JP2002286628A - Fluorescence measuring device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform high-speed and highly accurate measurement by simple operation, achieve compactness and portability, and perform fully automatic measurement. SOLUTION: This fluorescence measuring device is provided with a cell 2 for measurement obtained by incorporating a non-fluorescent carrier 1 made of a fiber body in a channel provided for a support made of a light-transmitting material, a cell holder 8 to which the cell 2 for measurement can be attached, a light emitting means 10 for irradiating a cell 7 for measurement with a light beam 9 to excite fluorescence in a fluorescent labeled body held by the non- fluorescent carrier 1 in the channel, and a detecting means 12 for detecting fluorescence 28 excited in the fluorescent labeled body.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence measuring apparatus for detecting and quantifying a substance contained in a sample from a fluorescence measurement utilizing a ligand-receptor reaction, or for analyzing the interaction between substances. The present invention relates to a fluorescence measuring device suitable for the above.

[0002]

2. Description of the Related Art Measurements utilizing a ligand-receptor reaction are being widely applied to the detection of trace substances in foods, industrial products, environmental samples, etc., mainly in the field of medicine, such as testing for drugs, bacteria, and diseases. The ligand-receptor reaction mentioned here is not limited to a general antigen-antibody reaction.
Broadly, it means a binding reaction between biological substances such as receptors and substances having affinity for them. The greatest advantage of measurement using the ligand-receptor reaction is that it utilizes the advanced interaction between substances in the living body to detect or quantify a target substance with selectivity that could not be achieved by physicochemical methods. Medical, food, drinking water,
It has the potential to detect a wide range of substances from low molecular weight compounds to biopolymers in various samples such as sewage and environmental samples.

[0003] For the above uses, many automatic measuring devices utilizing a ligand-receptor reaction have been proposed. Many of the devices use enzyme-linked immunosorbent assay (Enzyme-linke
d Immunosorbent assay) to automate the detection of the substance to be measured. In this method, generally, a substance such as an antigen or an antibody is immobilized on a 96-well microplate as a solid phase, an enzyme-labeled substance that binds to the solid phase substance is added to the sample, and the sample is reacted. This is a method for determining the presence or absence and unknown concentration of an unknown substance in a sample from the activity. At present, labels such as fluorescence and radioactivity are used in addition to enzyme labels. This method is simple and excellent in that it can analyze many samples at once.

[0004]

However, in the above-mentioned automatic measuring apparatus, since only the detection of the substance to be measured is automated, the dilution of the standard sample, the antibody or the receptor and the subsequent dispensing to the plate are performed. Work must be performed, and workability is poor. In addition, since a general-purpose plate is used, and detection is performed for all or individual holes on the plate, the apparatus cannot be downsized.

Further, in the enzyme-linked antibody method employed in the above-mentioned automatic measuring apparatus, (1) the reaction requires a long time, the measuring time is multi-step and long, and (2) the solid phase such as a plate is miniaturized. (4) the equilibrium dissociation constant is difficult to determine except in special cases, and (5) it is not possible to use many types of detection elements at once. There are points to be improved.
These improvements are attributable to the small effective area of solid phase immobilization in the enzyme-linked antibody method and the subsequent static measurement field where competitive reactions tend to occur.

[0006] For this reason, attempts have been made to improve these and thereby improve the drawbacks of the enzyme-linked antibody method. For example, instead of a microplate as a solid phase, test tube walls, microtubules,
A method using a particle group or the like and a method of continuously introducing a sample into a fixed film or a particle group as a dynamic measurement field are known. However, it is hard to say that these methods sufficiently improve the above problems.

Accordingly, an object of the present invention is to provide a fluorescence measurement apparatus for detecting and quantifying a substance contained in a sample from a fluorescence measurement utilizing a ligand-receptor reaction, or analyzing an interaction between substances. The present invention further provides a fluorescence measurement device that is easy to operate, can perform high-speed, high-accuracy measurement, can be miniaturized, can be portable, and can perform fully automatic measurement. With the goal.

[0008]

Means for Solving the Problems The present inventor has improved the above-mentioned main problems in the enzyme-linked antibody method and the like in that the effective area of solid phase immobilization is small and that a static measurement field is formed. As a result of intensive research, the non-fluorescent carrier with a fibrous structure is more immobilized per volume than the conventionally used solid phase, such as test tube walls, microtubules, particle groups, and fixed membranes. Effective area is remarkably large, excellent in light transmittance, and does not generate fluorescence, so it is suitable for measurement at various fluorescence wavelengths, and has excellent permeability of aqueous solution, quick flow rate, and uniform solid-liquid contact I found that I can do it. Therefore, if such a fibrous body is incorporated into a cell as a solid phase, the device becomes a very compact device while retaining the substance to be immobilized at a high density, and becomes a device capable of performing simple and accurate measurement. This has led to the present invention.

In order to achieve the above object, the fluorescence measuring apparatus according to the first aspect of the present invention is a measuring apparatus in which a non-fluorescent carrier made of a fibrous body is incorporated in a flow path provided in a support made of a light transmitting material. Cell, a cell holder in which the measurement cell can be mounted, a light emitting means for irradiating the measurement cell with a light beam to excite fluorescence in the fluorescent label held by the non-fluorescent carrier in the flow path, and a fluorescent label And a detecting means for detecting the fluorescence excited in the step (a). Here, the fibrous body is not limited to a fiber having a fine network structure such as a woven fabric, but has a network structure such as a fiber or a filament filled in a certain volume or simply entangled or a nonwoven fabric. This is a concept that includes fibers having a high density of fibers even if they are not formed. In particular, it is preferable that the fibers be as thin and dense as possible and have a uniform flow rate.

[0010] Therefore, since the carrier is made of a fibrous body, the immobilization effective area per volume can be significantly increased as compared with a conventional test tube wall or the like used as a solid phase. In addition, since the carrier is a fibrous body and has excellent light transmittance and the carrier is non-fluorescent and does not generate fluorescence,
It can be suitable for measurement at various fluorescent wavelengths. Further, since the carrier is a fibrous body, excellent permeability of the aqueous solution is obtained and a rapid flow rate is obtained, so that a dynamic measurement site can be realized and uniform solid-liquid contact can be achieved.

Thus, the amount of the substance such as an antigen immobilized on the non-fluorescent carrier can be extremely increased, so that the amount of the fluorescently labeled substance to be measured can be increased, and the amount of the substance to be measured can be measured by a fluorescence measuring device. The fluorescence intensity can be increased. Therefore, the accuracy of the fluorescence measurement by the fluorescence measurement device can be improved. At the same time, if the amount of the fluorescent label held by the non-fluorescent carrier is maintained at the same level as that of a conventional carrier, the size of the non-fluorescent carrier can be significantly reduced, and the size of the fluorescence measurement device can be reduced. .

In addition, since the amount of the substance such as an antigen fixed to the non-fluorescent carrier can be extremely increased, it is possible to sufficiently obtain a cumulative effect that is gradually accumulated by flowing the substance to be solidified. Become. For this reason, since the sensitivity of the measurement of the fluorescence measurement device can be increased, the measurement can be performed even on a thin sample which was not able to be measured conventionally with low sensitivity. As a result, the measurement object is expanded, and for example, milk, blood, and environmental samples can be measured at the same concentration.

Moreover, since a sufficient cumulative effect can be obtained,
Even when a sample containing a trace amount of a fluorescent label is allowed to flow, fluorescence can be measured by accumulating the fluorescent label. Here, since a sample containing a trace amount of the fluorescent label is not measured as it is, the fluorescence of the sample itself is not measured by measuring the fluorescence while flowing this sample. Fluorescence can be measured only for the fluorescent label. Therefore, since the fluorescence from the fluorescent label accumulated in the non-fluorescent carrier can be measured while flowing the sample,
The accuracy of the measurement can be improved by reducing the influence of the contaminants, and the measurement time can be shortened.

Further, since the amount of the substance such as an antigen fixed to the non-fluorescent carrier can be extremely increased, one non-fluorescent carrier can be used many times. For this reason, the frequency of replacing the carrier can be reduced, and the operability can be improved. In addition, multiple substances can be immobilized at the same time by increasing the immobilization area, and multiplex detection can be performed.

Furthermore, since the carrier is a fibrous body and has excellent light transmittance and the carrier is non-fluorescent and does not generate fluorescence, it can be suitable for measurement at various fluorescent wavelengths, and the versatility can be improved. Can be.

Since the non-fluorescent carrier is incorporated into a support to form a measuring cell, when reacting a substance with the non-fluorescent carrier, it is sufficient to introduce the substance into the measuring cell and the operability is good. Further, when the non-fluorescent carrier is replaced, it is sufficient to replace the entire measuring cell, so that the handling of the non-fluorescent carrier can be facilitated.

Further, since the cell holder capable of mounting the measuring cell is provided, the substance can react with the non-fluorescent carrier by mounting the measuring cell on the cell holder and introducing the substance into the measuring cell. . Therefore, the operability of the measurement can be improved.

According to a second aspect of the present invention, in the fluorescence measuring apparatus according to the first aspect, the cell holder covers at least a portion of the held measurement cell where the fluorescence is generated and darkens the fluorescence to such an extent that the fluorescence can be detected. In addition, a communication unit is provided to secure an optical path of a light beam from the light emitting unit to the measuring cell and a fluorescent optical path from the measuring cell to the detecting unit.

Therefore, since the portion of the measurement cell where the fluorescence is generated is darkened by the cell holder, even a minute fluorescence can be measured with high accuracy without being affected by external light. Further, since each optical path of light and fluorescence entering and exiting the measurement cell is secured by the communicating portion, fluorescence can be generated and detected while the portion where the fluorescence is generated is kept dark by the cell holder.

According to a third aspect of the present invention, in the fluorescence measuring apparatus of the second aspect, the support of the measuring cell has a convex lens portion for condensing a light beam from the light emitting means to the flow path. Like that. Therefore, since the light beam can be condensed on the flow channel, the measurement cell can be irradiated with the light beam to effectively excite the fluorescence in the fluorescent label held by the non-fluorescent carrier in the flow channel.

The invention described in claim 4 is the same as the claim 3.
In the fluorescence measurement device described above, the measurement cell and the cell holder have positioning means for making the optical axis of the convex lens portion coincide with the optical axis of the light emitting means. Therefore,
Since the optical axis of the convex lens portion and the optical axis of the light emitting means can be made to coincide with each other by the positioning means, the light beam can be surely focused on the flow path by a one-touch operation simply by mounting the measuring cell on the cell holder.

According to a fifth aspect of the present invention, in the fluorescence measuring device of the fourth aspect, the positioning means is formed on a positioning surface formed on an outer peripheral portion of a support of the measuring cell, and on a cell holder. The positioning surface of the measurement cell is provided with a reference surface which is positioned by contact.
Therefore, the optical axis of the convex lens portion and the optical axis of the light emitting means can be surely matched with a simple configuration.

The invention according to claim 6 is the invention according to claim 2.
6. The fluorescence measuring device according to any one of items 1 to 5, further comprising: an optical unit having a cell holder, a light emitting unit, and a detecting unit; A control unit that controls the operation of the optical unit and the supply and waste liquid means, and the supply and waste liquid means, a pump having a plurality of fluid reservoirs and a storage unit, a flow path unit having a plurality of mixing chambers and a rotary valve, A sample supply unit, and the sample supply unit and the plurality of fluid reservoirs are respectively connected to a pump so as to be switchable via a rotary valve, and the pump is switchable to a plurality of mixing chambers via the rotary valve. The plurality of mixing chambers are further connected to a measurement cell mounted on a cell holder in a switchable manner via a rotary valve. It is way.

Therefore, all the steps of supplying the sample to the measuring cell and measuring the fluorescence can be automated, so that the work of the measurement can be performed in comparison with the conventional case where only the detection of the substance to be measured is automated. The performance can be greatly improved.

According to a seventh aspect of the present invention, in the fluorescence measuring device according to the sixth aspect, the liquid in the fluid reservoir is supplied to the storage part of the pump via a rotary valve, and the liquid in the storage part is supplied through the rotary valve. To the mixing chamber,
A plurality of liquids are supplied to the mixing chamber to prepare a standard sample for a calibration curve, the standard sample is supplied from the mixing chamber to a reservoir of a pump via a rotary valve, and the standard sample is supplied from the reservoir to a rotary valve. The liquid is sent to the measurement cell via Therefore, in addition to supplying the sample to the measurement cell and measuring the fluorescence, preparation of the standard sample can be automated.

[0026]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The structure of the present invention will be described below in detail based on an embodiment shown in the drawings. 1 and 2 show an example of an embodiment of the fluorescence measurement device 7 of the present invention. As shown in FIG. 3, the fluorescence measuring device 7 includes a measurement cell 2 in which a non-fluorescent carrier 1 made of a fibrous body is incorporated in a flow path 4 provided in a support 3 formed of a light transmitting material.
A cell holder 8 to which the measuring cell 2 can be attached as shown in FIG. 1;
A light emitting unit 10 for exciting the fluorescent label held by the non-fluorescent carrier 1 therein to emit fluorescence, and a detecting unit 12 for detecting the fluorescence excited by the fluorescent label are provided. The non-fluorescent carrier 1 is made of a fibrous body. For this reason, the immobilization effective area per volume can be significantly increased as compared with a conventional test tube wall or the like used as a solid phase. Also,
Since the carrier 3 is non-fluorescent and does not generate fluorescence, it can be made suitable for measurement at various fluorescent wavelengths. Furthermore, excellent permeability of the aqueous solution and a rapid flow rate can be obtained, so that a dynamic measurement site can be realized and uniform solid-liquid contact can be achieved. Moreover, such a fibrous body has the property of being freely deformable and is insoluble in aqueous solvents. For these reasons, by using the non-fluorescent carrier 1 made of a fibrous body, the measurement time by the fluorescence measuring device 7 can be reduced to, for example, several minutes, and real-time measurement becomes possible. In addition, since a sufficiently high cumulative effect can be obtained, measurement can be performed while flowing the sample,
The measurement accuracy can be improved by reducing the influence of the contaminants.

The fibrous body forming the non-fluorescent carrier 1 has a fine network structure as shown in FIG. 4, for example. Examples of the material of the fibrous body include cotton (FIG. 4A),
Of synthetic or semi-synthetic materials such as natural materials such as silk and hemp, polyesters, aromatic polyamides, nylons and polyolefins (FIG. 4 (C)), or mixtures thereof, for example, polyesters and polyolefins (FIG. 4 (B)) Examples thereof include a mixture, but are not particularly limited as long as they have a large number of fibers at a high density. As the fibrous body, a cloth such as a woven fabric, a knitted fabric, or a nonwoven fabric is preferable because the filling operation of the measuring cell 2 into the support 3 or the like is easy.
Fibers or filaments may be filled in a certain volume or simply entangled.

Of these, natural fibers, especially fibrous bodies containing cotton, have a large specific surface area, with the fibers being as thin as about several μm,
It is preferable because it shows high immobilization efficiency, has high hydrophilicity, and is excellent in liquid permeability and the like. The appropriate range of the fineness and density of the fiber is not particularly limited since it depends on the target fluid to be circulated and the like, but in order to obtain a uniform flow rate and a high fixing efficiency, for example, 50 to 20
A fibrous body capable of obtaining a gas permeability of about 0 cm ³ / cm ² / sec is preferred.

The measuring cell 2 includes the support 3 formed of a light-transmitting material as described above, and the non-fluorescent carrier 1 which is a solid phase on which a substance is immobilized. The substance to be fixed to the non-fluorescent carrier 1 is a single substance or plural kinds of substances that react with the substance to be measured, or a single substance or plural substances to be measured.

After the substance is fixed to the non-fluorescent carrier 1 by the method described later, it is incorporated into the support 3 shown in FIG. The support 3 has a convex lens portion 5 for condensing an external light beam on the flow path. For this reason, since a light beam can be condensed on the flow channel 4, the light beam is irradiated on the measuring cell 2 to effectively excite the fluorescence in the fluorescent label held by the non-fluorescent carrier 1 in the flow channel 4. Can be.

In the fluorescence measuring device 7, the measuring cell 2 is detachable from the cell holder 8, so that the measuring cell 2 is mounted on the cell holder 8 and a substance is introduced into the measuring cell 2. Since the substance can react with the non-fluorescent carrier 1, the operability can be improved. Further, when the non-fluorescent carrier 1 is exchanged, the whole cell for measurement 2 may be exchanged, so that the handling of the non-fluorescent carrier 1 can be facilitated.

The measuring cell 2 and the cell holder 8 have positioning means 11 for making the optical axis of the convex lens 5 coincide with the optical axis of the light emitting means 10. For this reason, since the optical axis of the convex lens portion 5 and the optical axis of the light emitting means 10 can be matched by the positioning means 11, the light rays 9 can be surely condensed on the flow path 4.

In the present embodiment, the positioning means 11 is configured such that the positioning surface formed on the outer peripheral portion of the support 3 of the measuring cell 2 and the positioning surface of the measuring cell 2 formed on the cell holder 8 come into contact with each other. A reference surface to be positioned is provided. For example, in the present embodiment, the outer peripheral contour shape of the support 3 is a regular octagon, and at least one surface of the octagon is used as a positioning surface. On the other hand,
For example, as shown in FIG.
, And each block 25, 26
Are formed with concave portions 25a and 26a that form an octagonal hole 27 when the two blocks 25 and 26 are combined, and at least one surface of the concave portions 25a and 26a is used as a positioning surface. Then, the measurement cell 2 is positioned by aligning this positioning surface with the reference surface formed on the cell holder 8. Therefore, the optical axis of the convex lens portion 5 and the optical axis of the light emitting means 10 can be easily and reliably matched with a simple configuration.

The cell holder 8 covers at least a portion of the held measurement cell 2 where the fluorescence is generated and darkens the fluorescence so that the fluorescence can be detected. The optical path of the light beam 9 from the light emitting means 10 to the measurement cell 2 is also provided. And a communication section 29 for securing an optical path of the fluorescent light 28 from the measuring cell 2 to the detecting means 12. For this reason, since the portion of the measurement cell 2 where the fluorescence is generated (the non-fluorescent carrier 1 near the convex lens portion 5) is darkened by the cell holder 8, even minute fluorescence can be measured with high accuracy without being affected by external light. be able to. Further, each optical path of the light 9 and the fluorescent light 28 entering and exiting the measurement cell 2 is connected to the communication section 2.
9, the fluorescent light can be generated and detected while the cell generating portion is kept dark by the cell holder 8.

In the present embodiment, the two blocks 25 and 26 for holding the measuring cell 2 are both light-shielding members such as metal or opaque synthetic resin. For this reason, the measuring cell 2 accommodated in the hole 27 formed by combining the blocks 25 and 26 is entirely covered and kept dark enough to detect the fluorescence. In addition, a cover 30 that covers the entire cell holder 8 is provided. Therefore, the measurement cell 2 can be more effectively shielded from light.

Further, the light emitting means 10 and the detecting means 12
In the concave portion 25a of the block 25 installed on the side, a through-hole serving as a communication portion 29 for communicating the light emitting means 10 and the detecting means 12 with the measuring cell 2 is formed. In the present embodiment, the light 9 from the light emitting means 10 and the fluorescent light 28 from the measuring cell 2 pass through the same communication part 29,
However, the present invention is not limited to this, and a plurality of communicating portions 29 may be formed to allow the light 9 and the fluorescent light 28 to pass separately.

In the cell holder 8, the blocks 25 and 26 are provided so that the upper part of the block 25 and the hinge 24 can be opened and closed. Each of the blocks 25 and 26 is urged in a closed state by a closing spring 31 composed of a tension coil spring. For this reason, when the measuring cell 2 is mounted, the block 26 provided on the opposite side of the light emitting means 10 and the detecting means 12 is opened against the closing spring 31 so that the liquid supply tube 32 is connected to the measuring cell 2. Attach. Then, the measuring cell 2 is mounted in the concave portion 25 a of the block 25. At this time, by aligning each surface of the positioning means 11, the optical axis of the convex lens unit 5 and the optical axis of the light emitting means 10 are matched. When the block 26 is closed, the closed state is maintained by the closing spring 31. Therefore, attachment and detachment of the measurement cell 2 can be easily performed by one-touch operation.

In this embodiment, the positioning means 11 includes the positioning surface of the support 3 and the reference surface of the cell holder 8. However, the present invention is not limited to this, and the measuring cell 2 is positioned so as not to rotate with respect to the cell holder 8. Anything is fine. For example, the support 3 may have a cylindrical shape, and may have projections and depressions and a fitting portion for positioning. Also in this case, the positioning can be easily and reliably performed in the same manner as when the surfaces are aligned.

The sizes of the support 3 and the flow path 4 are not limited as long as the function of the measuring cell 2 is not impaired. In the drawing, reference numeral 6 denotes a connector press-fitted at both ends of the support 3 for introducing a sample into the measuring cell 2. With the connector 6 attached, the measuring cell 2 is inserted into the cell holder 8.
Attached to.

The constituent material of the support 3 is a plastic material such as glass, (meth) acrylic resin, polycarbonate, polyolefin, etc., which is excellent in light transmittance and generates a small amount of fluorescence, but has low fluorescence and light transmittance. Is not particularly limited as long as it indicates

The non-fluorescent carrier 1 having a fiber structure is incorporated in the light irradiation area of the flow path 4 at the center of the support 3. Therefore, the size and volume of the solid phase are determined by the width and length of the range of the light irradiation part, and the size and volume are not particularly limited.

When the non-fluorescent carrier 1 is incorporated into the support 3, it is important to realize uniform solid-liquid contact with the sample introduced from the opening of the flow channel 4. Solid-liquid contact is determined using the flow rate of the sample as an index, and specifically, for example, a flow rate of at least 5.0 ml / min, more preferably a flow rate of about 0.1 to 10.0 ml / min. That is possible.

When the fibrous body is a cloth, for example, the fibrous body can be wound and formed into a cylindrical shape, and can be inserted and arranged at a predetermined position in the flow path 4 of the support 3. In this case, without using a special fall prevention body such as a mesh screen,
There is no danger of the support 3 flowing out of the site.

Since the measuring cell 2 has a simple structure as described above and its manufacturing cost is low, it can be made a disposable, that is, a disposable product, and is accurate and quick. Measurement work can be performed.

The fluorescence measuring device 7 using the above-described measuring cell 2 will be described below. This fluorescence measuring device 7
Supply and waste liquid means 14 and 15 which are detachably connected to the optical unit 13 and the measurement cell 2 to supply a sample or the like and pass the liquid;
And a control unit 16. The optical section 13 has the cell holder 8, the light emitting means 10, and the detecting means 12.
The supply / drainage means 14 and 15 include a pump 18 having a plurality of fluid reservoirs 17 and a storage section, a flow path section 14 having a plurality of mixing chambers 19 and a rotary valve 20, and a sample supply section 15. In FIG. 1, a thin solid line connecting the elements indicates the liquid sending line 32, a thick line indicates an electrical connection, and a broken line indicates each part.

The sample supply section 15 is composed of a sample tank provided outside the fluorescence measurement device 7 and is for taking an actual sample into the fluorescence measurement device 7.

The function of the flow path section 14 is to send the standard sample and the actual sample in the fluorescence measuring device 7 and to drain the waste liquid from the measuring cell 2, and also to automatically prepare the standard sample.

In the flow path section 14, the sample supply section 15 and the plurality of fluid reservoirs 17 are respectively connected to a pump 18 via a rotary valve 20 so as to be switchable, and the pump 18 is connected to a plurality of pumps via the rotary valve 20. Are switchably connected to the mixing chambers 19. Further, the plurality of mixing chambers 19 are connected to a measurement cell 2 mounted on a cell holder in a switchable manner via a rotary valve 20 and a tube 32. Each operation of the rotary valve 20, the pump 18, and the mixing chamber 19 is controlled by the control unit 16. This makes it possible to automate all of the steps of supplying the sample to the measurement cell 2 and measuring the fluorescence, thereby improving the workability of the measurement as compared with the conventional case where only the detection of the substance to be measured is automated. Can be greatly improved.

Here, a procedure for preparing a standard sample using the flow path section 14 will be described.

The liquid in the fluid reservoir 17 is supplied to the reservoir of the pump 18 via the rotary valve 20, and the liquid in the reservoir is supplied to the mixing chamber 19 via the rotary valve 20. Supply liquid to prepare a standard sample for the calibration curve. Then, the standard sample is supplied from the mixing chamber 19 to the storage section of the pump 18 via the rotary valve 20, and the standard sample is sent from the storage section to the measurement cell 2 via the rotary valve 20. Therefore, in addition to the supply of the sample to the measurement cell 2 and the fluorescence measurement, the preparation of the standard sample can be automated.

In the present embodiment, the fluorescence measuring device 7 is
As shown in the figure, a reservoir 17a for the standard antigen, a reservoir 17b for the control antibody, a reservoir 17c for the buffer, and a reservoir 17d for the measurement antibody are provided. Then, by switching the flow path of the rotary valve 20, a certain amount of antigen, antibody, and buffer can be collected from each reservoir 17 into the syringe pump 18. In addition, the standard sample means a sample containing a measurement antibody and a measurement antigen, and the control sample means a sample containing a control antibody and a control antigen.

After the collection, the entire amount of the solution in the syringe pump 18 is transferred to one of the plurality of mixing chambers 19 by switching the flow path of the rotary valve 20. Next, a standard sample having a different antigen concentration is prepared in each mixing chamber 19 while changing the collection amount of the antigen and the buffer solution to be collected in the mixing chamber 19 and keeping the total amount in the mixing chamber 19 constant. The sample supply unit 1 is operated in the same manner.
A mixed solution of the actual sample from 5 and the antibody is also prepared and prepared in another mixing chamber 19. When a plurality of antibodies are used, a plurality of antibodies or antigens are added to the antibody solution and the antigen solution in advance. Alternatively, antibodies and antigens may be added to separate reservoirs 17 and a certain amount may be collected from each. The prepared standard sample and actual sample are sequentially sent from the syringe pump 18 to the measurement cell 2 at a constant flow rate by switching the flow path of the rotary valve 20. In the present embodiment, a standard sample is prepared as an example of preparing a sample using the flow path unit 14.
Of course, the sample that can be prepared by the method 4 is not limited to this, and a control sample and the like can also be prepared. In some cases, fluorescence measurement may be performed without preparing a standard sample.

On the other hand, the optical section 13 includes exchangeable excitation and emission filters in addition to the above-described cell holder 8, light emitting means 10, and detecting means 12. Therefore, corresponding to the wavelength of the light beam 9 to be excited and the fluorescence wavelength of the fluorescence 28,
The filter can be replaced. As a result, it is possible to cope with a wide range of fluorescence wavelengths, and it is possible to broaden the options of usable fluorescent dyes.

The detecting means 12 comprises, for example, a photodiode. Then, the detection means 12 can detect the fluorescence excited by the fluorescent label in real time or over time.

In the optical section 13, the measuring cell 2 is irradiated with a laser beam by the light emitting means 10 to optically excite a fluorescent dye labeled with an antibody captured in the measuring cell 2 by an antigen-antibody reaction. Then, the emitted fluorescence is detected by detecting means 1.
2 and converted into an electric signal for measurement. By the combination of the optical unit 13 and the measuring cell 2 having such a configuration,
The standard sample and the actual sample guided from the flow path unit 14 can be measured in batch or continuously in real time.

Since the optical section 13 has a structure having a replaceable fluorescent filter, a filter suitable for a fluorescent dye can be selected and mounted. Therefore, by exchanging the fluorescent filter, the interaction between substances can be analyzed simultaneously with the detection and quantification of one or more kinds of substances such as an organic compound or an inorganic compound containing a biopolymer such as a nucleic acid and a protein. In the present embodiment, the detecting means 12
However, the present invention is not limited to this, and a detector capable of performing other photoelectric conversion such as a photomultiplier may be used.

The control section 70 for controlling the operation of the flow path section 14 and the optical section 13 includes a circuit board 21 for defining the operation of each section, a voltmeter 22 for receiving a signal from the detection means 12, and an operation control section. It is composed of a communication board 23 capable of externally outputting results and software for controlling these. Circuit board 21
Is connected to the syringe pump 18 of the flow path section 14 and the mixing chamber 1.
9 and the operation of the rotary valve 20 are defined.

The control unit 70 controls the operation of the flow path unit 14 and reads out the detection value from the detection unit 12 through the communication board (communication board) 23 by software on an external computer. The control items include the positions of various reservoirs 17 and rotary valves 20 and the operation of the mixing chamber 19. In addition, a predetermined flow rate, for example, 0.25 to 5.0 ml, may be set in any direction between the reservoir 17, the syringe pump 18, and the mixing chamber 19.
The liquid can be sent at a set flow rate of / min.

When a sample is prepared and held in each of the plurality of mixing chambers 19, the flow path 14
Thus, the sample can be sent from each mixing chamber 19 to the measurement cell 2 in an arbitrary order and at an arbitrary flow rate setting within the above flow rate range. The fluorescence signal can be displayed by software in real time or at the end of measurement as time elapses, and at the same time, it can be displayed as a fluorescence intensity change value at any time. The data at the time of measurement
It can be recorded on a suitable medium such as a disk as the fluorescence intensity at regular intervals, for example, at one second intervals, or as a fluorescence intensity change value at every arbitrarily set time.

As described above, since the control section 16 controls the flow path section 14 and obtains the measured values from the optical section 13, a calibration curve is created based on the obtained data to further analyze the actual sample. The analysis can be performed fully automatically.

The miniaturization of the optical system by the disposable measuring cell 2 as described above and the mixing chamber 1 of the flow path system
By adopting the simplification of FIG. 9, the portable fluorescence measuring device 7 can be configured. For example, the size of the device 7 itself can be reduced to at least about 30 cm in length, 30 cm in width, 20 cm in height, and about 2 kg in weight excluding a power supply. Also, the reservoir 17
Further, depending on the number and volume of the mixing chamber 19, further miniaturization is possible.

Next, the fixing of the substance to the non-fluorescent carrier 1 will be described. The immobilizing substance defined in the present invention is an organic compound or an inorganic compound containing a biopolymer such as a nucleic acid, a protein or the like, alone or in combination. For example, immunoglobulins derived from various animals, low-molecular-weight organic compounds such as female hormones and vitamins, complexes of female hormones with serum globulin, and biological macromolecules that are bacterial toxin proteins. Thus, it can be used regardless of the molecular weight.

The method for immobilizing these substances on the non-fluorescent carrier 1 is not particularly limited as long as the substance itself is not denatured or inactivated. For example, a substance can be directly bound to a carrier using a natural adsorption method, an ion bonding method, a covalent bonding method, or the like. In addition to the method of directly bonding, it is also possible to indirectly bond and fix via an appropriate spacer substance. In addition, other known methods can be appropriately adopted.

When the above-described fluorescence measuring device 7 is used, a unique accumulation and binding equilibrium elimination effect is exhibited.
The feature is that detection and quantification of a substance contained in a sample or binding interaction between substances can be automatically analyzed in various kinds of measurement modes. These effects are due to the continuous flow of the sample through the flow path system and the creation of a dynamic measurement field by the measurement cell 2 equipped with the non-fluorescent carrier 1 which has excellent sample permeability in the optical system and has a high density substance fixing ability Can be achieved by In both modes, the fluorescence measurement is observed in real time, and the substance can be quantified by a change in the fluorescence intensity per unit time. Here, a measurement procedure common to both measurement modes will be described below.

When each of the standard sample and the actual sample is continuously supplied to the optical section 13 including the measuring cell 2, the measuring cell 2 fixes a high density and a large amount of substances by the non-fluorescent carrier 1 having a fine mesh structure. Therefore, the concentration of the label in the sample can be extremely reduced. Therefore, a linear increase in fluorescence intensity is measured in proportion to the liquid sending time (liquid sending amount under a constant flow rate). This is because the amount of the labeled substance captured and accumulated in the non-fluorescent carrier 1 with the passage of time (the amount of liquid to be sent) is clearly larger than the amount of the antibody in the liquid.
Therefore, the change in fluorescence intensity per unit time is a function of the concentration of the unbound antibody in the solution, and the concentration of the analyte in the sample or the reaction equilibrium between the substances is determined from the rate of change in the intensity through the unbound antibody concentration. Can be determined.

The sample to be provided to the fluorescence measuring device 7 according to the present invention is not particularly limited, but medical treatments (blood, urine, body fluid,
In addition to artificial cultures, drugs, and the like, foods (soft drinks, alcoholic beverages, milk, and the like), drinking water, sewage, environmental samples (river water, seawater, groundwater, and the like), and the like can be applied.

Next, the first method will be described. A large amount and high density of a substance which is considered to be present in the sample and which is reactive with the substance to be measured is fixed to the non-fluorescent carrier 1 in the measurement cell 2 using the method described above, and the measurement cell 2 is placed in the cell holder 8. To be attached to the fluorescence measuring device 7.

Next, the sample is mixed with a fluorescently labeled substance to be measured at a constant concentration. That is, a fluorescent label of a substance having the same reactivity as the substance to be measured or the same as the substance to be measured or immobilized on the non-fluorescent carrier 1 is added to the sample. The fluorescent label may be a method of labeling a substance using a direct ion binding method, a covalent bonding method, or the like, or may be labeled via a fluorescent-labeled secondary substance. In addition, any known dye can be used as the fluorescent dye.

Thereafter, the sample is continuously fed into the measuring cell 2 to bring the sample and the solid phase into contact. At this time, due to the solid-liquid contact, a competitive reaction between the substance to be measured and the labeling substance having reactivity with the immobilized substance occurs with the substance on the solid phase.
The degree of the competition reaction is observed as a change in the fluorescence intensity over a certain period of time. That is, if a high change is observed over time, it indicates that the labeling substance reacts more with the substance on the solid phase (small competitive reaction). Does not react well with the substance on the solid phase (competitive reaction is large). Therefore, at the time of sending the sample, the competitive reaction is measured in real time as a change in fluorescence intensity per time, and the degree of the competitive reaction is determined from the amount of the change. Since the amount of change is related to the amount of the label in the measurement cell 2, the unknown concentration of the analyte in the sample can be finally determined from the degree of competition.

More specifically, when the target substance to be measured is contained in a large amount in the sample, the degree of the reaction of the substance to be measured with the substance on the solid phase increases according to the concentration. On the other hand, the degree of reaction of a labeled substance of known concentration artificially mixed with a sample decreases. Therefore, the rate of change of the fluorescence intensity is small. If the target analyte is not contained in the sample, or if the concentration is extremely dilute, apparently reacting with the substance on the solid phase is a known concentration of the labeled substance mixed with the sample artificially. , A high change in fluorescence intensity is obtained. In this way, by observing the degree of the competition reaction with the change rate of the fluorescence intensity, the concentration of the unknown analyte in the sample can be finally determined.

The above-mentioned measuring cell 2 provides a reaction field which is indispensable for establishing the first method. Specifically, since the non-fluorescent carrier 1 can fix a high density and a large amount of substances, when the sample is continuously introduced into the measuring cell 2,
It can encourage more competitive reactions. In addition, since the reacted phosphor can be accumulated in the measuring cell 2 in proportion to the amount of the sample to be sent, the sensitivity for determining the unknown concentration of the diluted substance to be measured is realized. Therefore, it becomes possible to measure the concentration of a diluted substance to be measured such as milk.

Next, the second method will be described. A large amount and high density of a substance having the same reactivity as the substance to be measured or a substance to be added to the sample, which is considered to be present in the sample, is removed from the measurement cell 2 using the method described above. The cell 2 for measurement is fixed to the fluorescent carrier 1, and the cell for measurement 2 is attached to the fluorescence measuring device 7 with the cell holder 8.

The fluorescent label of a substance having a reactivity with an unknown concentration of the substance to be measured contained in the sample is mixed by the above-mentioned operation. The fluorescent label may be a method of labeling a substance using a direct ion binding method, a covalent bonding method, or the like, or may be labeled via a fluorescent-labeled secondary substance. In addition, any known dye can be used as the fluorescent dye. After the addition, the mixture is left for a certain period of time, and the reaction between the substance to be measured and the label reaches an equilibrium state.

Thereafter, the sample is continuously fed into the measuring cell 2 to bring the sample and the solid phase into contact. At this time, due to the solid-liquid contact, the unreacted label in the sample reacts with the substance on the solid phase, and the amount of the label on the solid phase can be detected in real time as the fluorescence intensity observed over time from the cell. Since the amount of the label on the solid phase is proportional to the concentration of the unreacted label in the sample, the concentration of the analyte in the sample can be determined from the change rate of the fluorescence intensity. That is, if a high change in fluorescence intensity is observed, it indicates that the labeling substance has reacted more with the substance on the solid phase, and at the same time, the labeling substance mixed in the sample in a certain amount in the reaction equilibrium state is measured. Indicates that it does not react well with the substance. If the change in fluorescence intensity is low, it indicates that the labeling substance has not reacted well with the substance on the solid phase,
At the same time, it shows that the labeled substance added to the sample in a certain amount in the reaction equilibrium state is well reacted with the analyte.

Therefore, the amount of the label on the solid phase is proportional to the concentration of the unreacted label in the sample, and the concentration of the substance to be measured can be determined by measuring the reaction equilibrium state in the sample from the change in fluorescence intensity. . At the same time, an equilibrium solution constant and an equilibrium binding constant which are indicators of a reaction equilibrium state in a sample can be determined, and a reaction interaction between substances can be analyzed.

Briefly, the reaction equilibrium state with the label determined by the concentration of the substance to be measured in the sample is determined by capturing the unreacted label on the solid phase in the measurement cell 2 and measuring its fluorescence. It is determined from the amount of the label that can be calculated from the change in intensity. Further, once the reaction equilibrium state is determined, the concentration of the substance to be measured in the unknown sample can be determined from the amount of the fixed amount of the label added.

To enable the above-mentioned measurement, the effect of accumulating a substance obtained by fixing a high density and a large amount of substance on the non-fluorescent carrier 1 and the uniform solid-liquid The equilibrium reaction exclusion effect of contact is essential.

Since the non-fluorescent carrier 1 can immobilize a high-density and a large amount of substances, when a sample is continuously introduced into the measuring cell 2, more reaction fields can be provided for unreacted label. Further, in the same continuous measurement with time, the label captured by the reaction can be accumulated in proportion to the amount of the sample sent. For this reason,
The amount of the label added to the sample can be extremely reduced, and a reaction equilibrium state with a more dilute analyte can be created. As a result, the analyte can be quantified to a dilute concentration.

Further, since the non-fluorescent carrier 1 can fix a high-density and large amount of substance and can perform uniform solid-liquid contact, the non-fluorescent carrier 1 requires only a short contact time between the sample and the solid-phase carrier in a continuous liquid sending field. A few percent of all the labeled substances in the solution can be continuously captured by reaction and accumulated in the measurement cell 2. Thus, an equilibrium reaction exclusion effect that can be analyzed without disturbing the binding equilibrium in the liquid is expected.

In the second method, it is also possible to use a combination of a measurement antibody (standard antibody) and a control antibody, which are particularly effective when analyzing an environmental sample or the like. this is,
This is effective when the reaction between the substance in the sample and the substance used as the detection element is non-specifically inhibited for some reason. This is because these inhibitions can be observed even when the substance is not contained in the sample, thus giving an incorrect quantitative value as a false positive reaction. However, when a measurement antibody and a control antibody are used in combination, the measurement antibody performs the detection itself and the control antibody performs the presence or absence of non-specific inhibition, estimating a false positive reaction, and quantifying the actual concentration of the analyte. There is a special feature that can be done.

That is, when fluorescence measurement is performed on a sample that does not cause a false positive reaction using the measurement antibody and the control antibody, accurate measurement results can be obtained for any of the antibodies. When a fluorescence measurement is performed on a sample that causes the above, an inaccurate measurement result is obtained due to a false positive reaction for any of the antibodies. However, by obtaining a calibration curve for the control antibody in advance, it is possible to calibrate an incorrect measurement result obtained by a false positive reaction. With reference to this calibration amount, an accurate measurement result can also be estimated for the measurement antibody. In addition, the calibration curve for the control antibody may be created together with the measurement of the measurement antibody, or may be prepared in advance as data.

In the above two types of measurement modes, the detection is not limited to the detection of a single substance, but the detection of a plurality of substances simultaneously and multiplely based on the same principle, or the detection of a plurality of substances. It is also possible to detect sequentially.

[0083]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will now be described with reference to the drawings, but these are for illustrating the present invention in more detail and not for limiting the present invention. In addition,
In the following examples, the materials and methods used are as follows.

(1) Non-fluorescent carrier 1, antibody, female hormone, vitamin and enterotoxin 17-β-estradiol, estriol and biotin were purchased from Wako Pure Chemical Industries. In addition, biotin, estril or a complex of estradiol and bovine serum albumin were purchased from Sigma, USA, respectively. Monoclonal mouse anti-estradiol antibody is U.S. Biospacific, Monoclonal mouse anti-estriol antibody is U.S. Biostride, monoclonal mouse anti-biotin antibody is Jackson, U.S.A., and enterotoxin B and monoclonal mouse anti-enterotoxin B antibody are U.S. toxin technology, Ig
purchased from en. The fibrous body was purchased from Japan Vilene.

(2) Immobilization of the substance on the non-fluorescent carrier 1 Examples of the substance for immobilization include a complex of estradiol and bovine serum albumin, a complex of estriol and bovine serum albumin, a complex of biotin and bovine serum albumin, or enterotoxin. B was used. All solid phase immobilization was performed by natural adsorption. 5 mg of each of the various fibrous bodies was placed alone in 1 ml of physiological saline containing 100 μg of the above substance, and shaken at 37 ° C. for 2 hours. After this, 100
A solution prepared by dissolving mg of bovine serum albumin in 1 ml of PBS was prepared, and 0.1 ml of this solution was added to the above suspension, followed by shaking at the same temperature for 2 hours. After shaking, the carrier was washed with physiological saline, inserted into a measuring cell, and then attached to the device.

(3) Fluorescent Labeling of Antibody and Complex and Preparation of Sample All fluorescent labels are CY5 manufactured by Amsham Pharmacia.
A labeling kit was used. Each label is appropriately 1.0 m
It was diluted with physiological saline containing g / ml bovine serum albumin before use. The estradiol solution was dissolved in dimethylsulfoxide at a concentration of 1 μM, and when further diluted, a physiological saline solution containing bovine serum albumin at a concentration of 1.0 mg / ml was prepared. Biotin was prepared directly using physiological saline containing bovine serum albumin at a concentration of 1.0 mg / ml. When enterotoxin B was used, it was diluted with commercially available milk. Paddy water around Abiko City, Chiba Prefecture, or lake water from Teganuma, Japan was used as an environmental sample. Since the dilution is performed using milk and environmental samples, these samples can be directly detected, and highly accurate measurement can be performed.

The antigen, antibody or complex solution prepared by the above procedure was mixed in equal amounts in various combinations for the measurement. In addition, in the fully automatic fluorescence measurement device 7, the antigen or the fluorescence-labeled antibody was added to bovine serum albumin by 1.0 mg.
/ Saline as a buffer in the reservoir 17c as a buffer, and a certain amount of antigen, antibody, 1.0 mg / ml from each reservoir 17 by switching the flow path of the rotary valve 20 into the syringe pump 18. Physiological saline solution was collected. Thereafter, by switching the flow path of the rotary valve 20, the entire amount of the solution in the pump 18 is discharged to the mixing chamber 1.
Move to 9. In this operation, the amounts of the antigen and the buffer solution were varied so that the constant amount of the antibody solution and the total amount were constant, and standard samples having different antigen concentrations were prepared in different mixing chambers 19. The prepared standard sample was sent to the measurement cell 2 at a constant flow rate through the syringe pump 18 by sequentially switching the flow path of the rotary valve 20.

(4) Measuring Method The above-mentioned fully automatic fluorescence measuring device 7 was used for the measurement. The measurement cell 2 containing a complex of estradiol and bovine serum albumin, a complex of estriol and bovine serum albumin, a complex of biotin and bovine serum albumin, or a fibrous solid phase on which enterotoxin B is immobilized is sequentially placed in the fluorescence measuring device 7. The sample was attached, and a prepared sample corresponding to each solid phase was sent. The flow rate was changed by experiment, but the flow rate was 0.25 ml.
/ Min. By the solid-liquid contact, the unreacted label in the liquid reacts with the substance on the solid phase, and the amount of the label on the solid phase can be detected as the fluorescence intensity.

At this time, the fluorescence intensity can be measured as an intensity change value per time. This change value indicates the amount of the labeled substance on the solid phase that is accumulated by a fixed amount over time, and is proportional to the concentration of the unreacted label substance in the sample. Can decide. As for the change value, the fluorescence signal (excitation wavelength; 620 nm, measurement wavelength; 670 nm) was recorded on a computer every 1 second, and thereafter, the signal change value was shown every 5 seconds.

Example 1 Determination of Substance Concentration in Sample Based on Fluorescence Intensity Change per Time Using a measurement cell 2 in which a complex of estradiol and bovine serum albumin was immobilized, a mouse antibody labeled with various concentrations was used. In the solution containing the estradiol antibody, the change in the fluorescence intensity per time was determined independently (FIG. 4).
(A)). Thereafter, the rate of change of the fluorescence intensity per second obtained from the mouse anti-estradiol antibody solutions with different fluorescent labels at different concentrations was plotted against the antibody concentration. A positive linear relationship was obtained (FIG. 4B). This linear relationship indicated that the concentration of the antibody not bound to the antigen in the sample was determined from the change rate, and the antigen could be quantified based on this.

Therefore, estradiol was mixed at a concentration of 7.8, 31.2, 125, 500, or 2000 pM with a solution containing a mouse anti-estradiol antibody fluorescently labeled at a concentration of 100 pM, and the rate of change in fluorescence intensity per second was determined. Thereafter, the rate of change obtained from the antibody solution containing no estradiol used as a control was taken as 100%, and the rate of change determined for the antibody solution containing estradiol was calculated as the relative fluorescence intensity change rate. The results are shown in FIG. 4C as the relationship between the relative fluorescence intensity change rate and the estradiol concentration. As a result, it was found that the estradiol concentration in the sample could be determined from the relative fluorescence intensity change rate.

Example 2 Various samples (saline,
Determination of substances in milk and environmental samples)
Biotin, enterotoxin B and estriol were added at the indicated concentrations, respectively, and the substance was quantified from the change in relative fluorescence intensity. In each measurement, a measurement cell 2 in which a complex of biotin and bovine serum albumin, a complex of enterotoxin B and estriol and bovine serum albumin was fixed was used.

As a result, biotin in physiological saline was in the range of 1 nM to 400 nM (FIG. 5 (A)), enterotoxin B in milk was in the range of 30 pM to 7 nM (FIG. 5 (B)), paddy water and lakes and marshes. Estriol in water was quantified in the range of 2 pM to 1 nM (FIG. 5 (C)).

(Example 3) Quantification of Substances by Continuous Liquid Feeding A CY5 label at a concentration of 100 pM was attached to a measuring cell 2 containing a non-fluorescent carrier 1 on which a complex of estradiol and bovine serum albumin fixed to a fluorescence measuring device 7 was attached. A solution in which estradiol was mixed at the indicated concentration with a sample containing an anti-estradiol antibody was automatically prepared by the method described above. After this,
Each solution was continuously treated with 0.1% estradiol in decreasing order of concentration.
The solution was sent at 25 ml / min, and the fluorescence intensity was measured. As a result, a solution having a lower estradiol concentration in the sample showed a higher change in fluorescence intensity per time (FIG. 6).
(A)).

Then, the rate of change obtained from the solution containing no estradiol was defined as 100%, and the rate of change determined for the antibody solution containing estradiol was calculated as the rate of relative fluorescence intensity change. The results are shown in FIG. 6B as the relationship between the relative fluorescence intensity change rate and the estradiol concentration. As a result, it was found that the estradiol concentration in the sample could be determined from the relative fluorescence intensity change rate even by continuous liquid feeding.

(Example 4) Substance quantification by continuous liquid feeding using a measurement antibody and a control antibody A fluorescent-labeled anti-biotin antibody was used as a control antibody to obtain a 40 μl sample.
Solution A containing M and biotin as control antigens at a concentration of 100 nM, and Solution B prepared by adding a fluorescently labeled anti-estradiol antibody to Solution A at a concentration of 100 pM as a measurement antibody were prepared. In addition, 0, 8, 24, 74, 222, 66
6. Seven kinds of C solutions to which estradiol was added as a measurement antigen at a concentration of 2000 pM, and finally seven kinds of D liquids to which a solution containing 100 nM of biotin and an anti-estradiol antibody at a concentration of 100 pM were added with estradiol at the same concentration as the C solution. A liquid was prepared.

Using a measuring cell 2 in which a complex of estradiol and bovine serum albumin and a complex of biotin and bovine serum albumin were immobilized in a multiplex manner, the solutions were sequentially and continuously fed in order from A to D, and finally buffered. The liquid was sent. In addition,
A and B always selected the same solution, and C and D selected the solution with the same estradiol concentration. A total of seven combinations (sets) were continuously fed in the order of A to D. In addition, the measuring cell 2 was re-installed every time the set solution was sent.

The solutions A to E were as follows. A: Fluorescently labeled anti-biotin antibody 40 pM + biotin 100 nM B: Fluorescently labeled anti-biotin antibody 40 pM + biotin 100 nM +
Fluorescently labeled anti-estradiol antibody 100pM C: Fluorescently labeled anti-biotin antibody 40pM + biotin 100nM +
Fluorescently labeled anti-estradiol antibody 100pM + estradiol 0-2000pM D: Biotin 100nM + fluorescently labeled anti-estradiol antibody
100 pM + estradiol 0-2000 pM E: buffer solution When each solution set was sequentially and continuously fed, the fluorescence intensity was traced in real time (FIG. 7). In the figure,
The estradiol concentration was 2000, 666, 222, 7 from the bottom of the solid line.
4, 24, 8, 0 pM. As a result, in all the sets, traces having almost the same fluorescence intensity were obtained for the solutions A and B. However, in C and D solutions,
For each set, the change in fluorescence intensity per time became smaller as the estradiol concentration increased. From the above, it was found that the measurement antibody and the control antibody can be used in combination.

Therefore, the change rate obtained when the solution D did not contain estradiol was set to 100%, and the change rate determined for the solution D containing estradiol was calculated as the relative fluorescence intensity change rate. FIG. 8 shows the relationship between the relative fluorescence intensity change rate and the estradiol concentration. As a result, it was found that the estradiol concentration in the sample could be determined from the relative fluorescence intensity change rate.

The changes in the fluorescence intensity per unit time obtained when the solutions A to D are sent are derived from the following reactions. A: reaction of control antibody when control antigen is always present at a constant concentration, B: reaction of control antibody when control antigen is always present at a constant concentration and reaction of measurement antibody when no measurement antigen is present, C: The reaction of the control antibody when the control antigen is always present at a constant concentration and the reaction of the measurement antibody when the unknown concentration of the measurement antigen is present. D: The reaction of the measurement antibody when the unknown concentration of the measurement antigen is present.

That is, if an appropriate control antigen is set and solution A and solution B are prepared with physiological saline or the like, the intensity change due to the reaction between the normal control antibody and the control antigen is obtained in solution A, and the solution B in solution B The intensity change due to the reaction between the normal control antibody and the control antigen and the reaction of the measurement antibody in the absence of the measurement antigen is obtained. These intensity changes are important for calibration, and are intensity changes obtained when an ideal antigen-antibody reaction occurs.

On the other hand, when the solution C and the solution D are prepared using a sample to be measured, in the solution C, the intensity change due to the reaction between the control antibody and the control antigen and the reaction between the control antibody and the measurement antibody in accordance with the concentration of the existing measurement antigen is obtained. In solution D, an intensity change due to the reaction with the measurement antibody in accordance with the concentration of the measurement antigen present is obtained. Here, when the change in intensity of D is subtracted from the change in intensity of C, the remaining change is derived from the reaction between the control antibody and the control antigen. If the antigen-antibody reaction of the control antibody occurs normally, the change in intensity of A Should match. That is, the change in D is equal to the change in intensity of C minus the change in intensity of A.

Then, when the change in D calculated indirectly from the changes in C and A was compared with the change in D determined directly,
Almost the same calibration curve was obtained (FIG. 8). In other words, when a similar measurement is performed using a sample, if a result in which the actual measurement value and the calculated value match is obtained, this indicates that the measurement is normally performed. Conversely, when the measured values do not match, it can be predicted that a normal antigen-antibody reaction has not occurred, and it can be determined that a substance that nonspecifically inhibits the antigen-antibody reaction is mixed in the sample. In other words, by the method using both the measurement antibody and the control antibody, the measurement antibody performs the detection itself, and the control antibody performs the presence or absence of non-specific inhibition. Can be performed.

[0104]

As is apparent from the above description, according to the fluorescence measuring device of the first aspect, since the carrier is made of a fibrous body, it can be compared with a conventional test tube wall or the like used as a solid phase. Thus, the immobilization effective area per volume can be significantly increased. Further, since the carrier is a fibrous body and has excellent light transmittance and the carrier is non-fluorescent and does not generate fluorescence, it can be suitable for measurement at various fluorescent wavelengths. Furthermore, since the carrier is a fibrous body, excellent permeability of the aqueous solution is obtained and a rapid flow rate is obtained, so that a dynamic measurement site can be realized and uniform solid-liquid contact can be achieved. For these reasons, the measurement time by the fluorescence measurement device can be reduced, and the measurement accuracy can be increased.

Since the measurement cell is formed by incorporating the non-fluorescent carrier into the support, when reacting the substance with the non-fluorescent carrier, it is sufficient to introduce the substance into the measurement cell and the operability is good. Further, when the non-fluorescent carrier is replaced, it is sufficient to replace the entire measuring cell, so that the handling of the non-fluorescent carrier can be facilitated. In addition, since the optical system can be miniaturized by employing the measuring cell, a portable and small fluorescence measuring device can be configured.

Further, by mounting the measurement cell on the cell holder and introducing the substance into the measurement cell, the substance can react with the non-fluorescent carrier, so that the operability can be improved.

According to the fluorescence measuring apparatus of the present invention, since the fluorescent light generating portion of the measuring cell is darkened by the cell holder, even minute fluorescent light can be measured with high accuracy without being affected by external light. can do. Further, since each optical path of light and fluorescence entering and exiting the measurement cell is secured by the communicating portion, fluorescence can be generated and detected while the portion where the fluorescence is generated is kept dark by the cell holder.

Further, according to the fluorescence measuring device of the third aspect, since the light beam can be focused on the flow channel, the fluorescent label held on the non-fluorescent carrier in the flow channel by irradiating the measuring cell with the light beam. The body can be effectively excited with fluorescence.

According to the fluorescence measuring apparatus of the fourth aspect, since the optical axis of the convex lens portion and the optical axis of the light emitting means can be made to coincide with each other by the positioning means, the light beam can be surely focused on the flow path. Become like

Further, according to the fluorescence measuring device of the fifth aspect, the optical axis of the convex lens portion and the optical axis of the light emitting means can be surely matched with a simple configuration.

According to the sixth aspect of the present invention, according to the fluorescence measuring apparatus, all the steps of supplying the sample to the measuring cell and measuring the fluorescence can be automated. The workability of the measurement can be greatly improved as compared with the case where only the detection of is automatically performed. In addition, the compactness of the optical system by the measurement cell and the simplification of the adoption of the mixing chamber of the flow path system make it possible to configure a portable and compact fluorescence measuring device.

Further, according to the fluorescence measuring apparatus of the present invention, in addition to the supply of the sample to the measuring cell and the measurement of the fluorescence, the preparation of the standard sample can be automated.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a main part of a fluorescence measuring device according to the present invention.

FIG. 2 is a schematic diagram showing one embodiment of a fluorescence measuring device.

FIG. 3 is a drawing showing a measuring cell, wherein (A) is AA
A central longitudinal sectional view along a line, (B) shows a front view, and (C) shows a side view.

FIG. 4 is a micrograph (magnification: 200 ×) as a drawing-substituting photograph showing the structure of a non-fluorescent carrier;
(B) shows a polyolefin + polyester, and (C) shows a polyolefin.

5A and 5B are graphs showing results of determination of a substance concentration in a sample based on a change in fluorescence intensity per unit time. FIG.
0, 40, 60, 80, 100, 150, 200 pM), (B) is (A)
And (C) is the relationship between the relative fluorescence intensity change and the antigen concentration.

FIG. 6 shows the results of substance quantification in various samples, (A)
Is the determination of biotin in physiological saline, (B) is the determination of enterotoxin B in milk, (C) is the determination of estradiol in lake water and paddy water (in the figure, ●; lake water, ▲;
Paddy water).

FIG. 7 shows the results of substance quantification by continuous liquid feeding, (A)
(A) is a time-dependent change in fluorescence intensity when a solution containing a fluorescent-labeled anti-estradiol antibody and estradiol at the indicated concentration is continuously fed, and (B) is a relationship between the estradiol concentration and the relative fluorescence intensity change rate.

FIG. 8 is a graph showing a change over time in fluorescence intensity during continuous liquid feeding using a measurement antibody and a control antibody.

FIG. 9 shows the results of estradiol quantification by continuous liquid feeding using a measurement antibody and a control antibody. In the figure, ●: measured values, ▲: calculated values.

[Description of Signs] 1 Non-fluorescent carrier 2 Measurement cell 3 Support 5 Convex lens unit 7 Fluorescence measurement device 13 Optical unit 14 Flow path unit 15 Sample supply unit 16 Control unit 17 Reservoir 18 Syringe pump 19 Mixing chamber 20 Rotary valve

Continuation of the front page (71) Applicant 599145122 Sapidine Instrument Inc. Sapidyne Instrument Inc. 83706-6700, Idaho, USA United States Park Center Boulevard # 445 967 967 E. Park Center Bl vd. # 445, Boise ID 83706-6700, USA (72) Inventor Naoya Omura 1646 Abiko, Abiko-shi, Chiba Japan Electric Power Research Institute Abiko Research Institute (72) Inventor Steve Lucky 83706-6700 Boise, Idaho United States of America Boise, E. Park Center Boulevard # 445 967 Sapidine Instrument Inc. F-term (reference) 2G043 AA01 BA16 CA03 DA02 DA05 DA09 EA01 GA07 GB01 GB05 GB17 HA01 LA01 MA01 NA11 2G057 AA04 AC01 BA01 DA01 DA06 DB01 DC07 FA05

Claims (7)

[Claims] 1. A measuring cell in which a non-fluorescent carrier made of a fibrous body is incorporated in a flow path provided in a support formed of a light-transmitting material; a cell holder to which the measuring cell can be attached; A light emitting unit that irradiates a light beam to the measurement cell to excite fluorescence in a fluorescent label held by the non-fluorescent carrier in the flow path, and a detecting unit that detects the fluorescence excited by the fluorescent label. A fluorescence measurement device comprising: 2. The cell holder covers at least a portion of the held measurement cell where the fluorescence is generated and darkens the fluorescence so that the fluorescence can be detected. 2. The fluorescence measuring apparatus according to claim 1, further comprising a communicating portion for securing an optical path of the light beam and an optical path of the fluorescence from the measurement cell to the detection unit. 3. The fluorescence measuring apparatus according to claim 2, wherein said support of said measuring cell has a convex lens portion for condensing a light beam from said light emitting means on said flow path. 4. The measurement cell and the cell holder,
4. The fluorescence measuring apparatus according to claim 3, further comprising a positioning unit that matches an optical axis of the convex lens unit with an optical axis of the light emitting unit. 5. The positioning means, wherein a positioning surface formed on an outer peripheral portion of the support of the measurement cell and a positioning surface formed on the cell holder and the positioning surface of the measurement cell abut on each other. The fluorescence measuring device according to claim 4, further comprising a reference surface. 6. An optical unit having the cell holder, the light emitting unit, and the detecting unit, a supply / drainage unit detachably connected to the measurement cell, for supplying a sample or the like and passing the liquid, and the optical unit. And a control unit for controlling the operation of the supply / drainage means, wherein the supply / drainage means is a pump having a plurality of fluid reservoirs and a storage unit, a flow path unit having a plurality of mixing chambers and a rotary valve, and a sample. A supply unit, and the sample supply unit and the plurality of fluid reservoirs are each switchably connected to the pump via a rotary valve, and the pump is connected to the plurality of mixing chambers via the rotary valve. The mixing chambers are switchably connected to each other, and the plurality of mixing chambers are switchably mounted on the cell holder via the rotary valve. The fluorescence measurement device according to any one of claims 2 to 5, wherein the fluorescence measurement device is connected to the measurement cell. 7. The liquid in the fluid reservoir is supplied to the reservoir of the pump through the rotary valve, and the liquid in the reservoir is supplied to the mixing chamber through the rotary valve. A plurality of liquids are supplied to prepare a standard sample for a calibration curve, the standard sample is supplied from the mixing chamber to the reservoir of the pump via the rotary valve, and the standard sample is supplied from the reservoir. The fluorescence measurement device according to claim 6, wherein the liquid is supplied to the measurement cell via the rotary valve.