the samples with the recombinant hCG of 20, 10, 5 and 2 IU / L, but the test line for the 0.2 IU / L sample. In addition, the fluorescent coloration of the control line could not be confirmed.
From the above results, the test strip using the mixed particles can detect a certain amount of the recombinant hCG, but if the amount of the recombinant hCG is very small, the target substance cannot be detected at all, and the target substance is detected. The sensitivity was low.
Unlike the composite particles of the present invention, when a mixed particle of colored particles and fluorescent particles is used, an antigen as a target substance binds to both the colored particles and the fluorescent particles. Therefore, the concentration of the antigen trapped on the surface per particle is lowered as compared with the composite particle of the present invention. Accordingly, the force with which the antibody on the test line captures the labeled particles is reduced, and the detection sensitivity of the target substance is reduced.

Example 2 (Detection evaluation of recombinant hCG using a test strip after standing for a long time)
Each of the test strips prepared using the conjugate pad containing the fluorescent latex particles prepared in Reference Example 2 and the test strip prepared using the conjugate pad containing the composite particles prepared in Production Example 4, Left at room temperature for 9 months.
When 100, 50, 20, 10, 5 or 3 IU / L of recombinant hCG was dropped on the test strip after standing for 9 months and the line was detected, the test strip containing the composite particles was found to contain the recombinant hCG. When the value was 50 IU / L or more, the color development of the test line and the control line could be confirmed visually. On the other hand, in the test strip containing fluorescent latex particles, the color development of the test line and the control line by visual observation is confirmed, and the color development of the test line and the control line by the fluorescence detector is observed regardless of the concentration of the recombinant hCG. We could not confirm both.

  From the above results, it can be seen that the test strip prepared using the conjugate pad containing the composite particles of the present invention is suitable for long-term storage.

Example 3 (Evaluation of whether or not line coloring can be confirmed after leaving a test strip for a long period of time for detection of recombinant hCG)
For each of the test strips produced using the conjugate pad containing the fluorescent latex particles produced in Reference Example 2 and the test strips produced using the conjugate pad containing the composite particles produced in Production Example 4, 100 μL of 50 IU / L recombinant hCG was dropped to develop the test line and the control line. The test strip was left at room temperature for 10 months.
As a result, the test strip produced using the conjugate pad containing the fluorescent latex particles could not confirm the color development or the color development of the control line and the test line by visual observation or by using a fluorescence detector. On the other hand, the test strip produced using the conjugate pad containing the composite particles could visually confirm the color of the control line and the test line.

  From the above results, it can be seen that the test strip prepared using the conjugate pad containing the composite particles of the present invention is suitable for storing the test strip after the test as a test result for a long period of time.

DESCRIPTION OF SYMBOLS 1 Test strip 2 Sample pad 3 Conjugate pad 4 Antibody fixed membrane 5 Absorption pad 6 Backing sheet 41 Test line 42 Control line

Claims (6)

  An immunochromatography having a structure in which the outer side of a fine particle made of metal is covered with at least one layer of silica containing at least one kind of fluorescent substance, and the surface is modified with a labeling substance that specifically recognizes the target substance Composite particles.   The composite particle for immunochromatography according to claim 1, wherein the metal substance is a substance that causes plasmon absorption.   The composite particle for immunochromatography according to claim 1 or 2, wherein the metal substance is at least one selected from the group consisting of gold, silver, copper, platinum, and a mixture thereof.   The immunochromatography according to any one of claims 1 to 3, wherein the fluorescent substance is an organic substance, and the difference between the maximum absorption wavelength of the fluorescent substance and the maximum plasmon absorption wavelength of the metal is 100 nm or more. Composite particles.   The target substance is specifically captured using the immunochromatographic composite particles according to any one of claims 1 to 4, and the target substance detection is determined by visually observing the color development of the metal of the composite particles capturing the target substance. And detecting the target substance and / or quantifying the target substance by observing the fluorescence of the fluorescent substance of the composite particles capturing the target substance with a fluorescence detection device. An immunochromatographic test strip comprising the immunochromatographic composite particles according to claim 1.

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