JP2005091054A - Method for optically measuring constituent in liquid specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for highly sensitively and highly accurately measuring a specific constituent in a liquid specimen capable of sufficiently lowering background noise owing to fluorescence arising from an optical waveguide itself and fluorescence caused by coexistent substances in the specimen when exciting a fluorescent material by evanescent wave to measure the specific constituent in the liquid specimen by means of the intensity of generated light emission. <P>SOLUTION: According to this method, excitation light is introduced into the optical waveguide and caused to internally reflect, thereby generating the evanescent wave. The fluorescent material, being locked in the vicinity of the surface of the optical waveguide and existing in a compound material with the specific constituent in the liquid specimen, is excited by the evanescent wave. The specific constituent in the liquid specimen is measured based on variation in the intensity of generated light emission. According to this method for optically measuring a constituent in a liquid specimen, the intensity of light emission is measured 10μsec after irradiating excitation light. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for optically measuring a component in a liquid sample. More specifically, the present invention relates to a method of exciting a fluorescent substance in a complex with a specific component in a liquid sample by an evanescent wave, and The present invention relates to a method for measuring specific components in a liquid sample, for example, proteins, peptides, nucleic acids, nucleobases and their derivatives, nucleotides, hormones, saccharides or other organic compounds, by increasing or decreasing.

  Conventionally, a fluorescence measurement method is known in which an evanescent wave is used to excite a fluorescent substance constrained by an antigen-antibody reaction and the intensity of the emitted light is measured to measure the degree of immune reaction. In this type of measurement method, an evanescent wave is generated by introducing excitation optics into the optical waveguide and propagating it through the optical waveguide while totally reflecting the excitation optics. Then, the fluorescent substance constrained in the vicinity of the optical waveguide by the antigen-antibody reaction is excited by the evanescent wave, the light generated from the excited fluorescent substance is emitted from the optical waveguide, and by measuring the intensity of the emitted light, The degree of immune response is measured.

  Patent Document 1 below discloses an example of this type of optical measurement method. Here, as a means for solving the problem that the measurement accuracy is lowered due to light generated by exciting the optical waveguide itself, a fluorescent material having an absorption peak near 650 nm is used, and excitation light having a wavelength near 635 nm is used. Used.

On the other hand, Patent Document 2 below discloses a method for measuring a plurality of fluorescent dyes. In the measurement method described in Patent Document 2, laser light is introduced into an optical waveguide of a device that detects a fluorescent dye in a sample. Then, the fluorescent dye contained in the sample is excited by the evanescent component of the laser light, and the fluorescence is detected. In the method described in Patent Document 2, when the wavelength of excitation light is 488 nm, an example of detecting fluorescence at a wavelength of 520 nm, and when detecting the organic dye CY5, measurement is performed when the wavelength of excitation light is 633 nm. An example in which the wavelength of fluorescence to be emitted is 670 nm is disclosed.
Japanese Patent No. 3362206 Japanese Patent No. 3187386

  The method described in Patent Document 1 has a problem that the wavelength difference (Stokes shift) between excitation light and fluorescence is not sufficiently large, and the measurement sensitivity and measurement accuracy cannot be sufficiently increased.

  On the other hand, in the method described in Patent Document 2, since the Stokes shift is small and CY5 has a short fluorescence lifetime, it is apt to be greatly influenced by the fluorescent substance coexisting in the optical waveguide and the sample. Therefore, the measurement sensitivity could not be sufficiently increased. When trying to measure a component in a sample with high sensitivity, in addition to the fluorescence generated from the optical waveguide itself, the presence of a substance that generates fluorescence in addition to the fluorescent dye as the label contained in the sample becomes a problem.

  In particular, when measuring components in blood samples, large fluorescence is generated by various proteins, nucleic acids, hemoglobin, etc., and in some cases by administered drugs, and the measurement sensitivity must be low. .

  In view of the current state of the prior art described above, an object of the present invention is to excite a fluorescent substance constrained in the vicinity of the surface of an optical waveguide by an evanescent wave generated by introducing excitation light into the optical waveguide and internally reflecting it. In the method of measuring a specific component in a liquid sample by the intensity of luminescence, the fluorescence generated from the optical waveguide itself and the fluorescence derived from substances other than the fluorescent substance as a label present in the sample can be reduced to a negligible level. Another object of the present invention is to provide an optical measurement method capable of measuring a fluorescent substance constrained near the surface of an optical waveguide with extremely high sensitivity.

  The present invention relates to a fluorescent substance in a complex with a specific component in a liquid sample that is constrained near the surface of an optical waveguide by an evanescent wave generated by introducing and internally reflecting excitation light into the optical waveguide. In a method for measuring a specific component in a liquid sample by excitation and increase / decrease in the generated emission intensity, the component in the sample is measured by measuring the emission intensity after 10 microseconds after irradiation with excitation light.

  In the optical measurement method of the present invention, a rare earth metal complex is preferably used as the fluorescent material.

  In the optical measurement according to the present invention, preferably, the rare earth metal is at least one selected from the group consisting of europium, terbium, samarium, dysprosium, gadolinium and neodymium.

  In the optical measurement method according to the present invention, preferably, the fluorescent substance is contained in polymer particles.

  In a more limited aspect of the optical measurement method according to the present invention, the emission intensity after 200 microseconds is measured after excitation light irradiation.

  In a specific aspect of the optical measurement method according to the present invention, the specific component in the liquid sample is glycohemoglobin.

  In the optical measurement method according to the present invention, the liquid sample may or may not be BF separated. In the method without BF separation, preferably, a plurality of samples are continuously measured on a single optical waveguide. .

  In the method for optically measuring a component in a liquid sample according to the present invention, the fluorescent substance in the complex with the component in the liquid sample constrained in the vicinity of the surface of the optical waveguide is excited by the evanescent wave, and the intensity of the generated light emission The component in the liquid sample is measured by the increase / decrease, but the component in the sample is measured by measuring the luminescence intensity after 10 microseconds after irradiation with the excitation optics. The lifetime of the fluorescence is relatively short due to substances other than the measurement component coexisting in the optical waveguide itself and the sample, and the generated intensity is rapidly attenuated by 10 microseconds. In the present invention, since the emission intensity after 10 microseconds is measured after the excitation light irradiation, the background noise due to the fluorescent substance coexisting in such an optical waveguide or sample can be sufficiently reduced. By this, the measurement sensitivity of a specific component in the liquid sample can be effectively increased.

  In the present invention, when a rare earth metal complex is used as the fluorescent material, the fluorescence life of the rare earth metal complex is long and the Stokes shift is large. Therefore, in the present invention, light is emitted after 10 microseconds after excitation light irradiation. By measuring the intensity, the fluorescence by the rare earth metal complex, which is a fluorescent substance bonded to a specific component in the sample, can be reliably measured.

  In particular, when the rare earth metal is at least one selected from the group consisting of europium, terbium, samarium, dysprosium, gadolinium and neodymium, the lifetime of the phosphor is further increased, and background noise according to the present invention. The light emission intensity derived from the measurement component can be measured in a state where the light emission is effectively reduced.

  Further, in the present invention, when the fluorescent substance is dispersed in the polymer particles, the fluorescence intensity due to the coexisting substance in the liquid sample is greater than when the rare earth metal complex is dissolved in the liquid sample. Is unlikely to occur. Therefore, the sensitivity can be further increased.

  In the present invention, when measuring the emission intensity after 200 microseconds after excitation light irradiation, the background noise can be further reduced.

  In the method for optical measurement of components in a liquid sample according to the present invention, components in various liquid samples can be measured. When the component is glycated hemoglobin, according to the present invention, for example, a blood sample or the like The intensity of glycohemoglobin in the liquid sample can be measured with high sensitivity.

  In the optical measurement method according to the present invention, when a plurality of samples are measured without performing BF separation using a single optical waveguide, components in the plurality of samples can be continuously and efficiently measured. it can. As described above, when the BF separation is not performed, the influence of the fluorescent substance coexisting in the liquid sample is increased, and the background noise is increased. However, in the present invention, the influence of background noise can be reduced by using a fluorescent material having a long fluorescence lifetime.

  Therefore, according to the present invention, the components in the liquid sample can be measured by a simple operation without performing BF separation. When BF separation is not performed, a plurality of samples can be continuously measured using a single optical waveguide, thereby efficiently measuring components in the liquid sample.

  Hereinafter, the present invention will be clarified by describing specific embodiments of the present invention.

  In the optical measurement method according to the present invention, an optical waveguide made of an appropriate material capable of generating an evanescent wave by introducing excitation light and internally reflecting it can be used as the optical waveguide. For example, a resin plate or a glass plate can be used as such an optical waveguide.

  When a glass plate is used as an optical waveguide, for example, short wavelength light in the vicinity of 350 nm may be incident on a glass plate having a thickness of 0.5 mm to several mm at an angle of total reflectance and propagated. The excitation light source is not particularly limited, and a monochromatic laser or LED can be used. The light generated from the light source may be coupled to the optical waveguide via a prism, for example.

  The fluorescent material constrained near the surface of the optical waveguide is excited by the evanescent field of the excitation light that has propagated. The surface of the optical waveguide is coated with a reactant specific to the first component contained in the liquid sample, the coated surface is exposed to the sample, and a composite layer formed by reaction with the first component is formed. . It can be known by measuring the fluorescence intensity that the complex and the second component in the liquid sample interacted with the complex.

For labeling with a fluorescent substance, the same component as the first component contained in the liquid sample may be fluorescently labeled, or the second component may be fluorescently labeled.

  The reactant on the optical waveguide may be provided at one place on the optical waveguide or may be provided at a plurality of places. When reactants are provided at a plurality of locations, the reactants may be provided at an arbitrary interval.

  Fluorescence generated from the fluorescent material excited by the evanescent field may be detected by a detector positioned at a predetermined position with respect to the optical waveguide. As the detector, for example, a photoelectric conversion device such as a photodiode array, a CCD array, or a photomultiplier tube can be used.

  The reaction on the surface of the optical waveguide may be an antigen-antibody reaction or DNA hybridization, and the mechanism is not particularly limited. In addition, various components may be mentioned as measurement components as the measurement target in the optical measurement method according to the present invention, such as proteins, peptides, nucleic acids, nucleobases and their derivatives, nucleotides, hormones, saccharides, and others. The organic compound of these is mentioned.

  Organic fluorescent dyes such as fluorescein and rhodamine emit a large quantum yield and strong fluorescence, but the wavelength difference between excitation light and fluorescence, that is, the Stokes shift is small, and the fluorescence lifetime is about several nanoseconds. Therefore, there is a problem that it is greatly affected by coexisting substances in the sample, fluorescence from the optical waveguide itself, and scattered light of excitation light.

  In contrast, in the present invention, a fluorescent material having a long fluorescence lifetime is used. That is, in the present invention, the emission intensity after 10 microseconds after the excitation light irradiation is measured, but a fluorescent material having a fluorescence lifetime of 10 microseconds or later is used. Such a fluorescent substance is not particularly limited as long as it has a long fluorescence lifetime. Examples of the fluorescent material having a long fluorescence lifetime as described above include rare earth metal complexes or long afterglow phosphorescent fluorescent particles.

  Rare earth metal complexes have a large wavelength difference between excitation light and fluorescence, and their fluorescence lifetime is several hundred microseconds or longer. Long-lasting phosphorescent phosphor particles have a longer fluorescence lifetime.

  As the rare earth metal constituting the rare earth metal complex, scandium or yttrium, or a metal belonging to the lanthanoid group, that is, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, Examples thereof include ytterbium and lutetium. Preferably, europium, samarium, terbium, or dysprosium can be used.

  The ligand that forms a complex with the rare earth metal is not particularly limited as long as it can form a rare earth metal complex capable of generating fluorescence together with the rare earth metal. For example, ethylenediaminetetraacetic acid ( EDTA) s, 4,7-bis- (chlorosulfophenyl) -1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), or 4,4′-bis (1 ″, 1 ″, 1 ", 2", 2 ", 3", 3 "-heptafluoro-4", 6 "-hexanedione-6" -yl) -chlorosulfo-o-terphenyl (BHHCT). Further, hexafluoroacetylacetone (HFA), triphenylphosphine oxide (TPPO), diphenyl sulfoxide (DPSO), 1,10-phenanthroline (Phen), and the like can be given.

In the present invention, preferably, the phosphor is contained in polymer particles. When the fluorescent substance is contained in the polymer particles, it is preferable that a decrease in fluorescence intensity due to the coexisting substance in the liquid sample can be suppressed as compared with the case where the fluorescent substance is dissolved in the liquid sample.

  The phosphor-containing photopolymer particles containing the phosphor can be prepared by a conventional method. For example, a method of radical polymerization by emulsifying a polymerizable monomer solution containing a rare earth metal complex as a phosphor, a polymerization initiator, and a stabilizer in water in the presence of an emulsifier can be mentioned. A method of performing suspension polymerization in the presence of various dispersants is also possible.

  In order to more effectively suppress the influence of coexisting substances in the liquid sample, the polymer particles preferably have a small particle size, and more specifically, it is desirable that the particle size is 100 nm or less. In order to reduce the particle size of the polymer particles, small monomer droplets may be formed at the time of polymerization, and can be achieved by using a homogenizer, an ultrasonic crusher, a micromixer, a microchannel, or the like. Moreover, it is desirable that the variation in the particle size of the polymer particles is small, for example, it is desirable that the CV is 10% or less.

  When using phosphor-containing polymer particles, the content of the fluorescent substance in the phosphor-containing polymer particles may be appropriately selected according to the composition of the fluorescent substance and the polymer. Usually, it is preferably 5 to 80% by weight, more preferably 15 to 60% by weight. If the amount is 5% by weight or less, the measurement sensitivity cannot be increased because a sufficient amount of the fluorescent substance is not contained. If the amount exceeds 80% by weight, the effect of including the phosphor in the polymer particles is sufficiently obtained. There may not be. The particle diameter of the phosphor-containing polymer particles is appropriately selected, but is usually preferably 0.04 to 5 μm, more preferably 0.03 to 0.2 μm.

  Examples of the polymer that forms the polymer particles include vinyl polymer, olefin polymer, polyester, polyamide, polyamideimide, polyimide, polysiloxane, polyacetal, polycarbonate, polysulfone, and polysulfide. The particles may be functional group-containing polymers.

  Particularly preferred polymer particles are vinyl polymer particles or functional group-containing vinyl polymer particles. Examples of the vinyl monomer constituting the main component of the vinyl polymer or the functional group-containing vinyl polymer include fragrances such as styrene, α-methylstyrene, o-vinyltoluene, m-vinyltoluene, p-vinyltoluene, and divinylbenzene. Group vinyl compounds; unsaturated carboxylic acids such as (meth) acrylic acid and crotonic acid; methyl (meth) acrylate, ethyl (meth) acrylate, n-propyl (meth) acrylate, i-propyl (meth) acrylate, n-butyl (Meth) acrylate, t-butyl (meth) acrylate, n-hexyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, cyclohexyl (meth) acrylate, (poly) ethylene glycol di (meth) acrylate, (poly) propylene Glycol di (meth) (Meth) acrylates such as acrylate and trimethylolpropane tri (meth) acrylate; vinyl cyanide compounds such as (meth) acrylonitrile and vinylidene cyanide; vinyl chloride, vinylidene chloride, vinyl fluoride, vinylidene fluoride, tetrafluoroethylene And the like, and the like. Of these vinyl monomers, aromatic vinyl compounds and (meth) acrylates are preferred.

  The vinyl monomers can be used alone or in admixture of two or more.

Moreover, examples of the functional group in the functional group-containing vinyl polymer include a carboxyl group, a hydroxyl group, a mercapto group, an amino group, a substituted amino group, and an epoxy group. In addition to the method of (co) polymerizing the unsaturated carboxylic acids, the functional group-containing vinyl polymer copolymerizes the vinyl monomer not containing a functional group with another vinyl monomer containing a functional group. It can be produced by a method, a method of introducing a functional group into the (co) polymer of the vinyl monomer other than unsaturated carboxylic acids, and the like.

  The measurement of the emission intensity by the optical measurement method of the present invention is performed by the above-described photoelectric conversion device. In this case, in order to reduce the influence of the fluorescence generated by the coexisting substance in the liquid sample and the optical waveguide itself, the emission intensity is measured after 10 microseconds after the excitation light irradiation. The light emission intensity may be measured over a certain period of time after 10 microseconds, and the light emission intensity may be obtained by an average value or an integral value. Although the elapsed time in this case can be set arbitrarily, the emission intensity may be measured usually after 10 microseconds to several hundred microseconds. More preferably, in order to more effectively suppress the background noise generated from the coexisting substance in the sample and the optical waveguide, the emission intensity may be measured after 200 microseconds after the excitation light irradiation.

  According to the optical measurement method of the present invention, various components contained in a liquid sample can be measured quickly and highly sensitively and more easily with an inexpensive apparatus. Therefore, the optical measurement method of the present invention can be suitably used for measuring various components in blood samples or monitoring environmental pollutants.

  Conventionally, examinations by clinical assay methods have been performed in examination centers and hospitals of medium-sized or larger, but the measurement method of the present invention makes it possible to measure quickly, with high sensitivity and with accuracy using an inexpensive device. Even a practitioner at the individual level will be able to test sufficiently. Therefore, it becomes possible for a patient who has missed the medical care to know the test result on the spot, to reduce the number of hospital visits and the time / cost required for the visit, and to reduce the medical expenses in the country.

  The measurement method of the present invention is also particularly suitable for a method for measuring glycated hemoglobin in a blood sample.

Glycohemoglobin (GHb) refers to a series of small amounts of hemoglobin components produced by the binding of various sugars (most commonly glucose) to hemoglobin molecules. Human erythrocytes are freely permeable to glucose. Within each red blood cell, GHb is produced at a rate that is directly proportional to the surrounding glucose concentration. The reaction between glucose and hemoglobin proceeds non-enzymatically, irreversibly and slowly so that only a portion of total hemoglobin is glycated during the life of the red blood cells (120 days). As a result, measuring GHb provides a weighted “moving” average of blood glucose levels that can be used to monitor long-term blood glucose levels and is an accurate indicator of average blood glucose levels over the past 2-3 months. . Blood glucose control in diabetic patients is the most important clinical application of GHb measurement.

Hemoglobin A1c (HbA1c) is a specific type of glycohemoglobin and the most important hemoglobin species for diabetes. The total hemoglobin level of HbA1c is about 3 to 6% in non-diabetic patients and 20% or more in diabetic patients who are not well managed. In HbA1c, glucose is bound to the amino terminal valine residue of one or both β chains of hemoglobin A. HbA1c can be separated from non-glycated hemoglobin by a method of separating molecules based on the difference in charge. Traditional methods for assessing glycemic control in diabetes, including urine and blood glucose levels, can vary, do not provide glucose information over time, and are of limited value because they are dramatically affected by diet . However, the measurement of GHb is an accurate index of the average blood glucose level of the subject over the past 2 to 3 months, and the diabetic patient can grasp the outline of achievement of the long-term goal for blood glucose level management.

  In clinical assays, GHb is separated from total hemoglobin based on either charge differences or structural features. This separation is generally performed by high pressure liquid chromatography (HPLC).

  Clinical assay methods by HPLC are widespread and are practiced in medium and large hospitals with laboratory centers and laboratories. In addition, immunoassay methods (immunoinhibition turbidimetry, latex agglutination, enzyme immunoassay) suitable for processing a large amount of specimens are known.

  The method for measuring glycohemoglobin by the optical measurement method according to the present invention is simple, inexpensive and high in a short time by using a small automatic device as compared with the measurement methods by various immunoassays described above. Sensitivity can be measured with high accuracy.

  Since there is a correlation between the measurement value obtained by the conventional immunoassay method and the measurement value obtained by the HPLC method, generally, the measurement value obtained by the immunoassay method is converted into the measurement value of the HPLC method by a correction formula. .

  Similarly, the measurement value obtained by the optical measurement method according to the present invention can be converted into the measurement value of the HPLC method.

  Next, specific examples will be described.

(Example 1)
1) Preparation of monoclonal antibodies against human hemoglobin and human glycated hemoglobin A1C Human hemoglobin and human glycated hemoglobin A1C were sufficiently dispersed in Freund's complete adjuvant, and 100 μl of this was immunized 4 times every 2 weeks with Balb / c mice. The spleen of this immunized mouse was removed and 106 spleen cells were obtained therefrom. It was fused and cultured with mouse myeloma cells in the presence of PEG. The supernatant of the proliferated cells was collected and examined for the presence of each antibody by ELISA. Cells positive for the antibody were tested by the limiting dilution method to confirm the cells producing each antibody. The cells thus obtained were cultured in large quantities and injected into the mouse abdominal cavity. Ascites was collected every 3 days from 2 weeks to obtain the target monoclonal antibody (IgG).

2) Preparation of fluorescent polymer particles and anti-human glycohemoglobin A1C antibody-adsorbed fluorescent polymer particles 80 parts by weight of water and 1.5 parts of sodium dodecyl sulfonate were dissolved in a glass flask. While stirring this, a monomer solution in which 20 parts by weight of styrene, 3 parts by weight of a rare earth metal complex and 0.15 parts by weight of AIBN were mixed was dropped and emulsified. While cooling this, it was dispersed with a clear mix and an ultrasonic crusher. Thereafter, after purging with nitrogen, the reaction solution was heated to 80 ° C. with stirring and heated to 80 ° C., and continued for about 8 hours. The phosphor polymer fine particles had a volume average particle size of 0.12 μm and a CV of 9%.

  Anti-human glycohemoglobin A1C antibody was dissolved in 0.05 M glycine buffer (pH 8.6) so as to be 1 mg / ml. The fluorescent polymer particles were washed three times with the same buffer solution, and then dispersed in the buffer solution so that the particle concentration was 1%. To 1 ml of this dispersion, 0.5 ml of the antibody solution was added and stirred at 35 ° C. for 1 hour. Thereafter, 1 ml of bovine serum albumin (BSA) 1% dissolved in phosphate buffer solution (pH 7.2) was added, stirred at room temperature for 1 hour, and then centrifuged (15000 rpm × 1 hour). Washed with liquid (pH 7.2). This centrifugation and washing were performed 3 times, 2 ml of the same phosphate buffer solution (pH 7.2) was added, and the particles were dispersed with an ultrasonic homogenizer.

3) Coating of optical waveguide A glass plate having a thickness of 1 mm was used as the optical waveguide. The glass plate was immersed in ethanol for 30 minutes and then washed with distilled water. 1 mg / ml of anti-human hemoglobin monoclonal antibody
It was dissolved in 0.1 M Tris-HCl buffer (pH 9.0) so as to be ml, immersed in the surface of a glass plate, and allowed to stand at 30 ° C. for 1 hour. This was immersed in the same Tris buffer and washed, and then 1% bovine serum albumin (BSA) dissolved in phosphate buffer (pH 7.2) was immersed in the same surface of the glass plate and allowed to stand at 30 ° C. for 1 hour. By this, the surface of the glass plate not coated with the antibody was coated. And after immersing and washing in the same phosphate buffer solution, it was stored at 4 ° C. in the same buffer solution until use.

4) Reaction with sample 0.05M phosphate buffer (pH 6.0) in which Triton X-100 is 0.1% by weight as a hemolytic reagent and tetrapolyphosphate is dissolved in 0.1% by weight as a reagent for removing unstable glycated hemoglobin. ) Was used.

  As a blood sample, 10 mg of sodium fluoride was added as a blood anticoagulant to 1 ml of whole blood immediately after blood collection. A solution obtained by diluting 3 μl with the above hemolytic reagent 150 times was used as a diluted blood sample.

  A predetermined amount of anti-human glycohemoglobin A1C monoclonal antibody-adsorbed fluorescent polymer particles was added to the diluted blood sample, mixed, and allowed to stand for 5 minutes. 50 μl of this mixed solution was dropped onto a glass plate coated with an anti-human hemoglobin monoclonal antibody.

5) Measurement The glass plate on which the sample was dropped was set as an optical waveguide in the apparatus. The 360 nm laser light is incident through the lens from the side of the glass plate at an angle at which the laser beam is totally reflected, and an evanescence field is generated in a portion where the sample is dropped. The amount of light from 10 microseconds to 110 microseconds was detected after irradiating the emitted light with a wavelength near 600 nm.

  The results are shown in Table 1 below.

(Example 2)
In the measurement, it was the same as Example 1 except that after the light irradiation, the light amount from 20 microseconds to 120 microseconds was measured.

  The results are shown in Table 1 below.

(Example 3)
At the time of measurement, it was the same as Example 1 except that the light quantity from 1000 microseconds to 2000 microseconds was measured after light irradiation.

  The results are shown in Table 1 below.

(Comparative Example 1)
In the measurement, it was the same as Example 1 except that after the light irradiation, the light amount from immediately after the light irradiation to 100 microseconds was measured.

  The results are shown in Table 1 below.

  The detected relative intensity in Table 1 is the relative intensity of light measured by the above example. Moreover, A1c value * in Table 1 is a value when the same sample is measured by the HPLC method.

  As is apparent from Table 1, it can be seen that there is a sufficient correlation between the values measured by the HPLC method and the detected relative intensities measured in the examples. Therefore, the detected relative intensity measured in the present Example can be converted into the A1c value * obtained by the HPLC method shown in Table 1 to obtain the A1c value in the measuring method according to the present invention.

Claims (7)

Generated by exciting a fluorescent substance in a complex with a specific component in a liquid sample, which is constrained near the surface of the optical waveguide, by evanescent waves generated by introducing and internally reflecting excitation light into the optical waveguide In a method for measuring a specific component in a liquid sample by increasing or decreasing emission intensity,
A method for optically measuring a component in a liquid sample, wherein the component in the sample is measured by measuring the luminescence intensity after 10 microseconds after the excitation light irradiation. The optical measurement method according to claim 1, wherein the fluorescent material is a rare earth metal complex. The optical measurement method according to claim 2, wherein the rare earth metal is at least one selected from the group consisting of europium, terbium, samarium, dysprosium, gadolinium and neodymium. The optical measuring method according to claim 1, wherein the fluorescent substance is contained in polymer particles. The optical measurement method according to claim 1, wherein the emission intensity after 200 microseconds is measured after the excitation light irradiation. The optical measurement method according to claim 1, wherein the specific component in the liquid sample is glycohemoglobin. The optical measurement method according to any one of claims 1 to 6, wherein a single optical waveguide is used and a plurality of samples are continuously measured without BF separation.