JP2006308412A - Fluorescence resonance detector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence resonance detector capable of reliable detection by a simple device by using near-field light. <P>SOLUTION: The fluorescence resonance detector 100 detects a substance to be detected in an inspection target 500 by utilizing fluorescence by the fluorescence resonance reaction between a first fluorescent label and a second one. The fluorescence resonance detector 100 comprises a light source 50; a reflection member 10 for reflecting light emitted from the light source 50; a holding section 22 to be inspected that is provided at a region in which near-field light emitted from a reflection surface 14 of the reflection member 10 is formed and holds the first and second fluorescent labels and the inspection target; and fluorescence detection sections 70, 80, 90 for detecting fluorescence. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a fluorescence resonance detection apparatus, and more particularly, to a fluorescence resonance detection apparatus that detects a detection target that is excited by near-field light (EFL) and performs fluorescence resonance.

In recent years, interest in daily examinations for food allergies, food poisoning, and adult diseases has increased, and small-scale medical institutions that use antigen-antibody reactions or the like, and small-sized examination devices for general households have attracted attention. Typical detection methods using this antigen-antibody reaction include ELISA (Enzyme-Linked Immunosorbent Assay) method, EFL method (see, for example, Patent Document 1), FRET (Fluorescence Resonance Energy Transfer) method, and the like. However, it has not been put into practical use as an inspection apparatus for general households because each of the inspections takes time, the reaction operation is complicated, and the inspection object is limited.
JP 2004-279041 A

  In an examination in a small-scale medical institution or a general household, it is important to achieve a quick determination time, simple operability, and downsizing of the apparatus in addition to the reliability of the examination result. In this respect, although the FRET method is an ideal detection method, it is difficult to say that the FRET method is suitable for measurement of a sample containing a large amount of impurities other than the measurement object such as blood (plasma) (Table 1). As a method for solving this problem, an improved FRET method using a metal complex has been devised, but there is a problem that an expensive and complicated optical mechanism such as an ultraviolet wavelength laser is required.

  In Table 1, each detection method has a merit (◯) and a demerit (×).

本発明は、上記の課題に鑑みてなされたものであって、近接場光を用いることにより、簡易な装置で信頼性の高い検出ができる蛍光共鳴検出装置を提供することを目的とする。

The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a fluorescence resonance detection apparatus that can perform highly reliable detection with a simple apparatus by using near-field light.

  In order to achieve the above object, the fluorescence resonance detection apparatus of the present invention detects fluorescence to detect a substance to be detected in a test object using fluorescence by a fluorescence resonance reaction between a first fluorescence label and a second fluorescence label. A resonance detecting device, provided in a region where a light source, a reflecting member that reflects outgoing light emitted from the light source, and a near-field light emitted from a reflecting surface of the reflecting member are formed, An inspection object holding unit that holds the fluorescent label, the second fluorescent label, and the inspection object, and a fluorescence detection unit that detects the fluorescence are provided. Thereby, highly reliable detection can be performed with a simple device.

  According to a second aspect of the present invention, in the fluorescence resonance detection apparatus according to the first aspect, the fluorescence detection unit is provided at a position facing the reflection surface of the reflection member. Thereby, it becomes difficult to be affected by the problem that the background fluorescence of impurities and the fluorescence from the detection target are attenuated, and more reliable detection can be performed with a simple apparatus.

  According to a third aspect of the present invention, in the fluorescence resonance detection apparatus according to the first or second aspect, the fluorescence detection unit includes a spectrum measuring unit that measures a spectrum of the fluorescence. Thereby, not only the presence (presence / absence) of the substance to be detected in the test object can be detected, but also quantitative detection can be performed.

  According to a fourth aspect of the present invention, in the fluorescence resonance detection apparatus according to any one of the first to third aspects, the reflection member and the inspection object holding unit are configured integrally and from the fluorescence resonance detection apparatus. It is provided detachably. Thereby, a some test object can be test | inspected in a short time by replacing | exchanging the reflecting member and test object holding | maintenance part comprised integrally.

  The invention according to claim 5 is the fluorescence resonance detection apparatus according to any one of claims 1 to 4, wherein the first fluorescent label and the second fluorescent label are such that the inspection target is the inspection target holding unit. Before being introduced into the inspection object holding portion. Thereby, the inspection can be performed only by introducing the inspection object to the inspection object holding unit, and the operation becomes simpler. In addition, when the first fluorescent label and the second fluorescent label are held in a lyophilized state, storage is easy.

  The present invention enables highly reliable detection with a simple device.

  Hereinafter, the configuration of the fluorescence resonance detection apparatus 100 according to the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a schematic cross-sectional view showing the configuration of the fluorescence resonance detection apparatus 100. The fluorescence resonance detection apparatus 100 includes a sample holding unit 40 including a light guide prism 10, a chamber plate 20, a cover plate 30, a light source 50, an excitation side collimator lens 60, a light reception side collimator lens 70, and a spectrometer 80. And a control unit 90 that performs analysis processing of measurement data from the spectrometer 80 and controls various components in the fluorescence resonance detection apparatus 100.

  In this embodiment, a blue light emitting diode (manufactured by Ocean Optics: Blue LED Excitation Sources LS-450) is used as the light source 50, and light from the light source 50 is optical fiber (manufactured by Ocean Optics: P400-2-UV-). VIS) 52 and led to an excitation side collimator lens (Ocean Optics, 74-VIS) 60. Since the light emitted from the optical fiber 52 spreads at an emission angle of 25 °, the light is converted into parallel light by the excitation-side collimator lens 60 and is incident on the light guide prism 10.

The incident surface 12 of the light guide prism 10 is formed to be inclined by θ (about 60 °) with respect to the reflection surface 14, and this angle θ is determined by the refractive index n _p of the light guide prism 10 and the light guide prism 10. It is derived based on Snell's law by the refractive index n _{s of the} sample 500 introduced on the reflecting surface 14. In the case of the present embodiment, assuming that the light guide prism 10 is an acrylic material and the sample 500 is water, the refractive index n _p of the acrylic material is larger than the refractive index n _{s of} water, so the critical angle θ _c is It is derived from Equation 1.

ここで、導光プリズム１０の屈折率ｎ_pに１．６を、サンプル５００の屈折率ｎ_sに１．３３を代入し、臨界角θ_cを算出すると、５６．３°となるので、サンプル５００の屈折率ｎ_sの変動や導光プリズム１０の作製のしやすさなどを考慮して、本実施の形態の導光プリズム１０はθを約６０°とした。

Here, substituting 1.6 for the refractive index n _p of the light guide prism 10 and 1.33 for the refractive index n _s of the sample 500 and calculating the critical angle θ _c gives 56.3 °. Considering the change in the refractive index n _s of 500 and the ease of manufacturing the light guide prism 10, the light guide prism 10 of the present embodiment has θ set to about 60 °.

  A chamber plate 20 and a cover plate 30 are arranged on the light guide prism 10 as shown in FIG. 2, and a measurement chamber 22 is provided at the center of the chamber plate 20. The sample 500 is injected into the cover plate 30 and the chamber plate 20 from the sample injection port 32 and the sample injection path 24 provided so as to penetrate therethrough, and the sample circulation that connects the measurement chamber 22 and the sample injection path 24 of the chamber plate 20 is connected. It is introduced into the measurement chamber 22 through the path 26. Although not shown, the sample 500 may be introduced by providing a pressurizing means such as a syringe on the sample injection side to supply pressure to the measurement chamber 22, or the exhaust path 28 communicating from the measurement chamber 22 to the outside or its An exhaust pump may be provided on the exhaust side, and the sample 500 may be sucked and fed to the measurement chamber 22 by being sucked by the exhaust pump.

Returning to FIG. 1, the dimension of the measurement chamber 22 is preferably larger than the area where the parallel light L _p passing through the light guide prism 10 hits the reflection surface 14. This is to prevent measurement noise from occurring due to irregular reflection at the edge 23 of the measurement chamber 22 when the dimension of the measurement chamber 22 is smaller than the area where the parallel light L _p hits the reflecting surface 14. is there. In the present embodiment, a circular cross-sectional dimension of the diameter 5mm parallel light L _p, the parallel light L because _p is incident perpendicularly to the plane of incidence 12, the major axis is 10mm the reflective surface 14, Reflects in an ellipse with a minor axis of 5 mm. Therefore, it is desirable to set the measurement chamber 22 to have a diameter of 10 mm or more. Further, it is better if the edge portion 23 is chamfered.

Two kinds of antibodies freeze-dried in advance are introduced into the measurement chamber 22. Of these two types of antibodies, one of the antibodies A is provided with a labeled FITC (Fluorescein isothiocyanate), and the other antibody B is provided with a labeled TAMRA (6-carboxy-tetramethyl-rhodamine). As shown in FIG. 3, when antigen C to be joined to antibody A and antibody B is introduced into measurement chamber 22, antibody A and antibody B are joined to antigen C. In this state, the excitation wavelength E _xf (peak wavelength: 494 nm) of FITC the 450nm light near is irradiated to the measuring chamber 22, FITC emits a 518nm as peak (E _mf), excitation wavelength of TAMRA E _xt Is 555 nm, TAMRA emits light with a peak (E _mt ) of 580 nm by FITC light emission.

  This phenomenon is a known technique as a fluorescence resonance reaction or a resonance energy transfer reaction (FRET), but excitation light transmitted through a sample 500 as shown in FIGS. 7A and 7B ( In a conventional detection apparatus using FRET that detects transmission excitation light) or fluorescence from a detection target that has passed through the sample 500 (transmission fluorescence), the sample 500 contains impurities such as protein, nucleic acid, or metal complex (eg, hemporphyrin). In this case, there is a problem that the S / N ratio is reduced by the background fluorescence of the impurities by the transmitted excitation light, or the transmitted fluorescence is attenuated in the sample 500.

  However, in the present invention, the portion of the sample 500 irradiated with the near-field light by the near-field light that the parallel light Lp that is the excitation light leaks from the reflection surface 14 of the light guide prism 10 (having several hundred nm from the reflection surface 14). FRET is caused in the range), and the fluorescence from the detection target is received by the light receiving surface 16 facing the reflection surface 14, so that it is not easily affected by the problem of the background fluorescence of impurities and the fluorescence from the detection target being attenuated. Therefore, measurement can be performed without using a light source such as an ultraviolet wavelength laser.

  In addition, FRET does not occur unless there is a primary fluorescent label (FITC) and a secondary fluorescent label (TAMRA) facing each other at a predetermined angle within a range of about 1 to 10 nm. Thus, the emission spectrum changes depending on the amount of antigen C. Accordingly, by providing the light receiving side with a spectrometer 80 capable of measuring a spectrum as in the present embodiment, it is possible to estimate not only the presence or absence of the antigen C but also the amount thereof.

  A light receiving side collimator lens 70 is disposed at a position facing the measurement chamber 22 via the light guide prism 10. It is efficient if the light receiving surface 16 and the reflecting surface 14 on which the light receiving side collimator lens 70 is arranged are parallel to each other. The light collected by the light-receiving side collimator lens 70 passes through an optical fiber (Ocean Optics: P400-2-UV-VIS) 82 and is used for a spectrophotometer (Ocean Optics: Plug-and-Play Fiber Optic Spectrometer USB2000). ) 80 and a spectrum measurement is performed.

  The parts such as the sample holding unit 40 and the collimator lenses 60 and 70 prevent external light from entering as noise, and prevents light reflected from the reflecting surface 14 of the light guide prism 10 from affecting the outside. It is desirable to arrange in a light-shielded casing (not shown). In this case, the casing is provided with an inlet / outlet with a lid through which the sample holder 40 is taken in and out, and the sample holder 40 into which the sample 500 is introduced from the sample inlet 32 to the measurement chamber 22 is placed inside the casing from the inlet / outlet of the casing. Fix in place. Then, by closing the entrance cover and pressing the measurement start button, a command to output light from the control unit 90 to the light source 50 is issued, and then a command to perform spectrum measurement from the control unit 90 to the spectrometer 80 is issued. By doing so, a series of measurements can be automatically performed.

Next, the configuration of the sample holding unit 140 according to the first embodiment of the present invention will be described in detail with reference to FIG. Figure 5 is an exploded perspective view showing a sample holder 140 for use in a sterile test of cell culture, in the present embodiment, the syringe 102, the primary fluorescent label is provided with antibody A ₁ that is lyophilized, secondary A sample 500 in which the antibody B ₁ provided with a fluorescent label and freeze-dried and the cell culture solution to be examined is introduced and mixed well from the syringe 102 are mixed into the sample inlet 132, the sample The liquid is pressurized and fed to the measurement chamber 122 via the injection path 124 and the sample flow path 126.

After the measurement, the sample holder 140 is removed from the fluorescence resonance detection apparatus 100, and the sample holder 140 is replaced. In order to prevent the arrangement position of the sample holding unit 140 from being shifted and the position where the parallel light L _p of the fluorescence resonance detection apparatus 100 hits the sample holding unit 140 from being shifted during replacement, a positioning notch (not shown) is not shown. Is provided on the bottom surface (light receiving surface 116) of the light guide prism 110, or the stage on which the sample holder 140 is placed is made a movable stage so that the position can be adjusted when the sample holder 140 is placed. Still good. Alternatively, the cleaning liquid may be introduced from the sample injection port 132 to clean the sample injection port 132, the sample injection channel 124, the sample flow channel 126, and the measurement chamber 122 so that the sample holding unit 140 can be used repeatedly.

  Next, the configuration of the sample holding unit 240 according to the second embodiment of the present invention will be described in detail with reference to FIG. FIG. 6 is an exploded perspective view showing a sample holder 240 used for blood testing. In this embodiment, the measurement chamber 222 is provided with a primary fluorescent label, and the freeze-dried antibody A2 and the secondary fluorescent label are provided. The antibody B2 provided and lyophilized is introduced in advance, and the sample 500 is sent from the sample injection port 232 to the measurement chamber 222 via the sample injection path 224 and the sample flow path 226. At this time, the sample 500 is sucked and fed into the measurement chamber 222 by making the pressure in the measurement chamber 222 negative by an air pump (not shown) connected to the exhaust passage 228.

  The sample holder 240 of this embodiment used for blood tests is provided with a 1 μm mesh filter 204 in the sample flow path 226, and solid components (red blood cells, white blood cells, platelets) in the blood are captured by the filter 204. The Then, only liquid components such as plasma and minute solid components that are not captured by the 1 μm mesh filter 204 are introduced into the measurement chamber 222 to be inspected. In the sample holding unit 240 with a filter as in this embodiment, the filter 204 is clogged. Therefore, it is more suitable to replace the sample holding unit 240 for each inspection object. The cleaning liquid needs to be introduced from the exhaust passage 228.

  The present invention is considered to be applicable to all tests that utilize nucleic acid complementation and enzyme activity, including antigen-antibody reaction tests of body fluids (blood, urine, digestive fluid, etc.) including pregnancy tests by urine. .

1 is a system configuration diagram schematically showing the configuration of a fluorescence resonance inspection apparatus according to the present invention. It is the disassembled perspective view which showed the structure of the sample holding part of the fluorescence-resonance inspection apparatus which concerns on this invention. It is the schematic diagram which showed the principle of the fluorescence resonance by an antigen antibody reaction. It is the schematic diagram which showed the difference in the output of the fluorescence resonance by an antigen antibody reaction. It is the disassembled perspective view which showed the structure of the sample holding part which concerns on one Example of this invention. It is the disassembled perspective view which showed the structure of the sample holding part which concerns on the other Example of this invention. It is the system block diagram which showed typically the conventional detection apparatus using FRET method.

Explanation of symbols

10, 110, 210 Light guiding prism 12, 112, 212 Incident surface 14, 114, 214 Reflecting surface 16, 116, 216 Light receiving surface 20, 120, 220 Chamber plate 22, 122, 222 Measuring chamber 23, 123, 223 Edge portion 24, 124, 224 Sample injection path 26, 126, 226 Sample flow path 28, 128, 228 Exhaust path 30, 130, 230 Cover plate 32, 132, 232 Sample inlet 40, 140, 240 Sample holder 50 Light source 52, 82 Optical fiber 60 Excitation side collimator lens 70 Receiving side collimator lens 80 Spectrometer 90 Control unit 100 Fluorescence resonance detector 102 Syringe 204 Filter 500 Sample

Claims (5)

A fluorescence resonance detection apparatus for detecting a substance to be detected in a test object using fluorescence by a fluorescence resonance reaction between a first fluorescent label and a second fluorescent label,
A light source;
A reflecting member that reflects the emitted light emitted from the light source;
An inspection object holding unit that is provided in a region where near-field light emitted from the reflection surface of the reflecting member is formed, and holds the first fluorescent label, the second fluorescent label, and the inspection object;
A fluorescence detection unit for detecting the fluorescence;
A fluorescence resonance detection apparatus comprising:   The fluorescence resonance detection apparatus according to claim 1, wherein the fluorescence detection unit is provided at a position facing the reflection surface of the reflection member.   The fluorescence resonance detection apparatus according to claim 1, wherein the fluorescence detection unit includes a spectrum measurement unit that measures the spectrum of the fluorescence.   4. The fluorescence resonance detection apparatus according to claim 1, wherein the reflection member and the inspection object holding unit are integrally configured and are detachably provided from the fluorescence resonance detection apparatus. 5. . 2. The first fluorescent label and the second fluorescent label are held in the inspection object holding unit before the inspection object is introduced into the inspection object holding unit. 5. The fluorescence resonance detection apparatus according to any one of 4 to 4.

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