JP2005156527A - Optical measurement device and optical measurement method of specific binder using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical measurement device capable of quickly and accurately measuring a specific binder without performing B/F separation. <P>SOLUTION: In this measurement device for optically measuring a specific binder in a fluid sample, a reaction part carrying a first specific binding member, which can form the specific bond with a measured substance bound with a second specific binding member associated with fluorescent labeling or light diffusion labeling to construct the specific binder, on one surface is fixed on one surface of a transparent waveguide, and a transparent intermediate layer and a light absorbing layer are sequentially layered on the other surface of the transparent waveguide having a light emission side edge face. When a refractive index of the waveguide is represented by n<SB>w</SB>, that of the intermediate layer is represented by n<SB>m</SB>, and that of the fluid sample is represented by n<SB>s</SB>, the respective refractive indexes satisfy the following formula 1: n<SB>w</SB>>n<SB>m</SB>≥n<SB>s</SB>. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to an optical measuring apparatus and a method for optically measuring a specific binding substance using the same, and in particular, propagates while reflecting fluorescence or scattered light converted or scattered by a fluorescent label or light scattering label. On the surface of the waveguide, the second specific binding member to which the fluorescent label or the light scattering label is bound is bound to the target substance, and further, the target substance is bound to the first specific binding member, Fluorescence or scattering emitted from the light emitting side edge surface of the waveguide by introducing light into the waveguide, which is converted or scattered by the fluorescent label or the light scattering label and irradiating the light beam. The present invention relates to an optical measurement apparatus and an optical measurement method for measuring a specific binding substance by measuring light, and consequently measuring a substance to be measured in a fluid sample.

Conventionally, the light from the light source is propagated while totally reflecting through the optical waveguide, and the fluorescent substance constrained near the surface of the optical waveguide by the antigen-antibody reaction is excited by the evanescent wave component, and the intensity of the resulting fluorescence is measured. Therefore, studies on fluorescent immunoassay devices that indirectly measure the degree of immune reaction have been conducted.

As a fluorescence immunoassay device with excellent measurement sensitivity and measurement accuracy, the antigen-antibody reaction is carried out on the surface of the optical waveguide that propagates while totally reflecting the introduced excitation light, and further a substance labeled with a fluorescent substance is reacted. Fluorescence immunoassay that measures the degree of immune reaction based on the fluorescence emitted from the optical waveguide by introducing the fluorescence emitted by the fluorescent material excited by the evanescent wave component of the excitation light into the optical waveguide and propagating it while totally reflecting it. A fluorescence immunoassay device characterized in that a fluorescent material having an absorption peak near 650 nm is used as the device and light having a wavelength near 635 nm is used as excitation light has been proposed (see, for example, Patent Document 1). ).
JP-A-7-63756

However, in the above-described fluorescence immunoassay apparatus, the light to be irradiated has to be irradiated with a laser beam having a specific wavelength at a specific angle (an angle at which the light is totally reflected in the waveguide), and an ordinary incandescent lamp, xenon lamp, etc. Since the light source cannot be used, the apparatus becomes large, the cost is high, and the measurement is difficult.

A method for detecting a specific binding analyte using different waveguides is a method for detecting the presence or amount of one or more specific binding analytes in a fluid sample, comprising: (a) (i) a fluid sample A transparent element having a higher refractive index, (ii) a light-receiving edge, and (iii) a first specific binding member of at least one cognate binding pair immobilized at a plurality of sites on the surface of the element A waveguide device (wherein the first specific binding member is optionally coupled via an intermediate cognate binding pair), wherein the first specific binding member is optionally immobilized via other intermediate sites. And (b) capable of specifically binding to at least one analyte), and (b) a sample suspected of containing said one or more analytes and a second cognate binding pair. Light scattering bound to specific binding members. The reaction surface is contacted with a bell (wherein the specific binding member of the second cognate binding pair is optionally coupled to the one or more analytes in the case of a sandwich assay via an intermediate cognate binding pair). An analyte in the sample, which can bind specifically, and in the case of a competitive assay, can bind to an immobilized first specific binding member of said first cognate binding pair) And (c) irradiating the light receiving edge of the waveguide with light effective to generate total internal reflection inside the waveguide. Irradiating all reaction surfaces simultaneously; (d) collecting scattered light substantially simultaneously from each site and non-site portion of the surface if scattered light is present; and (e)
The light scattering degree of each part is compared with (i) the light scattering degree of a non-part part, or (ii) the light scattering degree of another part, or both. Such methods have been proposed that involve correlating with the presence or amount of an analyte specific for a binding member (see, for example, Patent Document 2).
Japanese National Patent Publication No. 10-506190

In the above method, almost any electromagnetic energy source can be used as the light source, including energy in the visible, ultraviolet and near IR spectra, such as laser, light emitting diode, flash lamp,
An arc lamp, an incandescent bulb, a fluorescent discharge lamp, etc. are described.

However, the light is emitted from the light receiving edge of the waveguide device to the inside of the transparent element. Since the thickness of the transparent element is very thin, the light must be irradiated through a slit narrower than this thickness. The amount of light that reaches the surface decreases, and it is susceptible to noise.

In addition, if the surface accuracy of the light receiving edge of the waveguide device is low, not only the totally reflected light but also the light that does not totally reflect reaches the reaction surface, and this light enters the fluid sample and remains in the fluid sample. In this case, the light scattering label bound to the specific binding member of the second cognate binding pair is reached, and scattered light is generated, so that the measurement accuracy is deteriorated.

Furthermore, when the reaction surface is wide and the fluid sample is widely present, the measurement image for measuring the scattered light becomes large, the influence of noise becomes large, and the measurement sensitivity is lowered.

In the past, B / F separation was required to perform high-sensitivity and high-precision analysis.
Since the / F separation process requires complicated processing, there is a drawback that analysis cannot be performed in a short time.

The purpose of the present invention is to irradiate a substance to be measured with a light beam using an arbitrary light source in view of the above drawbacks,
Fluorescence or scattered light converted or scattered by a fluorescent label or light scattering label is introduced into the waveguide and propagated while being reflected in the waveguide, and fluorescence or scattered light that is not totally reflected is removed,
By measuring only the totally reflected fluorescence or scattered light at the end face of the waveguide, an optical measuring device capable of measuring a specific binding substance at high speed and with high sensitivity without performing B / F separation, and the same It is an object of the present invention to provide a method for optically measuring a specific binding substance using the above.

The optical measurement apparatus according to claim 1 is an apparatus for optically measuring a specific binding substance in a fluid sample, which is bound to a second specific binding member to which a fluorescent label or a light scattering label is bound. A transparent waveguide having a reaction part in which a first specific binding member capable of specifically binding to a substance to be measured and constituting the specific binding substance is fixed to one surface, and a light emitting side edge surface A transparent intermediate layer and a light absorption layer are sequentially laminated on the other surface of the substrate, where n _{w is} the refractive index of the waveguide, n _{m is} the refractive index of the intermediate layer, and n _s is the refractive index of the fluid sample. The rate satisfies the following formula (1).
n _w > n _m ≧ n _s (1)

An optical measuring apparatus according to claim 1 will be described with reference to the drawings. FIG. 1 is a cross-sectional view showing an example of an optical measuring apparatus according to claim 1.

In the figure, A is a transparent waveguide having a light output side edge surface 11, and a reaction portion 13 is formed on one surface of the waveguide A.
Is formed, and a transparent intermediate layer B and a light absorbing layer C are sequentially laminated on the other surface, and the optical measuring device 1
Is formed. Reference numerals 15 and 15 denote spacers for adhering the waveguide A and the light absorption layer C and maintaining a constant distance therebetween.

In the waveguide A, since the fluorescence or scattered light converted or scattered by the fluorescent label or the light scattering label is propagated while being totally reflected, a film, a sheet-like object or a plate-like object made of an optically transparent material is used. One side is a light-emitting side edge surface 11 and one surface is a reaction part 13.

Examples of the optically transparent material include glass, quartz, acrylic resin, fluorine resin, polycarbonate resin, aromatic polyester resin, polyarylate resin, epoxy resin, silicon resin, polystyrene resin, polyimide resin, and fluorine thereof. In particular, glass, quartz, acrylic resin, aromatic polyester resin, polycarbonate resin, and polystyrene resin are preferable.

Examples of the acrylic resin include polymethyl methacrylate (refractive index = 1.4
90), polybenzyl methacrylate (refractive index = 1.568), polyphenyl methacrylate (refractive index = 1.571), poly 1-phenylethyl methacrylate (refractive index = 1.5).
49), polycyclohexyl methacrylate (refractive index = 1.507), methyl methacrylate-benzyl methacrylate copolymer, methyl methacrylate-phenyl methacrylate copolymer, methyl methacrylate-1,1,2-trifluoroethyl methacrylate Examples thereof include a polymer and a methyl methacrylate-styrene copolymer.

Moreover, as said polyester resin, a polyethylene terephthalate etc. are mentioned, for example.

The refractive index n _w of the waveguide A must be greater than the refractive index n _{s of} the fluid sample, and if the fluid sample is an aqueous solution, the refractive index of water is 1.33, so n _w > 1.33. ,Preferably,
n _w > 1.49.

The thickness of the waveguide A is preferably 10 to 1000 times the wavelength of the fluorescence or scattered light converted or scattered by the fluorescent label or the light scattering label, generally 5 to 600 μm, more preferably 15 to 100 μm.

The reaction unit 13 includes a first substance that can specifically bind to a substance to be measured that is bound to a second specific binding member to which a fluorescent label or a light scattering label is bound. Specific binding members are immobilized.

The first specific binding member varies depending on the substance to be measured. For example, an enzyme, a microorganism, an antigen, an antibody, an antibody fragment, a lectin, a receptor, an ionophore, a proton pump, a biological membrane, an artificial biological element, DNA Or a molecule selected from the group consisting of RNA molecules, proteins, sugar chains, glycoproteins, metalloproteins, and mixtures thereof.

As a method for fixing the first specific binding member to the reaction part, any conventionally known method may be employed. For example, a reactive group capable of covalently binding to the first specific binding member on the reaction part surface. Covering the reaction part surface with a compound layer that can be adsorbed by the first specific binding member such as protein, and the like. Examples include a method of adsorbing the first specific binding member.

The fluorescent label is a substance that generates fluorescence by converting light when irradiated with light. For example, a compound that generates fluorescence when irradiated with light, a compound that generates fluorescence. Examples thereof include synthetic resin particles.

Examples of the compound generating fluorescence include fluorin isothiocyanate, fluorescein, fluorescein N-hydroxysuccinimide ester, 6-(((4- (
4,4-difluoro-5- (2-thienyl) -4--4-bora-3a, 4a-diaza-5-indacene-3-yl) phenoxy) acetyl) amino) hexanoic acid, succinimidyl ester, 4-acetamide -4'-isocyanatostilbene-2,2'-disulfonic acid,
7-amino-4-methylcoumarin, 7-amino-4-trimethylcoumarin, N- (4-anilino-1-naphthyl) maleimide, dansyl chloride, 4′6-diamidino-2-phenylindole, 5- (4 6-dichlorotriazin-2-yl) aminofluorescein, 4
, 4'-Diisothiocyanatostilbene-2,2'-disulfonic acid, eosin isothiocyanate, erythrosin B, fluoresamine, fluorescein-5 (6) -carboxyamidocaproic acid N-hydroxysuccinimide ester, 5-isothiocyanate (is
othiosyanante) diacetate, 4-methylumbelliferone, o-phthaldialdehyde, QFITC, rhodamine B isothiocyanate, rhodamine 101 acid chloride, tetramethylrhodamine isothiocyanate, 2 ', 7'-difluorofluorescein, cyanine dye, Examples thereof include rhodamine and rare earth metal complexes, and rare earth metal complexes having a long emission lifetime are preferably used.

Examples of the rare earth metal constituting the rare earth metal complex include scandium, yttrium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, and the like. And samarium, europium, terbium and dysprosium are preferably used.

The ligand constituting the rare earth metal complex together with the rare earth metal is not particularly limited as long as it can constitute a rare earth metal complex that reacts with the rare earth metal to generate fluorescence. For example, ethylenediaminetetraacetic acid, 4, 7-bis- (chlorosulfophenyl) -1,10-
Phenanthroline-2,9-dicarboxylic acid, 4,4′-bis-1 ″, 1 ″
, 1 ", 2", 2 ", 3", 3 "-heptafluoro-4", 6 "-hexanedione-6"
-Yl) -chlorosulfo-o-terphenyl, hexafluoroacetylacetone, triphenylphosphine oxide, diphenyl sulfoxide, 1,10-phenanthroline and the like.

Synthetic resin particles containing a compound that generates fluorescence are less likely to be reduced by coexisting substances in the fluid sample compared to when the compound that generates fluorescence is dissolved in the fluid sample. preferable.

Any conventional method may be employed as a method for causing the synthetic resin particles to contain the compound that generates fluorescence. For example, emulsion polymerization of a polymerizable monomer in the presence of a compound that generates fluorescence,
Examples thereof include a polymerization method by a polymerization method such as suspension polymerization.

Examples of the polymerizable monomer include aromatic vinyl compounds such as styrene, α-methylstyrene, o-vinyltoluene, m-vinyltoluene, and p-vinyltoluene; methyl (meth) acrylate, ethyl (meth) acrylate, n -Propyl (meth) acrylate,
acrylates such as i-propyl (meth) acrylate, n-butyl (meth) acrylate, t-butyl (meth) acrylate, n-hexyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, and cyclohexyl (meth) acrylate; Vinyl cyanide compounds such as (meth) acrylonitrile and vinylidene cyanide; vinyl halide compounds such as vinyl chloride, vinylidene chloride, vinyl fluoride, vinylidene fluoride and tetrafluoroethylene; vinyl esters such as vinyl acetate and vinyl propionate Is mentioned.

The polymerizable monomer may be a copolymer of a monofunctional monomer or a polyfunctional monomer that is generally copolymerized when these polymerizable monomers are polymerized. Examples of the monofunctional monomer include (meth) Acrylic acid, maleic anhydride, glycidyl acrylate and the like can be mentioned. Examples of the polyfunctional monomer include divinylbenzene, (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and trimethylol. Examples thereof include propane tri (meth) acrylate.

Since the content of the compound that generates fluorescence in the synthetic resin particles decreases, the amount of generated fluorescence decreases and the measurement sensitivity decreases. 5-80 weight% is preferable in a synthetic resin particle, More preferably, it is 15-60 weight%.

When the synthetic resin particle becomes larger, the synthetic resin particle becomes larger than the interval at which the first specific binding member is fixed, and the synthetic resin particle may be attached to all the first specific binding members. It becomes difficult. Further, if the synthetic resin particles attached to the first specific binding member are too close to each other and overlap, there is a possibility that the fluorescence interferes and quenches. On the other hand, if the synthetic resin particles are too small, the amount of light becomes small, so 10 μm or less is preferable, and more preferably 20 nm to 500 nm.
nm, more preferably 30 nm to 200 nm.

In order to synthesize such synthetic resin particles having a small particle diameter, it is only necessary to prepare and polymerize a fine droplet of a polymerizable monomer. For example, the polymerizable monomer and necessary components such as an emulsifier, a dispersant, and a polymerization initiator The mixed solution may be supplied to a homogenizer, a micromixer, a microchannel or the like to produce fine droplets and polymerized according to a conventional method.

The light scattering label is a compound or fine particles that can scatter light when irradiated with light, for example, colloidal fine particles of metal such as gold and silver, chalcogenite fine particles such as CdS and CdSe, polystyrene resin, polycarbonate Examples thereof include polymer fine particles such as resin and poly (meth) acrylic resin, inorganic oxide fine particles such as silica gel, alumina and titanium oxide, and core-shell fine particles formed by a combination of two or more of these. The polymer fine particles and inorganic oxide fine particles may be dyed, or fluorescent molecules or metal nanoparticles may be dispersed.

The size of the light scattering label particles is not particularly limited, but is generally 10 nm to 1 nm.
0 μm, preferably 50 nm to 500 nm, more preferably 70 to 200 n.
m.

The second specific binding member is capable of specifically binding to the analyte to be measured, and the analyte is sandwiched between the first specific binding member and the second specific binding member. Is something that can be done.

Accordingly, the second specific binding member differs depending on the substance to be measured and the first specific binding member, but for example, an enzyme, a microorganism, an antigen, an antibody, an antibody fragment, a lectin, a receptor, an ionophore, a proton Pumps, biological membranes, artificial biological elements, DNA molecules, RNA
1 selected from the group consisting of molecules, proteins, sugar chains, glycoproteins, and metalloproteins
Seeds or a mixture thereof.

Therefore, in the case of an immunoassay method, the substance to be measured is an antibody or an antigen, and an antigen or an antibody that reacts specifically with the first specific binding member and the second specific binding member is used. The

While the fluorescence or scattered light generated in the reaction unit 13 is reflected in the waveguide A and propagates to the light output side edge surface of the waveguide A, the fluorescence or scattered light not totally reflected in the waveguide A is removed. Therefore, it is preferable that as much fluorescent or scattered light that is not totally reflected is reflected in the waveguide A as much as possible.

Therefore, the distance from the reaction part 13 to the light-emitting side edge surface 11 is such that fluorescence or scattered light is in the waveguide (
A) It is preferably 2 times or more, more preferably 20 times or more of the distance of one round trip that propagates with total reflection inside.

The transparent intermediate layer B is a layer that is laminated on the other surface of the waveguide A and receives fluorescence or scattered light that does not totally reflect among the fluorescence or scattered light propagating through the waveguide A. Refractive index _nm
Needs to satisfy the following formula (1).
n _w > n _m ≧ n _s (1)

That is, when the refractive index n _m of the intermediate layer B and the refractive index n _{s of the} fluid sample are larger than the refractive index n _{w of the} waveguide A, all of the fluorescence or scattered light introduced into the waveguide A is in the waveguide A. Without totally reflecting
Become incident on the intermediate layer B, also the refractive index n _s of the fluid sample is greater than the refractive index n _m of the intermediate layer B, fluorescence or scattered light excited by the floating fluorescent label or light scattering label of the fluid sample This is because there may be total reflection in the waveguide A.

Examples of the material constituting the transparent intermediate layer B include a fluorinated epoxy resin, an acrylic resin, a fluorinated acrylic resin, and water.

Approximately the same when the refractive index n _m of the refractive index n _s and the intermediate layer B of the fluid sample is the same, light is not totally reflected in the waveguide A is the refractive index n _s of the fluid sample since all can be absorbed into the intermediate layer B A material having a refractive index of 5 is preferable, and water is preferably used.

Further, if the material constituting the intermediate layer B is a material capable of adhesively bonding the waveguide A and the light absorption layer C, the intermediate layer B is formed at the same time as the waveguide A and the light absorption layer C are adhesively bonded. Therefore, an acrylic resin adhesive, an acrylic resin adhesive film or a tape is preferably used.

When the material constituting the intermediate layer B is water, the spacer 15 is necessary, and a gap is formed between the waveguide A and the light absorption layer C, and water is injected to form the intermediate layer B. It is not necessary to use the material.

The thickness of the intermediate layer B is not particularly limited, but when it becomes thin, it becomes difficult to form an intermediate layer by injecting water, and an intermediate layer is formed by applying an acrylic resin adhesive film or tape. It is difficult to form an intermediate layer, for example, it is difficult to do so, and even if the thickness is increased, the effect of entering fluorescent light or scattered light that is not totally reflected is not improved, and the thickness of the optical measuring device is increased. 6 ~
1000 μm is preferred.

The light absorption layer C is a layer that absorbs the fluorescence or scattered light that has entered the intermediate layer B. By absorbing the light, the non-totally reflected fluorescence or scattered light in the waveguide A is eliminated. In particular, a synthetic resin plate colored in black is preferable.

Examples of the synthetic resin include olefin resin, vinyl chloride resin, polystyrene resin, acrylic resin, ABS resin, fluorine resin, polycarbonate resin, polyester resin, polyarylate resin, epoxy resin, silicon Thermoplastic resins such as epoxy resins and polyimide resins, thermosetting resins, and photocurable resins.

For coloring, a colorant may be kneaded into the synthetic resin, or a colorant layer may be laminated on the surface of the synthetic resin layer. Examples of the colorant include azo, phthalocyanine, selenium, and dyes. Organic colorants such as lakes; inorganic colorants such as carbon black, oxides, molybdenum chromates, sulfides / selenides, ferrocyanides, etc. Carbon blacks that can be colored black are preferred .

In the case where a metal is used for the light absorption layer C, the surface of which the surface is subjected to chemical conversion treatment, plating, anodic oxidation, electrolytic coloring, etc., and blackened can be used. For example, a black anodized aluminum plate can be used. Preferably, it is more preferable that the surface of the metal is matted (mechanical finish, chemical etching) before the blackening treatment because it becomes almost non-reflective.

The optical measuring device according to claim 2 is characterized in that a light blocking layer is laminated on the one surface of the waveguide except for the reaction part. FIG. 2 is a cross-sectional view showing an example of the optical measuring apparatus according to the second aspect.

In the figure, A is a transparent waveguide having a light output side edge surface 11, and a reaction portion 13 is formed on one surface of the waveguide A.
In addition, the light-shielding layer D is laminated on one surface except for the reaction part 13, and the transparent intermediate layer B and the light-absorbing layer C are sequentially laminated on the other surface to form the optical measuring device 1. Has been. In addition, 15 and 15 are spacers.

The light shielding layer D is a layer that shields one surface of the waveguide A and prevents light from entering the waveguide A from one surface, and improves the optical measurement accuracy of the optical measuring device 1. Contribute to.

The light blocking layer D may be any layer that can block light, but an aluminum film or sheet having excellent light blocking properties, the above-described black colored synthetic resin plate, or a black-treated metal plate is preferably used. The

The optical measuring apparatus according to claim 3, wherein a transparent second intermediate layer and a light shielding layer are sequentially laminated on the one surface of the waveguide except for the reaction portion. The optical measuring device described. FIG. 3 is a cross-sectional view showing an example of the optical measuring apparatus according to the third aspect.

In the drawing, A is a transparent waveguide having a light output side edge surface 11, and a reaction portion 13 is formed on the one surface of the waveguide A, and a second transparent portion except for the reaction portion 13 is formed on the one surface. The optical measuring device 1 is formed by laminating the intermediate layer B ′ and the light-shielding layer D and sequentially laminating the transparent intermediate layer B and the light absorbing layer C on the other surface. The intermediate layer B ′ stacked on one surface of the waveguide A and the intermediate layer B stacked on the other surface of the waveguide A are the same, and 15 and 15 are spacers.

The light-shielding layer D is a layer that shields one surface of the waveguide A and prevents light from entering the waveguide A from one surface, and the intermediate layer B ′ is similar to the intermediate layer B. Since the fluorescent light or scattered light that does not totally reflect among the fluorescent light or scattered light propagating through the waveguide A is incident, the fluorescent light or scattered light that does not totally reflect can be attenuated more, and the optical of the optical measuring apparatus 1 can be attenuated. Contributes to the improvement of measurement accuracy.

The optical measurement apparatus according to claim 4 is characterized in that a transparent second intermediate layer and a second light absorption layer are sequentially laminated on the one surface of the waveguide except for the reaction portion. Item 4. The optical measuring device according to Item 1. FIG. 4 is a cross-sectional view showing an example of the optical measuring apparatus according to the fourth aspect.

In the drawing, A is a transparent waveguide having a light output side edge surface 11, and a reaction portion 13 is formed on the one surface of the waveguide A, and a second transparent portion except for the reaction portion 13 is formed on the one surface. The optical measuring device 1 is formed by laminating the intermediate layer B ′ and the second light absorption layer C ′ and sequentially laminating the transparent intermediate layer B and the light absorption layer C on the other surface.

The intermediate layer B ′ and the light absorption layer C ′ laminated on one surface of the waveguide A and the intermediate layer B and the light absorption layer C laminated on the other surface of the waveguide A are the same, respectively. 15 and 15 are spacers.

Similarly to the intermediate layer B, the intermediate layer B ′ is a layer into which fluorescent light or scattered light that does not totally reflect among the fluorescent light or scattered light propagating through the waveguide A enters, and the light absorbing layer C ′ is a light absorbing layer. Intermediate layer B as in C
Since it is a layer that absorbs the fluorescence or scattered light that has entered ', it can further attenuate the fluorescence or scattered light that is not totally reflected, and contributes to the improvement of the optical measurement accuracy of the optical measuring device 1.

The optical measurement apparatus according to claim 5, wherein a transparent second intermediate layer, a second intermediate layer is formed on the one surface of the waveguide.
The optical measuring device according to claim 1, wherein the light absorption layer and the light shielding layer are sequentially laminated except for the reaction part. FIG. 5 is a cross-sectional view showing an example of the optical measuring apparatus according to the fifth aspect.

In the drawing, A is a transparent waveguide having a light output side edge surface 11, and a reaction portion 13 is formed on the one surface of the waveguide A, and a second transparent portion except for the reaction portion 13 is formed on the one surface. Middle layer B ′,
A second light absorption layer C ′ and a light blocking layer D are stacked, and a transparent intermediate layer B and a light absorption layer C are sequentially stacked on the other surface to form the optical measuring device 1.

The transparent second intermediate layer B ′ and the second light absorption layer C ′ stacked on the one surface of the waveguide A, and the intermediate layer B and the light stacked on the other surface of the waveguide A. The absorption layers C are the same.

Similarly to the intermediate layer B, the intermediate layer B ′ is a layer into which fluorescent light or scattered light that does not totally reflect among the fluorescent light or scattered light propagating through the waveguide A enters, and the light absorbing layer C ′ is a light absorbing layer. Intermediate layer B as in C
Since it is a layer that absorbs the fluorescence or scattered light that has entered the light, it can further attenuate the fluorescence or scattered light that is not totally reflected, and the light shielding layer D shields the light absorbing layer C from one surface. Waveguide A
As a result, it is possible to prevent the light from entering into the optical measurement apparatus 1, thereby contributing to the improvement of the optical measurement accuracy of the optical measurement apparatus 1.

In addition, a light shielding layer D may be laminated on the light absorbing layer C for the purpose of shielding the light absorbing layer C and preventing light from entering the waveguide A from the other surface side of the waveguide A.

The optical measurement device according to claim 6, wherein a plurality of reaction portions are formed on the one surface of the waveguide. is there. FIG. 6 is a plan view showing an example of the optical measuring apparatus according to the sixth aspect.

In the figure, reference numeral 1 denotes an optical measuring device having a substantially rectangular shape in a plan view.
.. Are formed, and reaction portions 13, 13... Are formed on the respective protrusions.
In this optical measuring device, six types of fluid samples can be measured simultaneously.

The method for optically measuring a specific binding substance according to claim 12, wherein a fluid sample containing a second specific binding member to which a fluorescent label or a light scattering label is bound and a substance to be measured is included. The second specific binding member to which a fluorescent label or a light scattering label is bound, and the second specific binding member, which is supplied to the reaction part of the optical measurement device according to any one of the above, and is immobilized on the reaction part via the substance to be measured After binding with the first specific binding member to form a specific binding product, the specific binding product is irradiated with light, and fluorescence or scattered light converted or scattered by the fluorescent label or light scattering label is guided. It is introduced into the waveguide (A), propagates while reflecting through the waveguide (A), and fluorescence or scattered light emitted from the light-exiting side edge surface is measured by a light receiving element.

Next, an optical measurement method for a specific binding substance according to claim 12 will be described with reference to the drawings. FIG. 7 is a schematic diagram showing an example of a method for optically measuring a specific binding substance using an optical measuring device, and FIG. 8 shows a second specific binding to which a fluorescent label or a light scattering label is bound. It is a schematic diagram which shows the state by which the specific binding material which the member, to-be-measured substance to-be-measured substance, and the 1st specific binding member couple | bonded is fixed to the reaction part surface.

In the figure, reference numeral 1 denotes an optical measuring device, which is formed by laminating a transparent waveguide A having a light output side edge surface 11, a transparent intermediate layer B, and a light absorption layer C. A reaction portion 13 in which the first specific binding member 5 is fixed is formed on one surface 12 in the vicinity of the light emitting side edge surface 11 and the opposing surface of the waveguide A. 15 is a spacer, which bonds the end of the other surface 14 of the waveguide A and the end of the light absorption layer C;
Water 16 is injected to form an intermediate layer B.

In the method for optically measuring a specific binding substance, the first step is to prepare a fluid sample containing a substance to be measured and a second specific binding member to which a fluorescent label or a light scattering label is bound.
A second specific binding member to which a fluorescent label or a light scattering label is bound is supplied to the reaction part of the optical measurement device according to any one of claims 1 and fixed to the reaction part via the substance to be measured First
The specific binding member is bound to form a specific binding substance.

That is, a larger amount of the second specific binding member 7 to which the fluorescent label or the light scattering label 8 is bound than the substance to be measured 6 contained in the fluid sample 2 and the fluid sample containing the substance to be measured 6 are reacted with the reaction unit 13. , The substance 6 to be measured specifically binds to the second specific binding member 7 to which the fluorescent label or the light scattering label 8 is bound, and the first specific binding immobilized on the reaction unit 13 The second specific binding member 7 to which the member 5 is specifically bound and the fluorescent label or the light scattering label 8 is bound, the specific substance 9 to which the analyte 6 and the first specific binding member 5 are bound. Is formed.

Since the first specific binding member 5 is fixed to the reaction part 13, the specific binding substance 9 is fixed to the reaction part 13. In addition, the second specific binding member 7 to which the excess fluorescent label or light scattering label 8 is bound is suspended in the fluid sample 2.

Next, the specific binding substance is irradiated with light, the fluorescence or scattered light converted or scattered by the fluorescent label or light scattering label 8 is introduced into the waveguide A, propagates while reflecting through the waveguide A, and exits the light. Fluorescence or scattered light emitted from the edge surface is measured by the light receiving element 4.

That is, when light is irradiated from the light source 3 onto the reaction unit 13 (fluid sample 2) as indicated by an arrow E, fluorescence or scattered light converted or scattered by the fluorescent label or the light scattering label 8 in the fluid sample 2 is generated.

Of the fluorescence or scattered light converted or scattered by the fluorescent label or light scattering label 8 bound to the second specific binding member 7 floating in the fluid sample 2, it is shallow enough to be totally reflected on the surface of the waveguide A A light beam having an incident angle is totally reflected on the surface of the waveguide A, and only a light beam having a deep incident angle that is not totally reflected on the surface of the waveguide A enters the waveguide A as shown by the light beam G. .

Since the light beam G incident on the waveguide A is a light beam having a deep angle that is not totally reflected on the surface of the waveguide A, the light beam G crosses the waveguide A and enters the intermediate layer B from the other surface 14 of the waveguide A. Light absorption layer C
Is partially absorbed and partially reflected.

The reflected light beam G enters the waveguide A again, is reflected by the one surface 12 of the waveguide A, enters the intermediate layer B from the other surface 14 of the waveguide A, and is partially absorbed by the light absorption layer C. And partly reflected. The light G repeats the above reflection and light absorption and is no longer attenuated.

On the other hand, the fluorescent or scattered light converted or scattered by the fluorescent label or the light scattering label 8 contained in the specific binding substance 9 fixed to the reaction unit 13 is also a light beam having a shallow angle of incidence and a deep angle incident angle. However, a light ray having a deep incident angle is repeatedly reflected and absorbed as in the case of the light ray G, and is not attenuated.

As shown by the light beam F, the light beam having a shallow incident angle propagates while being totally reflected by the waveguide A and emits light from the light-emitting side edge surface 11, so that the emitted fluorescent light or scattered light is received by the light receiving element 4. Receive light and measure by electrical processing. The emitted light beam may be received through a filter that selectively transmits fluorescence or scattered light.

As the light source, any known light source can be used, and examples thereof include lasers, light emitting diodes, flash lamps, arc lamps, incandescent lamps, fluorescent lamps, xenon lamps, mercury lamps, and the like. Usable, for example, visible light, ultraviolet light, infrared light, and the like.

As the light receiving element, any measuring device capable of sensing light can be used, but in general, a photodiode, a CMOS camera, a CCD camera, or the like is preferably used.

Since the configuration of the optical measurement apparatus of the present invention and the optical measurement method of a specific binding substance using the same is as described above, it can be easily irradiated using an arbitrary light source and fixed to the waveguide. Fluorescent or dispersed light converted or scattered by a fluorescent label or light-dispersible label other than the fluorescent label or light-dispersible label present in the specific binding product is attenuated when propagating through the waveguide and does not reach the optical measurement device Therefore, a specific binding substance can be measured with high sensitivity and accuracy at high speed without performing B / F separation.

In addition, it efficiently propagates only light rays having a shallow incident angle out of fluorescence or dispersed light converted or scattered by a fluorescent label or light scattering label in a specific binding substance fixed to the reaction part on the surface of the waveguide A. Therefore, the waveguide can be made thin, the optical measuring device can be thinned, the light beam to be measured becomes bright and the measurement sensitivity is excellent. Therefore, the substance to be measured in the fluid sample can be measured with high sensitivity and accuracy.

  Examples of the present invention will be described below, but the present invention is not limited to the following examples.

(Example 1)
Monoclonal antibody against human hemoglobin and human glycohemoglobin A1c
Preparation of Monoclonal Antibody Balb / c mice were immunized 4 times every 2 weeks with 100 μl of a solution (20 μg / ml) in which human hemoglobin and human glycohemoglobin A1c were sufficiently dispersed in Freund's complete adjuvant.

Two weeks after the end of immunization, the spleen of the immunized Balb / c mouse was excised and 106 splenocytes were obtained. The obtained splenocytes were fused and cultured in the presence of myeloma cells and PEG (polyethylene glycol). The supernatant of the proliferated cells was collected and examined for the presence or absence of each antibody by ELISA.

Cells positive for each antibody were tested by the limiting dilution method, and cells producing each antibody were confirmed. These cells are cultured in large quantities, injected into the peritoneal cavity of Balb / c mice, and ascites is collected every 3 days from 2 weeks, and a monoclonal antibody against human hemoglobin and human glycohemoglobin A are collected.
Monoclonal antibody against 1c was obtained.

Preparation of fluorescent synthetic resin particles 80 parts by weight of water and 1.5 parts by weight of sodium dodecyl sulfonate are supplied to a glass polymerizer and stirred to dissolve sodium dodecyl sulfonate, and then 20 parts by weight of styrene, rare earth metal complex (Hexafluoroacetylacetone / trioctylphosphine oxide ligand complex coordinated to europium) 4 parts by weight and azobisisobutyronitrile 0,15 parts by weight are supplied and dispersed at high speed with a homogenizer while cooling to obtain an emulsion. Got.

The obtained emulsion was purged with nitrogen while being dispersed at high speed with a homogenizer, heated to 80 ° C. with warm water, and polymerized at 80 ° C. for 8 hours to obtain fluorescent synthetic resin particles having a volume average particle diameter of 120 nm.

Preparation of anti-human glycohemoglobin A1c antibody-adsorbing fluorescent synthetic resin particles Anti-human glycohemoglobin A1c antibody was dissolved in 0.05 M glycine buffer (pH 8.6) to obtain an antibody solution having a concentration of 1 mg / ml.

The obtained fluorescent synthetic resin particles were washed three times with 0.05 M glycine buffer (pH 8.6), and then 0.05 M glycine buffer (so that the fluorescent synthetic resin particle concentration was 1.0% by weight ( 0.5 ml of the obtained antibody solution was added to 1 ml of the resulting dispersion, and the mixture was stirred and mixed at 35 ° C. for 1 hour.

Next, 1 ml of bovine serum albumin 1.0 wt% phosphate buffer solution (pH 7.2) was added, stirred and mixed at room temperature for 1 hour, centrifuged (15000 rpm, 1 hour), and phosphate buffer solution. Washed with liquid (pH 7.2). After performing this centrifugation and washing three times, 2 ml of a phosphate buffer solution (into the obtained anti-human glycohemoglobin A1c antibody-adsorbed fluorescent synthetic resin particles (
pH 7.2) was added, and the mixture was stirred with an ultrasonic homogenizer, and anti-human glycohemoglobin A1c was added.
A dispersed phosphate buffer solution of antibody-adsorbed fluorescent synthetic resin particles was obtained.

Production of reaction part As a waveguide (A), a transparent polymethyl methacrylate sheet having a length of 10 mm, a width of 1 mm and a thickness of 100 μm is used, and the position of the reaction part 13 of the polymethyl methacrylate sheet (waveguide (A)) is distilled. After washing with water, a buffer solution in which a monoclonal antibody against human hemoglobin and a monoclonal antibody against human glycohemoglobin A1c were dissolved in 0.1 M Tris-HCl buffer (pH 9.0) to a concentration of 1 mg / ml was prepared. The mixture was supplied to the surface of the reaction part 13 with an injection needle and allowed to stand at 30 ° C. for 1 hour.

Next, after washing with 0.1 M Tris-HCl buffer (pH 9.0), 1 of bovine serum albumin
. A 0 wt% phosphate buffer solution (pH 7.2) was supplied and allowed to stand at 30 ° C. for 1 hour to coat the surface of the reaction part 13 that was not covered with the above monoclonal antibody.

Next, after washing again with 0.1 M Tris-HCl buffer (pH 9.0), it was covered with 0.1 M Tris-HCl buffer (pH 9.0) and coated with a polymethyl methacrylate sheet (waveguide (A)
) The reaction part 13 was formed on the top.

A transparent polymethyl methacrylate sheet on which the reaction part 13 is produced is used as a waveguide (A) for producing an optical measuring device, and a light absorption layer (C) is made of ABS resin and carbon black and has a length of 10 m.
A black ABS resin sheet having a width of 3 mm, a width of 3 mm, and a thickness of 100 μm was used.

As shown in FIG. 1, a spacer 15 made of a black ABS resin sheet having a thickness of 0.5 mm is pasted around one surface of a black ABS resin sheet (light absorption layer (C)), and a reaction is performed thereon. A polymethylmethacrylate sheet (waveguide (A)) is pasted so that the part 13 side is up,
Water 16 was injected into the space formed by the both with a syringe to form an intermediate layer (B) to obtain a measuring device.

Preparation of blood sample for measurement Immediately after collecting the blood of a subject, sodium fluoride (blood anticoagulant) 10 per ml of whole blood
mg was added, and then 3 μl of blood was diluted 150-fold by adding a hemolysis reagent to obtain a diluted blood sample.

The hemolysis reagent was polyoxyethylene (10) octylphenyl ether (Pharm).
0.1% by weight of manufactured by Inc., Inc., trade name "TritonX-100")
A 0.05 M phosphate buffer (pH 6.0) obtained by dissolving 0.1% by weight of tetrapolyphosphate was used as a removal reagent.

The obtained blood sample was measured using an A1c dedicated measuring device (liquid chromatography, manufactured by Kyoto Daiichi Kagakusha,
Supplied to the trade name “Hi-AutoA1cHA-8121” and human glycohemoglobin A1
c concentration was measured, and based on the result, the hemolysis reagent was further added to the blood sample to prepare blood samples for measurement having human glycohemoglobin A1c concentrations of 3%, 5%, 10% and 12%.

Measurement To the obtained blood sample for measurement, an excessive anti-human glycohemoglobin A1c antibody-adsorbed fluorescent synthetic resin particle-dispersed phosphate buffer solution was mixed, and then allowed to stand for 5 minutes.
0 ml was supplied to the reaction unit 13 of the optical measuring device.

Using a xenon lamp as the light source 3 and irradiating a light beam of 0.1 J / Flash 1000 times at an interval of 2000 times / second, using a photodiode as the light receiving element 4, a waveguide (A)
The intensity of a light beam having a wavelength of about 650 nm emitted from the light emitting side edge surface 11 was measured.

When the obtained light intensity and the human glycohemoglobin A1c concentration were plotted on a graph, a linear graph was obtained, and it was found that the human glycohemoglobin A1c concentration can be measured with the above optical measuring device.

It is sectional drawing which shows an example of the optical measuring device of this invention by which the transparent intermediate | middle layer and the light absorption layer were laminated | stacked on the surface of the transparent waveguide. It is sectional drawing which shows an example of the optical measuring apparatus with which the light-shielding layer was laminated | stacked on the surface on the opposite side to the intermediate | middle layer of FIG. It is sectional drawing which shows an example of the optical measuring device with which the transparent intermediate | middle layer and the light-shielding layer were laminated | stacked on the surface on the opposite side to the intermediate | middle layer of FIG. It is sectional drawing which shows an example of the optical measuring device by which the transparent intermediate | middle layer and the light absorption layer were laminated | stacked on the surface on the opposite side to the intermediate | middle layer of FIG. It is sectional drawing which shows an example of the optical measuring device with which the transparent intermediate | middle layer, the light absorption layer, and the light-shielding layer were laminated | stacked on the surface on the opposite side to the intermediate | middle layer of FIG. It is a top view which shows an example of the optical measuring device in which the some reaction part was formed in the surface of a waveguide. It is a schematic diagram which shows an example of the optical measuring method of a specific binding material using an optical measuring device. A state in which a second specific binding member to which a fluorescent label or a light scattering label is bound, a substance to be measured, and a specific binding substance to which the first specific binding member is bound are immobilized on the reaction surface. It is a schematic diagram.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Optical measuring apparatus A Waveguide B Intermediate | middle layer C Light absorption layer D Light light shielding layer 2 Fluid sample 3 Light source 4 Light receiving element 5 1st specific binding member 6 Measured substance 7 2nd specific binding member 8 Fluorescent label Or light scattering label 9 Specific binding substance 11 Light emission side edge surface 13 Reaction part 15 Spacer 16 Water 17 Convex part

Claims (14)

An apparatus for optically measuring a specific binding substance in a fluid sample, comprising:
A substance to be measured bound to a second specific binding member to which a fluorescent label or a light scattering label is bound, and a first specific binding member that can specifically bind to form the specific bound substance, A transparent intermediate layer and a light absorbing layer are sequentially laminated on the other surface of the transparent waveguide having the reaction portion fixed on the surface and the light-exit-side edge, and the refractive index of the waveguide is n _w An optical measuring device characterized in that each refractive index satisfies the following formula (1), where n _{m is} the refractive index of the liquid and n _s is the refractive index of the flow material.
n _w > n _m ≧ n _s (1) 2. The optical measuring device according to claim 1, wherein a light shielding layer is laminated on the one surface of the waveguide except for a reaction portion. 2. The optical measurement apparatus according to claim 1, wherein a transparent second intermediate layer and a light-shielding layer are sequentially laminated on the one surface of the waveguide except for the reaction portion. 2. The optical measurement apparatus according to claim 1, wherein a transparent second intermediate layer and a second light absorption layer are sequentially laminated on the one surface of the waveguide except for the reaction portion. 2. The optical device according to claim 1, wherein a transparent second intermediate layer, a second light absorption layer, and a light shielding layer are sequentially laminated on the one surface of the waveguide except for the reaction portion. measuring device. 6. A plurality of reaction portions are formed on the one surface of the waveguide.
The optical measuring device according to any one of the above. The optical waveguide according to any one of claims 1 to 6, wherein the waveguide is made of a material selected from the group consisting of glass, quartz, acrylic resin, polyester resin, polycarbonate resin, and polystyrene resin. Measuring device. The optical measurement apparatus according to claim 1, wherein the intermediate layer is a substance having a refractive index of the fluid sample substantially equal to n _s . The optical measuring apparatus according to claim 8, wherein the intermediate layer is water or an acrylic resin adhesive. 10. The distance from the light output side edge surface of the waveguide to the reaction portion is at least twice as long as one round-trip distance in which fluorescence or scattered light propagates by total reflection in the waveguide. The optical measuring device according to any one of the above. A fluid sample containing a second specific binding member to which a fluorescent label or a light scattering label is bound and a substance to be measured is supplied to the reaction part of the optical measurement device according to any one of claims 1 to 10. And
The second specific binding member to which the fluorescent label or the light scattering label is bound is bound to the first specific binding member fixed to the reaction part via the substance to be measured to form a specific binding substance. After that, the specific binding substance is irradiated with light, the fluorescent or scattered light converted or scattered by the fluorescent label or the light scattering label is introduced into the waveguide, propagated while reflecting the waveguide, and the light emitting side edge surface A method for optically measuring a specific binding substance, which comprises measuring fluorescence or scattered light emitted from a light-receiving element. The specific binding according to claim 11, wherein the substance to be measured is an antibody or an antigen, and the first specific binding member and the second specific binding member are an antigen or an antibody that specifically binds thereto. Optical measurement method for objects. The method for optically measuring a specific binding substance according to claim 11 or 12, wherein the fluorescent label is a rare earth metal complex. 14. The method for optically measuring a specific binding substance according to claim 11, wherein the fluorescent label is a synthetic resin particle containing a compound that generates fluorescence.

Images