JP2012068226A - Chemiluminescence measuring device and method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device and a method capable of accurately measuring chemiluminescence intensity even when a test material has a light absorption band in a chemiluminescence wavelength region.SOLUTION: A chemiluminescence measuring device 1 includes a sample container 10, a measurement part 20, a memory part 30, and an operation part 40. The measurement part 20 receives chemiluminescence generated from a chemiluminescence reagent contained in a sample solution in the sample container 10 together with a test material, obtains a chemiluminescence intensity measurement value E according to intensity of the received chemiluminescence, and outputs the chemiluminescence intensity measurement value E as an electrical signal to the operation part 40. The operation part 40 corrects the chemiluminescence intensity measurement value E obtained by the measurement part 20 in accordance with a predetermined correction formula based on a normalized chemiluminescence spectrum i(λ) and an absorbance spectrum A(λ) of the test material, which are memorized by the memory part 30, to evaluate chemiluminescence intensity Eafter the correction.

Description

  The present invention relates to a chemiluminescence measuring apparatus and a chemiluminescence measuring method.

  Chemiluminescence measurement (see Patent Literatures 1 to 4 and Non-Patent Literature 1) is used for, for example, new food functionality evaluation using the immune response mechanism of human immune cells. The role of neutrophil cells, a type of white blood cell, which is the largest player of innate immunity, produces and releases active oxygen (superoxide) against the invasion of pathogens from outside, and attacks the pathogen by this superoxide Is responsible. Neutrophil cells that have been stimulated by the entry of foreign enemies take calcium ions inside and produce superoxide by using the calcium ions as a trigger.

  For detection of the amount of calcium ions in neutrophil cells, a fluorescent reagent for detecting calcium (for example, Fluo3-AM) is used. For the detection of the amount of superoxide produced by neutrophil cells, a chemiluminescence reagent for superoxide detection (for example, CLA, MCLA, etc.) is used. That is, the amount of calcium ions in neutrophil cells is detected based on the intensity of fluorescence generated from the fluorescent reagent, and the amount of superoxide produced in neutrophil cells is determined based on the intensity of chemiluminescence generated from the chemiluminescent reagent. Detected.

  When a test substance to be evaluated is added to such an assay system, the time-dependent changes in fluorescence and chemiluminescence intensity change variously due to the action of the test substance on the immune mechanism. By analyzing changes in light intensity using a plurality of stimulants, the action mechanism of the test substance can be estimated.

JP-A-6-217794 Japanese Patent No. 3290193 JP 2006-126152 A JP 2008-203088 A

Eugene H. Ratzlaff and S. R. Crouch, "Absorption-Corrected Chemiluminescence Measurements with aDual-Pathlength Spectroscopy", Anal. Chem., Vol.55, No.2, pp.348-352, 1983.

  Some analytes to be evaluated may have a light absorption band in the chemiluminescence wavelength region. In such a case, since chemiluminescence is absorbed by the test substance, the actually detected chemiluminescence intensity is smaller than the chemiluminescence intensity that should be detected originally. When the test substance has an action of removing active oxygen, the actually detected change in chemiluminescence intensity is caused by any change in the active oxygen removal ability of the test substance and the light absorption rate of the test substance. Cannot be distinguished.

  Among a plurality of chemiluminescent reagents, it is possible to select and use a reagent having an emission wavelength that is not easily affected by the light absorption of the test substance. However, when fluorescence and chemiluminescence are measured simultaneously, it is necessary to distinguish the fluorescence wavelength, chemiluminescence wavelength, and excitation light wavelength from each other, which greatly limits the selection of reagents.

  The present invention has been made to solve the above problems, and even when the test substance has a light absorption band in the chemiluminescence wavelength region, it is possible to accurately measure the chemiluminescence intensity. It is an object to provide an apparatus and method that can be used.

The chemiluminescence measuring apparatus of the present invention is an apparatus for measuring chemiluminescence generated from a chemiluminescent reagent contained in a sample liquid contained in a sample container together with a test substance, and (1) a sample contained in a sample container A measurement unit that measures the intensity of chemiluminescence reached from the liquid and obtains the measured chemiluminescence intensity value E; (2) a standardized chemiluminescence spectrum i _C (λ) of chemiluminescence produced by the chemiluminescent reagent; The chemiluminescence intensity measurement value obtained by the measurement unit based on the absorbance spectrum A (λ) of the test substance and the optical path from each light emission position in the sample liquid contained in the sample container to the light reception position of the measurement unit And an arithmetic unit that corrects E and obtains the corrected chemiluminescence intensity E ₀ .

The chemiluminescence measurement method of the present invention is a method for measuring chemiluminescence generated from a chemiluminescence reagent contained in a sample solution contained in a sample container together with a test substance, and (1) a measurement unit applies a sample to the sample container. The intensity of chemiluminescence reached from the contained sample solution is measured to obtain the chemiluminescence intensity measurement value E. (2) The chemiluminescence spectrum i normalized by the chemiluminescence reagent generated by the chemiluminescence reagent is obtained by the calculation unit. Acquired by the measurement unit based on _C (λ), the absorbance spectrum A (λ) of the test substance, and the optical path from each light emission position in the sample liquid contained in the sample container to the light reception position of the measurement unit The chemiluminescence intensity measurement value E is corrected, and the corrected chemiluminescence intensity E ₀ is obtained.

In the chemiluminescence measuring apparatus of the present invention, the calculation unit corrects the chemiluminescence intensity measurement value E acquired by the measuring unit with the depth of the sample container being d according to the following correction formula, and the corrected chemiluminescence intensity E _It is preferable to obtain ₀ . In the chemiluminescence measurement method of the present invention, the calculation unit corrects the chemiluminescence intensity measurement value E acquired by the measurement unit according to the following correction formula, where d is the depth of the sample container, and the chemiluminescence after the correction. it is preferred that determine the intensity E _0.

  The chemiluminescence measuring device of the present invention further comprises (a) an excitation unit that repeatedly and repeatedly irradiates excitation light for exciting a fluorescent reagent contained in a sample solution contained in a sample container together with a test substance and a chemiluminescent reagent. (B) When the excitation unit is not irradiated with excitation light by the excitation unit, the chemiluminescence intensity measurement value is obtained by measuring the intensity of the chemiluminescence reached from the sample liquid contained in the sample container, When excitation light is irradiated by the excitation unit, the total intensity of chemiluminescence and fluorescence that has reached from the sample liquid contained in the sample container is measured to obtain the total light intensity measurement value, and (c) the calculation unit However, based on the chemiluminescence intensity measurement value and the total light intensity measurement value acquired by the measurement unit, the fluorescence intensity measurement value representing the intensity of the fluorescence reaching the measurement unit from the test substance contained in the sample container is obtained. Is preferred.

  In the chemiluminescence measurement method of the present invention, (a) the excitation unit repeatedly irradiates the sample liquid contained in the sample container with excitation light that excites the fluorescent reagent contained together with the test substance and the chemiluminescent reagent in a pulsed manner. (b) When the excitation unit is not irradiated with excitation light, the measurement unit measures the intensity of chemiluminescence that has reached from the sample liquid contained in the sample container and obtains the measured chemiluminescence intensity value. When the excitation light is irradiated by the unit, the total intensity of chemiluminescence and fluorescence reached from the sample liquid contained in the sample container is measured to obtain the total light intensity measurement value, and (c) the calculation unit Based on the chemiluminescence intensity measurement value and the total light intensity measurement value obtained by the measurement unit, a fluorescence intensity measurement value representing the intensity of the fluorescence that has reached the measurement unit from the test substance contained in the sample container is obtained. Is preferred.

  According to the present invention, even when the test substance has a light absorption band in the chemiluminescence wavelength region, the chemiluminescence intensity can be accurately measured.

It is a lineblock diagram of chemiluminescence measuring device 1 of a 1st embodiment. It is a figure explaining derivation | leading-out of a correction formula. It is a diagram illustrating a normalized chemiluminescence spectrum i _{C (lambda)} in Example. It is a figure which shows the absorbance spectrum A ((lambda)) of the to-be-tested substance in an Example. It is a figure which shows the chemiluminescence spectrum I ((lambda)) after the absorption correction in an Example. It is a figure which shows the correction result in an Example. It is a block diagram of the chemiluminescence measuring apparatus 2 of 2nd Embodiment. It is a block diagram of the chemiluminescence measuring apparatus 3 of 3rd Embodiment. It is a figure which shows the relationship between a point light source and a micro surface. It is a figure explaining the 1st calculation example of 4th Embodiment. It is a graph which shows the result of having calculated | required the relationship between the light absorbency A and correction coefficient (delta) ₁ using the numerical integration by the 1st calculation example of 4th Embodiment. It is a figure explaining the 2nd calculation example of 4th Embodiment. It is a graph which shows the result of having calculated | required the relationship between the light absorbency A and correction coefficient (delta) ₂ using the Monte Carlo method by the 2nd calculation example of 4th Embodiment.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

  (First embodiment)

  FIG. 1 is a configuration diagram of a chemiluminescence measuring apparatus 1 according to the first embodiment. The chemiluminescence measurement device 1 according to the first embodiment includes a sample container 10, a measurement unit 20, a storage unit 30, and a calculation unit 40. The sample container 10 is a container that contains a sample solution containing immune cells (for example, neutrophil cells), a chemiluminescent reagent, and a test substance. This is an assay in which reactive oxygen produced when stimulating immune cells is detected with a chemiluminescent reagent, and studies how the test substance affects the ability of immune cells to produce active oxygen.

  The measurement unit 20 receives chemiluminescence that has arrived from the sample solution among the chemiluminescence generated from the chemiluminescence reagent contained in the sample solution contained in the sample container 10, and the chemiluminescence intensity measurement value corresponding to the received light intensity. E is acquired, and the chemiluminescence intensity measurement value E is output to the computing unit 40 as an electrical signal.

The storage unit 30 includes a chemiluminescence standardized chemiluminescence spectrum i _C (λ) generated by a chemiluminescence reagent contained in the sample solution in the sample container 10 and a test substance contained in the sample in the sample container 10. Is stored in advance. The normalized chemiluminescence spectrum i _C (λ) is normalized so that the result of integration with respect to the wavelength λ becomes a predetermined value (for example, value 1).

The calculation unit 40 receives the chemiluminescence intensity measurement value E acquired by the measurement unit 20, and the normalized chemiluminescence spectrum i _C (λ) stored in the storage unit 30 and the absorbance spectrum A (( λ). Then, the calculation unit 40 corrects the chemiluminescence intensity measurement value E acquired by the measurement unit 20 based on the normalized chemiluminescence spectrum i _C (λ) and the absorbance spectrum A (λ) according to the following correction formula, The corrected chemiluminescence intensity E ₀ is obtained. Here, the wavelength range λ1 to λ2 includes the chemiluminescence wavelength region.

Next, the derivation of the correction formula in the present embodiment will be described. FIG. 2 is a diagram illustrating the derivation of the correction formula. At a certain wavelength, the extinction coefficient of the sample liquid having light absorption is ε, the intensity of light incident on the sample liquid from the outside is I _in, and the light transmitted through the sample liquid and emitted to the outside The intensity is _Iout, and the optical path length in the sample liquid is d. At this time, the absorbance A in this sample solution is expressed by the following equation (3) from Lambert Beer's law.

Here, as shown in FIG. 2, it is assumed that the chemiluminescent reagent emits light with an intensity I ₀ at a position P at a depth z from the inner surface of the sample container 10 on the measurement unit 20 side. In this case, the light intensity I (z) observed by the measuring unit 20 is similarly expressed by the following equation (4) according to Lambert Beer's law. Assuming that the absorbance A is known, using the equation (3), the equation (4) becomes the following equation (5).

  Since chemiluminescence is considered to occur in the entire range in the depth direction, the chemiluminescence intensity I observed by the measurement unit 20 can be obtained by integrating equation (5) from 0 to d in the z (depth) direction. And is represented by the following formula (6).

Here, chemiluminescence intensity when analyte is not present, i.e., consider the chemiluminescence intensity I _C to be obtained originally, excluding the effect of the test substance. Since the test substance does not exist, the following formula (7) is obtained by substituting A = 0 into the formula (6).

Thus, Rewriting with _{I C} (6) where the following equation (8). From this equation (8), the original chemiluminescence intensity I _C when chemiluminescence I is observed in the solution containing the test substance is obtained by the following equation (9). This equation (9) is a correction equation for obtaining the original chemiluminescence intensity from the chemiluminescence intensity when the test substance exists.

Actually, since chemiluminescence and the absorption of the test substance have a certain wavelength width, I _C , I, and A are functions of the wavelength λ, and the spectra I _c (λ) and I (λ ), A (λ). Therefore, the expressions (8) and (9) are expressed as the following expressions (10) and (11), respectively.

  The signal intensity E actually output from the measurement unit 20 in the presence of the test substance is obtained by integrating the chemiluminescence spectrum I (λ) with the wavelength λ, and is expressed by the following equation (12). From the expression (10), the expression (12) becomes the following expression (13).

On the other hand, the signal intensity E _{0 to} be originally obtained after correcting the influence of the test substance is expressed by wavelength integration of the corrected chemiluminescence spectrum I _C (λ), and is expressed by the following equation (14).

Here, a standardized function i _C (λ) defined by the following equation (15) is introduced.

  Therefore, the following equation (16) is obtained from the equation (14). Substituting this equation (16) into equation (13) yields the following equation (17). From this equation (17), the correction equation of the above equation (2) is obtained.

The above equation (2) is obtained under the condition that there is a test substance that actually absorbs light based on the absorbance spectrum A (λ) and the normalized chemiluminescence spectrum i _C (λ) of the test substance measured in advance. It is shown that the correct chemiluminescence signal intensity E0 can be obtained by correcting the chemiluminescence signal intensity E.

As described above, in the present embodiment, the measurement unit 20 measures the intensity of chemiluminescence reached from the sample solution contained in the sample container 10 and acquires the measured chemiluminescence intensity value E. Then, the chemiluminescence intensity acquired by the measurement unit 20 based on the normalized chemiluminescence spectrum i _C (λ) of chemiluminescence generated by the chemiluminescence reagent and the absorbance spectrum A (λ) of the test substance by the calculation unit 30. The measured value E is corrected according to the correction formula (2), and the corrected chemiluminescence intensity E ₀ is obtained. By doing in this way, even if the test substance has a light absorption band in the chemiluminescence wavelength region, the chemiluminescence intensity can be measured accurately.

Next, examples will be described with reference to FIGS. Here, soybean soy sauce was used as the test substance, and MCLA was used as the chemiluminescent reagent. FIG. 3 is a diagram showing the standardized chemiluminescence spectrum i _C (λ) in the example. FIG. 4 is a graph showing the absorbance spectrum A (λ) of the test substance in the example. This figure shows the absorbance spectrum A (λ) when the concentration of the test substance (soybean soy sauce) is 0.03%, 0.01%, and 0.001%.

FIG. 5 is a diagram showing a chemiluminescence spectrum I (λ) after absorption correction in the example. The figure shows the chemiluminescence spectrum I (λ) after absorption correction when the concentration of the test substance (soybean soy sauce) is 0.03%, 0.01%, and 0.001%. The normalized chemiluminescence spectrum i _C (λ) is also shown. The value obtained by integrating the chemiluminescence spectrum I (λ) with respect to the wavelength is the corrected chemiluminescence intensity E ₀ .

  FIG. 6 is a diagram illustrating a correction result in the embodiment. The figure shows the emission intensity E when the concentration of the test substance (soybean soy sauce) is 0.03%, 0.01%, and 0.001%, corrected for constant absorbance, and The emission intensity E when corrected using the absorbance spectrum A (λ) of the test substance is shown. When the concentration of the test substance is 0.03%, it is accurately corrected using the emission intensity E when the absorbance of the test substance is assumed to be constant and the absorbance spectrum A (λ) of the test substance. An error of 15.5% was observed with the emission intensity E.

  (Second Embodiment)

  FIG. 7 is a configuration diagram of the chemiluminescence measuring apparatus 2 according to the second embodiment. The chemiluminescence measurement device 2 of the second embodiment includes a sample container 10, a measurement unit 20, a spectrometer 21, a storage unit 30, a calculation unit 40, and a light source 50. Compared with the configuration of the chemiluminescence measuring apparatus 1 of the first embodiment shown in FIG. 1, the chemiluminescence measuring apparatus 2 of the second embodiment shown in FIG. 7 further includes a spectrometer 21 and a light source 50. Is different.

  The light source 50 irradiates the sample container 10 with light. The wavelength range of the output light of the light source 50 includes the emission wavelength band of the chemiluminescent reagent contained in the sample solution contained in the sample container 10. The spectroscope 21 receives light output from the light source 50 and transmitted through the sample container 10, splits the input light, and outputs it to the measurement unit 20. The measuring unit 20 receives the light split and output by the spectroscope 21 and outputs the received light intensity measurement value to the computing unit 40 as an electrical signal.

The chemiluminescence measuring device 2 can also measure the absorbance spectrum A (λ) of the test substance and the chemiluminescence spectrum of the chemiluminescent reagent, and using these, the same as in the first embodiment, the formula (2) Accordingly, the corrected chemiluminescence intensity E ₀ can be obtained.

  The absorbance spectrum A (λ) of the test substance is measured as follows. A sample solution containing only the test substance is placed in the sample container 10. This sample solution does not contain a chemiluminescent reagent. Of the light output from the light source 50, the light transmitted through the sample container 10 is split by the spectroscope 21 and detected by the measurement unit 20. The measurement value of the received light intensity by the measurement unit 20 is given to the calculation unit 40 as an electrical signal. Thereby, the absorbance spectrum A (λ) of the test substance is obtained by the calculation unit 40 and stored in the storage unit 30.

The chemiluminescence spectrum of the chemiluminescent reagent is measured as follows. A sample solution containing immune cells (for example, neutrophil cells) and a chemiluminescent reagent is placed in the sample container 10. This sample solution does not contain the test substance. Then, the immune cells are stimulated to produce active oxygen, and the chemiluminescent reagent is caused to emit light. The chemiluminescence generated by the chemiluminescent reagent is dispersed by the spectroscope 21 and detected by the measuring unit 20. The measurement value of the received light intensity by the measurement unit 20 is given to the calculation unit 40 as an electrical signal. Thereby, the chemiluminescence spectrum of a chemiluminescent reagent is acquired by the calculating part 40, and also the normalized chemiluminescence spectrum i _C (λ) is obtained. The normalized chemiluminescence spectrum i _C (λ) is stored in the storage unit 30.

After the normalized chemiluminescence spectrum i _C (λ) and the absorbance spectrum A (λ) of the test substance are obtained and stored in the storage unit 30 as described above, the corrected chemiluminescence spectrum i _C (λ) is corrected in the same manner as in the first embodiment. A chemiluminescence intensity E ₀ is determined. That is, a sample solution containing immune cells, a chemiluminescent reagent, and a test substance is placed in the sample container 10. The measurement unit 20 measures the intensity of chemiluminescence that has arrived from the sample solution contained in the sample container 10 and acquires the measured value E of the chemiluminescence intensity. Then, based on the normalized chemiluminescence spectrum i _C (λ) and the absorbance spectrum A (λ) stored in the storage unit 30, the chemiluminescence intensity measurement value E acquired by the measurement unit 20 is calculated by the calculation unit 30. Correction is performed according to the correction formula (2), and the corrected chemiluminescence intensity E ₀ is obtained.

  In the second embodiment, the chemiluminescence spectrum, the absorption spectrum and the active oxygen assay can be performed by the same apparatus, and the chemiluminescence spectrum or the absorption spectrum is changed due to the influence of the pH of the sample solution. In this case, the chemiluminescence intensity can be accurately corrected.

  (Third embodiment)

  FIG. 8 is a configuration diagram of the chemiluminescence measuring apparatus 3 according to the third embodiment. The chemiluminescence measuring apparatus 3 of the third embodiment includes a sample container 10, a lens 22, an optical filter 23, a photomultiplier tube 24, a photon counter 25, a storage unit 30, a calculation unit 40, an excitation light source 60, a lens 61, and an optical filter. 62, a stirring unit 70 and a temperature adjustment unit 80 are provided.

  Compared with the configuration of the chemiluminescence measuring apparatus 1 of the first embodiment shown in FIG. 1, the chemiluminescent measuring apparatus 3 of the third embodiment shown in FIG. The difference is that the photon counter 25 is provided, and the lens 22, the optical filter 23, the excitation light source 60, the lens 61, the optical filter 62, the stirring unit 70, and the temperature adjusting unit 80 are further provided.

  The chemiluminescence measuring apparatus 3 includes a stirring unit 70 that stirs the sample solution in the sample container 10, so that the sample solution is distributed evenly even when the sample solution in the sample container 10 is a suspension. Can be maintained. The stirring unit 70 includes, for example, a magnetic stirrer placed in the sample container 10 and a magnetic stirrer controller that controls the magnetic stirrer.

  The chemiluminescence measuring apparatus 3 can maintain the sample solution in the sample container 10 at a predetermined temperature by including the temperature adjusting unit 80 that adjusts the temperature of the sample solution in the sample container 10. The temperature adjusting unit 80 includes, for example, a thermobus connected to the sample container 10 via a pipe.

  The chemiluminescence measuring device 3 can not only obtain the intensity of chemiluminescence generated in the sample container 10 but also an excitation light source 60 for exciting a fluorescent reagent contained in the sample liquid contained in the sample container 10. By providing, the intensity of the fluorescence generated in the sample container 10 can also be obtained.

  In addition, the chemiluminescence measuring apparatus 3 repeatedly irradiates the sample liquid contained in the sample container 10 with excitation light that excites the fluorescent reagent contained together with the test substance and the chemiluminescent reagent in a pulsed manner by the excitation light source 60. Chemiluminescence intensity and fluorescence intensity can also be measured substantially simultaneously.

  The sample solution contained in the sample container 10 includes immune cells (for example, neutrophil cells) into which a fluorescent reagent has been introduced, a chemiluminescent reagent, and a test substance. This is an assay that detects changes in intracellular calcium concentration when stimulating immune cells with a fluorescent reagent, and simultaneously detects active oxygen produced as a result of stimulation with a chemiluminescent reagent. The purpose of this study is to investigate how the intracellular calcium concentration changes and the ability to produce active oxygen in immune cells.

  Of the pulsed excitation light output from the excitation light source 60, the light of the specific wavelength component that has passed through the optical filter 62 is condensed and irradiated onto the sample liquid in the sample container 10 by the lens 61. Therefore, in the sample solution in the sample container 10, fluorescence derived from the fluorescent reagent is generated and chemiluminescence derived from the chemiluminescent reagent is generated.

  Fluorescence and chemiluminescence generated in the sample solution are received by the photomultiplier tube 24 through the lens 22 and the optical filter 23. Note that the excitation light scattered in the sample solution is blocked by the optical filter 23. When fluorescent or chemiluminescent photons are incident on the photomultiplier tube 24, current pulses are output from the photomultiplier tube 24 in response to the photon incident event, and the pulses are counted by the photon counter 25. The counting result is given to the calculation unit 40.

  Fluorescence is generated when the sample liquid is irradiated with excitation light, and chemiluminescence is generated regardless of whether the sample liquid is irradiated with excitation light or not. Therefore, when the excitation light is not irradiated by the photomultiplier tube 24 and the photon counter 25, the intensity of the chemiluminescence reached from the sample solution contained in the sample container 10 is measured, and the measured chemiluminescence intensity value E When the excitation light is irradiated, the total intensities of chemiluminescence and fluorescence reached from the sample liquid contained in the sample container 10 are measured, and the total light intensity measurement value is acquired.

Then, the calculation unit 40 subtracts the chemiluminescence intensity measurement value from the total light intensity measurement value, thereby obtaining a fluorescence intensity measurement value representing the intensity of the fluorescence that has reached the measurement unit from the sample solution contained in the sample container 10. It is done. Further, the corrected chemiluminescence intensity E ₀ is obtained by the calculation unit 40 as in the first embodiment.

  (Fourth embodiment)

In the first to third embodiments described above, it is assumed that the measurement unit 20 receives light traveling in one direction among the light generated in the sample liquid contained in the sample container 10. By using the correction formula based on such a premise, the chemiluminescence intensity E ₀ corrected accurately by a simple calculation can be obtained. However, in order to obtain the more accurately corrected chemiluminescence intensity E ₀ , the standardized chemiluminescence spectrum i _C (λ) of the chemiluminescence produced by the chemiluminescence reagent and the absorbance spectrum A (λ) of the test substance are used. In addition, correction is performed according to the correction equation (18) below using a correction coefficient δ that also takes into account the optical path from each light emission position in the sample liquid contained in the sample container 10 to the light receiving position of the measurement unit 20. Is preferred.

  In the fourth embodiment, correction is performed in consideration of the optical path from each light emission position in the sample liquid contained in the sample container 10 to the light receiving position of the measurement unit 20. The fourth embodiment corresponds to a generalization of the first to third embodiments. Below, although the sample container 10 and the measurement part 20 are demonstrated in detail, about the other component, it is the same as that of the case of the 1st-3rd embodiment.

As shown in FIG. 9, when the angle between a light beam from an arbitrary point light source having a radiation intensity I and the normal of the minute surface is θ and the distance between the point light source and the minute surface is r, the point light source emits a minute amount. The intensity E ₀ of light incident on the surface is expressed by the following equation.

When a light-absorbing test substance exists in the sample solution, the intensity of light reaching the measuring unit 20 is attenuated according to Lambert Beer's law. When the absorbance of the test substance A (lambda), the intensity E _A of the light reaching the measuring section 20 becomes the following equation.

In chemiluminescence measurement, the above point light source corresponds to chemiluminescent molecules in the sample liquid in the sample container 10, and the minute surface corresponds to a part of the light receiving surface of the light receiving unit 20. Therefore, the sum P _A chemiluminescence incident on the minute surface of the light receiving portion 20 is made as a three-dimensional region V (20) equation of the sample solution in the field of view of the micro surface of the light receiving portion 20 has a triple integral, by: expressed.

Furthermore, if the light receiving surface of the measuring unit 20 has a certain area, the sum P _A of light incident on the light receiving surface of the measuring unit 20, the area of the light receiving surface of the measuring unit 20 with respect to the infinitesimal surface mentioned above S It is a double integral with the following formula.

When there is a gap between the measurement unit 120 and the chemiluminescent reagent due to the thickness of the sample container 10 or the like, the optical path length related to absorption is shortened by this gap. If the length of the gap is u, the length of the optical path that passes through the gap is u / cos θ, and the length of the optical path that passes through the sample liquid is r− (u / cos θ). Therefore, the sum P _A of the light in consideration of the gap, the following expression obtained by transforming the equation (22).

This equation (23) is a general formula for obtaining the intensity of light observed when chemiluminescence from the sample liquid in the three-dimensional region V in the sample container 10 is detected by the light receiving surface of the surface region S of the measuring unit 20. It becomes. The correction coefficient chemiluminescence intensity for light absorbing analyte δ in the sample solution is calculated as the ratio between the light intensity P _A in the case where the light intensity P ₀ in a case where the test substance is absent, present, It is expressed by the following formula.

  When the mathematical formulas described so far are actually applied, an appropriate coordinate system is applied to each of the three-dimensional region V in the sample container 10 and the surface region S of the measurement unit 20, and each of the actual three-dimensional region V and the surface region S is applied. Find the corresponding integration interval and integrate. If the integral is difficult to solve analytically, it can be solved using numerical integration or Monte Carlo methods.

  Further, depending on the optical system from each light emitting position in the sample solution to the light receiving position of the measuring unit 20, it may take time to calculate the correction coefficient δ. In such a case, the relationship between the absorbance A and the correction coefficient δ is calculated with sufficient accuracy in advance, and a simpler approximate expression is obtained using this relation, and the actual correction coefficient is obtained using this approximate expression. Also good. Hereinafter, calculation examples of correction coefficients in some typical optical systems will be described. Note that the influence of light absorption and refractive index of the sample container 10 can be ignored.

  In the first calculation example, as shown in FIG. 10, a sample container 10 having a cubic volume of 1 cm on each side and a thickness of 0.1 cm is used, and the sample container 10 contains a chemiluminescent reagent. It is assumed that the sample liquid is filled, a point sensor is used as the measurement unit 20, and the measurement unit 20 is disposed in close contact with the center of a certain surface of the sample container 10. Even when the light receiving surface of the light receiving unit 20 has an area, a pin hole is disposed in close contact with the center of a certain surface of the sample container 10, and the light passing through the pin hole is transmitted by the light receiving unit 20. The first calculation example can also be applied to the case where light is received or when the light that has passed through the pinhole is dispersed by the spectroscope and then received by the light receiving unit 20.

In this case, when the calculation of the light intensity P _A may be used above (23) but, given the light receiving surface of the measuring section 20 is a point sensor and small area, the integration of the S region is unnecessary. The light receiving surface of the measurement unit 20 is set as the origin, and an arbitrary point in the integration region V is set as (x, y, z) in the xyz orthogonal coordinate system. Light intensity _{P A} can be calculated by the following equation (25). The integration region V is a region in the sample container 10 and is a region represented by the following expression.

The correction coefficient δ ₁ according to the first calculation example is calculated by the following equation. It is difficult to solve this equation analytically. FIG. 11 is a graph showing the results of obtaining the relationship between the absorbance A and the correction coefficient δ ₁ using numerical integration according to the first calculation example of the fourth embodiment.

  In the second calculation example, as shown in FIG. 12, a sample container 10 having a cylindrical body volume of 1 cm in diameter and 1 cm in height and having a thickness of 0.1 cm is used. A case is assumed in which a sample liquid containing a luminescent reagent is filled, a sensor having a circular light receiving part with a diameter of 1 cm is used as the measuring part 20, and the light receiving surface of the measuring part 20 is disposed in close contact with one bottom side of the sample container 10. .

In this case, since the light receiving surface of the light receiving unit 20 has an area, integration with respect to the S region in the above equations (22) and (23) is also required. The integration region V is a region in the cylindrical sample container 10. If the center of the light receiving surface of the light receiving unit 20 is the origin, any point in the integration region S is orthogonal coordinates (n, m), and any point in the integration region V is orthogonal coordinates (x, y, z), light intensity _{P A} can be calculated similarly to the above in the following equation (28). The integration regions V and S are expressed by the following equation.

The correction factor [delta] ₂ of the second calculation example is calculated by the following equation. Since the integral of this equation is a quintuple integral and the integrand is complex, it takes a long time to calculate when the numerical integration is actually performed. For such calculations, integration by the Monte Carlo method is suitable. FIG. 13 is a graph showing the results of determining the relationship between the absorbance A and the correction coefficient δ ₂ using the Monte Carlo method according to the second calculation example of the fourth embodiment.

  As a calculation example for obtaining the correction coefficient δ in consideration of the optical path from each light emitting position in the sample liquid contained in the sample container 10 to the light receiving position of the measuring unit 20, the first calculation example and the second calculation example will be described so far. However, the present embodiment is not limited to these. By correcting according to the correction formula (18) using such a correction coefficient δ, an accurate value of chemiluminescence can be obtained even when the test substance is a light absorbing substance.

  DESCRIPTION OF SYMBOLS 1-3 ... Chemiluminescence measuring apparatus, 10 ... Sample container, 20 ... Measuring part, 21 ... Spectroscope, 22 ... Lens, 23 ... Optical filter, 24 ... Photomultiplier tube, 25 ... Photon counter, 30 ... Memory | storage part, DESCRIPTION OF SYMBOLS 40 ... Calculation part, 50 ... Light source, 60 ... Excitation light source, 61 ... Lens, 62 ... Optical filter, 70 ... Stirring part, 80 ... Temperature adjustment part.

Claims (6)

An apparatus for measuring chemiluminescence generated from a chemiluminescent reagent contained in a sample solution contained in a sample container together with a test substance,
A measurement unit for measuring the intensity of chemiluminescence reached from the sample liquid contained in the sample container and obtaining the chemiluminescence intensity measurement value E;
Standardized chemiluminescence spectrum i _C (λ) of chemiluminescence generated by the chemiluminescence reagent, the absorbance spectrum A (λ) of the test substance, and each luminescence in the sample solution contained in the sample container A calculation unit that corrects the chemiluminescence intensity measurement value E acquired by the measurement unit based on the optical path from the position to the light receiving position of the measurement unit, and obtains the corrected chemiluminescence intensity E ₀ ;
A chemiluminescence measuring device comprising: The calculation unit corrects the chemiluminescence intensity measurement value E acquired by the measurement unit according to the following correction formula with the depth of the sample container as d, and obtains the corrected chemiluminescence intensity E ₀ .

The chemiluminescence measuring apparatus according to claim 1. An excitation unit that repeatedly irradiates the sample liquid contained in the sample container with pulsed excitation light that excites the fluorescent reagent contained together with the test substance and the chemiluminescent reagent;
When the measurement unit is not irradiated with excitation light by the excitation unit, the intensity of chemiluminescence reached from the sample liquid contained in the sample container is measured to obtain the chemiluminescence intensity measurement value, When excitation light is irradiated by the excitation unit, the total intensity of chemiluminescence and fluorescence reached from the sample liquid contained in the sample container is measured to obtain the total light intensity measurement value,
Fluorescence representing the intensity of fluorescence that has reached the measurement unit from the sample liquid contained in the sample container, based on the chemiluminescence intensity measurement value and the total light intensity measurement value acquired by the measurement unit. Find the strength measurement,
The chemiluminescence measuring apparatus according to claim 1 or 2, wherein A method for measuring chemiluminescence generated from a chemiluminescent reagent contained in a sample solution contained in a sample container together with a test substance,
The measurement unit measures the intensity of chemiluminescence reached from the sample solution contained in the sample container to obtain the chemiluminescence intensity measurement value E,
The calculation unit generates a standardized chemiluminescence spectrum i _C (λ) of chemiluminescence generated by the chemiluminescence reagent, an absorbance spectrum A (λ) of the test substance, and the sample liquid contained in the sample container. Based on the optical path from each light emitting position to the light receiving position of the measurement unit, the chemiluminescence intensity measurement value E acquired by the measurement unit is corrected, and the corrected chemiluminescence intensity E ₀ is obtained.
The chemiluminescence measuring method characterized by the above-mentioned. The calculation unit corrects the chemiluminescence intensity measurement value E acquired by the measurement unit according to the following correction formula, where d is the depth of the sample container, and obtains the corrected chemiluminescence intensity E ₀ .

The chemiluminescence measuring method according to claim 4. The excitation unit repeatedly and repeatedly irradiates the sample liquid contained in the sample container with excitation light that excites the fluorescent reagent contained together with the test substance and the chemiluminescent reagent,
When the excitation unit is not irradiated with excitation light by the measurement unit, the chemiluminescence intensity measurement value obtained by measuring the intensity of the chemiluminescence reached from the sample contained in the sample container, When excitation light is irradiated by the excitation unit, the total intensity of chemiluminescence and fluorescence reached from the sample liquid contained in the sample container is measured to obtain the total light intensity measurement value,
Based on the chemiluminescence intensity measurement value and the total light intensity measurement value acquired by the measurement unit by the calculation unit, fluorescence representing the intensity of the fluorescence that has reached the measurement unit from the sample liquid contained in the sample container Find the strength measurement,
The chemiluminescence measuring method according to claim 4 or 5, characterized in that.

Images