JP2007155548A - Quantitative analysis using infrared phosphor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of performing quantitative analysis with substantially high sensitivity. <P>SOLUTION: The method for performing the quantitative analysis of a test material by using an infrared phosphor radiating fluorescence having a wavelength in an infrared domain when being irradiated with excitation light having a wavelength in the infrared domain includes (1) a step of irradiating a mixture M<SB>1</SB>including the test material and the infrared phosphor or a mixture M<SB>2</SB>acquired by taking a part therefrom with the excitation light having the wavelength in the infrared domain, and acquiring fluorescence intensity I<SB>1</SB>relative to the fluorescence having the wavelength in the infrared domain radiated from the mixture M<SB>1</SB>or M<SB>2</SB>, and (2) a step of determining the quantity Q<SB>1</SB>of the test material from the fluorescence intensity I<SB>1</SB>acquired in the step (1) based on the correlation A between the fluorescence intensity I relative to a model mixture in the step (1) and the quantity Q of the test material, which is acquired beforehand by a model experiment using the infrared phosphor and the test material. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a quantification method using an infrared phosphor that emits fluorescence having an infrared wavelength when irradiated with excitation light having an infrared wavelength.

  Even in a very small amount of fluorescent material, the light emitted from the fluorescent material can often be detected, and the fluorescent material is used for various purposes. Usually, excitation light and fluorescence in the ultraviolet region to the visible region are used. However, light from the ultraviolet region to the visible region is greatly affected by light emission, absorption or scattering by various substances, and is also affected by disturbance light from fluorescent lamps etc. even indoors. Such an effect cannot be completely ignored in the quantitative analysis of the test substance using the. Therefore, in the quantitative analysis of the test substance, it is necessary to remove the substance existing around the test substance by washing or the like, which requires much labor and cost. In addition, there has been a problem that such an effect cannot be sufficiently suppressed, the substantial sensitivity cannot be increased, and the analytical instrument is increased in size due to the suppression of such an effect.

  On the other hand, the problem of the fluorescent substance itself used for such quantitative analysis still remains. For example, organic phosphors have low fluorescence stability. In addition, quantum dots, which are representative inorganic phosphors, generally have high toxicity.

  The present invention has been made to solve the above-described problems. An object of the present invention is to provide a method for quantitatively analyzing a test substance while suppressing the influence of substances existing around the test substance.

In order to solve the above problems, the present invention provides:
A method for quantitative analysis of a test substance using an infrared phosphor that emits fluorescence having a wavelength in the infrared region when irradiated with excitation light having a wavelength in the infrared region,
(I) The mixture M _{1 including the} test substance and the infrared phosphor, or the mixture M ₂ extracted from the mixture M ₁ is irradiated with excitation light having a wavelength in the infrared region, Obtaining fluorescence intensity I ₁ for the fluorescence in the infrared region emitted from the mixture M ₁ or M ₂ , and (ii) obtained in advance by a model experiment using an infrared phosphor and a test substance, Based on the correlation A between the fluorescence intensity I for the model mixture in step (i) and the amount Q of the test substance, the amount Q _{1 of the} test substance is calculated from the fluorescence intensity I ₁ obtained in step (i). A method comprising the step of determining.

In the method of the present invention, it is necessary that a correlation A (Q = fn _A (I)) exists between the fluorescence intensity I and the amount Q of the test substance. It is necessary to obtain in advance by a model experiment using a test substance.

In practice, there is generally a correlation B (P = fn _B (I)) between the fluorescence intensity I for the mixture M ₁ or M ₂ and the amount of infrared phosphor P contained in the mixture. Further, since the correlation A is necessary for the determination of the test substance based on the fluorescence intensity, the correlation C (between the infrared phosphor amount P and the test substance quantity Q in the mixture M ₁ or M ₂ is used. Q = fn _C (P)) is required. Therefore, if the correlations B and C are obtained, the amount Q _{1 of the} test substance can be obtained from the fluorescence intensity I ₁ obtained in the step (i). However, and if either or both of the correlation B or C is not obtained, absorption by components of the mixture M ₁ or M _2, emission, not is determined correlation A from simply correlated B and C by such scattering There is a case. In this case, it is necessary to directly obtain the correlation A between the fluorescence intensity I and the amount Q of the test substance. Usually, the correlation A is theoretically obtained directly by calculation or the like, and thus is obtained by “model experiment” using a “model mixture”. Further, even when the correlations B and C can be obtained, a “model experiment” using a “model mixture” is usually required for either or both of the correlations B and C. As described above, in the present invention, it is necessary to obtain the correlation A (Q = fn _A (I)) in advance using all or part of the “model experiment”.

The “model mixture” as used herein refers to a mixture containing at least an infrared phosphor and a test substance and having a clear ratio thereof (that is, a mixture serving as a model of the mixture M ₁ obtained in the step (i)), Or a mixture obtained by removing a part thereof (that is, a mixture serving as a model of the mixture M ₂ obtained in the step (i)), and all the components of the mixture M ₁ or M _{2 in} the step (i) are substantially the same. Although it is desirable to include an amount, it may include only the main components of the mixture M ₁ or M ₂ .

  On the other hand, the “model experiment” herein refers to, for example, when obtaining the correlation A, prepare several “model mixtures” in which the amount Q of the test substance is changed, measure each fluorescence intensity I, and calculate the calibration curve. It is an experiment for obtaining the correlation A by creating it. A similar experiment can be used for correlation B or C.

Fluorescence generated and the excitation light irradiating the mixture M ₁ or a mixture M ₂ in the method of the present invention has high transparency to such various materials in order to have a wavelength in the infrared region. Therefore, it is possible to suppress the influence of light emission, absorption or scattering by various substances (for example, dyes) existing around the infrared phosphor and the test substance. Accordingly, quantitative analysis can be performed with substantially high sensitivity while keeping the background low without removing such infrared phosphors and substances present around the test substance by washing or the like. In addition, when an infrared phosphor formed from a metal oxide is used, not only the fluorescence intensity does not substantially decrease even when irradiated with light, but a stable fluorescence intensity can be obtained, and the toxicity of the infrared phosphor itself is also increased. Since it is low, stable quantitative analysis can be performed under conditions safe for living bodies.

BEST MODE FOR CARRYING OUT THE INVENTION

  Hereinafter, the method of the present invention will be described in detail.

  The “infrared phosphor” used in this specification means a phosphor that emits energy of light having a wavelength in the infrared region when irradiated with excitation light having a wavelength in the infrared region. Therefore, upon irradiation with excitation light, light is emitted as “fluorescence” if light energy is emitted in a very short time, but light is emitted as “phosphorescence” when light energy is emitted over a long time. Thus, the “infrared phosphor” used in the method of the present invention substantially means a phosphor that emits “fluorescence” or “phosphorescence”.

  Further, in the present specification, the “test substance” will be described later with a specific example, but means a substance that is a target in general quantitative analysis, for example, industrial production, inspection, research, etc. Means a substance to be measured in the field. The “non-test substance” means a substance existing around the test substance and / or the infrared phosphor. For example, a substance (conventionally considered to reduce the sensitivity of quantitative analysis) In particular, substances that have been washed and removed during quantitative analysis using excitation light and fluorescence in the ultraviolet region to the visible region, and are selected from the group consisting of water, solvents, resins, additives, dyes, microparticles, and biological materials. Examples include at least one non-test substance.

FIG. 1 is a flowchart showing the steps of the method of the present invention. First, in step (i), forming a mixture M ₁ comprising a by mixing a test substance and the infrared phosphor, or which a further test substance by mixing with another substance with infrared phosphor To do. Further, if necessary, an operation such as separation is further performed to obtain a mixture M ₂ obtained by partially removing the mixture M ₁ .

  The infrared phosphor used is preferably in the form of particles (hereinafter, the particulate infrared phosphor is also referred to as “infrared phosphor particles”). In particular, when the infrared phosphor is used in powder form, in order to prevent variation in the results obtained, it is preferable that the infrared phosphor particles can be dissolved or dispersed uniformly in the liquid sample containing the test substance. The upper limit of the diameter of the infrared fluorescent particles is preferably 5 μm or less, more preferably 500 nm or less, still more preferably 100 nm or less. On the other hand, the lower limit of the diameter of the infrared fluorescent particles is determined depending on whether the production is possible and whether a detectable fluorescence intensity is obtained, and generally 2 nm or more is preferable. In view of the above, it is preferable that the infrared fluorescent particles have a particle diameter of 2 nm to 5 μm. The “particle size” here is obtained by, for example, randomly selecting 100 particles from an image magnified with an electron microscope or an optical microscope, reading the diameter of each particle, and averaging these. The particle size of However, if the diameter is not uniform, the average of the maximum and minimum diameters is determined as the diameter of each particle. It should be understood that the preferred particle size of such infrared fluorescent particles can vary depending on the shape and type of the test substance or infrared fluorescent particles.

  The infrared phosphor used in the method of the present invention may be formed from any material such as an inorganic material, an organic material, a composite material, or a complex. In particular, an infrared phosphor formed of an inorganic material is preferable as the infrared phosphor of the present invention because the decrease in fluorescence intensity due to irradiation with excitation light is small and the stability is excellent.

The infrared phosphor used in the method of the present invention is preferably excellent in safety or environmental aspects. For example, metal oxide-based infrared phosphors are generally suitable for the infrared phosphor of the present invention because of their high stability and low toxicity. Examples of the infrared phosphor made of a metal oxide include a compound containing a transition metal element, a phosphorus element, and an oxygen element. Typical examples of such compounds include Y · Nd · Yb · PO ₄ , Lu · Nd · Yb · PO ₄ and La · Nd · Yb · PO ₄ (wherein Y: yttrium element, Nd: neodymium element, Yb: Ytterbium element, Lu: lutetium element, La: lanthanum element, P: phosphorus element, O: oxygen element) and the like.

Among infrared phosphors composed of metal oxides, in particular, the general formula A _1-xy Nd _x Yb _y PO ₄ (wherein A is at least one selected from the group consisting of Y, Lu and La) Compounds represented by the following formulas are preferred: 0 <x ≦ 0.5; 0 <y ≦ 0.5 and 0 <x + y <1). Further, among the compounds represented by the general formula A _1-xy Nd _x Yb _y PO ₄ , compounds having an afterglow duration of 100 μs or more are particularly preferable. Here, “afterglow duration” refers to the time obtained by measuring the time until the fluorescence intensity after stopping the excitation light irradiation is reduced to 1/10.

  In the embodiment of the present invention, the test substance used in step (i) may be any kind of liquid, solid, dissolved substance, etc., as long as the correlation A can be obtained in advance by a model experiment. It may be a substance. Further, a liquid material may be gelled or solidified by drying or curing. Preferably, the test substance is at least one substance selected from the group consisting of a solvent, a resin, an additive, a solid additive, a curable liquid, and a biological substance. The test substance or infrared phosphor is preferably used in a state dissolved or dispersed in a liquid sample containing water, a solvent, etc., or dissolved or dispersed in a solid or gel. As long as the fluorescence measurement is possible, it may be separated by sedimentation or floating at the time of fluorescence measurement. For example, if a solution containing a test substance is placed in a flat bottom container and a precipitate is uniformly formed at the bottom of the container, irradiation with excitation light from the top or bottom of the container and observation of fluorescence from the top or bottom of the container will give quantitative results. Fluorescence measurement with In this case, for example, if excitation light is irradiated from the side surface, the quantitative property may not be obtained.

Mixture M ₁ of step (i) by mixing a liquid sample containing an analyte and an infrared phosphor, or which is further formed by mixing with another substance, preferably, be tested After adding the infrared phosphor to a container or the like containing a liquid sample containing a substance, operations such as shaking, stirring, kneading and / or standing are performed as necessary. Further, if necessary, the precipitate / phase-separated one or the solid content is separated, and any one of the extracted components is used as the mixture M ₂ for subsequent fluorescence measurement. Next, the obtained mixture M ₁ or M ₂ is irradiated with excitation light having a wavelength in the infrared region, and fluorescence having a wavelength in the infrared region is emitted from the mixture M ₁ or M ₂ due to the excitation light. Will be.

The excitation light and the resulting fluorescence that irradiates the mixture M ₁ or the mixture M ₂ have a wavelength in the infrared region that is highly transmissive to the test substance and the substances existing around it, and that emits, absorbs, or scatters less from those substances. It is what you have. Preferably, the peak wavelength of the excitation light spectrum and the fluorescence spectrum is in the near infrared region of 700 to 3000 nm. Light in a shorter wavelength range than this wavelength not only absorbs and emits more light in the visible region, but also scatters more than the wavelength of the test substance and its surrounding materials, This is because the infrared absorption by the mixture M ₁ or the mixture M ₂ is increased in light having a long wavelength range. In particular, when the mixture M ₁ or the mixture M ₂ contains moisture, the peak wavelengths of the excitation light spectrum and the fluorescence spectrum are in the near infrared region of 700 to 1300 nm where light absorption by water is small. It is more preferable. Furthermore, considering that the greater the difference between the excitation light wavelength and the fluorescence wavelength, the easier it is to cut the influence of excitation light when measuring fluorescence intensity using an infrared phosphor, the peak wavelength of the excitation light spectrum Is more preferably in the near infrared region range of 700 to 1100 nm and the peak wavelength of the fluorescence spectrum is in the near infrared region range of 850 to 1200 nm.

  If the difference between the peak wavelength of the excitation light spectrum and the peak wavelength of the fluorescence spectrum is 20 nm or less, it is difficult to separate the excitation light and the fluorescence by a spectral filter or the like, and even if they can be separated, the respective lights overlap. Therefore, the difference between the peak wavelength of the excitation light spectrum and the peak wavelength of the fluorescence spectrum is preferably 20 nm or more. More preferably, the difference between the peak wavelength of the excitation light spectrum and the peak wavelength of the fluorescence spectrum is 50 nm or more, and more preferably 100 nm or more.

  The “excitation light having a wavelength in the infrared region” used in the method of the present invention can be obtained by using a laser having a wavelength in the infrared region, and can also use light emitted from a light source such as a halogen lamp as an appropriate spectral filter. Can be obtained by threading.

In step (i), a fluorescence intensity I ₁ is obtained for fluorescence in the infrared region emitted from the mixture M ₁ or M ₂ due to the excitation light. In the method of the present invention, it is necessary that a correlation A (Q = fn _A (I)) exists between the fluorescence intensity I and the amount Q of the test substance. And it is necessary to obtain in advance by a model experiment using a test substance.

In fact, "fluorescence intensity" may, if other conditions are constant, contains a concentration of the infrared phosphor contained in the mixture M ₁ or M ₂ is subjected to the measurement or in a mixture M ₁ or M _2, since the is proportional to the general absolute amount of the infrared phosphor (see FIG. 2), and the fluorescence intensity I of the mixture M ₁ or M _2, the amount of infrared phosphor contained in the mixture M ₁ or M ₂ A correlation B (P = fn _B (I)) generally exists with P. Further, since the correlation A is necessary for the determination of the test substance based on the fluorescence intensity, the correlation C (between the infrared phosphor amount P and the test substance quantity Q in the mixture M ₁ or M ₂ is used. Q = fn _C (P)) is required, and a mixing method and a processing method to be used are selected so that the correlation can be obtained.

When the correlations B and C are obtained, the amount Q _{1 of the} test substance can be obtained from the fluorescence intensity I ₁ obtained in the step (i), but either or both of the correlations B and C are not obtained. In some cases, the correlation A cannot be obtained simply from the correlations B and C due to absorption, emission, scattering, and the like due to the components of the mixture. In this case, it is necessary to directly obtain the correlation A between the fluorescence intensity I and the amount Q of the test substance. Usually, the correlation A is theoretically difficult to obtain directly by calculation or the like, and is thus obtained by “model experiment” using a “model mixture”. For example, the correlation A can be obtained by preparing several “model mixtures” with different amounts of the test substance Q, measuring each fluorescence intensity I, and creating a calibration curve. Even when the correlations B and C can be obtained, a “model experiment” using a “model mixture” is usually required for either or both of the correlations B and C. When the correlation B or C is not affected by the components in the mixture, and either of the correlations B or C is theoretically calculated, etc., either of these is calculated theoretically. Alternatively, the other may be obtained by a model experiment, and the correlation A may be obtained.

As described above, it is necessary in the present invention to obtain the correlation A (Q = fn _A (I)) in advance using all or part of the “model experiment”, and the correlation A is used. Thus, the amount Q _{1 of the} test substance can be obtained from the fluorescence intensity I ₁ obtained in the step (i).

The “model mixture” here includes at least an infrared phosphor and a test substance (in the case of using several model mixtures, a part of the mixture in which the amount of the test substance = 0 may be included). and obvious mixtures thereof ratios, or a mixture obtained by extracting a part from them, it is desirable to include about the same amount of all components of the mixture M ₁ or M ₂ step (i), the mixture M ₁ or it may be one containing only the main component of M _2. However, since components having absorption, emission, scattering, etc. in the infrared region affect the fluorescence intensity to be obtained, it is most preferable to contain the same or close amount as the mixture M ₁ or M _{2 in} (i). Further, when the process of obtaining the mixture M ₁ or M ₂ affects the fluorescence intensity I ₁ , it is desirable to obtain the model mixture through a similar process.

  Unless otherwise specified, the fluorescence intensity can be obtained from the fluorescence emitted in the step (i) by using a fluorometer generally used in the field of instrumental analysis.

  In the method of the present invention, excitation light / fluorescence in the infrared region is used, and since it is highly permeable to various substances, even if other substances exist around the infrared phosphor and the test substance. This is advantageous in that it is not necessary to remove such substances by washing or the like. Therefore, in the method of the present invention, quantitative analysis is performed without removing substances that had to be washed and removed by conventional quantitative analysis (that is, quantitative analysis using excitation light and fluorescence from the ultraviolet region to the visible region). it can. As a result, the washing step can be omitted or reduced, and the time and labor required for quantitative analysis can be reduced. In addition, the number of necessary containers can be reduced, and in some cases, it is possible to use only one container. In addition, when the specimen is a solid, it is often possible to perform quantitative analysis nondestructively. Furthermore, it is possible not only to provide a lid on a sample container containing a test substance or to seal it with a film made of paraffin or polyolefin or the like, but also for such a sample to contain a dye, fine particles, fibers and / or blood. It is also possible to carry out a sample to which an additive such as a biological substance has been added or to harden the sample with a resin having low transparency because it has little influence on the method of the present invention.

Next, an apparatus suitable for the method of the present invention will be described. Such a device is
A light source that emits excitation light including wavelengths in the infrared region,
A light receiving sensor for detecting fluorescence in the infrared region emitted from a mixture M ₁ comprising an infrared phosphor and a test substance or a mixture M ₂ partially extracted therefrom; and a light source and a light receiving sensor. Comprising means for holding or passing the mixture M ₁ or the mixture M ₂ in the optical path between,
This is an apparatus capable of quantifying a test substance based on the obtained fluorescence intensity.

  As described above, in carrying out the method of the present invention, a combination of small parts such as a combination of a laser and a photodiode may be used, and since the influence of disturbance light is small, strict light shielding is not necessary, so that the apparatus can be downsized. Is planned.

  As the “light source” used in the apparatus suitable for the method of the present invention, any type of light source may be used as long as it emits excitation light including wavelengths in the infrared region. For example, a light source (for example, a laser or a halogen lamp) used in a general analytical instrument can be used. A spectral filter that can extract excitation light having a wavelength in the infrared region from light emitted from the light source is preferably provided.

The “light-receiving sensor” of the apparatus suitable for the method of the present invention is any type as long as it can detect fluorescence in the infrared region emitted from the mixture M ₁ or the mixture M ₂ by irradiation with excitation light. It may be a sensor. For example, a light receiving sensor such as a photodiode, an avalanche photodiode, or a CCD used in a general analytical instrument can be used.

The “means for holding or passing the mixture M ₁ or the mixture M ₂ ” of the apparatus suitable for the method of the present invention is capable of providing the mixture M ₁ or the mixture M ₂ in the optical path between the light source and the light receiving sensor. Any type of means can be used. In other words, any material that supports or allows the mixture M ₁ or the mixture M ₂ to pass through the position where the excitation light having the wavelength in the infrared region is irradiated. For example, the means used for holding or moving the measurement sample in a general analytical instrument can be used.

Incidentally, the insertion between, between such mixtures M ₁ or a mixture M ₂ and the light-receiving sensor or a suitable filter both, of a mixture M ₁ or a mixture M ₂ and the light source including the test substance and the infrared fluorescent However, it is preferable that excitation light generated by the light source does not reach the light receiving sensor.

  Hereinafter, although an example is given and explained concretely, the present invention is not limited to this example.

<Synthesis of infrared fluorescent particles>
Infrared fluorescent particles (hereinafter, also referred to as “infrared fluorescent particles A”) to which “functional groups or substances to which a test substance can bind” are not immobilized according to Example 1 of Japanese Patent Publication No. 3336572 Synthesized. Specifically, raw materials composed of Nd ₂ O ₃ : 3.5 g, Yb ₂ O ₃ : 4.0 g, Y ₂ O ₃ : 18.0 g and H ₃ PO ₄ : 60.0 g are mixed thoroughly to obtain alumina. After filling a crucible with a lid made of the product, it was put in an electric furnace, heated from room temperature to about 700 ° C. at a constant heating rate over 2 hours, and then fired at 700 ° C. for 6 hours. Immediately after the completion of firing, the product was taken out from the electric furnace and allowed to cool in air. Next, hot water at 100 ° C. was put into the crucible and boiled. The resulting fluorescent particles were removed from the crucible, washed with 1N nitric acid, washed with water, and dried. Through the above operation, infrared fluorescent particles A represented by the general formula Nd _0.1 Yb _0.1 Y _0.8 PO ₄ were obtained. In this infrared fluorescent particle A, “functional group or substance capable of binding to the test substance” is not immobilized. In the infrared fluorescent particles A, the peak wavelength of the fluorescence spectrum of 980 nm was obtained when the excitation light having the peak wavelength of the excitation light spectrum of about 810 nm was irradiated.

<Preparation of sample used for fluorescence intensity measurement test>
Next, a sample used in the following fluorescence intensity measurement test was prepared by using the infrared fluorescent particles A and other materials.

First, the following samples were prepared as examples.
Example 1 Preparation of Sample A
Sample A was prepared by uniformly dispersing the infrared fluorescent particles A in water so as to be 10 ppm.

(Example 2 ... Preparation of sample B)
Sample B was prepared by uniformly dispersing the infrared fluorescent particles A in water so as to be 10 ppm, and then mixing 50 μL thereof with 950 μL of ethyl alcohol.

Example 3 Preparation of Sample C
Sample C was prepared in the same manner as in Example 2, except that ethyl alcohol was changed to a 1% by weight heated solution of agarose and cooled to room temperature after mixing to obtain a slightly cloudy gel.

Example 4 Preparation of Sample D
Sample D was prepared in the same manner as in Example 2 except that ethyl alcohol was changed to a hemoglobin solution.

(Example 5: Preparation of sample E)
Sample E was prepared by irradiating the sample A obtained in Example 1 with excitation laser light for 5 minutes.

Next, the following samples were prepared as comparative examples corresponding to the respective examples (excluding comparative example 2 corresponding to example 2).
(Comparative Example 1 ... Preparation of Sample A ')
Sample A ′ was prepared in the same manner as in Example 1 except that the visible fluorescent dye FITC (Fluorescein isothiocyanate) was used instead of the infrared fluorescent particle A.

(Comparative Example 3 ... Preparation of Sample C ')
Sample C ′ was prepared in the same manner as in Example 3 except that the visible fluorescent dye FITC was used instead of the infrared fluorescent particle A.

(Comparative Example 4 ... Preparation of Sample D ')
Sample D ′ was prepared in the same manner as in Example 4 except that the visible fluorescent dye FITC was used instead of the infrared fluorescent particle A.

(Comparative Example 5 ... Preparation of sample E ')
Sample E ′ was prepared in the same manner as in Example 5 except that the visible fluorescent dye FITC was used instead of the infrared fluorescent particle A.

Samples obtained in Examples and Comparative Examples are summarized in Table 1.

<Fluorescence intensity measurement test>
The fluorescence intensity was measured for the samples obtained in Examples 1 to 5 and Comparative Examples 1 to 5 (excluding Comparative Example 2).

  Various laser light sources, sample containers, silicon light receiving elements and filters were arranged side by side for measurement. In the measurement using the sample of the example, a bandpass filter that can extract a wavelength centered at 810 nm using a 810 nm laser is inserted on the light source side, and a band that can extract a wavelength centered on 980 nm is inserted on the light receiving element side. A path filter was inserted. In the measurement using the sample of the comparative example, a bandpass filter capable of extracting a wavelength centered on 495 nm using a 495 nm laser is inserted on the light source side, and a wavelength centered on 520 nm is inserted on the light receiving element side. A removable bandpass filter was inserted.

  The results of fluorescence intensity are shown in Table 2. The fluorescence intensity is expressed using the signal intensity (dB) of the detection light, and the signal intensity at the time of shading in Example 1 and Comparative Example 1 is normalized to 0 (dB). In addition, “light shielding” in the table substantially means “there is disturbance light”, whereas “no light shielding” substantially means “no disturbance light”.

The results in Table 2 showed that:

-When comparing the result of Example 1 using an infrared phosphor with the result of Comparative Example 1 using a visible phosphor, Example 1 detects light with respect to the conditions of "light shielding" / "no light shielding". Signal intensity difference between the two is small and the influence of ambient light is small;

When the result of Example 1 is compared with the result of Example 2 (Sample B of Example 2 has a lower infrared phosphor concentration than Sample A of Example 1), the change in concentration of the infrared phosphor Is observed as a change in the signal intensity of the detected light, and if a calibration curve is created based on this change, quantitative analysis is possible;

When comparing the results of Example 3 and the results of Comparative Example 3, the signal intensity of the detected light is lower in Example 3 using the infrared phosphor than in Comparative Example 3 using the visible phosphor. , And infrared light is more transmissive than visible light;

-When the result of Example 4 and the result of Comparative Example 4 are compared, in the case of Example 4 using an infrared phosphor, fluorescence can be detected even in the presence of hemoglobin having lower transparency. In the case of Comparative Example 4 using a visible phosphor, almost no fluorescence can be detected;

-When comparing the result of Example 5 with the result of Comparative Example 5, compared to the fluorescent dye of Comparative Example 5 in which the fluorescence intensity is reduced by the irradiation of excitation light, the metal oxide infrared phosphor of Example 5 Is stable with almost no decrease in fluorescence intensity.

  From the above results, it can be seen that in the example, not only stable fluorescence intensity can be obtained, but also quantitative analysis can be performed with little influence of disturbance light even when the transparency of the sample is low. It was shown that quantitative analysis can be performed with substantially high sensitivity by using a low background.

  When the infrared phosphor is used, the method of the present invention can be used for quantification of the mixing ratio and existence ratio of the test substance in industrial production, inspection, research, etc., measurement of the film thickness including the test substance, and the like.

FIG. 1 is a flowchart showing the steps of the method of the present invention. FIG. 2 is a graph schematically showing the correlation used in the method of the present invention.

Claims (10)

A method for quantitative analysis of a test substance using an infrared phosphor that emits fluorescence having a wavelength in the infrared region when irradiated with excitation light having a wavelength in the infrared region,
(I) The mixture M _{1 including the} test substance and the infrared phosphor, or the mixture M ₂ extracted from the mixture M ₁ is irradiated with excitation light having a wavelength in the infrared region, Obtaining fluorescence intensity I ₁ for the fluorescence in the infrared region emitted from the mixture M ₁ or M ₂ , and (ii) obtained in advance by a model experiment using an infrared phosphor and a test substance, Based on the correlation A between the fluorescence intensity I for the model mixture in step (i) and the amount Q of the test substance, the amount Q _{1 of the} test substance is calculated from the fluorescence intensity I ₁ obtained in step (i). A method comprising the step of determining.   In the step (i), by irradiating excitation light whose excitation light spectrum has a peak wavelength in the near infrared region, fluorescence having a fluorescence spectrum peak wavelength in the near infrared region is obtained. The method according to claim 1.   In the step (i), by irradiating excitation light having a peak wavelength of the excitation light spectrum in the range of 700 to 1100 nm, fluorescence having a peak wavelength of the fluorescence spectrum in the range of 850 to 1200 nm is obtained. Item 3. The method according to Item 2.   The method according to claim 2 or 3, wherein the difference between the peak wavelength of the excitation light spectrum and the peak wavelength of the fluorescence spectrum is 50 nm or more.   The method according to claim 1, wherein a non-test substance exists around the infrared phosphor and / or the test substance.   6. The non-test substance according to claim 5, wherein the non-test substance is at least one kind of non-test substance selected from the group consisting of water, a solvent, a resin, an additive, a dye, fine particles, and a biological substance. The method described.   The method according to claim 1, wherein the infrared phosphor is a particulate substance having a particle size of 2 nm to 5 μm.   The method according to claim 1, wherein the infrared phosphor is formed from a metal oxide.   9. The method according to claim 8, wherein the metal oxide comprises a transition metal element, a phosphorus element and an oxygen element. The infrared phosphor is represented by the general formula A _1-xy Nd _x Yb _y PO ₄ (wherein A is at least one element selected from the group consisting of Y, Lu and La; 0 < The method according to claim 9, wherein the metal oxide is represented by the following formula: x ≦ 0.5; 0 <y ≦ 0.5 and 0 <x + y <1.

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