JP2009287999A - Fluorescence detecting system and concentration measuring method using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence detecting system capable of correcting the quantity of fluorescence, which changes in dependence on a use time or an external temperature, in a real time, and a concentration measuring method using it. <P>SOLUTION: A fluorescence analyzing system 100 includes a fluorescence analyzing optical multiplexer/demultiplexer 56 equipped with a long-pass filter with a cut-off wavelength λ(λ<SB>1</SB><λ<λ<SB>2</SB>) for guiding the light from an incident end 56a to an emitting end 56b and guiding the light from the emitting end 56b to an emitting end 56c, the light source 53 for exciting light connected to the incident end 56a and emitting exciting light with a main wavelength λ<SB>1</SB>, the detector 54 connected to the emitting end 56c to detect fluorescence with a main wavelength λ<SB>2</SB>, a lens 40 for condensing exciting light incident from one end to the sample in a flow channel 204 to irradiate the sample and condensing the fluorescence incident from the other end, and an optical fiber 103 connected to the emitting end 56b and one end of the lens 40. The optical fiber 103 guides the light caused by exciting light to the emitting end 56b simultaneously with fluorescence. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a fluorescence detection system and a concentration measurement method using the same.

  Conventionally, there has been known a method for measuring the concentration of a measurement substance contained in a sample solution by irradiating the measurement substance with excitation light by a fluorescence detection system as shown in FIG. 9 and measuring the fluorescence emitted from the measurement substance at that time. (For example, refer to Patent Document 1).

  However, when the fluorescence is measured with the fluorescence detection system in a state where external light enters, light other than the fluorescence to be detected (hereinafter referred to as “base light”) is also guided to the detector simultaneously with the fluorescence. . That is, as shown by the solid line in FIG. 10, the detection signal obtained in the fluorescence measurement is in a state where the detection signal of the fluorescence to be measured is on the detection signal of the base light indicated by the dotted line. For this reason, fluorescence detection cannot be performed with high sensitivity. This problem can be remedied by installing the detector in a dark room so that the base light does not enter the detector.

On the other hand, external light other than excitation light may enter the substance to be measured, and fluorescence generated thereby may become the base light. Since this base light cannot be removed even if the detector is installed in the dark room as described above, the detection signal (dotted line 1101 in FIG. 11) obtained in the fluorescence measurement is the fluorescence generated by the external light. The detection signal of the fluorescence that is the measurement target is put on the detection signal of the base light (thick line 1102 in FIG. 11) generated by the above. As a method for removing such base light, a method is known in which excitation light is modulated and only a signal synchronized with the modulation is detected by a lock-in amplifier.
JP 2005-30830 A

  However, when an LD or LED is used as the excitation light source, the amount of excitation light varies depending on the light emission time and the external temperature. Further, the detection sensitivity of the light receiving element in the detector that receives the fluorescence also varies depending on the use time and the external temperature.

  For example, as shown in FIG. 12, even if the set value of the excitation light amount is maintained, the excitation light amount 1201 obtained immediately before the start of measurement can change to the excitation light amount 1200 after a predetermined time has elapsed. In this case, the detection signal fluctuates even if the detector is installed in the dark room and the intensity of the excitation light is modulated by the lock-in amplifier, and the fluorescence measurement of the sample made of the fluorescent material having the same concentration is performed. End up. Specifically, the detection signal obtained by the fluorescence measurement when the excitation light amount 1200 is irradiated is indicated by a dotted line 1202, whereas the detection signal obtained by the fluorescence measurement when the excitation light amount 1201 is irradiated is a dashed line. This is indicated by 1203.

  Conventionally, in order to carry out accurate fluorescence detection measurement in consideration of such fluctuations, if more accuracy is required every predetermined time, a correction operation called calibration for the excitation light source and detector is performed for each measurement. It took a lot of time to measure.

  An object of the present invention is to provide a fluorescence detection system capable of correcting in real time the amount of fluorescence that varies depending on the use time and external temperature, and a concentration measurement method using the same.

To achieve the above object, a fluorescent detection system of claim 1, wherein analyzes the dominant wavelength lambda ₂ of the fluorescence dominant wavelength lambda ₁ of the excitation light is generated from the sample irradiated (λ _2> λ ₁₎ fluorescent In the detection system, an optical multiplexer / demultiplexer that guides the light from the incident end to the first exit end, the light from the first exit end to the second exit end, and the entrance end, and An excitation light source that emits excitation light, a detector connected to the second emission end and receiving the fluorescence, and the excitation light incident from one end is condensed and applied to the sample and from the other end A lens for condensing the incident fluorescence; an optical fiber connected to the first emission end; and one end of the lens; and the detector receives light caused by the excitation light simultaneously with the fluorescence. It is characterized by doing.

  The fluorescence detection system according to claim 2 is the fluorescence detection system according to claim 1, wherein the optical multiplexer / demultiplexer includes a filter interposed between two lenses, and the diameter of the two lenses is equal to that of the filter. It is characterized by being larger than the diameter.

  The fluorescence detection system according to claim 3 is the fluorescence detection system according to claim 1, wherein the optical multiplexer / demultiplexer is a fusion coupler.

  The fluorescence detection system according to claim 4, wherein the optical fiber guides light caused by the excitation light to the first emission end simultaneously with the fluorescence. And

  The fluorescence detection system according to claim 5 is the fluorescence detection system according to claim 4, wherein a reflection element is introduced into a part of the optical fiber, and light caused by the excitation light is transmitted by the reflection element. A part of the excitation light is reflected light.

  A fluorescence detection system according to a sixth aspect is the fluorescence detection system according to the fifth aspect, wherein the reflective element is an element utilizing reflection at an end face thereof.

  The fluorescence detection system according to claim 7 is the fluorescence detection system according to claim 6, wherein the reflection at the end face is a planar reflection of glass.

The fluorescence detection system according to claim 8 is the fluorescence detection system according to any one of claims 4 to 7, wherein the optical multiplexer / demultiplexer is a long path having a cutoff wavelength λ (λ ₁ <λ <λ ₂ ). A filter comprising a filter is provided.

Fluorescence detection system according to claim 9, wherein, in the fluorescence detection system of claim 8, wherein the transmittance at a wavelength lambda ₁ of the filter is -20dB or less.

The fluorescence detection system according to claim 10 is the fluorescence detection system according to any one of claims 4 to 7, wherein the optical multiplexer / demultiplexer is a short circuit having a cutoff wavelength λ (λ ₁ <λ <λ ₂ ). A filter comprising a pass filter is provided.

Fluorescence detection system according to claim 11, wherein, in the fluorescence detection system according to claim 10, characterized in that the reflectance at a wavelength lambda ₁ of the filter is -20dB or less.

  The fluorescence detection system according to claim 12 is the fluorescence detection system according to any one of claims 8 to 11, wherein the filter is interposed between two lenses, and the diameter of the two lenses is It is larger than the diameter of the filter.

  A fluorescence detection system according to a thirteenth aspect is the fluorescence detection system according to any one of the fourth to seventh aspects, wherein the optical multiplexer / demultiplexer is a fusion coupler.

  The fluorescence detection system according to claim 14 is the fluorescence detection system according to claim 4, wherein the light caused by the excitation light is Raman scattered light from the optical fiber.

  A fluorescence detection system according to a fifteenth aspect is the fluorescence detection system according to the fourteenth aspect, characterized in that the Raman scattered light is due to Stokes scattering.

  In order to achieve the above object, the concentration measurement method according to claim 16 is the concentration measurement method using the fluorescence detection system according to any one of claims 1 to 15, wherein the first concentration is set at a reference time. A first acquisition step of irradiating the substance to be measured with the excitation light and acquiring the light guided to the detector at that time as a first detection signal; and a second concentration at the reference time A second acquisition step of irradiating the substance to be measured with the excitation light and acquiring the light guided to the detector at that time as a second detection signal; and at the reference time, the excitation light A third acquisition step of acquiring a third detection signal when only the light resulting from is guided to the detector, and a creation step of generating a calibration curve based on the first to third detection signals; , A predetermined time has elapsed since the reference time A fourth acquisition step of irradiating the substance to be measured having an unknown concentration with the excitation light and acquiring the light guided to the detector at that time as a fourth detection signal; Sometimes, when only light caused by the excitation light is guided to the detector, a fifth acquisition step of acquiring a fifth detection signal, and the third detection signal and the fifth detection signal A correction step for correcting the fourth detection signal, and a calculation step for calculating the value of the unknown concentration by applying the corrected fourth detection signal to the generated calibration curve. And

According to the fluorescence detection system of claim 1, in the fluorescence detection system for analyzing the fluorescence (λ ₂ > λ ₁ ) of the main wavelength λ ₂ generated from the sample irradiated with the excitation light of the main wavelength λ ₁ , the incident end An excitation light source that emits excitation light by connecting the light from the first output end to the first output end, the optical multiplexer / demultiplexer that guides the light from the first output end to the second output end, and the input end A detector for receiving fluorescence, condensing and irradiating the sample with excitation light incident from one end, and condensing the fluorescence incident from the other end; The detector is equipped with an output end and an optical fiber connected to one end of the lens, and the detector receives the light caused by the excitation light simultaneously with the fluorescence, so that the amount of fluorescence that varies depending on the usage time and external temperature can be measured in real time. Can be corrected.

  According to the fluorescence detection system of claim 2, the optical multiplexer / demultiplexer includes a filter interposed between the two lenses, and the diameter of the two lenses is larger than the diameter of the filter, which is caused by the excitation light. As light, light from the optical fiber connected to one end of the lens and / or light from the light source for excitation light can be reliably guided to the detector via the optical multiplexer / demultiplexer.

  According to the fluorescence detection system of the third aspect, since the optical multiplexer / demultiplexer is a fusion (melting type) coupler, the light from the optical fiber connected to one end of the lens and / or the light caused by the excitation light The light from the excitation light source can be reliably guided to the detector via the optical multiplexer / demultiplexer.

  According to the fluorescence detection system of claim 4, since the optical fiber guides the light caused by the excitation light to the first emission end simultaneously with the fluorescence, the light caused by the excitation light is reliably detected simultaneously with the fluorescence. Can be guided to the vessel.

  According to the fluorescence detection system of claim 5, a reflection element is introduced into a part of the optical fiber, and the light resulting from the excitation light is a light in which a part of the excitation light is reflected by the reflection element. Therefore, the light resulting from the excitation light can be reliably guided to the detector simultaneously with the fluorescence.

  According to the fluorescence detection system of the sixth aspect, since the reflection element is an element using reflection at the end face, the reflection element can be easily created.

  According to the fluorescence detection system of the seventh aspect, since the reflection at the end face is a flat reflection of glass, a reflecting element can be easily produced by processing the end face of the optical fiber or the like.

According to the fluorescence detection system of the eighth aspect, since the optical multiplexer / demultiplexer includes a filter composed of a long-pass filter having a cutoff wavelength λ (λ ₁ <λ <λ ₂ ), the reflection element introduced into the optical fiber The partially reflected excitation light can be guided to the detector through the optical multiplexer / demultiplexer.

According to the fluorescence detection system of the ninth aspect, since the transmittance at the wavelength λ ₁ of the filter composed of the long pass filter is −20 dB or less, the excitation light partially reflected by the reflection element introduced into the optical fiber is The light can be reliably guided to the detector through the optical multiplexer / demultiplexer.

According to the fluorescence detection system of claim 10, since the optical multiplexer / demultiplexer includes a filter composed of a short-pass filter having a cutoff wavelength λ (λ ₁ <λ <λ ₂ ), the reflecting element introduced into the optical fiber Thus, the excitation light partially reflected by can be reflected by the optical multiplexer / demultiplexer and guided to the detector.

According to the fluorescence detection system of claim 11, since the reflectance at the wavelength λ ₁ of the filter composed of the short pass filter is −20 dB or less, the excitation light partially reflected by the reflective element introduced into the optical fiber. Can be prevented from being excessively guided to the detector.

  According to the fluorescence detection system of claim 12, the filter is interposed between two lenses, and the diameter of the two lenses is larger than the diameter of the filter. Excitation light from the incident end can also be guided to the detector through the optical multiplexer / demultiplexer.

  According to the fluorescence detection system of the thirteenth aspect, since the optical multiplexer / demultiplexer is a fusion coupler, the excitation light from the incident end also passes through the optical multiplexer / demultiplexer as the light caused by the excitation light. Then, the light can be guided to the detector.

  According to the fluorescence detection system of claim 14, since the light caused by the excitation light is Raman scattered light from the optical fiber, the light caused by the excitation light is reliably guided to the detector simultaneously with the fluorescence. be able to.

  According to the fluorescence detection system of claim 15, since the Raman scattered light is due to Stokes scattering, the wavelength of the Raman scattered light can be substantially the same as the wavelength of the fluorescence, which is caused by the excitation light. As light, it can be more reliably guided to the detector simultaneously with fluorescence.

  According to the concentration measuring method of the sixteenth aspect, in the concentration measuring method using the fluorescence detection system, at the reference time, the substance to be measured having the first concentration is irradiated with the excitation light, and then introduced to the detector. The emitted light is acquired as a first detection signal, and at the time of reference, the substance to be measured having the second concentration is irradiated with excitation light, and the light guided to the detector at that time is converted into the second detection signal. Obtained as a detection signal, and at the time of reference, when only light caused by the excitation light is guided to the detector, a third detection signal is obtained, and a calibration curve is created based on the first to third detection signals. At the time of measurement after a predetermined time from the reference time, the measured substance having an unknown concentration is irradiated with excitation light, and the light guided to the detector at that time is acquired as a fourth detection signal. The fifth detection when only the light caused by the excitation light is guided to the detector Is obtained, the fourth detection signal is corrected based on the third detection signal and the fifth detection signal, and the corrected fourth detection signal is applied to the generated calibration curve, and the value of the unknown concentration is obtained. Therefore, based on the detection signal obtained by the fluorescence detection system, the amount of fluorescence that varies depending on the use time and the external temperature can be corrected in real time.

Fluorescent present inventors to analyze result of extensive studies to achieve the above object, the dominant wavelength lambda ₂ of the fluorescence dominant wavelength lambda ₁ of the excitation light is generated from the sample irradiated (λ _2> λ ₁₎ In the detection system, an optical multiplexer / demultiplexer that guides light from the incident end to the first exit end, and light from the first exit end to the second exit end, and the entrance end are connected to the excitation light. A light source for excitation light to be emitted, a detector connected to the second emission end, and receiving fluorescence, and condensing and irradiating the sample with excitation light incident from one end and condensing fluorescence incident from the other end And an optical fiber connecting one end of the lens and the first emission end, and when the detector receives the light caused by the excitation light simultaneously with the fluorescence, the amount of fluorescence that varies depending on the use time and the external temperature Has been found to be corrected in real time.

  The present invention has been made based on the above findings.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a diagram showing a schematic configuration of a fluorescence analysis system according to an embodiment of the present invention.

  In FIG. 1, a fluorescence analysis system 100 includes a fluorescence analysis optical module 100a and an optical fiber with a lens (hereinafter referred to as a “probe”) that collects excitation light on a sample solution in a flow path 204 inside the fluorescence analysis chip 20. ) 50 and a sample stage 21 on which the fluorescence analysis chip 20 is placed.

  The sample stage 21 includes a moving mechanism (not shown) that moves relative to the probe 50 to position the sample. In the embodiment of the present invention, the sample stage 21 is provided with a moving mechanism. However, the present invention is not limited to this as long as the sample can be positioned, and the probe 50 is also relative to the sample stage 21. A moving mechanism that moves may be provided.

Fluorescence analysis optical module 100a includes a pumping light source 53 composed of an LED for emitting a dominant wavelength lambda ₁ of the excitation light, the excitation light of the main wavelength lambda ₂ generated from the sample by irradiating through the probe 50 fluorescent Fluorescence analysis optical multiplexer / demultiplexer 56 that transmits (λ ₂ > λ ₁ ), detector 54 that receives this fluorescence, excitation light source 53, and incident end 56 a of fluorescence analysis optical multiplexer / demultiplexer 56 are connected. The optical fiber 106 to be connected, the optical fiber 103 having a length of 30 cm connecting the probe 50 and the emission end 56b of the fluorescence analysis optical multiplexer / demultiplexer 56, and the detector 54 and the emission end 56c of the fluorescence analysis optical multiplexer / demultiplexer 56 are provided. And an optical fiber 108 to be connected. By configuring the optical module for fluorescence analysis 100a as described above, the excitation light can be guided to the sample and the fluorescence from the sample can be guided to the optical multiplexer / demultiplexer 56 for fluorescence analysis.

Further, when an LD is used instead of an LED as the excitation light source 53 as in the present embodiment, the return excitation light λ ₁ from the fluorescence analysis optical multiplexer / demultiplexer 56 is used as the excitation light source 53. An isolator may be provided to prevent entry. Thereby, the output stability as a light source of LD can be raised.

  Thus, since the optical module for fluorescence analysis 100a uses an optical fiber to transmit light between the above-described devices constituting the module, the module can be simplified and reduced in size.

  The probe 50 includes a ferrule 104 that holds the tip 103a at the other end of the optical fiber 103, a lens 40 that is optically connected to the tip 103a of the optical fiber 103, and a tube 105 that fixes the ferrule 104 and the lens 40. It consists of. The distal end portion 103a of the optical fiber 103 is processed so as to reflect a part of the excitation light. Thereby, a reflective element can be easily and reliably introduced into the optical fiber 103. For example, when the optical fiber 103 is made of silica glass, approximately 3.5% of the excitation light returns into the optical fiber 103 with the above configuration. On the other hand, when the tip portion 103 a of the optical fiber 103 is obliquely polished and has a non-reflective coating, only about 0.01% of the excitation light returns into the optical fiber 103.

  The excitation light source 53 is connected to a lock-in modulation circuit 109, and the lock-in modulation circuit 109 performs On-Off modulation (for example, rectangular modulation) of the excitation light source 53 at 100 Hz to 10 KHz. Thereby, the detection sensitivity can be reliably increased.

  The lock-in modulation circuit 109 is connected to a lock-in amplifier, and performs detection by synchronizing with the modulation (lock-in) of the excitation light source 53. Thereby, measurement accuracy can be raised more.

  In the embodiment of the present invention, the lock-in modulation circuit 109 and the lock-in amplifier are used. However, the present invention is not limited to this as long as it is a light modulation mechanism that can increase the detection sensitivity by removing the influence of external light. For example, FFT may be used.

  FIG. 2 is a schematic cross-sectional schematic diagram of the fluorescence analyzing optical multiplexer / demultiplexer 56 in FIG.

  In FIG. 2, an optical multiplexer / demultiplexer 56 for fluorescence analysis includes a rod lens 500, a filter 501 deposited thereon, and a rod lens 502 fixed to the filter 501 with an adhesive in order from the output ends 56a and 56b. Are arranged in series, and these are integrally formed. Thereby, the optical multiplexer / demultiplexer 56 for fluorescence analysis can be made into a bonded structure, and can be made compact. Alternatively, the filter 501 may be formed on a glass substrate, and the glass substrate may be disposed between the rod lens 500 and the rod lens 502.

  The rod lenses 500 and 502 are gradient index cylindrical rod lenses provided with a refractive index gradient so that the refractive index decreases from the center toward the outside. As a result, the two end surfaces of the entrance surface and the exit surface can be made into a plane perpendicular to the optical axis direction, and assembly such as lens coupling can be facilitated. In addition, since the rod lenses 500 and 502 are cylindrical, they can be easily stored in a cylindrical holder, and the optical axis can be easily aligned.

The filter 501 is a dielectric multilayer film in which a layer (L) made of SiO ₂ or the like having a low refractive index and a layer (H) made of TiO ₂ , ZrO ₂ , Ta ₂ O ₅ or the like having a high refractive index are laminated in multiple layers. The cutoff wavelength λ is larger than λ _{1 and} smaller than λ ₂ . That is, the filter 501 is a so-called long pass filter. More specifically, the filter 501 according to the present embodiment is characterized in that the transmittance of light having a wavelength λ ₁ is −20 dB (about 1%) or less and the transmittance of light having a wavelength λ ₂ is −3 dB. Or more (97 to 50%). More preferably, the transmittance of the wavelength lambda ₁ of light than -30 dB, more preferably not less than -35 dB.

Here, the entrance end 56a and the exit end 56c are disposed on the upper side of the rod lens 500 in FIG. 2, and the exit end 56b is disposed on the lower side. Here, in the fluorescence analyzing optical multiplexer / demultiplexer 56, when the filter 501 is not interposed, light travels along the optical path indicated by the dotted line 503 from the incident end 56a, and light travels along the optical path indicated by the solid line 504 from the exit end 56b. However, in this embodiment, since the excitation light having the principal wavelength λ ₁ is incident from the incident end 56a, about 99% of the light is reflected by the filter 501 and guided to the output end 56b. Part of the transmitted excitation light exits from the output end 56d through an optical path indicated by a dotted line 503. With this configuration, it is possible to reliably block the light from the excitation light source 53 from directly entering the detector 54 via the fluorescence analysis optical multiplexer / demultiplexer 56.

The light incident from the output end 56b via the probe 50 is composed of fluorescence generated from the sample and excitation light partially reflected by the tip portion 103a of the optical fiber 103. Of these, the dominant wavelength of the fluorescence Λ ₂ , 97% to 50% of the light is transmitted through the filter 501 and guided to the output end 56c. On the other hand, since the main wavelength of the portion reflected excitation light is lambda _1, while its approximately 99% by the filter 501 is guided to the incident end 56a is reflected, from about 1% remaining filters 501 The light is transmitted and guided to the output end 56c. Thereby, the fluorescence is guided to the detector 54, and at the same time, a part of the excitation light (hereinafter referred to as “excitation base light”) as light other than the fluorescence to be detected can be guided to the detector 54. it can.

  With the above configuration, the fluorescence analysis system 100 can secure the intensity of the fluorescence detection signal, and can guide the excitation base light to the detector 54 simultaneously with the fluorescence. That is, in this system, when a sample that does not contain a fluorescent substance, which is an object to be measured, is passed through the flow path 204 and irradiated with excitation light, it is obtained when no measurement is performed as shown in FIG. A detection signal 300 indicated by a dotted line in which a detection signal 303 of excitation base light is placed on a detection signal 301 (hereinafter referred to as “BKG signal”) is measured. On the other hand, when a sample containing a fluorescent substance as a measurement object flows through the flow path 204 and is irradiated with excitation light, the BKG signal 301 is detected as a sum of fluorescence and excitation base light as shown in FIG. A detection signal 302 indicated by a dotted line on which the signal 304 is carried is measured.

  Here, the detection signals 300 and 302 can be removed from the BKG signal 301 by performing measurement in synchronization with the excitation light using a lock-in amplifier. Hereinafter, the signal obtained by removing the BKG signal 301 from the detection signal 300 in FIG. 3 is referred to as a “base signal”. This base signal is attributed only to the excitation base light.

In the present embodiment, the transmittance of the filter 501 with respect to the wavelength λ ₁ is controlled to be higher than usual. However, the present invention is not limited to this as long as a part of the excitation light can be guided to the output end 56c. is not.

For example, a filter 501 whose cutoff wavelength λ is shifted to the shorter wavelength side may be used. Further, as shown in FIG. 13, the filter 501 made of a long-pass filter may be changed to a filter 503 made of a short-pass filter, and the connection may be reversed. Here, the reverse connection is that the excitation light source 53 is connected to the output end 56c of the fluorescence analyzing optical multiplexer / demultiplexer 56, the probe 50 is connected to the output end 56b, and the detector 54 is connected to the input end 56a. To do. In this case, the reflectance at the wavelength λ ₁ of the filter 503 formed of a short pass filter with respect to the excitation light partially reflected from the distal end portion 103 a of the optical fiber 103 is −20 dB (about 1%) or less. Thereby, it is possible to prevent the excitation light partially reflected by the distal end portion 103 a of the optical fiber 103 from being excessively guided to the detector 54. More preferably, the reflectivity at the wavelength lambda ₁ of the filter 503 is above -30 dB, more preferably not less than -35 dB. As a result, a part of the excitation light as the excitation base light can be reflected on the detector 54 by the optical multiplexer / demultiplexer 56 for fluorescence analysis and reliably guided. Further, a band pass filter or a notch pass filter may be used instead of the filter 501 made of the long pass filter and the filter 503 made of the short pass filter.

  A branching coupler may be used as the optical multiplexer / demultiplexer for fluorescence analysis. As this branching coupler, a fusion coupler 1400 (FIG. 14) prepared by fusing and stretching two optical fibers, or an optical waveguide coupler in which an optical circuit is formed on a Si substrate or a quartz substrate is used. be able to. In this case, the excitation base light guided to the detector 54 via the fusion (melting) coupler 1400 includes not only the excitation light partially reflected by the distal end portion 103a of the optical fiber 103 but also the excitation light source. The excitation light guided directly from 53 to the fusion (melting) coupler 1400 is also included.

  Further, in the present embodiment, the front end portion 103a of the optical fiber 103 is flat-polished, but a reflective element is introduced into a part of the optical fiber 103, whereby a part of the excitation light is guided to the output end 56b. The configuration of the reflective element and the introduction location thereof are not limited to the present embodiment as long as the configuration can emit light.

  As described above, according to the fluorescence detection system 100 having the configuration shown in FIGS. 1, 2, 13, and 14, it is partially reflected by the tip 103 a of the optical fiber 103 simultaneously with the fluorescence generated from the sample. It is possible to guide the light caused by the excitation light composed of the excitation light and the like to the detector 54, and as a result, use a part of the excitation light guided to the detector 54 as the excitation base light. The amount of fluorescence that varies depending on the external temperature can be corrected in real time as described below.

  FIG. 4 is a graph used to explain a method for correcting the detection signal at the time of fluorescence measurement in real time. (A) shows the relationship between the lock-in detection signal and the excitation light amount at each FITC concentration. Indicates the relationship between the FITC fluorescence signal and the base signal at each FITC concentration. In this embodiment, the excitation light amount refers to the output of excitation light emitted from the excitation light source 53.

  In the following measurement, the sample in the channel 204 has a FITC (Fluorescein isothiocyanate) concentration (hereinafter referred to as “FITC concentration”) of 0.1 μmol / L as a fluorescent substance and a FITC concentration of 1 μmol / L. The ones used were used. In order to measure the base signal, a buffer solution that does not emit fluorescence was also used as a sample having a FITC concentration of 0.0 μmol / L. As long as a substance that emits fluorescence is not included, the buffer solution may not be used as the sample having the FITC concentration of 0.0 μmol / L. However, the use of a cleaning solution or a buffer solution is preferable in that the number of man-hours does not increase at the time of measurement because it can be performed between measurements.

  Next, the output value (mV) (hereinafter referred to as “lock-in detection signal”) from the lock-in amplifier detected when the above samples were irradiated with excitation light was measured while changing the excitation light amount. As a result of this measurement, as shown in FIG. 4A, it has been found that the lock-in detection signals of all the samples increase as the excitation light intensity increases.

  Here, since the lock-in detection signal of the sample having the FITC concentration of 0.0 μmol / L represents the base signal caused only by the above-described excitation base light, such a result is naturally obtained. However, the lock-in detection signal of the samples having FITC concentrations of 1.0 μmol / L and 0.1 μmol / L is a signal obtained by removing the BKG signal 301 from the detection signal 304 of FIG. 3, and is the sum of fluorescence and excitation base light. Signal. Further, the fluorescence depends not only on the amount of excitation light but also on the FITC concentration. Therefore, for each excitation light quantity, the base signal is subtracted from the lock-in detection signal of the samples having FITC concentrations of 1.0 μmol / L and 0.1 μmol / L, and a signal dependent on only the fluorescence generated from the sample (hereinafter referred to as “FITC fluorescence signal” And a signal ratio when the FITC fluorescence signal at the FITC concentration of 1.0 μmol / L is divided by the FITC fluorescence signal at the FITC concentration of 0.1 μmol / L (hereinafter referred to as “FITC signal ratio”). As a result, as shown in FIG. 4B, in the base signal at an excitation light amount of 4 μW or more, the FITC fluorescence signal is proportional to the base signal, and the FITC signal ratio becomes a substantially constant value regardless of the base signal. I understood.

From the above results, in the base signal at an excitation light amount of 4 μW or more, the FITC fluorescence signal is proportional only to the base signal and the FITC concentration, and therefore, the following relationship (1) is established.
F = b × d × C (1)
F: FITC fluorescence signal b: base signal d: FITC concentration C: constant On the other hand, it was found that the FITC signal ratio varied in the base signal at an excitation light amount of less than 1 μW. This is presumably because the FITC fluorescence signal with an excitation light amount of less than 1 μW cannot be measured with high accuracy because it is electrically influenced by a lock-in amplifier or the like.

  Further, it is necessary to secure the light amount of the excitation base light to such an extent that the relationship of FIG. 4A that the base signal is proportional only to the excitation light amount can be obtained. This is because if the light amount of the excitation base light is very small, it cannot be measured accurately due to the electrical influence from the lock-in amplifier, etc. as in the case described above. Conversely, the light amount of the excitation base light is very large. This is because the light receiving element is saturated and measurement cannot be performed with high accuracy.

  Based on the above results, a method for obtaining the concentration of the sample from the FITC fluorescence signal obtained for the sample of unknown concentration at the time of measurement will be described.

  First, the amount of excitation light is set to A (for example, A> 4) μW, fluorescence measurement is performed on a buffer solution and a plurality of samples having a known FITC concentration, the horizontal axis is FITC concentration, and the vertical axis is FITC fluorescence of each sample. Create a calibration curve as a signal.

  Next, without changing the setting of the excitation light amount, fluorescence measurement is performed on the buffer solution and the sample having an unknown FITC concentration when a certain time has elapsed after the calibration curve is created. Here, since the excitation light source 53 is an LED, the excitation light amount varies depending on the light emission time and the external temperature even if the setting of the excitation light amount is maintained. The influence on the FITC fluorescence signal caused by this fluctuation is corrected by the following method.

First, base signals obtained at the time of calibration curve creation and sample measurement are b ₀ and b ₁ , respectively, and lock-in detection signals at unknown FITC concentrations (concentration d ₁ ) are S ₀ , S ₁ , and concentration d _{1, respectively.} The FITC fluorescence signals at are _denoted by F ₀ and F ₁ , respectively. Since the FITC fluorescence signal is a value obtained by subtracting the base signal from the lock-in detection signal,
F ₀ = S ₀ −b ₀ (2)
F ₁ = S ₁ −b ₁ (3)
The relationship is obtained. Moreover, if it substitutes in said Formula (1),
F ₀ = S ₀ −b ₀ = b ₀ × d ₁ × C (4)
F ₁ = S ₁ −b ₁ = b ₁ × d ₁ × C (5)
The relationship is obtained.

Of these, S ₁ , b ₀ , b ₁ can be obtained as measured values, and therefore F ₁ can be calculated from equation (3). Furthermore, d ₁ × C can be calculated as F ₁ / b ₁ from the calculated value F ₁ , the measured value b ₁ and the equation (5).

Next, when d ₁ × C (= F ₁ / b ₁ ) calculated in Expression (4) is substituted, F ₀ = F ₁ × b ₀ / b ₁ is calculated.

Therefore, the concentration of d ₁ can be obtained with high accuracy by applying the value of F ₀ to the prepared calibration curve.

  In the above method, the FITC fluorescence signal obtained by subtracting the base signal from the sample lock-in detection signal is used. However, a FITC fluorescence signal ratio obtained by dividing the sample lock-in detection signal by the base signal may be used.

In this case, when the relationship between the base signal and the FITC fluorescence signal ratio is graphed based on the lock-in detection signal at each FITC concentration obtained in FIG. 4A, as shown in FIG. 5, the excitation light quantity is 4 μW or more. In the case of the base signal, the FITC fluorescence signal ratio is substantially constant without depending on the base signal. That is, since the FITC fluorescence signal ratio is proportional only to the FITC concentration, it was found that the following formula (6) is established.
F ′ = d × C ′ (6)
F ′: FITC fluorescence signal ratio d: FITC concentration C ′: constant Based on this result, a method for obtaining the concentration of a sample having an unknown FITC concentration using the FITC fluorescence signal ratio will be described.

  First, the amount of excitation light is set to A (for example, A> 4) μW, fluorescence measurement is performed on a buffer solution and a plurality of samples having known FITC concentrations, the horizontal axis is FITC concentration, and the vertical axis is lock-in of each sample. A calibration curve is created as a detection signal.

In this modification, since the FITC fluorescence signal ratio is a value obtained by dividing the lock-in detection signal by the base signal, the FITC fluorescence signal ratio at the concentration d ₁ obtained when the calibration curve is created and when the sample is measured is F ′ _0. , F ′ ₁ ,
F ′ ₀ = S ₀ / b ₀ (7)
F ′ ₁ = S ₁ / b ₁ (8)
The relationship is obtained. Moreover, when the value at the time of measurement is substituted into the above equation (6),
F ′ ₀ = d ₁ × C ′ (9)
F ′ ₁ = d ₁ × C ′ (10)
The relationship is obtained.

Of these, S ₁ , b ₀ , b ₁ can be obtained as measured values, and therefore F ′ ₁ can be calculated from equation (8). Therefore, d ₁ × C ′ can be calculated as S ₁ / b ₁ from Equation (10).

Substituting the calculated d ₁ × C ′ into the equation (9), the relationship S ₀ = S ₁ · b ₀ / b ₁ is obtained.

Therefore, the concentration of d ₁ can be obtained with high accuracy by applying the value of S ₀ to the created calibration curve.

  In the fluorescence analysis system 100, a part of the excitation light is guided to the detector 54 by adjusting the filter characteristics of the fluorescence analysis optical multiplexer / demultiplexer 56. However, the present invention is not limited to this configuration. .

  For example, the diameters of the rod lenses 500 and 502 may be made larger than the diameter of the filter 501, and a part of the excitation light may be guided to the detector 54. In this case, since the excitation light from the incident end 56a side takes an optical path that deviates from the theoretical optical path that bypasses the outside of the filter 501, a part of the excitation light is guided to the output end 56c. That is, in the case of this correction method, the excitation base light guided to the detector 54 is not only for excitation light partially reflected by the distal end portion 103a of the optical fiber 103 but also for excitation light entering from the incident end 56a. Excitation light from the light source 53 is also included.

  Therefore, when using this type of optical multiplexer / demultiplexer for fluorescence analysis, an excitation base that is guided to the detector 54 by using a conventional non-reflective treatment on the tip 103a of the optical fiber 103 is used. The light may be only the excitation light from the excitation light source 53 entering from the incident end 56a.

  FIG. 6 is a diagram showing a schematic configuration of a modification of the fluorescence analysis system according to the embodiment of the present invention.

In the fluorescence analysis system 100 ′ according to this modification, the fiber length is 7 m instead of the optical fiber 103 having a fiber length of 30 cm used in the above embodiment, and the tip portion 103a ′ has a non-reflection treatment. The optical fiber 103 ′ that is (obliquely polished + AP-coated) is used, and the optical fiber 108 has an edge filter 57 that uses a wavelength λ ′ where λ ₁ <λ ′ <λ ₂ as a cutoff wavelength. And the transmittance at λ ₁ of the filter 501 ′ of the fluorescence analyzing optical multiplexer / demultiplexer 56 ′ is −30 dB or less. The other elements of the present modification are the same as the corresponding ones in the above-described embodiment, and thus the same reference numerals are given and duplicate descriptions are omitted.

  In this modification, Raman scattered light generated by components constituting the optical fiber 103 ′ is used as excitation base light.

  Here, the scattered light includes Rayleigh scattered light having the same frequency as the incident light and Raman scattered light having a frequency different from the incident light. The deviation of the frequency of the Raman scattered light from the incident light is caused by the transfer of energy when the light incident on the material collides with the atoms and molecules of the material. Of these, the frequency is small. What becomes (wavelength becomes longer) is called Stokes scattered light, and on the contrary, light whose frequency becomes larger (wavelength becomes shorter) is called anti-Stokes scattered light. In this modification, Rayleigh scattered light is the same wavelength as the reflected light of the excitation light used in the above embodiment in that it has the same wavelength as the excitation light, so it is not used and only Raman scattered light is considered. Of the Raman scattered light, the anti-Stokes scattered light is designed to be removed by the filter 501 'of the fluorescence analyzing optical multiplexer / demultiplexer 56'. On the other hand, since the wavelength of Stokes scattering becomes longer, it overlaps with the wavelength of fluorescence and cannot be removed by the filter 501 '. In this modification, Raman scattering is used as excitation base light used for correction of the calibration curve in order to use the feature of Stokes scattering in reverse.

Next, although an example and a comparative example explain the present invention, the present invention is not limited to this.
Example 1
The base signal and the sample lock-in detection signal were measured by the system shown in FIG.

  Specifically, NSPB300B manufactured by Nichia Corporation is used as the excitation light source 53 composed of LEDs, SLW18 (0.4 pitch) manufactured by Nippon Sheet Glass is used as the lens 40, and Edmond is manufactured as the optical fiber 103. A quartz optical fiber having a ratio of core diameter / cladding diameter of 200/220 and NA of 0.22 was used. Further, an optical multiplexer / demultiplexer manufactured by Nippon Sheet Glass is used as the optical multiplexer / demultiplexer for fluorescence analysis 56, and SLW18 (0.25 pitch) manufactured by Nippon Sheet Glass is used as the lenses 500, 502 in the optical multiplexer / demultiplexer for fluorescence analysis 56. used. Further, PMT (H5784-20) manufactured by Hamamatsu Photonics was used as the detector 54.

  The tip portion 103a of the optical fiber 103 was polished at 0 ° and was not coated with an AR coat.

  In such a system configuration, a fluorescence measurement of a sample having a FITC concentration of 0.1 μmol / L was performed in a state where the excitation light amount emitted from the probe 50 was 40 μW as the average value of the blinking signal, and a lock-in amplifier output value was obtained.

  Thereafter, the excitation light amount of the excitation light source 53 was adjusted by the system shown in FIG. 7 so that the lock-in amplifier output value obtained above was obtained. The amount of excitation light obtained by this adjustment was 2 pW. That is, in the system 100 of FIG. 1, the total amount of fluorescence and excitation base light generated from the sample was 2 pW.

In the same manner, the buffer solution is irradiated with the excitation light to obtain a lock-in amplifier output value, and the excitation light source 53 in the system shown in FIG. 7 is excited so that the obtained lock-in amplifier output value is obtained. The amount of light was adjusted. The amount of excitation light obtained by this adjustment was 1 pW. That is, in the system 100 of FIG. 1, the amount of excitation base light was 1 pW.
(Comparative Example 1)
The difference from Example 1 was that the tip portion 103a of the optical fiber 103 was polished 10 degrees and the AR coating was applied, and all other measurements were performed under the same conditions.

  As a result, the amount of fluorescence from the sample was 1 pW, whereas the amount of excitation base light was 0.01 pW or less, which is a value less than the measurement limit.

  From the above results, the amount of excitation base light in Example 1 and the amount of fluorescence light are substantially the same. As a matter of course, if the concentration of the sample changes, the amount of fluorescence output from the sample changes, so that the amount of light of only the excitation base light may be smaller than the total amount of fluorescence and excitation base light. However, the light amount of the excitation base light obtained in the example is a value less than the measurement limit like the light amount of the excitation base light obtained in the comparative example, and the influence of noise must be taken into consideration. Not a small value. Therefore, it was found that the light partially reflecting the excitation light can be used as the excitation base light.

On the other hand, since the amount of saturation of the light receiving element in the detector 54 is 100 pW, it was found that the excitation base light detected in the example can be used without any problem.
(Example 2)
The length of the optical fiber 103 of the fluorescence analysis system 100 ′ of FIG. 6 was adjusted, and the adjusted Raman scattered light was detected.

  Specifically, NSPB300B manufactured by Nichia Corporation is used as the excitation light source 53 composed of LEDs, SLW18 (0.4 pitch) manufactured by Nippon Sheet Glass is used as the lens 40, and Edmond is manufactured as the optical fiber 103. A quartz optical fiber having a ratio of core diameter / cladding diameter of 200/220 and NA of 0.22 was used. Further, an optical multiplexer / demultiplexer manufactured by Nippon Sheet Glass is used as the optical multiplexer / demultiplexer for fluorescence analysis 56, and SLW18 (0.25 pitch) manufactured by Nippon Sheet Glass is used as the lenses 500, 502 in the optical multiplexer / demultiplexer for fluorescence analysis 56. used. Further, PMT (H5784-20) manufactured by Hamamatsu Photonics was used as the detector 54.

  Further, the tip portion 103a of the optical fiber 103 was polished 10 degrees and an AR coating was applied.

  In such a system configuration, the length of the optical fiber 103 was changed, and the intensity of Raman scattered light was measured.

  As a result, as shown in FIG. 8, the intensity of Raman scattered light detected when the fiber length was less than 1 m was less than 0.01 pW, which was less than the measurement limit.

  On the other hand, when the fiber length is 5 m, the Raman intensity is 0.5 pW, and when it is 7 m, it is about 1 pW, which is almost the same as the value of the FITC fluorescence signal. It was.

  Therefore, when the fiber length is 5 m or longer, more preferably 7 m or longer, the FITC concentration of the sample can be reliably obtained in the system shown in FIG. 6 by using Raman scattering as the excitation base light. all right.

  On the other hand, Raman scattering occurs when the excitation light passes through the optical fiber, and naturally occurs even in the case of the first embodiment. However, since the optical fiber length (= 30 cm) of the probe portion of Example 1 is 1 m or less, only Raman scattering below the measurement limit occurs. Therefore, it was found that Raman scattering cannot be used as excitation base light in a system such as Comparative Example 1.

1 is a diagram showing a schematic configuration of a fluorescence analysis system according to an embodiment of the present invention. FIG. 2 is a schematic cross-sectional schematic diagram of the optical multiplexer / demultiplexer for fluorescence analysis in FIG. 1. It is a graph used for explaining the detection signal measured by the fluorescence analysis system. It is a graph used for demonstrating the method of correct | amending the detection signal at the time of a fluorescence measurement in real time, (a) shows the relationship between the lock-in detection signal and excitation light quantity in each FITC density | concentration, (b) is each FITC density | concentration. 2 shows the relationship between the FITC fluorescence signal and the base signal. It is a graph which shows the relationship between the FITC fluorescence signal ratio calculated by the method different from FIG.4 (b), and a base signal. It is a figure which shows schematic structure of the modification of the fluorescence analysis system which concerns on embodiment of this invention. It is a figure which shows schematic structure of the system which adjusts the excitation light quantity of the light source for excitation light used in Example 1. FIG. It is a graph which shows the intensity | strength of the Raman scattered light detected with the fluorescence analysis system of FIG. It is a figure which shows schematically the structure of the conventional fluorescence detection system. It is a figure used for demonstrating the fluorescence measured with the fluorescence detection system of FIG. It is a figure used for demonstrating the fluorescence measured with the fluorescence detection system of FIG. It is a figure used for demonstrating the fluorescence measured with the fluorescence detection system of FIG. It is a figure which shows schematic structure of the modification of the fluorescence analysis system which concerns on embodiment of this invention. It is a figure which shows schematic structure of the modification of the fluorescence analysis system which concerns on embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Fluorescence analysis system 20 Fluorescence analysis chip 40 Lens 53 Excitation light source 54 Detector 56 Fluorescence analysis optical multiplexer / demultiplexer 103 Optical fiber 103a Tip 109 Lock-in modulation circuit

Claims (16)

In fluorescence detection system for analyzing a dominant wavelength lambda ₂ of the fluorescence emitted from a sample dominant wavelength lambda ₁ of the excitation light is irradiated (λ _2> λ _1),
An optical multiplexer / demultiplexer that guides light from the incident end to the first exit end, and guides light from the first exit end to the second exit end;
A light source for excitation light connected to the incident end and emitting the excitation light;
A detector connected to the second emission end and receiving the fluorescence;
A lens that condenses and irradiates the sample with the excitation light incident from one end, and condenses the fluorescence incident from the other end;
An optical fiber connected to the first emission end and one end of the lens;
The fluorescence detection system, wherein the detector receives light caused by the excitation light simultaneously with the fluorescence.   2. The fluorescence detection system according to claim 1, wherein the optical multiplexer / demultiplexer includes a filter interposed between two lenses, and the diameter of the two lenses is larger than the diameter of the filter.   The fluorescence detection system according to claim 1, wherein the optical multiplexer / demultiplexer is a fusion coupler.   The fluorescence detection system according to claim 1, wherein the optical fiber guides light resulting from the excitation light to the first emission end simultaneously with the fluorescence.   A reflection element is introduced into a part of the optical fiber, and the light resulting from the excitation light is light in which a part of the excitation light is reflected by the reflection element. 4. The fluorescence detection system according to 4.   6. The fluorescence detection system according to claim 5, wherein the reflection element is an element using reflection at an end face thereof.   The fluorescence detection system according to claim 6, wherein the reflection at the end face is a planar reflection of glass. The fluorescence detection system according to claim 4, wherein the optical multiplexer / demultiplexer includes a filter including a long-pass filter having a cutoff wavelength λ (λ ₁ <λ <λ ₂ ). The fluorescence detection system according to claim 8, wherein the transmittance of the filter at a wavelength λ ₁ is −20 dB or less. The fluorescence detection system according to claim 4, wherein the optical multiplexer / demultiplexer includes a filter including a short-pass filter having a cutoff wavelength λ (λ ₁ <λ <λ ₂ ). . The fluorescence detection system according to claim 10, wherein a reflectance of the filter at a wavelength λ ₁ is −20 dB or less.   The fluorescence detection system according to claim 8, wherein the filter is interposed between two lenses, and a diameter of the two lenses is larger than a diameter of the filter.   The fluorescence detection system according to any one of claims 4 to 7, wherein the optical multiplexer / demultiplexer is a fusion coupler.   The fluorescence detection system according to claim 4, wherein the light resulting from the excitation light is Raman scattered light from the optical fiber.   15. The fluorescence detection system according to claim 14, wherein the Raman scattered light is due to stochastic scattering. In the concentration measuring method using the fluorescence detection system according to any one of claims 1 to 15,
A first acquisition step of irradiating the substance to be measured having a first concentration with the excitation light at a reference time and acquiring the light guided to the detector at that time as a first detection signal;
A second acquisition step of irradiating the substance to be measured having a second concentration with the excitation light at the reference time and acquiring the light guided to the detector at that time as a second detection signal; ,
A third acquisition step of acquiring a third detection signal when only the light caused by the excitation light is guided to the detector at the reference time;
A creation step for creating a calibration curve based on the first to third detection signals;
During measurement after a predetermined time has elapsed from the reference time, the substance to be measured having an unknown concentration is irradiated with the excitation light, and light guided to the detector at that time is acquired as a fourth detection signal. A fourth acquisition step;
A fifth acquisition step of acquiring a fifth detection signal when only the light caused by the excitation light is guided to the detector during the measurement;
A correction step of correcting the fourth detection signal based on the third detection signal and the fifth detection signal;
A concentration measuring method comprising: applying the corrected fourth detection signal to the created calibration curve, and calculating a value of the unknown concentration.

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