JP2005180963A - Optical analysis device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the sensitivity using a light transmitter by perfectly separating an excited light from a fluorescence and by eliminating the background due to the excited light in a chemical sensor. <P>SOLUTION: Light is guided to a cylindrical optical waveguide which is a light-transmitting part for transmitting the excited light from a light source/an optical system, and a fluorescent pigment adhered to the outer side surface of the optical waveguide is excited with an evanescent light (electric field), caused by the excited light propagating in the optical waveguide out of the cylindrical optical waveguide for propagating the excited light. The resulting fluorescence is reflected by a reflector out of the optical waveguide, and detected by a light sensor going through the intermediary of the optical waveguide. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention generally relates to an optical analyzer for chemical and biochemical analysis and an analysis method thereof, and more particularly to an optical analyzer for chemical and biochemical analysis based on an evanescent wave (electric field).

  There are multiple markers in blood for specific diseases such as cancer and hepatitis. When affected, the concentration of a specific protein increases during normal times. It is expected as a next-generation medical technology because it is possible to detect a serious disease early by monitoring these from normal times. One method for analyzing raw and unpurified proteins is based on sensors that use biological ligand-receptor interactions to identify specific compounds. Those using such techniques include optical fiber evanescent wave sensors and surface plasmon resonance sensors.

  An optical fiber evanescent wave sensor uses an evanescent wave (electric field) effect. The evanescent wave (electric field) effect is that an electromagnetic wave that passes through a substance and is reflected at a dielectric interface generates an electric field that decays exponentially in a second substance on the opposite side of the interface. Say. The evanescent wave region, which is the depth of penetration into the second material, is a small part of the wavelength, but still has a size of reporter molecules that produce light or fluorescence, light absorbing or scattering molecules, and colloidal particles and microspheres. Larger than such optical label material. These labels can be used to generate or monitor optical changes in the evanescent region, or to change the propagation of light in adjacent dielectrics, to detect target materials near the surface Can do.

  Optical fiber evanescent wave sensors are described in Japanese Patent Publication No. 5-21185 and Japanese Patent Application Laid-Open No. 2002-257732.

  The evanescent wave sensors described in these documents attach a labeled antigen-antibody complex to the side wall of an optical fiber whose one end is a reflecting surface, and guide excitation light in the axial direction from the other end of the optical fiber. Yes. Due to the evanescent wave effect of the excitation light guided to the optical fiber, the antigen-antibody complex labeled on the side wall of the optical fiber is excited to generate fluorescence. Part of the emitted fluorescence is guided to the optical fiber, returns to the optical system together with the reflected light of the excitation light, and is detected by the optical sensor. The optical fiber need not be an optical fiber composed of a core layer having a high refractive index for communication and a clad layer having a low refractive index. The reason is that, in the optical fiber, a fiber formed of a single material serves as a core layer and is in contact with a liquid having a refractive index lower than that of the core layer, and this liquid has a function of a cladding layer.

On the other hand, although not directly related to the present invention, capillary electrophoresis as shown in Japanese Patent Application Laid-Open Nos. 09-119809 and 2003-329590 has been devised as a method for examining the base sequence of DNA. ing. In these techniques, DNA sequences are separated in a capillary by mass, introduced into a glass part as a detection part, and the base sequence is examined by reading the labeled dye of each base. These inventions are techniques for using a labeled DNA fragment present in each capillary and adjusting the center position of the capillary to the focus of the detection device.
Japanese Patent Publication No.5-221185 JP 2002-257732 A JP 09-119809 A JP 2003-329590 A

  However, in Japanese Patent Publication No. 5-21185 and Japanese Patent Application Laid-Open No. 2002-257732, the fluorescence emitted from the antigen antibody and the excitation light reflected from the end face are derived from the optical fiber together. For this reason, when measurement is performed using fluorescence, it is necessary to separate excitation light and fluorescence from light derived from the optical fiber end. In the method disclosed above, separation is performed by using a filter that cuts excitation light and allows fluorescence to pass. However, it is substantially difficult to manufacture a filter that cuts 100% of the excitation light and passes 100% of the fluorescence. Since the excitation light has a higher light intensity than fluorescence, the excitation light is detected by the optical sensor as a large background noise. If the emission intensity of the excitation light is not stable, fluorescence is detected as a minute change on the background. For this reason, it is necessary to extremely stabilize the excitation light, which is the background generation source, in order to detect fluorescence with high sensitivity with these configurations, and there is a problem that it is very expensive when used as an industrial product. .

In the present invention, the detection target is excited by the light propagation body that propagates light inside the light propagation body and the excitation light that propagates through the propagation body when the detection target adheres to the outer surface of the light propagation body. An analyte light detection apparatus comprising: a directing unit for directing generated fluorescence in a predetermined direction; and a light detection unit facing the directing unit and positioned in the predetermined direction. The directing means is a reflector,
The cross-sectional shape of the reflector is preferably a parabolic shape defined by the following formula (1).

y = (1 / 4p) x ² (1)
(P is the focal position of the reflector and is expressed as the distance from the origin of the two-dimensional coordinate system. The origin is the central axis of the optical waveguide)
The surface of the reflector is preferably at least a metal or a dielectric multilayer film, and the outer surface of the light propagating body has an outer surface to which a capturing component for capturing a detection target having a fluorescent material can be attached. preferable.

  The binding between the substance to be detected and the capture component of the present invention is due to an antigen-antibody reaction or a DNA hybridization reaction.

  The present invention provides an optical analyzer using evanescent waves (electric field) that is capable of spatially separating fluorescence to be measured from excitation light and inspecting only the fluorescence, and is more sensitive than before. it can.

  The present invention guides a fluorescent dye that is guided to a cylindrical optical waveguide serving as a light propagation part for propagating excitation light from a light source / optical system and adheres to the outer side wall of the cylindrical optical waveguide. The excitation light propagating in the optical waveguide is excited by evanescent light (electric field) generated outside the cylindrical optical waveguide that becomes a light propagation portion for propagating the excitation light, and the resulting fluorescence is emitted from the cylindrical optical waveguide. The light is reflected by a reflector (reflector) on the outside, and is detected by an optical sensor without using a cylindrical optical waveguide.

The reflector is preferably a reflector of a type that condenses parallel light at the focal point, and further preferably a cylindrical optical waveguide is disposed at the focal point. More preferably, the reflector is a reflector defined by a function shape of y = (1 / 4p) x ² with respect to the focus position p.
(First embodiment)
A first embodiment of the present invention will be described with reference to FIG. In the present embodiment, the reflector also serves as a part of the flow path for flowing the specimen and the cleaning liquid.

  A cylindrical optical waveguide 17 is disposed between the caps 16 in a flow path constituted by the cap 16 provided with the support 20, the reflector 11, the specimen or cleaning liquid inlet, or the outlets 21 and 22. A light source 19 is disposed at one end of the cylindrical optical waveguide 17, and excitation light emitted from the light source 19 is collected by the lens 18 and guided to the cylindrical optical waveguide 17. The fluorescent dye attached to the outer side wall of the cylindrical optical waveguide 17 of the cylindrical optical waveguide 17 is excited by an evanescent wave by the excitation light guided to the cylindrical optical waveguide 17 and emits fluorescence. The fluorescence is collected by the reflector 11 and transmitted through the support 20 to be emitted. The emitted transmitted light (fluorescence) is removed by the optical filter 12 after unnecessary wavelength components are collected, collected by the condenser lenses 13 and 14, and guided to the optical sensor 15.

The reflector 11 is preferably a reflector that collects parallel light at the focal point, and more preferably, the cylindrical optical waveguide 17 is disposed at the focal point. More preferably, the reflector 11 is a reflector defined by a function shape of y = (1 / 4p) x ² with respect to the focus position p.

  In the present embodiment, the cylindrical optical waveguide 17 is installed at the focal point of the reflector 11, the excitation light from the light source / optical systems 18 and 19 is guided to the cylindrical optical waveguide 17, and the fluorescent dye attached to the side wall Is excited by the evanescent light, and the light emitted as a result is reflected by the reflector 11 toward the optical system 12, 13, 14 by the reflector 11, and is efficiently collected in the optical system 12, 13, 14. It is characterized by being detected by the optical sensor 15 through the optical systems 12, 13, and 14.

  Since the excitation light propagates while being totally reflected in the optical waveguide, leakage to the outside of the optical waveguide hardly occurs.

  The reflector 11 has a parabolic shape, a semi-cylindrical shape, or a flat plate shape, and a mirror surface is made of a metal film such as Al, Ag, Au, or Cr or a dielectric multilayer reflective film (each interface of the multilayer film). The phase of the reflected light is matched to obtain a high reflectance by the interference effect). Furthermore, a passivation film made of a dielectric serving as a protective film may be provided on the metal film. In this case, it is more preferable to provide a ½ wavelength film silicon monoxide (SiO) or a dielectric multilayer reflective film as the passivation film.

  In this configuration, a parabolic reflector or a total reflection cylindrical concave mirror in which a dielectric multilayer film is deposited as a passivation film on Al or Ag is preferable. Further, in this configuration, a configuration in which the reflector also serves as a part of the flow path for flowing the sample and the cleaning liquid is preferable.

  As the optical filter 12, a color glass filter, a dichroic filter, a gelatin filter, a long pass filter, a visible light block filter, and an IR pass filter can be used. In this configuration, it is preferable to use a dichroic filter or a colored glass filter. Dichroic filters and colored glass filters are superior in wavelength separation and are excellent in supplying durability as industrial products.

  As the condensing lenses 13 and 14, a plano-convex cylinder lens, a plano-concave cylinder lens, an aspheric lens, a plano-convex or biconvex spherical lens group, a microscope objective lens, and a Selfoc lens can be used. In this configuration, since the condensed fluorescence becomes parallel light, it is preferable to use a plano-convex cylinder lens or a plano-concave cylinder lens capable of condensing the fluorescence on the surface.

  As the optical sensor 15, a photodiode or a photomultiplier tube can be used. In this configuration, a photodiode and a photomultiplier tube are appropriately selected depending on the concentration of the specimen. As the cap 16, a material such as a plastic resin, a semiconductor, or a metal can be processed and used. In this configuration, it is preferable to use a plastic resin or semiconductor colored black.

  The cylindrical optical waveguide 17 is preferably made of a material with little optical loss with respect to excitation light, and polystyrene (PS), polymethyl methacrylate (PMMA), and polycarbonate (PC) are preferably used.

  As the collimating lens 18, an aspherical lens, a Selfoc lens, a plano-convex or biconvex lens group, a microscope objective lens, or the like can be used. In this configuration, it is preferable to use a Selfoc lens or an aspheric lens.

  As the light source 19, a laser diode or a gas laser having a wavelength in the range of 200 nm to 1000 nm is selected and used. In this configuration, it is preferable to use a wavelength of 670 nm or less.

The support 20 is preferably made of a material with little light loss with respect to fluorescence, and plastic resin, various glasses, semiconductors, and the like are appropriately selected and used. In this configuration, it is preferable to use plastic resin or various glasses. The inlet / discharge ports 21 and 22 preferably have a configuration in which a through hole is formed in the cap 16 and a pipe is inserted to extend the tube.
(Second Embodiment)
A second embodiment of the present invention will be described with reference to FIG. In the present embodiment, the flow path and the reflector are separated.

  The same elements as those shown in the first embodiment are indicated by the same reference numerals. However, even if the constituent elements are the same, if they are elements of different shapes and materials, they will be described with different reference numerals.

  In the present embodiment, the cylindrical optical waveguide 17 is disposed inside the flow path 25, and the flow path 25 is provided with a cleaning liquid inlet or outlet 26, 27. One end of the cylindrical optical waveguide 17 is located outside the flow path 25, the excitation light emitted from the light source 19 is collected by the lens 18, and the excitation light is guided from the end to the cylindrical optical waveguide 11. The

As in the first embodiment, the reflector 23 is preferably a reflector of a type that focuses parallel light on a focal point, and it is preferable that the cylindrical optical waveguide 17 is disposed at the focal point. More preferably, the reflector 11 is a reflector defined by a function shape of y = (1 / 4p) x ² with respect to the focus position p.

  In the second embodiment, the cylindrical optical waveguide 11 integrated with the flow path 25 is installed at the focal position of the reflector 23. Excitation light from the light sources / optical systems 18 and 19 is guided to the cylindrical optical waveguide 17 and the fluorescent dye adhering to the side wall is excited by evanescent light. As a result, the emitted fluorescence is independent of the flow path 25. The light 23 that can not be captured by the optical systems 12, 13, 14 is reflected by the optical systems 12, 13, 14 by the reflector 23 provided in the optical system 12, 13, 14, and is efficiently collected in the optical systems 12, 13, 14. Then, the light sensor 15 detects the light.

  As the reflector 23, a parabolic, semi-cylindrical or flat plate shape is used, and the phase of reflected light at each interface of a metal film or multilayer film such as Al, Ag, Au, Cr is used on the mirror surface. A dielectric multi-layer reflective film is obtained as a reflective film, which achieves a high reflectivity by the interference effect by matching the above. Furthermore, a passivation film made of a dielectric serving as a protective film may be provided on the metal film. In this case, it is more preferable to provide a ½ wavelength film silicon monoxide (SiO) or a dielectric multilayer reflective film as the passivation film.

  In this configuration, a parabolic reflector or a total reflection cylindrical concave mirror in which a dielectric multilayer film is deposited as a passivation film on Al or Ag is preferable. As the cap 24, a material such as a plastic resin, a semiconductor, or a metal can be processed and used. In this configuration, it is preferable to process and use a plastic resin or semiconductor. As the flow path 25, a cylindrical capillary or tube is used, but a cylindrical capillary is preferable. As for the inlet / outlet 26 and 27, a configuration in which a through hole is formed in the flow path 25 and a pipe is inserted and the tube is extended is preferable.

  The present invention is not limited to the above-described embodiment, and various changes and modifications can be made without departing from the gist of the present invention.

Examples of the present invention will be described below, but the present invention is not limited only to the contents described herein.
(Example 1)
This will be described with reference to FIG. Reflector 11 is defined by a function-shaped y = (1 / 4p) x 2. p is the focal position of the reflector, and is expressed as a distance from the origin of the two-dimensional coordinate system. Here, p = 5 (mm) was set for trial manufacture. After processing the glass into a parabolic shape, the surface was mirror-polished, Al was deposited, and a SiO protective film was further coated. As an optical filter for No. 12, a dichroic filter (Asahi Spectroscopy, Cy5-Emisson filter) was used. As the cylindrical lenses 13 and 14, two plano-convex cylinder lenses (Sigma light machine, 20 mm x 40 mm: 1 sheet, 20 mm x 40 mm: 1 sheet) were used. A photodiode (Hamamatsu Photonics, S2833-01, plano-convex cylinder lens) was used as the 15 optical sensor. As the cap No. 16, black ABS resin was used so that extra stray light did not enter from the outside. In addition, a transparent PS product was used for 20 supports.

  The reflector 11, the cap 16 and the support 20 are bonded with an adhesive so that the specimen and the cleaning liquid do not leak out. As the cylindrical optical waveguide 17, a PS rod having a length of 40 mm and a diameter of 1 mmφ was used. In order to reduce the transmission loss of light, a tapered shape was provided in a range of 5 mm from the end face of the incident light. The shape of the taper is 1 mm in diameter at a point where the diameter of the end face is 1.5 mm and 5 mm from the end face. The cylindrical optical waveguide 17 is inserted from a hole provided in the cap 16 and is devised so that the specimen and the cleaning liquid do not leak out by the O-ring and the flange. As the light source 19, a laser diode (Sanyo Electric, DL3038-033) was used. As the collimating lens 18, two plano-convex lenses (Sigma optical machine, plano-convex lens 5 mmφ) were used in combination.

The optical waveguide was immersed in a Cy5 (Amersham Biosciences (US product fluorescent dye)) solution having a concentration of 1 × 10 ⁻⁷ mol / L, and then placed in the completed optical system. When laser light (wavelength: about 638 nm, effective intensity: 3 mW, modulated with a 135 Hz rectangular wave) is introduced from the laser diode, the fluorescence on the surface of the optical waveguide is excited by the evanescent light and efficiently collected by the reflector, and the photodiode It was confirmed that it was detected with high sensitivity.

Next, detection of PSA antibody (prostate specific antigen) known as a marker for prostate cancer is attempted. First, streptavidin is attached to the surface of the optical waveguide. Then, biotin-modified PSA antibody is adsorbed, and an immunosensor is prepared and set in a fluorescence analyzer. Then do the following protocol.
(1) A PSA antibody fluorescently labeled with Cy5 dye is introduced into the flow path and incubated for 5 minutes.
(2) The labeled antibody is extracted and washed with a phosphate buffer.
(3) A solution mixed with an antigen protein is introduced into the flow path and incubated for 5 minutes.
(4) Remove the antigen solution and wash with phosphate buffer.
(5) The labeled antibody is introduced into the channel and incubated for 5 minutes.
(6) The labeled antibody is extracted and washed with a phosphate buffer.
(7) A phosphate buffer solution is injected into the flow path.

When laser light was introduced after the operation of (7) and the concentration of PSA protein was measured, it was confirmed that the measurement was very sensitive to 0.1 ng / mL.
(Example 2)
Using the optical system used in Example 1, DNA hybridization was measured. Streptavidin was attached to the surface of the optical waveguide, biotin-modified 20-mer DNA was adsorbed, and an immunosensor was prepared. The following protocol was then performed.
(1) A complex obtained by fluorescently labeling a capture DNA (target T1, 20-mer) corresponding to a fixed DNA (probe) with a Cy5 dye and a 20-mer DNA (target) having a base sequence that differs by one Prepare a sample solution in which a complex labeled with a Cy3 dye (T2, 20-mer) is mixed.
(2) The sample liquid is introduced into the flow path and then incubated for 5 minutes.
(3) Remove the specimen and wash with phosphate buffer.
(4) A phosphate buffer solution is injected into the flow path.

  When laser light was introduced after the operation of (4) and the fluorescence intensity was measured, it was confirmed that the DNA concentration was measured with high sensitivity up to 1 nM. When the fluorescence spectrum is measured with a spectroscope (not shown), it is confirmed that only a dye having a peak near 670 nm emits fluorescence, and that only T1 DNA is specifically bound. It was done.

It is a figure showing one of the embodiment of this invention. It is a figure showing one of the embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11: Reflector 12: Optical filter 13, 14: Condensing lens 15: Optical sensor 16: Cap 17: Cylindrical optical waveguide 18: Lens 19: Light source 20: Support body 21, 22: Injecting or discharging of specimen or cleaning liquid Outlet 23: Reflector 24: Channel 25: Channel 26, 27: Sample or cleaning liquid inlet or outlet

Claims (6)

A light propagator for propagating light inside the light propagator,
Directing means for directing fluorescence generated by excitation of the detected substance in a predetermined direction by excitation light propagating through the propagating body when the detected substance adheres to the outer surface of the light propagating body;
An optical analysis apparatus for a substance to be detected, comprising: a light detection means for detecting fluorescence directed in a predetermined direction by the directing means. The directing means is a reflector,
The optical analyzer according to claim 1, wherein a cross-sectional shape of the reflecting surface of the reflecting plate is a parabolic shape defined by the following formula (1).
y = (1 / 4p) x ² (1)
(P is the focal position of the reflector and is expressed as the distance from the origin of the two-dimensional coordinate system. The origin is the bottom (minimum point) of the reflector.)   3. The optical analyzer according to claim 2, wherein the surface of the reflecting surface of the reflecting plate is made of at least a metal or a dielectric multilayer film. The optical analyzer according to any one of claims 1 to 3, wherein an outer surface of the light propagating body has an outer surface to which a capturing component for capturing a target substance bound to a fluorescent substance can be attached. .   5. The optical analyzer according to claim 4, wherein the binding between the substance to be detected and the capture component is due to an antigen-antibody reaction.   5. The optical analyzer according to claim 4, wherein the binding between the substance to be detected and the capture component is due to a DNA hybridization reaction.

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