JP6011246B2 - Fluorescence measurement method, fluorescence measurement kit, and fluorimeter - Google Patents

Description

  The invention of the present application relates to a technique for identifying or quantifying a sample by measuring fluorescence intensity, and particularly relates to a technique for identifying or quantifying a sample by fluorescence measurement using an antibody antigen reaction.

As one field of light measurement, a fluorescence measurement technique for measuring fluorescence emitted from a substance is known. Material analysis by fluorescence measurement (fluorescence analysis method) is characterized by high sensitivity and high selectivity compared to absorptiometry and the like, and is effective when performing identification or quantification of a sample.
Sample identification and quantification by fluorescence measurement is limited to the case where the target substance is a fluorescent substance. However, in recent years, fluorescent labeling methods for labeling target substances with reagents (fluorescent reagents) made of fluorescent dyes have been developed, and fluorescent reagents are commercially available for various substances. For this reason, various target substances can be identified and quantified by fluorescence measurement, and their application in many fields such as research and development of new drugs and new materials, process monitoring in plants, and environmental evaluation are being studied. .

  For example, Patent Document 1 is an application field of clinical diagnosis, basic research, environmental research, and the like. An antibody is labeled with a fluorescent dye, and a change in fluorescence intensity due to elimination of quenching is used as an index to measure antigen in a liquid phase. A technique for measuring the concentration and visualizing the antigen is disclosed. Patent Document 2 discloses a technique for quantifying methamphetamine, which is a prohibited drug, by fluorescence measurement using an immune reaction. A technique for measuring the methamphetamine concentration of a sample by introducing a sample (a substance suspected of methamphetamine) into an anti-methamphetamine antibody solution and measuring the change in fluorescence intensity before and after the injection is disclosed in this publication. . In this specification, “fluorescence” is a broader concept and includes phosphorescence.

International Publication WO2011 / 061944 Japanese Patent Laid-Open No. 10-19892

It should be noted in the quantification and identification of samples by such fluorescence measurement that fluorescence may be emitted from other than the target substance. If no fluorescence is emitted from anything other than the target substance, the output of the detector that captures the fluorescence may be the same as the fluorescence intensity of the target substance. However, it is not usual that only the target substance is put into the fluorometer, and it is put into the fluorometer together with other materials such as a buffer solution. In this case, if the other material contains a fluorescent substance, the fluorescence from the target substance and the fluorescence from the other material are captured by the detector, and the fluorescence intensity from the other material is noise. It becomes. Therefore, unless such noise (fluorescence emitted from a substance other than the target substance) is removed from the measurement result, the measurement accuracy is lowered, and identification and quantification are erroneous.
In some cases, fluorescence from other than the target substance can be removed by a filter. In other words, the filter can be removed by arranging a filter that can transmit only light having a fluorescence wavelength from the target substance on the optical path in front of the detector. However, when the wavelength range of fluorescence from the target substance and the wavelength range of fluorescence from other than the target substance overlap, it cannot be sufficiently removed by the filter.

On the other hand, if the measurement work and the operation of the instrument become complicated due to excessive demand for high measurement accuracy, it may be a drag on the application of fluorescence measurement. For example, it is anticipated that the measurement of methamphetamine disclosed in Patent Document 2 will be performed at a prohibited drug enforcement site such as customs. When the officer in charge of such a measurement makes a measurement, if the operation of the fluorometer is complicated, it is easy to make a mistake in the measurement, or the use of the fluorometer is given up because it is too complicated. Is expected to. Therefore, it is necessary to prevent the measurement work and the operation of the fluorometer from becoming complicated.
The invention of the present application has been made in consideration of the above-described circumstances, and complicates the measurement operation and the operation of the photometer in the fluorescence measurement that uses the antibody reaction to identify or quantify the sample. The problem to be solved is to enable identification or quantification with high accuracy.

In order to solve the above-mentioned problem, the invention according to claim 1 of the present application is a fluorescence measurement method for measuring fluorescence of a sample that seems to contain an antigen,
A first measurement step of measuring the intensity of fluorescence from the first test solution by irradiating the first test solution containing an antibody against the antigen with excitation light in a state where a sample is not charged;
After the first measurement step, a second measurement step for measuring the intensity of fluorescence from the liquid phase object by irradiating the liquid phase object obtained by putting the sample into the first test solution with excitation light. When,
A calculation step for performing calculation for identification or quantification of the sample from the measurement results in the first and second measurement steps,
Arithmetic step includes a correlation ratio R which is previously obtained for the first test solution, the value F ₁ of the fluorescence intensity measured by the first measurement step, the measured value of fluorescence intensity in the second measurement step A step of performing an operation according to F ₂ ,
The correlation ratio R is the fluorescence intensity that will be obtained from the unreacted antibody in the first test solution and the fluorescence intensity that will be obtained from components other than the unreacted antibody in the first test solution. The ratio of
Calculation steps, _{Q 1} fluorescence intensity with antibodies before sample loading, when the fluorescence intensity of antibodies after sample loading was _Q ^{1 _*,} and _{F 1 = Q 1 + R ·} Q 1, and _{F 1} on the basis of this In this configuration, Q ₁ ^* / Q ₁ is obtained from the value of F ₂ .
In order to solve the above-mentioned problem, the invention according to claim 2 is the configuration according to claim 1, wherein the second measurement step comprises mixing the sample with a second test solution, and then It is a step of measuring the intensity of the fluorescence by putting it in a test solution, and the second test solution has at least a component that emits fluorescence that cannot be ignored among components other than antibodies in the first test solution. It has the composition of being.
In order to solve the above-mentioned problem, the invention according to claim 3 is the configuration according to claim 1, wherein the second measuring step is performed by mixing the sample with the second test solution and then the first measuring step. A step of measuring the intensity of the fluorescence by putting it in a test solution, wherein the antibody is fluorescently labeled with a fluorescent reagent, and the second test solution is other than the antibody and the fluorescent reagent in the first test solution It has a configuration in which at least components that emit fluorescence that cannot be ignored are common.

In order to solve the above problems, the invention according to claim 4 is a fluorescence measurement kit used in the fluorescence measurement method according to any one of claims 1 to 3, and has a display unit for the correlation ratio R. doing.
Moreover, in order to solve the said subject, invention of Claim 5 is a fluorescence measuring kit used for the fluorescence measuring method of Claim 2, Comprising: Said 1st test solution and said 2nd test solution The sample container has a structure that allows the second test liquid to be mixed with the first test liquid after the sample is put into the second test liquid. Have
The stored second test solution has a configuration in which at least components that emit fluorescence that cannot be ignored among components other than antibodies in the stored first test solution are in common.
Moreover, in order to solve the said subject, invention of Claim 6 is a fluorescence measuring kit used for the fluorescence measuring method of Claim 3, Comprising: Said 1st test solution and said 2nd test solution The sample container has a structure that allows the second test liquid to be mixed with the first test liquid after the sample is put into the second test liquid. Have
The antibody in the first test solution accommodated is fluorescently labeled with a fluorescent reagent,
The stored second test solution has a configuration in which at least a component that emits fluorescence that cannot be ignored among components other than antibodies and fluorescent reagents in the stored first test solution is common. Have.

In order to solve the above problems, the invention according to claim 7 includes a light source that emits excitation light that excites a fluorescent substance,
A detector that detects the intensity of the fluorescence emitted by the fluorescent material;
An optical system that guides excitation light from the light source to the liquid phase object contained in the sample container and guides fluorescence from the liquid phase object to the detector;
An arithmetic processing unit that processes fluorescence intensity data output from the detector,
The arithmetic processing unit applies the first fluorescence intensity measurement value F ₁ that is the result of measuring the fluorescence intensity using the first test solution previously stored in the sample container as a liquid phase object, and the first test solution. The second fluorescence intensity measurement value F ₂ that is the result of fluorescence measurement on the liquid phase object obtained by putting it in the sample, and the correlation ratio R acquired in advance for the first test solution and performing the calculation process And
The first test solution contains antibodies against the antigen that the sample is supposed to contain,
The correlation ratio R is the fluorescence intensity that will be obtained from the unreacted antibody in the first test solution and the fluorescence intensity that will be obtained from components other than the unreacted antibody in the first test solution. The ratio of
The arithmetic processing unit assumes that F ₁ = Q ₁ + R · Q ₁ when the fluorescence intensity due to the antibody before sample introduction is Q ₁ and the fluorescence intensity due to the antibody after sample introduction is Q ₁ ^*, and based on this F ₁ And Q ₂ ^* / Q ₁ from the value of F ₂ .
In order to solve the above-mentioned problem, the invention according to claim 8 is the configuration of claim 7, wherein the sample container is provided as included in a fluorescence measurement kit, and the fluorescence measurement kit Has a display unit displaying the correlation ratio R,
It has a configuration in which a means for reading the correlation ratio R displayed on the display unit is provided.

As described below, according to the invention described in claim 1, 4 or 7 of the present application, since the correlation ratio R is introduced when calculating the fluorescence enhancement ratio Q ₁ ^* / Q ₁ , the measurement is complicated. Sufficient measurement accuracy can be obtained while avoiding this.
Further, according to the invention described in claim 2 or 5, in addition to the above effects, the measurement work is simplified without causing a decrease in accuracy even when components other than the antibody cannot be regarded as zero due to a large amount of fluorescence emission. The effect that it can be obtained.
Further, according to the invention of claim 3 or 6, in addition to the above-described effects, even when components other than the antibody and the fluorescent reagent have a large amount of fluorescent light emission and cannot be regarded as zero, the measurement work can be performed without causing a decrease in accuracy. The effect that it can be simplified is acquired.
Further, according to the invention described in claim 8, in addition to the above-described effect, since the reading means for the correlation ratio R displayed on the display unit is provided, there is no need to read the correlation ratio R and manually input it. The effect of becoming is obtained.

It is the figure which showed the schematic of the fluorescence measuring method of embodiment of this invention. FIG. 2 is a schematic perspective view showing a fluorometer suitably used for the fluorescence measurement method shown in FIG. 1. FIG. 3 is a schematic front sectional view of the fluorometer shown in FIG. 2. It is the schematic of the fluorescence measurement kit used for the fluorometer of embodiment shown in FIG.2 and FIG.3. FIG. 5 is a schematic perspective view of a sample container included in the fluorescence measurement kit of FIG. 4. FIG. 4 is a block diagram of the fluorometer shown in FIGS. 2 and 3. It is the figure which showed schematic structure and the operation principle of the code reader shown in FIG. It is the schematic shown about the example of the format of the liquid information code part shown in FIG. It is the schematic shown about the example of the table. It is the flowchart which showed the outline of the measurement program which implements the fluorescence measuring method shown in FIG.

Next, modes for carrying out the present invention (hereinafter referred to as embodiments) will be described.
FIG. 1 is a diagram showing a schematic diagram of a fluorescence measurement method according to an embodiment of the present invention. The method of the embodiment is a method for identifying or quantifying a sample using an antibody reaction.
In this method, the target substance for identification or quantification is an antigen, and the substance that becomes this antibody is used as a reagent. A reagent means a material used for measurement. The antibody itself is not necessarily a fluorescent substance, but in this embodiment, an antibody labeled with a fluorescent reagent is used. At this time, the molecule of the fluorescent dye bound to the antibody molecule is in a quenched state (quenched), and quenching is eliminated by the reaction with the antigen to generate fluorescence (or fluorescence is enhanced). A fluorescent reagent and an antibody having such properties are used.

  On the other hand, fluorescence measurement is often performed as an object in a liquid phase state from the viewpoint of stably obtaining sufficient intensity of fluorescence with good reproducibility (in this specification, “object” is referred to as an object of fluorescence intensity measurement). Meaning). In a liquid phase state, it is common to use a buffer solution in which an antibody or a fluorescent reagent can be dissolved. Also in this embodiment, the above-described antibody or fluorescent reagent is dissolved in an appropriate buffer and used for measurement. That is, an antibody labeled with a fluorescent reagent dissolved in a buffer is used as a test solution. The test liquid means a liquid phase material used for measurement.

  In the present embodiment, a test solution (hereinafter referred to as a first test solution) 81 in which an antibody labeled with a fluorescent reagent is dissolved is used, and as a first measurement step, first, a sample is not put in. Fluorescence measurement is performed (FIG. 1 (1)). As shown in FIG. 1 (1), the fluorescence measurement is performed by irradiating the first test solution 81 with excitation light from the light source 1 and capturing the fluorescence generated by the excitation with the detector 3. The wavelength of the excitation light is selected according to the characteristics of the fluorescent dye labeled with the antibody. In the commercially available fluorescent reagent, the wavelength of the excitation light is determined, and the wavelength of the light from the light source 1 is selected using a filter or the like accordingly, and the first test solution 81 is irradiated. .

Next, as shown in FIG. 1 (2), the sample S is put into the first test solution 81. At this time, the sample S may be put into the first test solution 81 as it is, but it is necessary to make the sample S as uniform as possible in the first test solution 81, and characteristics such as the ph value can be obtained. The sample S is dissolved in the test solution (hereinafter referred to as second test solution) 82 after being adjusted to a predetermined value, and then is supplied to the first test solution 81.
Then, as shown in FIG. 1 (3), a second measurement step is performed. The second measurement step is a step in which fluorescence measurement is performed using the first test solution 83 in a state in which the sample S is loaded as an object (the first test solution after the sample is loaded is the first sample that is not loaded). Hereinafter, in order to distinguish it from the test solution, it is referred to as a liquid phase object). Similarly, excitation light is irradiated to the liquid phase object 83 from the light source 1, and fluorescence from the liquid phase object 83 is captured by the detector 3.

After performing the first and second measurement steps in this way, the method of the present embodiment performs a calculation process for identifying or quantifying a sample from each measurement result (FIG. 1 (4)). This calculation process as shown in FIG. 1 (4), the fluorescence intensity F ₁ measured at the first measuring step, and a second fluorescence intensity F ₂ as parameters, ultimately according to a predetermined relationship This is a calculation process for obtaining Q ₁ ^* / Q ₁ . Here, Q ₁ is the fluorescence intensity of the antibody in the first test solution 81 before the sample S is introduced. Q ₁ ^* is the fluorescence intensity of the antibody after the sample S is introduced (after the reaction). The antibody content means fluorescence corresponding to the amount of antibody labeled with a fluorescent reagent. When the sample contains an antigen, quenching is eliminated by the reaction with the antibody as described above, so that Q ₁ ^* > Q ₁ is satisfied. For Q ₁ ^* enhancement ratio with respect to Q _1, by setting an appropriate threshold value, it can be determined that contain the antigen in the sample S (i.e., can be identified). Further, the correlation between the fluorescence enhancement rate and the amount of the antigen is examined in advance, calibration data is created, and the antigen in the sample S can be quantified by applying it to Q ₁ ^* / Q ₁ .

The method of this embodiment is characterized by introducing a correlation ratio R, which is a characteristic value of the first test solution 81, when calculating Q ₁ ^* / Q ₁ from F ₁ and F ₂ as described above. Is a point. Hereinafter, this point will be described in detail.
When Q ₁ ^* / Q ₁ is obtained from F ₁ and F ₂ , it cannot simply be assumed that Q ₁ = F ₁ and Q ₁ ^* = F ₂ . This is because there is a problem of “fluorescence from other than the target substance” described above. Specifically, for example, for F ₁ , in the first test solution 81 in which the sample S is not charged, in addition to the fluorescence from the antibody labeled with the fluorescent dye (fluorescence from the target substance), the buffer solution May fluoresce. In addition, a material added to a buffer for a specific purpose may fluoresce.

For example, the ph value needs to be within a predetermined range for the fluorescent dye to function properly, or the ph value needs to be within a predetermined range for an antibody-antigen reaction to occur properly. There is. In such a case, it is necessary to adjust the ph value of the test solution, and a ph adjusting agent is added. In addition, an additive (for example, BSA, bovine serum albumin) that prevents the antibody from adhering to the wall surface of the container may be added. Furthermore, antibodies and fluorescent dyes are often proteins, and thus preservatives are often added. Such various additives may emit fluorescence, and the fluorescence wavelength may overlap with the fluorescence from the antibody or may be close to the wavelength. For this reason, Q ₁ cannot be set to F ₁ .

また、測定された蛍光強度Ｆ_２をＦ_１とともに扱う場合、蛍光強度としての光度は単位体積当たりの発光強度であるため、第一の検査液と試料投入済みの第二の検査液との混合比ｋ（０＜ｋ＜１）を導入する必要がある。第二の測定ステップでの液相対象物における単位体積において、第一の検査液８１がｋの量で存在していれば、試料投入済みの第二の検査液９２は１−ｋの量で存在しているから、測定された蛍光強度Ｆ_２は、以下の式で表される。

式２で、Ｑ_３やＱ_４は、液相対象物８３における「目的物質以外からの蛍光」の強度に相当しており、Ｑ_３は試料が蛍光物質を含んでいる場合（例えば抗原が蛍光物質である場合）の当該蛍光物質からの蛍光強度、Ｑ_４は第二の検査液が蛍光物質を含んでいる場合の当該蛍光物質からの蛍光強度である。 On the other hand, for F ₂ as well, when the buffer solution or additive that is a component of the first test solution 81 is a fluorescent substance, these are included in the value and cannot be directly used as Q ₁ ^* . In the present embodiment, the second test solution 82 is further used, and the components in the second test solution 82 may emit fluorescence.
Here, with respect to the measured fluorescence intensity F ₁ , if the fluorescence intensity of a part emitted from a substance other than the fluorescently labeled antibody (the target substance here) is Q ₂ , F ₁ is expressed by the following formula 1. expressed.

Further, when the measured fluorescence intensity F ₂ is handled together with F ₁ , the luminous intensity as the fluorescence intensity is the emission intensity per unit volume, so that the first test solution and the second test solution that has been loaded with the sample are mixed. It is necessary to introduce the ratio k (0 <k <1). If the first test liquid 81 is present in an amount of k in the unit volume of the liquid phase object in the second measurement step, the sample-added second test liquid 92 is in an amount of 1-k. Since it exists, the measured fluorescence intensity F ₂ is expressed by the following equation.

In Equation 2, Q ₃ and Q ₄ correspond to the intensity of “fluorescence from other than the target substance” in the liquid phase object 83, and Q ₃ is a case where the sample contains a fluorescent substance (for example, the antigen is fluorescent). Q ₄ is the fluorescence intensity from the fluorescent material when the second test solution contains the fluorescent material.

Now, in the above formula 1 and formula 2, the correlation ratio R is introduced in this embodiment. The correlation ratio R, in a first test solution 81 is other than the ratio of the fluorescence intensity Q ₂ of components for the fluorescence intensity to Q ₁ antibody fraction fluorescently labeled. The correlation ratio R is examined in advance when the first test solution 81 is manufactured, and is applied to Formula 1 and Formula 2 as a constant.
As a method for obtaining the correlation ratio R, the fluorescence intensity of the first test solution 81 is first measured for materials other than the fluorescent reagent and the antibody. For example, the fluorescence intensity is obtained without adding a fluorescent reagent and an antibody by adding them to predetermined additives such as a ph adjusting agent, a vessel wall adhesion preventing agent, and an antiseptic and a buffer solution. Next, after adding a predetermined amount of the fluorescent reagent and the antibody, the fluorescence intensity is measured again, and the correlation ratio R can be obtained from these results. If a material other than the fluorescent reagent and the antibody is specified as a fluorescent material, the fluorescence intensity of only that material may be measured to obtain the correlation value R. For example, if only the buffer solution is a fluorescent substance, the fluorescence intensity of only the buffer solution is measured without an additive, and the correlation value R may be obtained from the result.

式４において、ｋを０．７〜０．８程度の大きな値とすると、第２項（１−ｋ）（Ｑ_３＋Ｑ_４）は、ゼロと見なし得る場合がある。抗体が蛍光物質でない場合やあっても少ない場合、（１−ｋ）Ｑ_３はゼロであるとみなして良い。特に、試料Ｓについては第二の検査液８２に対して微量投入すれば良い場合が多いから、試料Ｓからの蛍光は無視できる場合が多い。また、Ｑ_４についても、第二の検査液８２が蛍光物質でなかったり、蛍光の発光が少ないものであったりする場合、ゼロとみなし得る。各々ゼロとみなし得るとすると、式４は、以下の式５となる。

When the correlation ratio R is introduced, Equations 1 and 2 become Equations 3 and 4 below.

In Equation 4, when k is a large value of about 0.7 to 0.8, the second term (1-k) (Q ₃ + Q ₄ ) may be regarded as zero. If the antibody is not a fluorescent substance or if there is little, (1-k) Q ₃ may be considered to be zero. In particular, since the sample S may be added in a small amount to the second test solution 82, the fluorescence from the sample S is often negligible. Q ₄ can also be regarded as zero when the second test liquid 82 is not a fluorescent material or has little fluorescence emission. Assuming that each can be regarded as zero, Expression 4 becomes Expression 5 below.

即ち、第一の測定ステップでの測定結果Ｆ_１と、第二の測定ステップでの測定結果Ｆ_２と、各常数ｋ及びＲから、式６に従い、目的とする蛍光増強比Ｑ_１ ^＊／Ｑ_１が求められることになる。 Therefore, Q ₁ ^* / Q ₁ can be derived from Equation 3 and Equation 5 as follows.

That is, from the measurement result F _{1 in the first} measurement step, the measurement result F ₂ in the second measurement step, and the constants k and R, the target fluorescence enhancement ratio Q ₁ ^* / Q according to Equation 6 ₁ will be required.

よって、式３及び式７から、以下のようにＱ_１ ^＊／Ｑ_１を導出することができる。

即ち、第一の測定ステップでの測定結果Ｆ_１と、第二の測定ステップでの測定結果Ｆ_２と、各定数ｋ及びＲから、式８に従い、目的とする蛍光増強比Ｑ_１ ^＊／Ｑ_１が求められることになる。尚、式８の場合、式６と比べると、右辺第二項全体をゼロとみなす必要がないので、その分で測定精度が高くなる。 Even if the entire second term on the right-hand side of Equation 4 cannot be regarded as zero, it is possible to derive Q ₁ ^* / Q ₁ with high accuracy by using the second term in common with the component of the test liquid 82. For example, a component obtained by removing the antibody and the fluorescent reagent from the first test solution 81 is used as the second test solution 82. Not all may be shared, but only the fluorescent component may be shared. For example, in the first and second test solutions 81 and 82, if only the device wall adhesion preventing agent (for example, BSA) is a fluorescent substance other than the antibody and the fluorescence reagent, the same device wall adhesion preventing agent is used in common. Like that. In this way, the fluorescence intensity Q ₄ corresponding to the second test solution 82 coincides with Q ₂ in the first test solution, which is R · Q ₁ . Therefore, Expression 4 becomes Expression 7 below.

Therefore, Q ₁ ^* / Q ₁ can be derived from Equation 3 and Equation 7 as follows.

That is, from the measurement result F _{1 in the first} measurement step, the measurement result F ₂ in the second measurement step, and the constants k and R, the target fluorescence enhancement ratio Q ₁ ^* / Q according to Equation 8 ₁ will be required. In the case of Expression 8, compared to Expression 6, it is not necessary to consider the entire second term on the right side as zero, so that the measurement accuracy increases accordingly.

Next, a fluorometer that is preferably used in the fluorescence measurement method of the present embodiment will be described. The following description is also a description of an embodiment of the fluorometer invention.
2 is a diagram showing a fluorometer suitably used in the fluorescence measuring method shown in FIG. 1, FIG. 2 is a schematic perspective view, and FIG. 3 is a schematic front sectional view.
The fluorometer shown in FIGS. 2 and 3 is a portable type in consideration of fluorescence measurement at various places as described above. As shown in FIG. 2, the fluorometer of the present embodiment is a flat, substantially rectangular parallelepiped box shape as a whole. Since it is portable, its size is about the size of a person's palm or slightly larger.
As shown in FIG. 3, the fluorometer includes a light source 1, an optical system 2, a detector 3, a container mounting portion 4, and the like. The light source 1, the optical system 2, the detector 3, etc. are housed in a flat, substantially rectangular parallelepiped casing 5 as shown in FIG.

This fluorometer is designed to measure fluorescence by putting a sample in a dedicated container. Hereinafter, this container is referred to as a sample container. Each test solution is stored in the sample container in advance, and is provided to the measurer as a fluorescence measurement kit.
4 is a schematic view of a fluorescence measurement kit used in the fluorometer of the embodiment shown in FIGS. 2 and 3, and FIG. 5 is a schematic perspective view of a sample container included in the fluorescence measurement kit of FIG.
In the fluorescence measurement kit, a sample container 91 is enclosed in an individual bag 90 so as not to be soiled or mixed with foreign substances. The sample container 91 is a vertically long and narrow container as shown in FIG. The inside of the individual bag 90 may be degassed under reduced pressure or filled with nitrogen to prevent deterioration of the kit.

The sample container 91 includes a main body part 911, a first cell part 912 provided below the main body part 911, a second cell part 913 provided in the main body part 911, and the like. In the first cell portion 912, the first test solution 81 is stored in advance. In the second cell portion 913, the second test solution 82 is stored in advance.
The second cell part 913 is partitioned by a partition wall 914 with respect to the lower space including the first cell part 912. The partition wall 914 can be broken by a jig (not shown), and the partition wall 914 is broken when the second test solution 82 is mixed with the first test solution 81. A container lid 95 is provided at the upper end of the sample container 91. When the sample is charged, the container lid 95 is opened, and the sample is first charged into the second test solution 82 in the second cell portion 913 and mixed. Thereafter, the partition wall 914 is broken and the sample is put into the first inspection liquid 81 together with the second inspection liquid 82.

Such a sample container 91 (particularly the first cell portion 912) is formed of a material that sufficiently transmits excitation light and fluorescence. Specifically, a glass container such as borosilicate glass, quartz, or sapphire, or a resin such as PMMA (acrylic resin), polystyrene, or COC (cyclic olefin copolymer) is used as the sample container 91. Note that when a lot of fluorescence is emitted from the sample container 91 itself when the excitation light is irradiated, it is difficult to distinguish the fluorescence from the liquid phase object. Those with less (fluorescence emitted by themselves) are selected.
In addition, from the viewpoint of preventing measurement accuracy from being deteriorated due to contamination, the sample container 91 is preferably disposable (used for one-time measurement). From this viewpoint, the material of the sample container 91 is preferably a resin such as PMMA in terms of cost.

On the other hand, as shown in FIGS. 2 and 3, the casing 5 has a part of the upper surface portion serving as an opening / closing lid 51. When the opening / closing lid 51 is opened, the insertion hole 40 is exposed as shown in FIG. The insertion hole 40 is an upper end opening of the container mounting portion 4. The container mounting portion 4 is disposed in the casing 5 so as to extend downward from the opening / closing portion of the opening / closing lid 51.
As shown in FIG. 2, the insertion hole 40 is adapted to the cross-sectional shape of the main body portion 911 of the sample container 91, and includes a circumferential portion and a linear portion (hereinafter, linear portion) 401. Consists of.
When the sample container 91 is attached to the fluorometer, the opening / closing lid 51 is opened and the sample container 91 is inserted into the insertion hole 40 as shown in FIG. At this time, the flat surface portion 92 on the side surface of the main body portion 911 is brought into a state of being aligned with the linear portion 401 of the insertion hole 40. In this state, the sample container 91 is pushed down as it is and mounted on the container mounting portion 4. Thereafter, the opening / closing lid 51 is closed.
In this fluorometer, the sample can be loaded with the sample container 91 mounted on the container mounting portion. That is, since the container lid 95 is opened with the sample container 91 mounted on the container mounting portion 4, the sample is put in this state, the partition wall 914 is broken with a jig (not shown), and then the container lid 95 and the open / close state are opened. The lid 51 may be closed.

The light source 1 emits light (excitation light) that can excite a fluorescent substance in a liquid phase object to emit fluorescence. In the present embodiment, an LED lamp is used as the light source 1. Any lamp can be used as long as it emits light including excitation light. However, in this embodiment, an LED lamp is used in consideration of cost advantage and power saving. For example, LEDs emitting green light having a wavelength of 525 nm are commercially available from various companies, and those having a lens and an output of about 2 mW can be suitably employed.
The optical system 2 guides excitation light from the light source 1 into the first cell portion 912 of the sample container 91 and guides fluorescence from the liquid phase object in the first cell portion 912 to the detector 3. . In the present embodiment, the optical system 2 includes a condenser lens 21 that collects light from the light source 1, a dichroic mirror 22 for bending the optical path and selecting light, a filter 23 disposed on the optical path, 24 etc.

As shown in FIG. 3, the dichroic mirror 22 is disposed at a position substantially the same height as the first cell portion 912 of the sample container 91 mounted on the container mounting portion 4. The dichroic mirror 22 is disposed at an oblique angle of 45 °, and the light source 1 is disposed above the dichroic mirror 22. The light source 1 is configured to emit light downward. The dichroic mirror 22 reflects light having a wavelength of excitation light and transmits light having a wavelength of fluorescence to be measured.
Further, the detector 3 is disposed at a position opposite to the container mounting portion 4 with the dichroic mirror 22 in between. The first cell portion 912, the dichroic mirror 22 and the detector 3 of the sample container 91 mounted on the container mounting portion 4 are located at the same height, and have a horizontal optical axis (detection optical axis). Is placed on top. On the other hand, the optical axis (excitation optical axis) extending downward from the light source 1 is bent vertically by the dichroic mirror 22 and reaches the first cell portion 912. The container mounting portion 4 has an opening so as not to block excitation light and fluorescence.

As the filter, an excitation light filter 23 and a fluorescence filter 24 are arranged. The excitation light filter 23 selectively transmits light having a wavelength serving as excitation light, and is disposed on the optical path between the light source 1 and the dichroic mirror 22. For example, as described above, when 525 nm green light is used as excitation light, the light that transmits light in the wavelength range of about 510 to 545 nm and reflects light in other wavelength ranges is used as the excitation light filter 23. Is done.
The fluorescence filter 24 selectively transmits light having a fluorescence wavelength to be measured, and is disposed between the dichroic mirror 22 and the detector 3. For example, when the wavelength of fluorescence is 550 to 630 nm, a filter that transmits light in the wavelength range of about 570 to 610 nm and reflects light in other wavelength ranges is used as the filter 24 for fluorescence.

  Since the filters 23 and 24 are used for excitation light and fluorescence as described above, a half mirror having no wavelength selectivity may be used instead of the dichroic mirror 22. However, in the case of a half mirror, the amount of light is halved, so the dichroic mirror 22 is more advantageous. When the transmission wavelength range of the excitation light filter 23 and the fluorescence filter 24 is the above-described example, the dichroic mirror 22 transmits light having a wavelength range of, for example, 570 nm or more and reflects light having a wavelength range of 545 nm or less. Those having characteristics (in the case of 45 ° incidence) can be used.

The condensing lens 21 is used to irradiate the liquid phase object in the first cell unit 912 with a thin beam of light from the light source 1. The LED lamp as the light source 1 is preferably used with a small beam divergence angle. However, it is still wide enough to irradiate the small first cell portion 912. I try to irradiate. The numerical aperture NA of the condenser lens 21 does not need to be so large, and may be about 0.5.
The condensing position by the condensing lens 21 (the position where the beam becomes the thinnest) is the center of the first cell portion 912. The beam diameter is about 0.5 to 1.5 mm at the narrowest position. The condensing lens 21 is also disposed for the purpose of collecting the fluorescence emitted from the fluorescent material in the liquid phase object and causing it to enter the detector 3.

The detector 3 is appropriately selected from those using photodiodes or phototubes. In this embodiment, the one using a silicon photodiode is employed.
As shown in FIG. 2, a display 52 that displays the operation state of the photometer and measurement results and several operation buttons 531 to 536 are provided on the front surface of the casing 5. One of the operation buttons 531 to 536 is a measurement button (a button for operating the light source 1 to acquire the output of the detector 3). As the display 52, a liquid crystal display can be employed, and a touch panel can be employed. In addition, a power switch (not shown) is provided on the side surface of the casing 5.

Next, the signal processing system of the fluorometer shown in FIGS. 2 and 3 will be described. FIG. 6 is a block diagram of the fluorometer shown in FIGS. 2 and 3.
As shown in FIG. 3, a control box 60 is provided in the casing 5. In the control box 60, there is provided a control unit 6 that controls each part and performs signal processing. As shown in FIG. 6, the controller 6 includes a processor 61 that performs arithmetic processing, a memory 62 that stores data and programs, and the like.
The detector 3 is a photoelectric conversion unit (silicon photodiode in this example) 31 that receives fluorescence, an amplifier 32 that amplifies the output signal of the photoelectric conversion unit 31, and a fluorescence intensity signal that is output based on the amplified signal. Output circuit 33. The output circuit 33 includes a calibration circuit for displaying the light intensity (fluorescence intensity) as an absolute value as necessary.

In addition to the output from the detector 3, operation signals from the operation buttons 531 to 536 and signals from the power switch are input to the control unit 6. Further, a display 52 is connected to the control unit 6 via an interface (not shown).
As shown in FIG. 2, a battery case 8 is provided in the casing 5. The battery case 8 is equipped with a battery that supplies necessary voltages to the light source 1, the detector 3, the control unit 6, and the like.
Although not shown in FIG. 3, the fluorometer includes a temperature sensor 64. As shown in FIG. 6, the temperature sensor 64 is connected to the control unit 6, and constantly transmits a signal of the ambient temperature in the casing 5 to the control unit 6.

Such a fluorometer obtains information on the test solutions 81 and 82 previously stored in the sample container 91, and optimizes the measurement using this information. This information includes the correlation ratio R described above.
First, as shown in FIG. 5, the sample container 91 included in the fluorescence measurement kit is provided with a liquid information code section 93 that encodes information on the stored test liquids 81 and 82. In the present embodiment, the liquid information code portion 93 is a barcode as shown in FIG. The liquid information code portion 93 is provided on the flat surface portion 92 on the side surface of the sample container 91. For example, the liquid information code part 93 can be obtained by printing a barcode on a sticker and pasting it on the flat surface part 92.

On the other hand, the fluorimeter includes a code reader 7 that reads such a liquid information code section 93. Since the liquid information code part 93 is a bar code, the code reader 7 is a bar code reader. As shown in FIG. 3, the code reader 7 is attached to the container mounting portion 4.
The container mounting part 4 has a reading opening 41 at a midway height. A reader mounting portion 42 is formed so as to protrude obliquely upward from the edge of the reading opening 41. The reader attachment part 42 is a short cylindrical part. The code reader 7 is fitted and held in the reader attachment portion 42, and can be irradiated with light or received through the reading opening 41.

As shown in FIG. 3, the reading opening 41 and the reader mounting portion 42 are formed on the side surface on the center side. “Center side” means the center side of the fluorometer. Therefore, the code reader 7 also reads from the center side.
FIG. 7 is a diagram showing a schematic configuration of the code reader 7 shown in FIG. 3 and its operating principle. (1) in FIG. 7 shows a state where the code reader 7 is reading the liquid information code portion 93, and (2) shows a state where the code reader 7 is operating as a sensor for detecting the mounting of the sample container 91. As shown in FIG. 7, the code reader 7 includes a light emitter 71 that emits light to the liquid information code unit 93, a light receiver 72 that receives light from the liquid information code unit 93, and an output signal from the light receiver 72. And a binarizing element 73 for obtaining a digital signal.

  As described above, the sample container 91 is inserted from the insertion hole 40 of the casing 5. At this time, since the linear portion 401 of the insertion hole 40 is aligned with the flat surface portion 92, the flat surface portion 92 faces the center side during insertion. When the sample container 91 is pushed down in this state, as shown in FIG. 2 and FIG. 3, the liquid information code portion 93 passes in front of the reading opening 41. 93 is read.

  More specifically, in the present embodiment, the position of the code reader 7 is fixed, and the liquid information code section 93 moves relative to this position. The light emitter 71 irradiates a certain area with light, and the liquid information code part 93 passes through this light irradiation area. Therefore, the liquid information code | cord | chord part 93 reaches a light irradiation area | region, and light irradiation is performed for the lower end part, and an upper part is light-irradiated sequentially with a movement. Then, when the movement is completed (when the attachment is completed), the light irradiation has been completed up to the upper end portion. That is, the light irradiation region is relatively scanned (scanned) by the movement of the sample container 91.

As shown in FIG. 7A, during scanning, light from the liquid information code portion 93 irradiated with light enters the light receiver 72, and the binarization element 73 processes the output. The light that enters the light receiver 72 reflects the brightness of the barcode, and the binarization element 73 outputs a digital signal accordingly. As shown in FIG. 3, the code reader 7 of this embodiment irradiates light from an oblique direction, but light is diffused or scattered on the surface of the liquid information code portion 93 and the light is captured by the light receiver 72. It will be. In many cases, it is recommended that the light receiving device 72 does not receive strong reflected light from the light emitting device 71. In this embodiment, the code reader 7 is attached obliquely in consideration of this.
As such a code reader 7, for example, RPU813T manufactured by Okaya Electric Industry Co., Ltd. can be used. Moreover, what used the laser for the light-emitting device 71 may be used, for example, H0A6480 etc. of Honeywell company can be used.

  In the present embodiment, the code reader 7 also serves as a container sensor that detects completion of the mounting of the sample container 91. That is, as shown in FIG. 7, the liquid information code portion 93 has a white relatively large rectangular portion (hereinafter, rectangular portion) 94 following the portion where the inspection liquid information is converted into a barcode. The rectangular portion 92 is provided as a portion that is read by the code reader 7 in order to detect the completion of the mounting of the sample container 91 (hereinafter, a mounting detection identifying portion). That is, the rectangular portion 94 is a portion having a high reflectance, and when the sample container is correctly positioned at the mounting position, a position where the code reader 7 emits light and receives the reflected light as shown in FIG. It has become. The attachment detection identification unit may be a black pattern or a pattern other than a rectangle.

  As shown in FIG. 6, the control unit 6 includes an attachment confirmation unit 63 that holds information indicating that the attachment state of the sample container 91 is confirmed. A signal output when the code reader 7 reads the rectangular portion 94 is sent to the attachment confirmation portion 63. When the information is stored in software, the mounting confirmation unit 63 is a specific storage area of the memory, but may be stored in hardware, for example, a holding circuit, a photocoupler, or the like. May hold that the mounting confirmation is on.

Next, specific fluorescence measurement using such a fluorometer will be described. The following description is also a more specific description of the embodiment of the fluorescence measurement method shown in FIG.
FIG. 8 is a schematic diagram showing an example of the format of the liquid information code portion shown in FIG. As shown in FIG. 8, in this example, a 10-digit number is barcoded. Among these, the first one digit is the test solution specifying information, the next six digits are the expiration date information, and the next three digits are the R value.

  In the present embodiment, since the first and second two test solutions are used, the test solution specifying information specifies a combination of the two test solutions. The optimal test liquid is selected as the first and second test liquids according to the target substance to be identified and quantified, and the ID number indicating the combination is coded as the test liquid specifying information. If this fluorometer is capable of identifying or quantifying, for example, about four different target substances, an optimal combination of test liquids is set for each target substance, and 0 to 3 tests are performed for each test liquid combination. Liquid ID is given.

  Regarding the expiration date information, the first two digits are abbreviations of the Western calendar (13 in 2013), the next two digits are the month, and the next two digits are the day. If it is 130420, it means that April 20, 2013 is the expiration date. When the expiration dates are different for the first and second test solutions, the shorter expiration date is selected and coded as expiration date information.

  As described above, the R value means the correlation ratio R in the first test solution. In the present embodiment, a three-digit number is assigned as the R value. In this example, the first number of the R value means the one's place and the last two numbers mean the decimal point. That is, as shown in FIG. 8, if “124” is an R value, it means that the correlation ratio R is 1.24.

The test solution information coded in such a format is read by the code reader 7 as described above and sent to the control unit 6. Then, it is used for a measurement program executed by the control unit 6. At this time, it is difficult to code the test solution information in the form of data that can be directly handled, and the read data is converted into another data for use. The memory 62 stores a table (correspondence table) for performing such conversion. FIG. 9 is a schematic diagram showing an example of this table.
As shown in FIG. 9, in the table, a target substance name, a calculation formula ID, reference data, and the like are associated with each detection liquid ID. The target substance name is the name of the target substance that can be identified or quantified by the test liquid combination specified by the test liquid ID. The calculation formula ID specifies the formula for calculating the above-described fluorescence enhancement ratio Q ₁ ^* / Q ₁ . In the present embodiment, calculation is performed according to the above-described Expression 6 or Expression 8. The calculation formula ID “1” means Equation 6 and “2” means Equation 8.

The reference data is data that is referred to when identification or quantification is performed from the calculated fluorescence enhancement ratio Q ₁ ^* / Q ₁ . In the case of identification, it is determined whether or not the sample contains the target substance depending on whether a certain fluorescence enhancement ratio Q ₁ ^* / Q ₁ exceeds a certain threshold value. Therefore, the threshold value is referred to as reference data. For quantification, or a correspondence table of the amount of the target substance corresponding to the fluorescence enhancement ratio Q _{1 *} ^/ Q _1, or a calculation formula for calculating the amount of the target substance from the fluorescence enhancement ratio Q _{1 *} ^/ Q ₁ .

Next, a measurement program using the read test solution information and the table shown in FIG. 8 will be described. FIG. 10 is a flowchart showing an outline of a measurement program for implementing the fluorescence measurement method shown in FIG.
When the power switch of the fluorometer is turned on, the main program is automatically started and each operation menu is displayed on the display 52. When the measurement menu is selected here, the measurement program shown in FIG. 10 is started.

The measurement program first confirms the mounting of the sample container 91. Normally, since the sample container 91 is not attached, the output signal of the attachment confirmation unit 63 is OFF, and a signal indicating that no container is present is input to the control unit 6. After confirming the signal indicating no container, the main program displays on the display 52 an initial screen that prompts the user to attach the sample container 91. This is a screen displaying a message such as “Please attach the sample container”.
As an exception, a signal indicating that there is a container may be sent from the code reader 7. This is a case where the sample container 91 is already mounted and the code reader 7 is reading the rectangular portion 94. Whether the sample container 91 used in the previous measurement remains attached or whether the power switch is turned on after the sample container 91 is attached. In any case, since the test solution information necessary for the measurement is not acquired, the measurement program displays a screen for remounting the sample container 91. The screen is such as “The sample container is still attached. Please reattach the sample container.”

  When the sample container 91 is inserted from the insertion hole 40 and is correctly attached to the container attachment part 4, the liquid information code part 93 is read by the code reader 7 using the movement at that time as described above, and the inspection liquid information Is sent to the control unit 6. The control unit 6 stores the test solution information in a predetermined area of the memory 62.

The measurement program refers to the memory 62 and confirms whether the test solution information has been acquired. If not acquired, the display state of the above-described screen (screen for prompting mounting of the sample container) is maintained. Further, the measurement program refers to the output of the mounting confirmation unit 63 and checks whether the sample container 91 is correctly mounted. If it is not confirmed, the display state of the screen is similarly maintained.
When the test liquid information can be acquired and the mounting of the sample container 91 is confirmed, as shown in FIG. 9, the measurement program processes the test liquid information, and in accordance with the format shown in FIG. 8, the test liquid ID, expiration date information, R Get each value.

  Next, the measurement program calls the table shown in FIG. 8 from the memory 62, and acquires the target substance name for the detected liquid combination according to the detected liquid ID. The target substance name is displayed on the display 52 for confirmation. For example, a display such as “Check that the sample container is installed. The target substance is XX” is displayed. At this time, an OK button and a cancel button are displayed on the display 52. When the OK button is pressed, the program is continued as it is, and when the cancel button is pressed, the program is stopped. The cancel button is for when you accidentally use a fluorescence measurement kit for a different target substance.

  Next, the measurement program calls the system time (the real-time clock held by the processor as internal information), compares it with the expiration date information, and determines whether the expiration date has passed. If the expiration date has passed, error processing is performed. That is, although not shown in FIG. 9, an error message to that effect is displayed on the display 52. At this time, a screen asking whether to continue the measurement is displayed and either the continue button or the cancel button is pressed. If the continue button is pressed on this screen, the measurement program proceeds to the next step. If the cancel button is pressed, the measurement program is terminated without performing measurement.

After the expiration date check, the measurement program displays on the display 52 a screen for performing the first measurement step. That is, the measurement program displays on the display 52 a message such as “Please press the measurement button without first loading the sample”. When the light source 1 is operated by pressing the measurement button and the detector 3 captures the fluorescence and outputs the fluorescence intensity, the measurement program stores the value in a memory variable. This value is F ₁ in Equation 6 or Equation 8 described above.

When this is completed, the measurement program reads “Remove the sample container and load the sample. When loading the sample, shake the container lightly, then break the partition wall and use the first test solution for the second test. The message “Please mix with the liquid. Then, reload the container and press the measurement button.” Is displayed. Next, when the measurement button is pressed and the fluorescence intensity is similarly output from the detector 3, the measurement program stores the value in another memory variable. This value is F ₂ in Equation 6 and Equation 8 described above.

Next, the measurement program refers to the obtained calculation formula ID, and calculates the fluorescence enhancement ratio Q ₁ ^* / Q _{1 using} either formula 6 or formula 8 according to the calculation formula ID. Next, referring to the reference data, the final measurement result is obtained and displayed on the display 52. The example shown in FIG. 9 is a measurement program for identifying, and it is determined whether reference data is applied and the threshold value is exceeded. And when it exceeds, a display such as “judgment result: Yes (sample contains target substance XX)” is displayed. If not, a message such as “Judgment result: No (sample does not contain target substance XX)” is displayed.

  In the case of quantification, the reference data is applied to calculate the concentration of the target substance in the liquid phase object. Then, the content of the target substance in the sample is determined according to the mixing ratio k and the amount of the sample put into the second test solution. In addition, the amount of the sample put into the second test solution is defined as a common amount, for example, one cup of prescribed medicine. Further, the mixing ratio k is also determined in advance as a constant, and quantitative determination is performed while substituting these numerical values.

Although the measurement program is not shown in FIG. 10 at the time of the measurement, monitoring of the output of the attachment confirmation unit 63 is started after the first measurement button is pressed. If it is confirmed that the output of the attachment confirmation unit 63 is turned off before the second measurement button is pressed, the measurement program displays an error message. This error message is a message such as “The sample container has been taken out before the measurement in the sample loading state. Please repeat the measurement from the state in which the sample has not been loaded.” When this error message is displayed, the measurement program returns to the state immediately after the measurement program is started, and a screen for prompting the mounting of the sample container 91 without the sample is displayed on the display 52 again.
If the output of the attachment confirmation unit 63 is not turned off until the second measurement button is pressed, the measurement program selects the calculation formula as described above, and performs identification or quantification by applying the reference data.

  Although not shown in FIG. 8, information on the usable temperature range is registered in the table for each test liquid combination specified by the test liquid ID. When the measurement program shown in FIG. 9 processes the test liquid information and acquires the test liquid ID, the measurement program refers to the table and acquires information on the usable temperature range for the test liquid ID. Then, with reference to the temperature data sent from the temperature sensor 64, it is determined whether or not the ambient temperature is within the usable range. If it is within the usable range, the measurement program is continued. If it is not, an error operation for displaying a continue button and a cancel button is performed. If the continue button is pressed here, the measurement program is continued, and if the cancel button is pressed, the measurement program is canceled.

  Such a fluorescence measurement method will be described using a more specific example. As a more specific example, it is assumed that fluorescence measurement is performed for the control of prohibited drugs as described above. For example, it is assumed that the above-described fluorometer can detect four of cocaine, heroin, stimulant and cannabis. Therefore, it is planned that any one of cocaine, heroin, stimulant, and cannabis is used as the test solution.

  For example, suppose that white powder, which seems to be a prohibited drug, is attached to a package during customs inspection. The clerk decides that an inspection is necessary and temporarily collects the baggage before collecting the powder. First, a fluorescence measurement kit for cocaine is used and fluorescence measurement is performed without introducing a sample, and then a part of the powder is introduced into the sample container 91 to perform fluorescence measurement again. If it is determined as a result of the measurement that it is not cocaine, it is displayed on the display 52. Next, measurement is performed in the same manner using a fluorescence measurement kit for heroin. And when the result that it is not heroin is displayed similarly, the fluorescence measurement kit for stimulants is used next and it measures similarly. If the result is that it is not a stimulant, then further measurement is performed using a cannabis fluorescence measurement kit. In this way, it is possible to identify whether or not a sample is a specific prohibited drug by sequentially using dedicated fluorescence measurement kits.

  Besides the control of prohibited drugs at customs, the fluorometer of this embodiment can be used. For example, when a prohibited drug is detected at a crime investigation site, a chemical substance left at the crime scene is identified and used as evidence. In addition to these, the fluorometer of the present embodiment can also be used in a doping test performed in, for example, a sports competition. In this case, a small amount of urine from the subject may be collected and used as a sample.

As a more specific example in terms of materials, for example, for methamphetamine known as a typical stimulant, a technique for producing a monoclonal antibody as an anti-methamphetamine antibody by culturing a cell line obtained by immunizing an animal Are disclosed (Japanese Patent Laid-Open Nos. 1-96198, 5-7497, 6-261784, etc.). Further, as fluorescent dyes for methamphetamine, those composed of a pentamethine cyanine derivative (JP-A-6-66725) and those composed of a merocyanine derivative (JP-A-8-92211) are known.
Therefore, as a test solution for methamphetamine, an appropriately selected anti-methamphetamine antibody is labeled by binding an appropriately selected fluorescent labeling dye, and this is used as a PBS solution (phosphate physiological saline). What was dissolved in water) can be used.

According to the fluorescence measurement method of the embodiment and the fluorometer of the embodiment, since the correlation ratio R is introduced when calculating the fluorescence enhancement ratio Q ₁ ^* / Q ₁ , avoiding complication of measurement. Sufficient measurement accuracy can be obtained. When the correlation ratio R is not introduced, there is no means for grasping in advance the intensity of “fluorescence from a substance other than the target substance” (here, fluorescence from a substance other than the fluorescently labeled antibody). If this is the case, there is no choice but to measure them individually. Specifically, the fluorescence reagent and the antibody are obtained by first measuring the test solution in a state where the fluorescent reagent and the antibody are not added (a buffer solution in which an additive such as a ph adjusting agent is added) to obtain the fluorescence intensity. And measure again as the state of the first test solution to obtain the fluorescence intensity. Then, after the sample is added to the first test solution, measurement is performed again, and then the values are substituted into the calculation formula to obtain the fluorescence enhancement ratio Q ₁ ^* / Q ₁ . The measurement will be repeated three times. On the other hand, according to the method of the embodiment, two measurements are sufficient as described above.

In the present embodiment, since the mixing ratio 1-k of the second test solution is set to a value of about 0.2 to 0.4, a fluorescent material is contained in the sample or the second test solution. However, it is considerably smaller than the fluorescence intensity from the quenching-dissolved antibody as the target substance and can be ignored (can be regarded as zero) in many cases. For this reason, even when the fluorescence enhancement ratio Q ₁ ^* / Q ₁ is obtained by Equation 6, a decrease in accuracy is less likely to be a problem. Although it is more effective to make the mixing ratio 1-k smaller than 0.2, as a result of the sample amount being too small, a sufficient antibody reaction does not occur even if an antigen is present, and There is a possibility that the chin-removal action does not occur sufficiently. Therefore, the mixing ratio 1-k is preferably 0.2 or more. Further, when the mixing ratio 1-k is 0.4 or more, there are many cases where it cannot be ignored when the sample or the second test solution contains a fluorescent substance, which may cause a problem.

  Further, as described above, if at least the component that is a fluorescent substance is matched between the component obtained by removing the fluorescent reagent and the antigen from the first test solution and the component of the second test solution, the formula 8 can be satisfied. it can. For this reason, even if the components other than the antibody and the fluorescent reagent have a large amount of fluorescent light emission and cannot be regarded as zero, the effect of simplifying the measurement work can be obtained without causing a decrease in accuracy.

In most fluorescent materials, the amount of fluorescence generated increases as the temperature increases. However, the manner of increase is constant for many fluorescent materials, and therefore the correlation ratio R is often almost constant regardless of temperature. As long as the correlation ratio R is constant regardless of the temperature, the result does not differ regardless of the temperature within the operating temperature range of each test solution.
If the correlation ratio R varies greatly depending on the temperature within the operating temperature range, the rate of change may be measured in advance and stored in the memory 62 as one piece of reference data. The correlation value R acquired from the liquid information code unit 93 is corrected according to the temperature data sent from the temperature sensor 63 and the change rate data read from the memory 62, and the corrected correlation value R is applied to obtain the fluorescence enhancement ratio Q _1. ^* / _Q may be performed to calculate the _1.

  The simplification of the measurement work as described above is not performed by a specialized engineer in the laboratory, but when a person other than the engineer performs a simple measurement outside the laboratory, such as a prohibited drug enforcement site. Of particular significance. Making a non-technical person who does not have specialized knowledge perform complicated work is likely to cause measurement errors, and if the measurement work is complicated and time consuming, it is not suitable for the purpose of controlling prohibited drugs. turn into. Since the method of the present embodiment ensures sufficient accuracy without incurring complications, it is suitable for a case where a non-technical person simply measures outside the laboratory, etc. This method is very suitable for the field where simplicity is required.

  Further, according to the fluorometer of the embodiment, the information encoded by the liquid information code unit 93 provided in the sample container 91 includes the correlation ratio R, and is read and substituted into the calculation formula. This is also a simple method. For example, only the name of the first test solution 81 may be displayed on the sample container 91. In this case, a paper on which a correlation table of correlation ratios R for the first test solution 81 is printed is included in the fluorescence measurement kit, and the measurer uses the correlation ratio R as a fluorometer while looking at the paper during measurement. A configuration where manual input is also possible. However, such an operation is very troublesome, making measurement complicated and prone to errors. Further, when there is a variation in the correlation ratio depending on the production lot of the test solution, it is almost impossible to perform the measurement while compensating for it.

The point that the fluorometer of the embodiment includes the code reader 7 also makes the measurement easier. When the liquid information code unit 93 is a bar code as described above, it can be implemented even if the fluorometer does not include a code reader and is connected to a general-purpose bar code reader. However, it is necessary to prepare a fluorometer and a barcode reader at the measurement site, and it is necessary to connect the barcode to the fluorometer and acquire data for each measurement. The fluorometer in the embodiment is free from such complications.
The correlation ratio R may be displayed on the individual bag 90 shown in FIG. 4 in addition to the case displayed on the sample container 91. When displaying on the sample container 91, the correlation ratio R may be displayed as a numerical value as it is, or may be displayed with another information code such as a QR code (registered trademark of DENSO WAVE INCORPORATED). The fluorometer may be provided with means for reading

  In addition, the method and the fluorometer of the embodiment have another meaningful configuration in that measurement is performed by a person who is not an expert. That is, as described above, the measurement program monitors that the sample container 91 remains attached after the first measurement step is completed, and if the output of the attachment confirmation unit 63 is turned off by any chance, Since the sample container 91 is taken out, the first measurement step is performed again. This point takes into account measurement errors caused by the sample container 91 being mixed, changes in measurement conditions caused by taking out the sample container 91, and the like.

  As described above, in the method of the embodiment, a fluorescence measurement kit is prepared for each target substance for identification and quantification. Therefore, it is assumed that many sample containers 91 are prepared at the measurement site such as the control of prohibited drugs. In the fluorometer of the embodiment, after performing the first measurement step, the sample container 91 is taken out, the sample is put in a state of being removed from the photometer, the sample container 91 is remounted, and the second measurement is performed. It is also possible to perform a measurement step. However, if it does in this way, the sample container 91 is likely to be mixed up. After first attaching the sample container 91 for the substance A to the fluorometer and performing the first measurement step, the sample container 91 for the substance B was mistaken for the nearby sample container 91 for the substance B. When a sample (suspected to be substance A) is put in and this sample container 91 is mounted and the second measurement step is performed, it is natural that each test solution is not for substance A, so the measurement is wrong. Become. In this case, if it is detected that the sample container 91 has been removed after the first measurement step, an error message is displayed, and the liquid information code unit 93 reads again when the first measurement step is performed again. Since the target substance is displayed again on the display 52, the user is aware of the mistake. This prevents an erroneous measurement result from being obtained.

The above problems can also arise when many different samples have to be measured. For example, when identifying some substances discovered at the crime scene in an incident investigation, or when quantifying samples collected at several different places in an environmental investigation,
It is necessary to perform fluorescence measurement so that the sampling points are not mistaken. For example, it is assumed that there are samples A, B, and C collected at three points A, B, and C, and a fluorescence measurement kit is prepared for each sample (the target substances are the same). In this case, after the sample container A is mounted and the first measurement step is performed, if the sample container A is removed and the sample is loaded, the sample container B loaded with the sample B is mistaken. It is easy to make a mistake that the second measurement step is performed in this state. In this case, the first test solution that has acquired the fluorescence intensity F1 in the _first measurement step is different from the first test solution in which the sample is introduced in the second measurement step. That is, the fluorescence enhancement ratio before and after the reaction with the antigen is not measured for the same antibody. For this reason, the reliability of a measurement result falls.

  In addition, since the fluorometer of this embodiment is portable, it can be assumed that there are several fluorometers. In such a situation, the photometers are likely to be mixed up. It is assumed that after the first measurement step is first performed using the photometer A, when the sample is loaded and the sample container is remounted, the photometer B is erroneously mounted. In this case, even if the test solution is the same, since the photometers are different, measurement conditions such as the intensity of excitation light from the light source and the sensitivity of the detector may differ. Also, when the optical system is soiled (cloudy) due to use over time, the measurement conditions vary greatly. Therefore, a mistake in the photometer tends to directly lead to a decrease in measurement accuracy and a decrease in reliability of measurement results.

Such measurement mistakes are unlikely to occur in specialized measuring institutions that are measured by specialized engineers, but are more likely to occur when a non-specialist, such as a customs officer. In addition, if the sample container is removed after the first measurement step, the sample container is exposed to direct sunlight or strong room light, resulting in denaturation or deterioration of the internal test solution, which reduces the measurement accuracy. In some cases. In the present embodiment, in consideration of such points, as described above, when the sample container 91 is removed after the first measurement step, an error operation is performed.
It should be noted that the structure is such that the sample can be loaded with the sample container 91 mounted, and an error display (warning) is displayed if the sample container 91 is removed after the first measurement step and before the second measurement step. The point that the measurement is performed or the measurement is stopped has an independent meaning separate from the introduction of the correlation ratio R, and can be considered as a separate independent invention.

  From the viewpoint of preventing changes in measurement conditions, it is also effective to limit the time from the first measurement step to the second measurement step. For example, when the test solution is of a nature that gradually denatures when it comes into contact with air, the sample container 91 is provided in a degassed individual bag 90. In this case, after taking out the sample container 91 from the individual bag 90, it is necessary to complete the measurement in a short time. In addition to the denaturation of the test solution, there may be changes in the measurement conditions such as a slight decrease in the output of the light source due to battery consumption or a change in the ambient temperature due to some factor. Therefore, it is preferable to stop the measurement or display an error message when a certain time or more has elapsed after the first measurement step is completed. For example, it is about 5 minutes.

  In the above-described measurement operation, in order to remove background noise from the output value of the detector 3, the measurement may be initially performed without the sample container 91. For example, when the power switch is turned on, the output of the detector 3 is monitored by automatically operating the light source before displaying a screen prompting the mounting of the sample container, and this value is subtracted from each measured value as background noise. Like that. As background noise in this case, for example, when the wavelength of the excitation light and the wavelength of the fluorescence to be measured partially overlap, the excitation light becomes stray light and passes through the fluorescence filter and enters the detector 3. There may be noise.

In the present invention, it is not essential to use the second test solution. In some cases, the sample is directly put into the first test solution for measurement. In particular, when the components other than the fluorescent reagent and the antibody in the first test solution are the same as those in the second test solution, the second test solution may not be used. When the sample itself is in a liquid phase, it may be weighed as it is and put into the first test solution. In these cases, the effect of introducing the correlation ratio R can be obtained in the same manner.
Also, when the second test solution is used, the second test solution may not be stored in advance in the same sample container as the first test solution. That is, the fluorescence measurement kit may have a sample container containing the first test solution and another sample container containing the second test solution.

Next, a more specific example in which the fluorescence enhancement ratio Q ₁ ^* / Q ₁ is actually obtained in accordance with the method of the above-described embodiment will be described.
In this example, morphine was the target substance (antigen). As the first test solution, a morphine antibody was labeled with a fluorescent reagent and dissolved in a buffer solution. TAMRA was used as the fluorescent reagent. As the buffer solution, PBS (phosphate physiological saline) having a ph-adjusting action added with 1 vol% BSA as a vessel wall adhesion inhibitor and 0.05 vol% Tween 20 as a protein stabilizer is used. It was. The concentration of the morphine antibody in the first test solution is about 8 nM (nanomol).

An experiment for obtaining the correlation ratio R was performed on such a first test solution. First, the fluorescence intensity was measured in a state where no morphine antigen and fluorescent reagent were added, and it was 5.3 (arbitrary unit). Next, when the morphine antigen and the fluorescent reagent were added and the fluorescence intensity was measured, it was 7.1. Therefore, the fluorescence intensity of the morphine antigen and the fluorescent reagent was 1.8, and the correlation ratio R was 5.3 / 1.8 = 2.94. The fluorescence intensity of 7.1 is F ₁ described above.
On the other hand, the second test solution was exactly the same component as the buffer solution in the first test solution. That is, PBS with 1 vol% BSA and 0.05 vol% Tween 20 was used. The second test solution was charged with morphine as a sample so that the concentration was about 100 nM.

0.25 ml of the second test solution containing such a sample (morphine) was mixed with 0.75 ml of the first test solution to obtain 1 ml of a liquid phase object as a whole. Therefore, the mixing ratio k is 0.75. When such a liquid phase object was measured for fluorescence in the same manner, the fluorescence intensity (F ₂ described above) was 23.7. Since the fluorescence emission from the antigen for morphine is negligible, the light enhancement ratio Q ₁ ^* / Q ₁ was 13.6 (no unit) when calculated by applying the obtained F ₁ and F ₂ to Equation 8. It was. That is, the fluorescence intensity was enhanced by 13.6 times, and it was confirmed that the fluorescence intensity was greatly enhanced by the elimination of quenching associated with the antigen-antibody reaction.

DESCRIPTION OF SYMBOLS 1 Light source 2 Optical system 3 Detector 4 Container mounting part 5 Casing 52 Display 6 Control part 7 Code reader 81 First test liquid 82 Second test liquid 91 Sample container 912 First cell part 913 Second cell part 93 Liquid information code part S Sample

Claims (8)

A fluorescence measurement method for measuring fluorescence of a sample that seems to contain an antigen,
A first measurement step of measuring the intensity of fluorescence from the first test solution by irradiating the first test solution containing an antibody against the antigen with excitation light in a state where a sample is not charged;
After the first measurement step, a second measurement step for measuring the intensity of fluorescence from the liquid phase object by irradiating the liquid phase object obtained by putting the sample into the first test solution with excitation light. When,
A calculation step for performing calculation for identification or quantification of the sample from the measurement results in the first and second measurement steps,
Arithmetic step includes a correlation ratio R which is previously obtained for the first test solution, the value F ₁ of the fluorescence intensity measured by the first measurement step, the measured value of fluorescence intensity in the second measurement step A step of performing an operation according to F ₂ ,
The correlation ratio R is the fluorescence intensity that will be obtained from the unreacted antibody in the first test solution and the fluorescence intensity that will be obtained from components other than the unreacted antibody in the first test solution. The ratio of
Calculation steps, _{Q 1} fluorescence intensity with antibodies before sample loading, when the fluorescence intensity of antibodies after sample loading was _Q ^{1 _*,} and _{F 1 = Q 1 + R ·} Q 1, and _{F 1} on the basis of this A fluorescence measurement method, which is a step of obtaining Q ₁ ^* / Q ₁ from the value of F ₂ .   The second measurement step is a step in which the sample is mixed with a second test solution and then charged into the first test solution to measure the intensity of the fluorescence. 2. The fluorescence measuring method according to claim 1, wherein at least components that emit fluorescence that cannot be ignored among components other than antibodies in the first test solution are common.   The second measuring step is a step in which the sample is mixed with a second test solution and then introduced into the first test solution to measure the intensity of the fluorescence. The antibody is fluorescently labeled with a fluorescent reagent. The second test solution is characterized in that at least a component that emits non-negligible fluorescence among components other than the antibody and the fluorescent reagent in the first test solution is common. The fluorescence measuring method as described.   The fluorescence measurement kit used in the fluorescence measurement method according to any one of claims 1 to 3, further comprising a display unit for the correlation ratio R. A fluorescence measurement kit used in the fluorescence measurement method according to claim 2, comprising a sample container containing the first test solution and the second test solution, wherein the sample container is a sample And the second test solution after mixing the second test solution with the first test solution,
The second test solution contained in the first test solution contains at least a component that emits fluorescence that cannot be ignored among components other than antibodies in the first test solution contained therein. Measurement kit. A fluorescence measurement kit used in the fluorescence measurement method according to claim 3, comprising a sample container containing the first test solution and the second test solution, wherein the sample container is a sample And the second test solution after mixing the second test solution with the first test solution,
The antibody in the first test solution accommodated is fluorescently labeled with a fluorescent reagent,
The second test solution contained is at least a component that emits fluorescence that cannot be ignored among components other than the antibody and the fluorescent reagent in the first test solution contained. A fluorescence measurement kit. A light source that emits excitation light to excite the fluorescent material;
A detector that detects the intensity of the fluorescence emitted by the fluorescent material;
An optical system that guides excitation light from the light source to the liquid phase object contained in the sample container and guides fluorescence from the liquid phase object to the detector;
An arithmetic processing unit that processes fluorescence intensity data output from the detector,
The arithmetic processing unit applies the first fluorescence intensity measurement value F ₁ that is the result of measuring the fluorescence intensity using the first test solution previously stored in the sample container as a liquid phase object, and the first test solution. The second fluorescence intensity measurement value F ₂ that is the result of fluorescence measurement on the liquid phase object obtained by putting it in the sample, and the correlation ratio R acquired in advance for the first test solution and performing the calculation process And
The first test solution contains antibodies against the antigen that the sample is supposed to contain,
The correlation ratio R is the fluorescence intensity that will be obtained from the unreacted antibody in the first test solution and the fluorescence intensity that will be obtained from components other than the unreacted antibody in the first test solution. The ratio of
The arithmetic processing unit assumes that F ₁ = Q ₁ + R · Q ₁ when the fluorescence intensity due to the antibody before sample introduction is Q ₁ and the fluorescence intensity due to the antibody after sample introduction is Q ₁ ^*, and based on this F ₁ Q ₁ ^* / Q ₁ is obtained from the values of F ₂ and F ₂ . The sample container is provided as included in a fluorescence measurement kit, and the fluorescence measurement kit has a display unit displaying the correlation ratio R,
8. The fluorometer according to claim 7, further comprising means for reading the correlation ratio R displayed on the display unit.

Images