JP2013007726A - Reflector and capillary electrophoretic analyzer having the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly versatile reflector which allows detection sensitivity of a fluorescence detection part to be easily improved at low cost, exerting no influence on electrophoresis and electroosmotic flow; and a capillary electrophoretic analyzer having the same.SOLUTION: A reflector encloses a part of a capillary and includes an excitation light incident part through which excitation light passes, an excitation light reflection part which reflects the incident excitation light coming through the excitation light incident part and allows fluorescent light to pass through, and a reflection part which reflects the excitation light and the fluorescent light. The reflector is provided at the fluorescent light detection part of a capillary electrophoretic analyzer.

Description

  The present invention relates to a reflector that can be suitably implemented as a fluorescence detector of a capillary electrophoresis analyzer and a capillary electrophoresis analyzer including the reflector.

  Capillary electrophoresis analyzers are used in a wide range of research from the chemical and biological fields to the medical field, and include relatively low-molecular organic compounds such as drugs, amino acids, neurotransmitters, oligosaccharides and other sugar chains, nucleic acids, and proteins. It is used for separation and analysis of biopolymers such as

  The capillary electrophoresis analyzer includes a hollow capillary made of fused silica having an inner diameter of about 10 to 200 μm, a sample introduction side electrophoresis tank provided at one end of the capillary, and a discharge side electrophoresis tank provided at the other end of the capillary as an electrolyte. After filling with the electrophoresis solution that is a solution and introducing the sample to be analyzed into the tip of the sample introduction side capillary, a high voltage is applied between the electrophoresis tanks to separate the analyte in the sample.

  In the vicinity of the other end of the capillary, the liquid to be analyzed containing the analyte in the capillary is irradiated with light serving as excitation light, thereby exciting the separated analyte and generating fluorescence. By detecting the generated fluorescence with a fluorescence detector, it is an apparatus that can analyze whether or not an analyte exists in the liquid to be analyzed and the amount of the analyte.

  A fluorescence detection unit of a typical capillary electrophoresis analyzer is disclosed in Patent Document 1. According to the technique disclosed in Patent Document 1 (hereinafter referred to as “prior art 1”), the fluorescence detection unit of the capillary electrophoresis analyzer includes excitation light generating means for emitting excitation light, and excitation light on the capillary side. And a collector that reflects and condenses the fluorescence induced by the analyte in the liquid to be analyzed to the fluorescence detector side, and a light receiving portion that receives the fluorescence, and is received by this light receiving portion. And a fluorescence detector for detecting the intensity of the fluorescence.

  By the way, it is necessary to improve the detection sensitivity of the fluorescence detector for the analysis of trace substances. As a method for improving the detection sensitivity of the fluorescence detector, it is conceivable to increase the distance from when the excitation light enters the capillary until it exits, and to irradiate the analyte with much excitation light.

  However, in the prior art 1, since the excitation light is irradiated in a direction perpendicular to the axis of the capillary, the distance at which the excitation light is irradiated on the capillary has a length depending on the inner diameter of the capillary, and the capillary It will be dominated by the shape of.

  Patent Document 2 discloses a technique for increasing the distance from when the excitation light enters the capillary until it exits. In the technique disclosed in Patent Document 2 (hereinafter referred to as “Prior Art 2”), the capillary of the portion irradiated with the excitation light is formed into a bent shape, also referred to as a crank shape, and thereby the excitation light in the capillary is irradiated. By increasing the distance of the section, the irradiation amount of the excitation light to the analyte in the capillary is increased.

JP-A-9-119809 JP-A-4-363655

  However, in the prior art 2, since a small-diameter capillary made of fused silica having a melting point of about 1300 ° C. must be bent and processed, the processing of the capillary requires a large amount of cost, and the manufacturing cost of the capillary is high. In addition, it is necessary to change the shape of the detection window.

Further, in the prior art 2, since a part of the capillary is bent, electrophoresis (abbreviation: EP) and electroosmotic flow (abbreviation: EOF) are disturbed at the bent portion of the capillary. End up. As a result, the analyte is diffused, so that not only the peak shape of the analyte in the fluorescence detector becomes small, but also the peak shape is disturbed and the separation efficiency is lowered.
Further, the bent portion of the capillary may be damaged by heat generated when a high voltage is applied.

  In addition, since it is necessary to change the structure of the capillary electrophoresis analyzer in accordance with the shape of the capillary, it cannot be easily used in an existing capillary electrophoresis analyzer and has low versatility. Therefore, in the prior art 2, the detection sensitivity of the fluorescence detection unit cannot be improved inexpensively and easily.

  Therefore, an object of the present invention is to provide a highly versatile reflector that does not affect electrophoresis and electroosmotic flow, and that can improve the detection sensitivity of a fluorescence detection unit easily and inexpensively, and a capillary electric device including the same. An electrophoretic analyzer is provided.

The present invention comprises a capillary for electrophoresis of an analyte,
One end of the capillary is connected, and an introduction-side storage tank in which the electrophoresis solution is stored;
The other end of the capillary is connected, and a discharge-side storage tank that stores waste liquid flowing out from the capillary,
Electrodes provided respectively in the introduction-side storage tank and the outlet-side storage tank;
A power supply for applying a voltage between the electrodes;
A reflector provided in a fluorescence detection unit of a capillary electrophoresis apparatus including a fluorescence detection unit that irradiates a capillary with excitation light and detects fluorescence generated by an analyte in the capillary,
The reflector is provided so as to surround a part of the capillary,
An excitation light incident part through which the excitation light is transmitted;
An excitation light reflecting part that reflects the excitation light incident through the excitation light incident part and transmits fluorescence;
A reflector that reflects excitation light and fluorescence.

According to the present invention, the internal space of the reflector is filled with a refractive liquid.
Moreover, this invention is a capillary electrophoresis apparatus provided with the said reflector.

  According to the present invention, the reflector is provided so as to surround a part of the capillary, reflects the excitation light incident part through which the excitation light is transmitted, and the excitation light incident through the excitation light incident part and transmits the fluorescence. An excitation light reflection part and a reflection part that reflects excitation light and fluorescence are included.

  Thereby, the reflector can reflect the excitation light incident from the excitation light incident part by the reflection part. Therefore, since the capillary can be irradiated with excitation light a plurality of times, the amount of excitation light incident on the capillary can be increased, and the analyte can be excited reliably.

  In addition, the fluorescence emitted from the analyte by being irradiated with the excitation light is transmitted through the excitation light reflecting portion and guided to the light receiving portion of the fluorescence detector. Therefore, the fluorescence emitted from the analyte can be reliably emitted to the light receiving portion of the fluorescence detector.

  Thus, according to the present invention, the amount of excitation light incident on the capillary can be increased, and the fluorescence emitted from the analyte can be reliably emitted to the light receiving portion of the fluorescence detector. Therefore, the detection sensitivity of the fluorescence detector can be improved.

  In addition, the reflector may be attached so as to surround the existing capillary, so that it does not negatively affect electrophoresis and electroosmotic flow.

  Further, unlike the prior art, it is not necessary to change the shape of the capillary, so that the detection sensitivity of the fluorescence detection section can be improved easily and inexpensively. Moreover, since the reflector can be attached to an existing capillary electrophoresis apparatus, the versatility is high.

  Therefore, it is possible to realize a highly versatile reflector that can easily and inexpensively improve the detection sensitivity of the fluorescence detection unit without affecting electrophoresis and electroosmotic flow.

According to the invention, the internal space of the reflector is filled with a refractive liquid.
As a result, reflection of the excitation light incident on the capillary surface from the excitation light incident portion can be reduced. Therefore, the analyte can be irradiated with more excitation light, so that the detection sensitivity of the fluorescence detector can be further improved.

According to the invention, a capillary electrophoresis apparatus includes the reflector.
Thereby, a capillary electrophoresis apparatus with high detection sensitivity can be realized.

1 is a system diagram showing a configuration of a capillary electrophoresis apparatus 1 including a reflector 12 according to an embodiment of the present invention. 3 is a cross-sectional view schematically showing a configuration of a fluorescence detection unit 8. FIG. 3 is a perspective view showing a configuration of a reflector 12. FIG. 3 is a flowchart showing a manufacturing process of the reflector 12. It is a flowchart which shows the attachment process of the reflector. It is a graph which shows the measurement result of the fluorescence received light quantity by the fluorescence detector 13 for confirming that the detection sensitivity of the capillary electrophoresis apparatus 1 improves by installation of the reflector 12. It is a perspective view which shows the structure of 12 A of reflectors of other embodiment of this invention. It is sectional drawing of 12 A of reflectors seen from the cut surface line VI-VI of FIG.

  FIG. 1 is a system diagram schematically showing a configuration of a capillary electrophoresis apparatus 1 including a reflector 12 according to an embodiment of the present invention.

  The capillary electrophoresis apparatus 1 according to this embodiment includes a reflector 12 and is used to detect the presence of a biological substance or a chemical substance that requires quantification in a sample as an optical change due to fluorescence. .

  The capillary electrophoresis apparatus 1 includes a capillary 2 through which a liquid material containing an analyte is guided by electrophoresis, an introduction-side storage tank 3 in which one end of the capillary 2 is connected, and the electrophoresis solution is stored; Are connected to each other, and a discharge side storage tank 4 for storing the liquid waste liquid flowing out from the capillary 2, electrodes 5a and 5b provided in the introduction side storage tank 3 and the discharge side storage tank 4, respectively. A power source 6 that applies a DC voltage between the electrodes 5a and 5b, a thermostat 7 that keeps the temperature of the capillary 2 constant, and a downstream side of the thermostat 7 that irradiates the capillary 2 with excitation light; And a fluorescence detection unit 8 that detects fluorescence emitted from the analyte in the capillary 2.

  The capillary 2 is made of, for example, fused silica and is a cylindrical body having an inner diameter of 10 to 200 μm and an outer diameter of 150 to 400 μm. One end of the capillary 2 is connected to an introduction-side storage tank 3 in which the electrophoresis solution is stored. The other end of the capillary 2 is connected to a lead-out storage tank 4 that stores the liquid material that has flowed down the capillary 2.

  The introduction-side storage tank 3 and the outlet-side storage tank 4 are provided with electrodes 5a and 5b, respectively. In the present embodiment, the electrodes 5 a and 5 b are made of platinum and are connected to the power supply unit 6. A DC voltage of about 5000 V to 30000 V is applied between the electrode units 5 a and 5 b by the power supply unit 6.

  A sample containing an analyte is supplied to the capillary 2. Specifically, one end of the capillary 2 is inserted into a sample tank in which a sample containing an analyte is stored, a voltage is applied between the sample tank and the outlet-side storage tank 4, and the capillary 2 is subjected to electrophoresis. A sample is introduced in the length of about several mm to several cm. The introduction of the sample into the capillary 2 is not limited to this. For example, a drop is provided at the installation position between the sample tank and the outlet-side storage tank 4, and the movement of the electrophoretic liquid due to this drop is used to move the capillary 2 into the capillary 2. A method of introducing the sample into the tube or a method of pressurizing and injecting the sample by pressurizing the introduction side storage tank 3 side of the capillary 2 can be considered.

  After the sample is introduced into the capillary 2, one end of the capillary 2 is returned to the introduction-side storage tank 3, and a voltage is applied between the electrodes 5 a, 5 b, whereby the electrophoresis solution and the sample are extracted by electrophoresis. Move to the 4th side.

  At this time, a liquid that is a mixture of the analyte separated from the sample and the electrophoresis liquid flows down in the capillary 2 and moves to the outlet-side storage tank 4. Hereinafter, the direction in which the analyte moves in the capillary 2 will be referred to as the moving direction, and the introduction-side storage tank 3 will be referred to as the upstream side, and the outlet-side storage tank 4 will be described as the downstream side.

  A thermostatic chamber 7 is provided on the downstream side of the introduction-side storage tank 3. The thermostat 7 keeps the ambient temperature in the thermostat 7 at about 10 ° C. to 50 ° C. by forcibly circulating a gas such as an electrically insulating liquid or air kept at a constant temperature. Accordingly, it is possible to prevent the temperature of the liquid in the capillary 2 from fluctuating undesirably, so that the refractive index, electroosmotic flow rate, and analyte accompanying the temperature change of the capillary 2 and the liquid in the capillary 2 can be prevented. It is possible to prevent the measurement accuracy from deteriorating due to variations in the fluorescence intensity of the analyte and the integrated value (area) of detection peaks due to the change in the electrophoresis speed. A fluorescence detection unit 8 is provided in the vicinity of the thermostat 7 on the downstream side of the thermostat 7.

  FIG. 2 is a cross-sectional view schematically illustrating the configuration of the fluorescence detection unit 8, and FIG. 3 is a diagram schematically illustrating the configuration of the reflector 12. In FIG. 3, detailed configuration other than the reflector 12 is omitted for easy understanding.

  The fluorescence detection unit 8 includes an excitation light emitting unit 10 that emits the excitation light L, a condenser lens 11 that collects the excitation light L emitted by the excitation light emission unit 10, a reflector 12, and a fluorescence detector 13. And a collector 14 that reflects light to the fluorescence detector 13 and a stage 17.

  The excitation light emitting means 10 emits excitation light L for exciting the analyte. The wavelength of the excitation light L may be selected according to the analyte. For example, when the analyte is sodium fluorescein described later, the wavelength of the excitation light L is selected to be 475 nm. The excitation light emitting means 10 is realized by a semiconductor laser device, for example. The excitation light emitting means 10 is not limited to a semiconductor laser device, and a xenon lamp, a light emitting diode (abbreviated as “LED”), and the like can be used, for example.

  The sodium fluorescein is a fluorescent dye. In other embodiments of the present invention, the non-fluorescent material can be labeled with a fluorescent dye. In addition to sodium fluorescein, aminomethylcoumarin (AMCA); tetramethylrhodamine (TAMRA); diethylaminomethylcoumarin (DEAC); carboxy-fluorescein (FAM); carboxy-tetramethylrhodamine (TAMRA); carboxy-X- Rhodamine (ROX); Cascade Blue (CB); Fluorescein isothiocyanate (FITC); Oregon Green (OG); Alexa 488 (A488); Rhodamine Green (RGr); Carboxy-Rhodamine 6G (R6G); Texas Red (TxR); Cy3.5; Cy5; Cy5.5; Cy7; fluorescent labels such as carboxynaphthofluorescein (CNF) and Bodipy® dyes, or biotin ( IO); digoxigenin (DIG); and hapten labeling such as 2,4-dinitrophenyl (DNP), or one or more species selected from the group of green fluorescent protein (GFP) fusion protein, etc. it can.

  The capillary 2 may be configured such that the liquid flow is enhanced by capillary action, such as by coating the inner peripheral surface of the capillary 2. The coating for improving the fluidity of the capillary 2 by capillary action may be realized by subjecting the inner peripheral surface of the capillary 2 to corona discharge treatment, or a material having high water repellency on the capillary 2 itself, for example, clear polystyrene. , Polyester (PE), polymethylpentene (PMP), and PETG may be used.

  The condensing lens 11 condenses the excitation light L emitted from the excitation light emitting means 10 on the axis of the capillary 2. When a xenon lamp is used as the excitation light emitting unit 10, an optical filter 15 is provided between the excitation light emitting unit 10 and the condenser lens 11. The optical filter 15 transmits light having a predetermined wavelength. By providing the optical filter 15, only the excitation light L having a wavelength corresponding to the analyte can be transmitted and guided to the condenser lens 11. In another embodiment of the present invention, a diffraction grating may be used in place of the optical filter 15.

  As the optical filter 15, it is preferable to use an interference filter. As the interference filter, an interference filter having transmission and reflection characteristics necessary for an arbitrary wavelength band can be manufactured. For this reason, if an interference filter is used as the optical filter 15, it is easy to select the transmittance and the reflectance, so that light can be transmitted and reflected efficiently without loss.

  Here, the interference filter refers to a multilayer structure formed by laminating an arbitrary dielectric thin film or metal thin film in an arbitrary order in order to give a desired transmission characteristic.

The dielectric thin film is a hydrophilic or non-hydrophilic inorganic compound such as TiO 2 (titanium dioxide), Ta ₂ O ₅ (tantalum pentoxide), ZrO ₂ (zircon oxide), A1 ₂ O ₃ (aluminum oxide). ), Nb ₂ O ₅ (niobium pentoxide), SiO ₂ (silicon oxide), MgF ₂ (magnesium fluoride), and the like. Each film thickness of the dielectric multilayer thin film layer is set to several tens nm to several hundreds nm.

  Further, the “metal thin film layer” refers to a very thin film structure of a metal that is hardly oxidized and has a metallic luster such as gold, silver, and platinum. The method for forming these dielectric thin film and metal thin film is not particularly limited, but is preferably formed by vapor deposition or sputtering. Such an interference filter is formed on the surface of a substrate made of transparent glass or plastic.

  The fluorescence detector 13 includes a diffusing lens 20, a light receiving unit 21, and a signal processing unit 22. The diffusion lens 20 diffuses the fluorescence A emitted from the analyte and guides it to the light receiving unit 21. The light receiving unit 21 receives and detects the fluorescence A guided from the diffusing lens 20, generates a detection signal based on this, and transmits the detection signal to the signal processing unit 22. The signal processing unit 22 receives a detection signal from the light receiving unit 21, generates a detection result in response to the received detection signal, transmits the detection result to a display device (not shown), and the analyte is visualized as a detection image. It can be displayed as a spectrum representing the received light intensity with respect to the wavelength.

  Specifically, the signal processing unit 22 performs analog / digital conversion on the detection signal acquired from the light receiving unit 21 to generate, for example, 8-bit fluorescent image data, and to the display device realized by a liquid crystal display panel or the like. Output and display image. For example, when the analyte is sodium fluorescein, in the image displayed in this manner, a stained material having fluorescence of sodium fluorescein is displayed as a spectrum representing the received light intensity with respect to the visualized wavelength.

  On the light receiving surface of the light receiving unit 21, wavelength conversion is performed to convert the incident light having the wavelength λ1 from the diffusing lens 20 to the wavelength λ2 suitable for the light receiving characteristics of the light receiving element behind, that is, the highest light receiving sensitivity. An element may be provided. The wavelength conversion element includes a wavelength conversion substance and a fixing material that disperses and fixes the wavelength conversion substance.

  Examples of the wavelength converting material include organic phosphors such as rhodamine 6G and fluorescein, inorganic phosphors such as YAG phosphors doped with cerium (Ce), BAM phosphors doped with europium (Eu), and the like.

  The collector 14 is installed on the stage 17 with the opening facing the diffusion lens 20 in order to totally reflect the excitation light L and the fluorescence A and guide them to the diffusion lens 20 of the fluorescence detector 13. In the present embodiment, the collector 14 has a reflective layer 14a formed on the inner peripheral surface.

  The reflective layer 14a has a reflectance of 95% or more with respect to the light having the wavelengths of the excitation light L and the fluorescence A, and is preferably formed so that the transmittance is 0%. The collector 14 is formed so that the analyte is irradiated with the excitation light L and the fluorescence A is reflected by the fluorescence detector 13.

  Here, the reflectance and transmittance in this specification refer to the reflectance and transmittance defined in JIS H 0201: 1998, and can be measured in accordance with JIS-K-7375.

  The collector 14 is provided so as to contain the reflector 12. The collector 14 has an axis parallel to the axis of the insertion portion of the capillary 2, and a cross section perpendicular to the axis, that is, a cross section perpendicular to the paper surface of FIG. 2 is substantially U-shaped. The collector 14 is configured such that the inner peripheral surface thereof faces the diffusion lens 20 of the fluorescence detector 13 via the reflector 12 and guides the reflected light of the reflector 12 to the diffusion lens 20.

  The reflector 12 reflects the excitation light L and the fluorescence A, is provided in the first reflection part 30 having a substantially U-shaped cross section, and the first reflection part 30, and transmits the excitation light L and reflects the fluorescence A. A rectangular plate-shaped excitation light incident portion 31 and one end portion of the first reflection portion 30 are connected to the other end portion, are provided to face the first reflection portion 30, reflect the excitation light L, and transmit the fluorescence A. Second and third reflecting portions 33a and 33b, which are provided at both ends in the longitudinal direction of the excitation light reflecting portion 32 and the first reflecting portion 30, respectively, reflect the excitation light L and the fluorescence A, and are substantially rectangular in a plan view. It is comprised including. In this embodiment, the reflector 12 is formed in a hollow quadrangular prism shape. In another embodiment of the present invention, the reflector 12 may be formed in a substantially semicircular shape in plan view.

  The first reflecting unit 30 is realized by forming an arbitrary dielectric thin film or metal thin film on the outer surface of the glass, and has a reflectance of 95% or more with respect to the excitation light L and the fluorescence A. Have.

  The excitation light incident part 31 is formed on a part of the first reflection part 30. The excitation light incident part 31 can be realized by forming a region where the dielectric thin film or the metal thin film is not formed in a part of the region where the first reflection unit 30 is provided.

  The excitation light reflection part 32 is realized by forming a dielectric thin film or a metal thin film on the outer surface part of glass, and reflects 99% or more of the excitation light L having a wavelength of 450 nm to 485 nm. And a reflectance of 5% or less with respect to the fluorescence A having a wavelength of 495 nm or more.

  The second and third reflecting portions 33a and 33b are realized by forming a dielectric thin film or a metal thin film on the outer surface of the glass as a base, and for the excitation light L having a wavelength of 450 nm to 485 nm. It has a transmittance of 99% or more and a transmittance of 5% or less with respect to the fluorescence A having a wavelength of 495 nm or more.

  In addition, insertion holes 34a and 34b for inserting the capillary 2 are formed in the second and third reflecting portions 33a and 33b, respectively. The capillary 2 is inserted into the reflector 12 through the insertion hole 34a, passes through the internal space of the reflector 12, and is guided out of the reflector 12 through the insertion hole 34b.

  A refractive liquid is sealed in the internal space of the reflector 12. As the refracting liquid, a liquid having a refractive index substantially equal to the refractive index of the capillary 2 is selected. As the refractive liquid, for example, a refractive liquid manufactured by Cargill (trade name: phased silica matching liquid) can be used.

  In this embodiment, since the capillary 2 is formed of fused silica, the refractive liquid having a refractive index of 1.4587nd to 1.4592nd is selected.

  By enclosing the refractive liquid in the internal space of the reflector 12, the critical angle at the boundary surface between the outer peripheral surface of the capillary 2 and the internal space of the reflector 12 is increased. Therefore, reflection of the excitation light L incident on the capillary 2 from the excitation light incident part 42 on the surface of the capillary 2 can be reduced. As a result, the analyte can be irradiated with more excitation light L.

  FIG. 4 is a flowchart showing a manufacturing process of the reflector 12, and FIG. 5 is a flowchart showing an attaching process of the reflector 12. Hereinafter, the manufacturing method and attachment method of the reflector 12 will be described.

<Manufacturing method>
In step S0, a hollow quadrangular prism body made of transparent glass or synthetic resin as a base material of the base is prepared, and the manufacture of the reflector 12 is started. In step S1, the longitudinal direction of the hollow quadrangular prism body is started. At both ends, through holes 34a and 34b are respectively formed using a punching device, and a base is manufactured. When the base is thus manufactured, the process proceeds to step S2.

  In step S2, a base cleaning process is performed. In this base cleaning process, using an air brush or the like, the perforated waste generated by the formation of the insertion holes 34a and 34b in the base molding process in step S1 is removed, and the inner peripheral portion and the outer surface portion of the base are cleaned. . When the cleaning of the base is completed, the process proceeds to step S3.

  In step S3, a reflection part formation process is performed. In the reflection portion forming step, the first reflection portion 30 and the second and third reflection portions 33a and 33b are formed. An excitation light incident part 31 is also formed.

  Specifically, among the four main surface portions of the base, the three main surface portions and each end surface portion of the base have a wavelength of 5% or less with respect to excitation light L having a wavelength of 450 nm to 485 nm and fluorescence A having a wavelength of 495 nm or more. A dielectric thin film or a metal thin film is formed by vapor deposition or sputtering so as to have transmittance, thereby forming the first reflecting portion 30 and the second and third reflecting portions 33a and 33b.

  At this time, masking is performed on a predetermined region on one main surface portion of the three main surface portions where the first reflecting portion 30 is to be formed. In the masked region, the above-described dielectric thin film or metal thin film is not formed.

  Thus, after forming the 1st reflection part 30, the 2nd and 3rd reflection parts 33a and 33b, the excitation light incident part 31 is formed in the said area | region by removing masking. If the 1st reflection part 30 and the 2nd and 3rd reflection parts 33a and 33b are formed, it will shift to the following step S4.

  In step S4, an excitation light reflecting portion forming step is performed. In the excitation light reflecting portion forming step, the excitation light reflecting portion 32 is formed.

  Specifically, of the four main surface portions of the base, the main surface portion where the first reflecting portion 30 is not formed has a reflectance of 99% or more with respect to the excitation light L having a wavelength of 450 nm to 485 nm. The excitation light reflecting portion 32 is formed by forming a dielectric thin film or a metal thin film by a vapor deposition method or a sputtering method so as to have a reflectance of 5% or less with respect to the fluorescence A having a wavelength of 495 nm or more. When the excitation light reflecting portion 32 is formed, the manufacturing process of the reflector 12 is finished (step S5).

<Installation process>
In the attachment process, the reflector 12 is prepared and the attachment process of the reflector 12 is started (step T0).

  In step T1, the capillary 2 is inserted from the insertion hole 34a, inserted from the insertion hole 34b, and the capillary 2 is inserted into the reflector 12. When the capillary 2 is inserted through the reflector 12, the process proceeds to the next step T2.

  In step T <b> 2, the refractive liquid is dropped into the internal space of the reflector 12. Specifically, using a syringe, the refractive liquid is injected from the insertion hole 34a to fill the internal space of the reflector 12 with the refractive liquid. When the refractive space is filled in the internal space of the reflector 12, the process proceeds to the next step T3.

  In step T 3, the capillary 2 is moved to move the reflector 12 onto the stage 17, the excitation light incident portion 31 is provided so as to face the condenser lens 11, and the optical axis of the excitation light L is aligned with the axis of the capillary 2. Adjust to match. When this process is completed, the attachment process of the reflector 12 is completed (step T4).

  According to the present embodiment, the reflector 12 reflects the excitation light L and the fluorescence A, is provided in the first reflection part 30 having a substantially U-shaped cross section, and the first reflection part 30 and transmits the excitation light L. In addition, a rectangular plate-shaped excitation light incident part 31 that reflects the fluorescence A and one end part of the first reflection part 30 are connected to the other end part, and are provided to face the first reflection part 30 to reflect the excitation light L. In addition, the excitation light reflecting part 32 that transmits the fluorescence A and the first reflection part 30 are provided at both ends in the longitudinal direction, respectively, reflect the excitation light L and the fluorescence A, and are substantially rectangular in the plan view. 3 reflection parts 33a and 33b.

  By adopting such a configuration, the reflector 12 converts the excitation light L that has entered the internal space from the excitation light incident unit 31 into the first reflection unit 30, the second and third reflection units 33a and 33b, and the excitation. The light can be reflected by the light reflecting portion 32.

  As a result, the capillary 2 can be irradiated with the excitation light L a plurality of times, so that the incident amount of the excitation light L into the capillary 2 can be increased, and the analyte can be reliably excited. .

  Further, the fluorescence A emitted from the analyte by being irradiated with the excitation light L is reflected by the first reflection unit 30, the second and third reflection units 33a and 33b, and the excitation light incident unit 31, and the excitation light. The light is transmitted through the reflecting portion 32 and guided to the light receiving portion 21 of the fluorescence detector 13. Therefore, the fluorescence A emitted from the analyte can be reliably emitted to the light receiving unit 21 of the fluorescence detector 13.

  Thus, by providing the reflector 12, the incident amount of the excitation light L into the capillary 2 can be increased, and the fluorescence A emitted from the analyte can be reliably received by the fluorescence detector 13. Since it can be made to radiate | emit to the part 21, the detection sensitivity of the fluorescence detector 13 can be improved.

  Further, the reflector 12 may be attached so as to surround the existing capillary 2, so that the electrophoresis and electroosmotic flow are not disturbed.

  Further, unlike the prior art, since it is not necessary to change the shape of the capillary 2, the detection sensitivity of the fluorescence detector 13 can be improved easily and inexpensively. Moreover, since the reflector 12 can be attached to an existing capillary electrophoresis apparatus, the versatility is high.

  Furthermore, one of the second and third reflecting portions 33a and 33b of the reflector 12 is not formed, but is formed in a rectangular tube shape whose one end in the longitudinal direction is opened, and the insertion hole 34a or 34b is not formed. The reflector 12 can also be used in ordinary cells used in micro LC detectors, nano LC detectors, and the like. Accordingly, the reflector 12 is highly versatile because it can improve the detection sensitivity of a fluorescence detection unit such as a micro LC detector, a nano LC detector, a microchip electrophoresis apparatus, or a DNA chip.

  Furthermore, since the reflector 12 has the 1st reflection part 30, the function of the collector 14 can also be exhibited.

  FIG. 6 is a graph showing the measurement result of the amount of received fluorescence by the fluorescence detector 13. In order to confirm that the detection sensitivity of the capillary electrophoresis apparatus 1 is improved by the installation of the reflector 12, the amount of fluorescence received by the fluorescence detector 13 is measured, and the measurement result is shown in FIG. In FIG. 6, the vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the concentration of fluorescein sodium (μM).

<Measurement conditions>
Equipment: JASCO Product name: FP-6200 type spectrofluorometer Measurement temperature: Room temperature (23 ° C)
Sample: Fluorescein sodium aqueous solution Excitation wavelength: 475 nm
Fluorescence wavelength: 511 nm

Niobium oxide (Nb ₂ O ₅ ) and silicon oxide (SiO ₂ ) are alternately stacked on a standard cell (quartz glass cell T-3-UV-10, manufactured by Tosoh Quartz Co., Ltd.) by a vacuum deposition method. A wave pass filter was formed, and the first and second reflection portions and the excitation light reflection portion were formed.

  Further, the surface other than the surface facing the light receiving portion of the cell as a collector (mirror) was surrounded by a rusted aluminum foil. In order to transmit the excitation light, a part of the excitation light reflecting portion was cut into a 7 mm × 7 mm square on one surface where the excitation light was incident in the region surrounded by the aluminum foil, thereby forming the excitation light incident portion. . In Table 1, the glossy inner side indicates the result when the aluminum foil is surrounded with the glossy side of the aluminum foil facing the cell, and the cloudy inner side is the opposite side of the glossy side of the aluminum foil. The result when the aluminum foil is enclosed with the surface facing the cell is shown. The results are shown in Table 1.

The characteristics of a normal cell based on the measured values shown in Table 1 above are given by the following approximate expression:
y = 117.35x-4.1877 (1)
Where R ² = 0.99991
The characteristics of the mirror (inner gloss) are indicated by the following approximate expression:
y = 330.81 + 4.70747 (2)
Where R ² = 0.9999
The characteristics of the mirror (cloudy inside) are indicated by
y = 436.47x + 10.31 (3)
Where R ² = 0.9996
Indicated by.

  As shown in Table 1, the detection sensitivity was about twice that of the normal cell on the glossy inner side, and the detection sensitivity was about 2.5 times that of the normal cell on the cloudy inner side. In this way, by providing the reflector 12 in the capillary electrophoresis apparatus, the amount of excitation light applied to the cell can be increased, thereby increasing the amount of fluorescence generated and improving the detection sensitivity. confirmed.

  As a modification of the present embodiment, an example in which the first reflecting unit 30 is not formed is conceivable. As described above, the collector 14 totally reflects the excitation light L and the fluorescence A. Therefore, even if the first reflection part 30 is not formed, the same effect as the first reflection part 30 can be obtained by providing the collector 14. In this case, it is not necessary to form a dielectric thin film or a metal thin film in a region corresponding to the first reflecting portion 30 in the manufacturing process.

  FIG. 7 is a perspective view showing a configuration of a reflector 12A according to another embodiment of the present invention, and FIG. 8 is a cross-sectional view of the reflector 12A as seen from the section line VI-VI in FIG. Note that parts corresponding to those of the above-described embodiment are denoted by the same reference numerals.

  The reflector 12 </ b> A reflects the excitation light L and the fluorescence A, is connected to the semicircular arc-shaped first reflection part 40, one end part of the first reflection part 40 to the other end part, and faces the first reflection part 40. A rectangular plate-shaped excitation light reflecting portion 41 that reflects the excitation light L and transmits the fluorescence A, and an excitation light incident that is provided in the excitation light reflection portion 41 and transmits the excitation light L and reflects the fluorescence A. Part 42, and provided at both ends in the longitudinal direction of the first reflecting part 40, respectively, reflect the excitation light L and the fluorescence A, and include second and third reflecting parts 43a and 43b that are semicircular in a plan view. Composed. In the present embodiment, the reflector 12A is formed in a hollow substantially semi-cylindrical shape.

  Insertion holes 45a and 45b through which the capillary 2 is inserted are formed in the second and third reflecting portions 43a and 43b, respectively. The insertion holes 45 a and 45 b are formed in a shape slightly larger than the outer diameter of the capillary 2.

  A holding portion 46a protruding inward of the reflector 12A is formed on the inner surface portion of the second reflecting portion 43a. The holding part 46a has an insertion hole 47a whose central axis coincides with the central axis of the insertion hole 45a, and has a disk-shaped internal space 48a. The holding portion 46a accommodates an annular seal member 49a in the internal space 48a, and holds the seal member 49a in contact with the second reflecting portion 43a. Thereby, the airtightness and liquid tightness between the insertion hole 45a and the capillary 2 are maintained.

  Similarly, a holding portion 46b that protrudes inward of the reflector 12A of the second reflecting portion 43b is formed. The holding portion 46b has an insertion hole 47b whose central axis coincides with the central axis of the insertion hole 45b, and has a disk-shaped internal space 48b. The holding portion 46b accommodates an annular seal member 49b in the internal space 48b and holds the seal member 49b in contact with the second reflecting portion 43b. Thereby, the airtightness and liquid tightness between the insertion hole 45b and the capillary 2 are maintained.

  In this embodiment, the 1st reflection part 40 is implement | achieved by the dielectric thin film, and has a reflectance of 99% or more with respect to the excitation light L and the fluorescence A. FIG.

  The excitation light reflecting portion 41 is realized by a dielectric thin film, has a reflectance of 99% or more with respect to the excitation light L having a wavelength of 450 nm to 485 nm, and 5% or less with respect to the fluorescence A having a wavelength of 495 nm or more. Has reflectivity. When the excitation light is incident on the light receiving unit 21 of the fluorescence detector 13, the background light increases, which causes a decrease in detection sensitivity and a decrease in detection accuracy of the fluorescence detector 13. Therefore, the transmittance of the excitation light L in the excitation light reflection unit 41 needs to have a 0D value of −6 or more.

  The second and third reflecting portions 43a and 43b are realized by a dielectric thin film or a metal thin film, have a transmittance of 99% or more with respect to the excitation light L having a wavelength of 450 nm to 485 nm, and have a wavelength of 495 nm or more. It has a transmittance of 5% or less with respect to A.

  A refractive liquid is sealed in the internal space of the reflector 12A. As the refracting liquid, a liquid having a refractive index substantially equal to the refractive index of the capillary 2 is selected. As the refractive liquid, for example, a refractive liquid manufactured by Cargill (trade name: phased silica matching liquid) can be used.

  In this embodiment, since the capillary 2 is formed of fused silica, the refractive liquid having a refractive index of 1.4587nd to 1.4592nd is selected.

By encapsulating the refractive liquid in the internal space of the reflector 12A, the critical angle at the boundary surface between the outer peripheral surface of the capillary 2 and the internal space of the reflector 12 is increased. Therefore, reflection of the excitation light L incident on the capillary 2 from the excitation light incident part 42 on the surface of the capillary 2 can be reduced. As a result, the analyte can be irradiated with more excitation light L.
Further, the reflector 12 </ b> A can achieve the same effect as the reflector 12.

DESCRIPTION OF SYMBOLS 1 Capillary electrophoresis apparatus 2 Capillary 3 Inlet side storage tank 4 Outlet side storage tank 5a, 5b Electrode 8 Fluorescence detection part 12, 12A Reflector 13 Fluorescence detector 14 Collector 20 Diffusing lens 21 Light receiving part 22 Signal processing part 30 1st reflection Part 31 Excitation light incident part 32 Excitation light reflection part 33a Second reflection part 33b Third reflection part 40 First reflection part 41 Excitation light reflection part 42 Excitation light incidence part 43a Second reflection part 43b Third reflection part 45a, 45b Insertion Hole 46a, 46b Holding part 47a, 47b Insertion hole 48a, 48b Internal space 49a, 49b Seal member

Claims (3)

A capillary for electrophoresis of the analyte;
One end of the capillary is connected, and an introduction-side storage tank in which the electrophoresis solution is stored;
The other end of the capillary is connected, and a discharge-side storage tank that stores waste liquid flowing out from the capillary,
Electrodes provided respectively in the introduction-side storage tank and the outlet-side storage tank;
A power supply for applying a voltage between the electrodes;
A reflector provided in a fluorescence detection unit of a capillary electrophoresis apparatus including a fluorescence detection unit that irradiates a capillary with excitation light and detects fluorescence generated by an analyte in the capillary,
The reflector is provided so as to surround a part of the capillary,
An excitation light incident part through which the excitation light is transmitted;
An excitation light reflecting part that reflects the excitation light incident through the excitation light incident part and transmits fluorescence;
A reflector that reflects excitation light and fluorescence.   The reflector according to claim 1, wherein an internal space of the reflector is filled with a refractive liquid.   A capillary electrophoresis analyzer comprising the reflector according to claim 1.

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