WO2014167792A1 - Fluorescence detection device and fluorescence detection method - Google Patents
Fluorescence detection device and fluorescence detection method Download PDFInfo
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- WO2014167792A1 WO2014167792A1 PCT/JP2014/001711 JP2014001711W WO2014167792A1 WO 2014167792 A1 WO2014167792 A1 WO 2014167792A1 JP 2014001711 W JP2014001711 W JP 2014001711W WO 2014167792 A1 WO2014167792 A1 WO 2014167792A1
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- fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
Definitions
- the present invention relates to a detection technique for detecting a detection target that emits fluorescence based on a fluorescence method.
- a technology for detecting a detection target such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid) or protein with high sensitivity is required.
- a fluorescence method is generally known.
- the molecule to be detected or the molecule that specifically binds and / or reacts with the molecule to be detected is labeled with the fluorescent substance.
- the labeled molecule emits fluorescence under irradiation of excitation light from the outside.
- the observer can detect fluorescence emission and observe the process of the biospecific reaction of the molecule to be detected. Alternatively, the observer can detect fluorescence emission and perform quantification processing on the detection target.
- RNA detection is used to study the activity of viruses, pathogens and genes.
- Examples of RNA detection techniques include RT-PCR (Reverse Transcription Polymerase Chain Reaction) method and microarray method. The above-described fluorescence method is also used for these detection techniques.
- a DNA amplification method In the field of biotechnology, a DNA amplification method called a PCR method is often used.
- the PCR method utilizes an enzyme reaction called DNA polymerase.
- the PCR method makes it possible to selectively amplify only a desired region in DNA.
- the PCR method is also used for the above-mentioned RT-PCR method and microarray method. If the observer uses the PCR method, the DNA can be amplified. Therefore, even under conditions where the amount of RNA in the biological sample is very small and fluorescence detection is difficult, the observer can detect the fluorescence by amplifying the DNA.
- the fluorescence method and the PCR method are very useful.
- the PCR method has problems such as complicated primer design, increase in reagents, strict temperature control and amplification bias. If the observer uses a micro-microchip detector suitable for disposable use (so-called ⁇ TAS (Micro Total Analysis Systems)), the above-mentioned problem is notable due to the limited space of the detector. appear.
- ⁇ TAS Micro Total Analysis Systems
- a fluorescence observation apparatus (total reflection illumination fluorescence microscope) that enables dynamic and real-time observation of biomolecule activity from a single molecule has already been put into practical use.
- the total reflection illumination fluorescence microscope is different from the excitation light incident technique used in the epi-illumination microscope, and it exceeds the critical angle from a material having a high refractive index (for example, quartz) to a material having a low refractive index (for example, an aqueous solution).
- the evanescent light generated by the total reflection of the light incident on is used as excitation light. As the distance from the interface where total reflection occurs increases, the intensity of the evanescent light attenuates exponentially.
- the irradiation volume irradiated with the excitation light is very small compared to when the observer uses an epi-illumination microscope.
- water Raman scattering and various other background lights are greatly reduced. This results in a greatly improved detection sensitivity.
- Patent Document 1 addresses the realization of an irradiation volume smaller than that of excitation light using total reflection evanescent light.
- Patent Document 1 proposes to improve the sensitivity of fluorescence detection by using evanescent light generated by a nano-aperture as excitation light.
- excitation light is applied to a thin film in which nano openings having a diameter of about 200 nm shorter than the wavelength of excitation light are formed.
- Evanescent light leaking from the nano-aperture is used for fluorescence observation.
- the irradiation volume of the evanescent light leaking from the nano aperture depends on the size of the nano aperture. Therefore, the technique of Patent Document 1 can achieve an irradiation volume that is much smaller than the irradiation volume of total reflection evanescent light.
- Non-Patent Document 2 discloses an illumination technique using evanescent light leaking from a nano-aperture. According to Non-Patent Document 2, DNA polymerase is immobilized on the bottom surface in the nano-opening. The phosphor-labeled dCTP is incorporated into the immobilized DNA polymerase.
- Patent Document 2 proposes to increase the fluorescence intensity by utilizing the electric field enhancement effect generated by surface plasmon resonance.
- Patent Document 3 proposes to use obliquely incident excitation light and a low-refractive index film disposed between a film in which a nano-aperture is formed and a substrate. The technique of Patent Document 3 enables excitation light to be incident efficiently.
- the evanescent light leaking from the nano aperture is weak, and the detection target may not be sufficiently fluorescent. Therefore, the above-described technique has a problem in terms of detection sensitivity.
- JP 2004-163122 A JP2013-002986A International Publication No. 2011/002010 International Publication No. 2012/165400
- An object of the present invention is to provide a fluorescence detection technique capable of achieving high detection sensitivity.
- a fluorescence detection apparatus includes a light shielding film in which at least one flow path for guiding a plurality of detection targets and at least one slit that is sterically intersected by the at least one flow path are formed. And at least one intersection where the at least one flow path sterically intersects the at least one slit is irradiated with excitation light to generate fluorescence from the plurality of detection targets, and the fluorescence And a measuring unit that measures the passage amounts of the plurality of detection targets that have passed through the at least one intersecting portion based on intensity.
- the excitation light includes linearly polarized light along the extending direction of the at least one slit.
- the fluorescence detection method includes a step of moving a plurality of detection targets in at least one flow path that sterically intersects at least one slit, and the at least one slit is the at least one slit. Irradiating excitation light including linearly polarized light along the extending direction of the at least one slit to at least one intersecting portion intersecting the flow path to generate fluorescence from the plurality of detection targets; To measuring the passage amounts of the plurality of detection objects that have passed through the at least one intersection.
- the above-described fluorescence detection technology can achieve high detection sensitivity.
- FIG. 1 It is a conceptual diagram of the fluorescence detection apparatus of 1st Embodiment. It is a schematic flowchart of the fluorescence detection method of 2nd Embodiment. It is the schematic of the fluorescence detection apparatus of 3rd Embodiment. It is a schematic sectional drawing of the slit of the fluorescence detection apparatus shown by FIG. 1 (4th Embodiment). It is a schematic sectional drawing of the flow path of the fluorescence detection apparatus shown by FIG. 1 (5th Embodiment). It is a schematic sectional drawing of the microchip of the fluorescence detection apparatus shown by FIG. 3 (6th Embodiment). It is a schematic sectional drawing of the microchip of the fluorescence detection apparatus shown by FIG. 3 (6th Embodiment).
- FIG. 1 It is a schematic block diagram of the measurement part of the fluorescence detection apparatus shown by FIG. 1 (7th Embodiment). It is a timing chart of the detection signal which the measurement part shown in Drawing 7 generates. It is a schematic flowchart showing the process which the measurement part shown by FIG. 7 performs. It is the schematic of the fluorescence detection apparatus of 8th Embodiment. It is the schematic of the fluorescence detection apparatus of 9th Embodiment. It is the schematic of the fluorescence detection apparatus shown by FIG. 3 (10th Embodiment). 13 is a timing chart of detection signals generated by the fluorescence detection device shown in FIG. It is a schematic flowchart showing the process which the fluorescence detection apparatus shown by FIG. 12 performs.
- FIG. 1 11th Embodiment
- FIG. 7 12th Embodiment
- FIG. 7 12th Embodiment
- FIG. 7 12th Embodiment
- FIG. 7 13th Embodiment
- FIG. 14 14th Embodiment
- FIGS. 19A and 19B 20 is a schematic flowchart showing a manufacturing process of the microchip shown in FIGS. 19A and 19B. It is a schematic top view of the microchip which has many intersections (16th Embodiment).
- the detection target is fluorescently colored using light that has passed through a minute opening. Since the light that has passed through the minute opening is selectively used for detection of the detection target, the background light is effectively reduced. On the other hand, since the light used for detecting the detection target is weak, the detection target may not emit light strongly. This results in low detection sensitivity.
- a technique for causing a detection target to emit light strongly and achieving high detection sensitivity will be described.
- FIG. 1 is a conceptual diagram of the fluorescence detection apparatus 100 according to the first embodiment. With reference to FIG. 1, the fluorescence detection apparatus 100 will be described.
- the fluorescence detection apparatus 100 includes a flow path 200, a light shielding film 300, an irradiation unit 400, and a measurement unit 500.
- a slit 310 is formed in the light shielding film 300.
- the channel 200 guides a plurality of detection targets DO.
- the detection target DO moves along the flow path 200.
- the detection target DO may be DNA, RNA, or protein. The principle of this embodiment is not limited to a specific type of detection target DO.
- the slit 310 extends in a direction different from the flow path 200.
- the channel 200 intersects the slit 310 three-dimensionally. As a result, the intersection 110 is defined.
- the channel 200 may be perpendicular to the slit 310.
- the flow path 200 may intersect the slit 310 at other intersection angles.
- the principle of the present embodiment is not limited to a specific crossing angle between the flow path 200 and the slit 310.
- the irradiation unit 400 irradiates the intersection 110 with excitation light PL.
- a part of the excitation light PL sequentially passes through the slit 310 and the flow path 200.
- the excitation light PL reaches the detection target DO that flows along the flow path 200.
- Another part of the excitation light PL is shielded by the light shielding film 300. Therefore, the background light is sufficiently reduced.
- the light shielding film 300 may be a metal film deposited on a substrate (not shown). Alternatively, the light shielding film 300 may be formed of another material that is opaque to the excitation light PL emitted from the irradiation unit 400. If the light shielding film 300 is formed of a material that is opaque to the excitation light PL, the light shielding film 300 can completely shield the excitation light PL.
- the light shielding film 300 is a metal film, the light shielding film 300 has a high extinction coefficient. Since the light shielding film 300 can have high light shielding characteristics, the designer may set the thickness of the light shielding film 300 to a very small value.
- Various metal materials such as aluminum, gold, silver, chromium, platinum, germanium, and tungsten may be used for forming the light shielding film 300.
- the light shielding film 300 may be an alloy film formed from a plurality of types of metal materials. The principle of the present embodiment is not limited to a specific composition of the light shielding film 300.
- the irradiation unit 400 may make the excitation light PL enter substantially perpendicular to the light shielding film 300. Alternatively, the irradiation unit 400 may set another incident angle of the excitation light PL with respect to the light shielding film 300.
- the principle of the present embodiment is not limited to a specific incident angle of the excitation light PL with respect to the light shielding film 300.
- the irradiation unit 400 may include an ion laser light source.
- the irradiation unit 400 may be a light source having a wide band such as an LED light source.
- the irradiation unit 400 may include a white light source if fluorescence emission from the detection target DO is obtained.
- the principle of this embodiment is not limited to a specific structure of the irradiation unit 400.
- the wavelength of the excitation light PL is appropriately set so that fluorescence emission from the detection target DO can be obtained.
- the excitation light PL may include light components having a plurality of wavelengths.
- the shortest wavelength of the excitation light PL may be set in a range from 350 nm to 750 nm. In this case, the fluorescence FL in the visible light range is detected efficiently.
- the principle of this embodiment is not limited to a specific wavelength of the excitation light PL.
- the detection target DO is labeled with a phosphor.
- the detection target DO can receive the excitation light PL and emit fluorescence FL.
- the fluorescence FL propagates to the measurement unit 500.
- the measuring unit 500 detects the fluorescence FL and measures the amount of the detection target DO that has passed through the intersection 110 from the light amount of the fluorescence FL. Therefore, the passing amount of the detection target DO is appropriately measured without the immobilization technique. This facilitates the reuse of a microchip (not shown) in which the channel 200 and the slit 310 are formed.
- the passing amount of the detection target DO may be expressed as the number of detection target DOs. Alternatively, the passing amount of the detection target DO may be expressed as the concentration of the detection target DO.
- the principle of the present embodiment is not limited to the expression for specifying the passage amount of the detection target DO.
- optical techniques may be used to detect the fluorescence FL.
- the principle of this embodiment is not limited to a specific technique for detecting fluorescence FL.
- the excitation light PL includes linearly polarized light along the extending direction of the slit 310. Therefore, the detection target DO is strongly irradiated with the excitation light PL.
- the excitation light PL may include other light components in addition to the above-described linearly polarized light.
- the irradiation unit 400 may have a light source that emits linearly polarized light.
- the irradiation unit 400 may include a light source and an optical element (for example, a wavelength plate or a polarization filter) that linearly polarizes light from the light source.
- an optical element for example, a wavelength plate or a polarization filter
- the principle of this embodiment is not limited to a specific optical technique for obtaining linearly polarized light.
- the excitation light PL may be parallel light. Alternatively, the excitation light PL may be convergent light. Further alternatively, the excitation light PL may be diverging light.
- the flow path 200 may have a rectangular cross section.
- the channel 200 may have other cross-sectional shapes (eg, circular, oval or trapezoidal).
- the corners of the cross section of the channel 200 may be curved.
- the principle of this embodiment is not limited to a specific shape of the flow path 200.
- the slit 310 may have a rectangular cross section.
- the slit 310 may have other cross-sectional shapes (eg, circular, oval or trapezoidal).
- the corners of the cross section of the slit 310 may be curved.
- the principle of this embodiment is not limited to a specific shape of the slit 310.
- the detection target is appropriately detected.
- a fluorescence detection method is described.
- FIG. 2 is a schematic flowchart of the fluorescence detection method of the second embodiment. With reference to FIGS. 1 and 2, the fluorescence detection method will be described.
- Step S110 In step S ⁇ b> 110, the observer supplies the detection target DO after the labeling process to the flow path 200. As a result, the detection target DO moves along the flow path 200.
- An observer may use a pump (not shown) for supplying the detection target DO to the flow path 200.
- the observer may move the detection target DO within the flow path 200 using a known electrophoresis technique.
- the principle of the present embodiment is not limited to a specific technique for moving the detection target DO in the flow path 200.
- Step 120 is executed after the supply of the detection target DO to the flow path 200.
- Step S120 the excitation light PL is irradiated to the intersection 110. Since the linearly polarized light component of the excitation light PL efficiently leaks from the slit 310, the detection target DO that passes through the intersection 110 can emit fluorescence FL strongly. Step S130 is performed after irradiation of the excitation light PL.
- Step S130 In step S ⁇ b> 130, the measurement unit 500 detects the fluorescence FL of the detection target DO passing through the intersection 110 and measures the amount of passage of the detection target DO.
- Step S120 may be performed in parallel with step S110.
- FIG. 3 is a schematic diagram of the fluorescence detection apparatus 100A of the third embodiment.
- the fluorescence detection apparatus 100A will be described with reference to FIGS.
- the fluorescence detection device 100A includes a microchip 120, a light source 410, a measurement device 510, and a pump 600.
- the light source 410 corresponds to the irradiation unit 400 described with reference to FIG.
- the measuring device 510 corresponds to the measuring unit 500 described with reference to FIG.
- the microchip 120 includes an upper substrate 210, a lower substrate 320, and a light shielding film 300A.
- the light shielding film 300 ⁇ / b> A is sandwiched between the upper substrate 210 and the lower substrate 320.
- the light shielding film 300A corresponds to the light shielding film 300 described with reference to FIG.
- the lower substrate 320 may be made of glass.
- the lower substrate 320 may be another material transparent to the excitation light PL (for example, an inorganic material such as quartz or a transparent resin such as polydimethylsiloxane (PDMS)).
- PDMS polydimethylsiloxane
- the lower substrate 320 includes a front surface 321, a rear surface 322 opposite to the front surface 321, a left surface 323 extending between the front surface 321 and the rear surface 322, and a right surface 324 opposite to the left surface 323.
- a slit 310A extending from the front surface 321 to the rear surface 322 is formed in the light shielding film 300A.
- the slit 310A corresponds to the slit 310 described with reference to FIG.
- the lower substrate 320 is transparent to the excitation light PL. Therefore, the excitation light PL propagates through the lower substrate 320 to the slit 310A. The excitation light PL that has reached the light shielding film 300A around the slit 310A is blocked by the light shielding film 300A, while the excitation light PL that has reached the slit 310A propagates toward the upper substrate 210.
- the upper substrate 210 may be made of glass. Alternatively, the upper substrate 210 may be another material transparent to the fluorescent FL (for example, an inorganic material such as quartz or a transparent resin such as polydimethylsiloxane (PDMS)).
- the upper substrate 210 may be formed from the same material as the lower substrate 320. Alternatively, the upper substrate 210 may be formed from a different material than the lower substrate 320. The principle of this embodiment is not limited to a specific material of the upper substrate 210.
- Upper substrate 210 defines front surface 211, rear surface 212 opposite to front surface 211, left surface 213 extending between front surface 211 and rear surface 212, right surface 214 opposite to left surface 213, and flow path 200A. And a flow path wall 215 that performs.
- the channel 200A extends between the left surface 213 and the right surface 214. Accordingly, the flow path 200A three-dimensionally intersects the slit 310A.
- the channel 200A corresponds to the channel 200 described with reference to FIG.
- the pump 600 supplies a plurality of detection objects DO to the flow path 200A, and moves the detection objects DO from the left surface 213 to the right surface 214 along the flow path 200A.
- the moving unit is exemplified by the pump 600.
- Various surface treatments such as surface coating treatment, hydrophobic treatment, and hydrophilic treatment may be applied to the flow path wall 215.
- the surface treatment makes it difficult for the detection object DO to adhere to the flow path wall 215.
- the principle of this embodiment is not limited to a specific surface treatment for the flow path wall 215.
- the detection target DO may be miRNA (microRNA: microribonucleic acid). miRNAs are biomolecules that exist in cells. miRNA has about 18-20 bases. miRNA recognizes specific mRNA (messenger RNA). miRNAs cause poly A chain degradation and translational repression. For this reason, miRNA is considered to be involved in life phenomena such as cancer development and memory formation.
- miRNA miRNA: microribonucleic acid
- MiRNA alone does not emit fluorescence. Therefore, if miRNA is used as the detection target DO, a double strand formed by the miRNA and the artificial nucleic acid can be used.
- the artificial nucleic acid recognizes miRNA and binds as a double strand. Fluorescence emission is obtained only when the artificial nucleic acid is bound to miRNA as a double strand.
- WO 2008-111485 discloses various artificial nucleic acids that can be used for fluorescence emission of miRNA.
- the light source 410 may be an Ar ion laser light source that emits light having a wavelength of 514 nm.
- the Ar ion laser light source may emit the excitation light PL as continuous light.
- the Ar ion laser light source may emit pulse-modulated light as excitation light PL. If the excitation light PL is pulse-modulated light, the period during which the detection target DO is illuminated is shortened. Therefore, the fading of the fluorescence of the detection target DO is less likely to occur.
- the principle of this embodiment is not limited to a specific emission pattern of the excitation light PL.
- the excitation light PL that has reached the slit 310A propagates toward the upper substrate 210.
- the detection target DO flowing through the flow path 200A is illuminated by the excitation light PL.
- the detection target DO illuminated by the excitation light PL emits fluorescence FL.
- the light source 410 emits light having a wavelength of 514 nm as excitation light PL
- the fluorescence FL may have a wavelength of about 540 nm.
- the fluorescence FL propagates toward the measuring device 510.
- Measuring device 510 detects fluorescence FL.
- the measuring device 510 generates a detection signal representing the intensity of the fluorescence FL.
- the measuring device 510 includes an objective lens 511, an optical filter 512, an imaging lens 513, a detector 514, and an arithmetic device 515.
- the objective lens 511 condenses the fluorescence FL and the excitation light PL that has reached the objective lens 511.
- the optical filter 512 blocks the excitation light PL while allowing the fluorescence FL to pass therethrough.
- the imaging lens 513 images the fluorescence FL on the detector 514.
- the detector 514 generates a detection signal indicating the intensity of the fluorescence FL. The detection signal is output from the detector 514 to the arithmetic device 515.
- the arithmetic device 515 acquires information on the amount of passage of the detection target DO (for example, the number of detection target DOs passing through the intersection of the flow path 200A and the slit 310A and the concentration of the detection target DO) from the detection signal.
- the signal generation unit is exemplified by the detector 514.
- the acquisition unit is exemplified by the arithmetic device 515.
- the designer may remove the optical filter 512 if the light shielding film 300A has high light shielding characteristics.
- the designer may extract the fluorescence FL using a spectroscopic technique instead of the optical filter 512.
- the principle of this embodiment is not limited to a specific structure of the measuring device 510.
- ⁇ Fourth embodiment> If the slit dimensions are set appropriately, the excitation light can remain in the vicinity of the slit. As a result, the designer can design the measurement unit without incorporating an optical filter for shielding the excitation light. In the fourth embodiment, the design principle of the slit will be described.
- FIG. 4 is a schematic sectional view of the slit 310. The design principle of the slit 310 will be described with reference to FIGS. 1 and 4.
- the depth SD of the slit 310 may be defined as the thickness of the light shielding film 300.
- the width SW of the slit 310 may be defined as a dimension in a direction orthogonal to the thickness direction of the slit 310.
- these definitions regarding the cross-sectional dimension of the slit 310 do not limit the principle of this embodiment at all.
- FIG. 4 shows the shortest wavelength WL of the excitation light PL.
- the width SW of the slit 310 is set to a value smaller than the shortest wavelength WL. If the shortest wavelength WL of the excitation light PL is set in the range of 350 nm or more and 750 nm or less, the width SW of the slit 310 may be set to 200 nm. In this case, the excitation light PL can locally exist in the vicinity of the slit 310 without completely passing through the light shielding film 300.
- the principle of the present embodiment is not limited to a specific value of the width SW of the slit 310.
- the width SW of the slit 310 approximates the value of the shortest wavelength WL of the excitation light PL, a plurality of detection objects DO are easily exposed to the excitation light PL simultaneously. This means a decrease in detection accuracy. If the width SW of the slit 310 is set to an excessively small value, the excitation light PL localized in the vicinity of the slit 310 may not sufficiently cover the cross section of the channel 200. This means an increase in the detection target DO that passes through the intersection 110 without emitting fluorescence. Therefore, the designer may set the width SW of the slit 310 in consideration of the height dimension of the flow path 200.
- the depth SD of the slit 310 may be appropriately set in consideration of the light shielding characteristics of the light shielding film 300. If the light shielding film 300 is excessively thin, the light shielding film 300 cannot sufficiently shield the excitation light PL. This causes fluorescence emission in a region other than the intersection 110. Fluorescence emission in a region other than the intersection 110 increases noise included in the detection signal generated by the measurement unit 500. If the light shielding film 300 is excessively thick, the excitation light PL localized in the vicinity of the slit 310 does not reach the flow path 200 sufficiently.
- the thickness of the light shielding film 300 may be set in a range of 20 nm to 200 nm. If the light shielding film 300 is made of aluminum, the designer may set the thickness of the light shielding film 300 to 60 nm.
- Patent Document 1 a solution layer containing a biomolecule as a detection target is formed on an opening film in which a minute opening is formed.
- the evanescent field leaking from the minute aperture is used for fluorescence detection of biomolecules. Since the excitation light illuminates only the vicinity of the minute aperture, the observation probability of the biomolecule is unstable. This causes a decrease in biomolecules and an increase in detection time. Therefore, the prior art requires a calibration curve for quantitative evaluation of biomolecules. In quantitative evaluation using a calibration curve, it is difficult to detect the number of biomolecules with high accuracy.
- the solution layer is formed between the opening film and the cover glass. There may be a plurality of biomolecules in the region of the solution layer on the opening membrane. Therefore, the background light may not be sufficiently reduced.
- Patent Documents 2 and 3 disclose a technique for immobilizing a detection target in a minute opening.
- the prior arts of Patent Documents 2 and 3 require that a reactive substance that specifically reacts with the detection target be immobilized in the vicinity of the minute opening. This increases the manufacturing cost of the fluorescence detection device. In addition, it becomes difficult to reuse the fluorescence detection apparatus.
- Patent Document 4 makes it possible to reduce the irradiation volume using Raman scattered light.
- the technique of Patent Document 4 further reduces the background light by further using a minute aperture.
- the technique of Patent Document 4 requires that the minute aperture and the protrusion structure used for electric field enhancement be aligned with high accuracy (several nm level).
- the protrusion structure itself needs to be formed with high accuracy (several nm level). This increases the manufacturing cost and manufacturing difficulty of the fluorescence detection device.
- the flow path described in relation to the first embodiment is appropriately designed, a plurality of detection targets can flow one by one.
- the measurement unit detects the detection target flowing through the flow channel one by one, and can accurately detect the passage amount of the detection target without requiring a calibration curve.
- the detection target may not be fixed. Therefore, the detection of the detection target takes only a short time.
- the preparation required for detecting the detection target is simplified.
- the design principle of the flow path will be described.
- FIG. 5 is a schematic cross-sectional view of the flow path 200. The design principle of the flow path 200 will be described with reference to FIGS.
- the height CH of the flow path 200 may be defined as the dimension of the slit 310 in the propagation direction of the excitation light PL.
- the width CW of the flow channel 200 may be defined as a dimension in a direction orthogonal to the height direction of the flow channel 200.
- these definitions regarding the cross-sectional dimension of the flow path 200 do not limit the principle of this embodiment at all.
- the height CH of the channel 200 may be set to a value smaller than the width SW of the slit 310.
- the excitation light PL localized near the slit 310 can cover the cross section of the flow path 200.
- the height dimension is exemplified by the height CH.
- the height CH of the channel 200 may be set in a range of 30 nm or more and 150 nm or less. If linearly polarized light having a minimum wavelength of 350 nm or more and 750 nm or less is used as the excitation light PL, the height CH may be set to 100 nm.
- the width CW of the flow path 200 may be set based on the size of the detection target DO. If the value of the width CW of the flow path 200 is set in the range of 10 nm to 500 nm, the user can supply various biological cells to the flow path 200 as the detection target DO.
- 6A and 6B are schematic cross-sectional views of the microchip 120 described in relation to the third embodiment. 6A and 6B represent a cross section of the microchip 120 on the center line of the flow path 200A.
- the excitation light PL shown in FIG. 6A is linearly polarized light oriented in the extending direction of the slit 310A.
- the excitation light PL shown in FIG. 6B is linearly polarized light oriented at right angles to the extending direction of the slit 310A.
- Each of the plurality of curves shown in FIGS. 6A and 6B is a contour line representing the same light intensity. A simulation performed by the present inventors will be described with reference to FIGS. 3, 6A, and 6B.
- the inventors set the width of the slit 310A to 200 nm in accordance with the design principle described in relation to the fourth embodiment. In addition, the present inventors set the thickness of the light shielding film 300A to 60 nm.
- the present inventors set the height and width of the flow path 200A to 100 nm in accordance with the design principle described in relation to the fifth embodiment.
- the light shielding film 300A appropriately shields the excitation light PL.
- a part of the excitation light PL enters the slit 310A.
- the excitation light PL localized in the vicinity of the slit 310A is represented by contour lines. From the excitation light PL localized near the slit 310A in FIGS. 6 and 6B, the three-dimensional intersection structure of the flow path 200A and the slit 310A formed in the light shielding film 300A can reduce the irradiation volume of the excitation light PL. I understand. Therefore, the principle of the above-described embodiment makes it possible to detect the detection target DO under a high S / N ratio.
- FIG. 6A is compared with FIG. 6B, FIG. 6A shows more contour lines entering the flow path 200A from the slit 310A than in FIG. 6B.
- the excitation light PL can strongly illuminate the flow path 200A if the direction of linearly polarized light is along the extending direction of the slit 310A. Therefore, the designer may set the direction of the linearly polarized light of the excitation light PL to be substantially parallel to the extending direction of the slit 310A.
- the cross-sectional area of the flow path 200A formed according to the design principle described in relation to the fifth embodiment is very small. Therefore, the detection target DO flowing through the flow path 200A is also very small. Even if a small detection object DO is irradiated with weak excitation light as shown in FIG. 6B, only fluorescence insufficient for fluorescence detection can be obtained. If strong excitation light as shown in FIG. 6A is irradiated to a small detection object DO, strong fluorescence emission sufficient for fluorescence detection can be obtained. Therefore, the measuring device 510 can accurately count the detection target DO that has passed through the intersection between the flow path 200A and the slit 310A.
- the measurement unit of the fluorescence detection apparatus may have various data processing structures for finding the passage amount of the detection target from the fluorescence intensity.
- an exemplary structure of the measurement unit will be described.
- FIG. 7 is a schematic block diagram of the measurement unit 500. The structure of the measuring unit 500 will be described with reference to FIGS.
- the measurement unit 500 includes a signal generation unit 520 and an acquisition unit 530.
- the fluorescence FL is incident on the signal generation unit 520.
- the signal generation unit 520 generates a detection signal DS representing the intensity of the fluorescence FL in response to the incidence of the fluorescence FL.
- the detection signal DS is output from the signal generation unit 520 to the acquisition unit 530.
- the signal generation unit 520 corresponds to the detector 514 described with reference to FIG.
- the acquisition unit 530 includes a comparison unit 531 and a determination unit 532.
- the comparison unit 531 compares the detection signal DS with a preset threshold value, and generates data CRD representing the comparison result.
- the data CRD is output from the comparison unit 531 to the determination unit 532.
- the determination unit 532 determines the passage amount of the detection target DO that has passed through the intersection 110 by referring to the data CRD.
- the data PSD representing the passage amount determined by the determination unit 532 may be output to a computer (not shown) or other external device (not shown) connected to the fluorescence detection device 100 so as to be communicable.
- the acquisition unit 530 corresponds to the arithmetic device 515 described with reference to FIG.
- FIG. 8 is a timing chart of the detection signal DS. With reference to FIGS. 1, 7, and 8, calculation processing executed by the measurement unit 500 will be described.
- the detection signal DS may have a high signal value. If the intensity of the fluorescence FL is low, the detection signal DS may have a low signal value.
- the signal value may be the magnitude of the signal voltage. Alternatively, the signal value may be the magnitude of the signal current.
- the comparison unit 531 may generate a pulse signal having a pulse that rises while the signal value exceeds the threshold value TH as the data CRD.
- the determination unit 532 counts the number of pulses in the pulse signal. As shown in FIG. 8, if there are three pulses between time T1 and time T2, the determination unit 532 determines that the three detection target DOs cross the intersection 110 during the period from time T1 to time T2. You may determine that it has passed.
- FIG. 9 is a schematic flowchart showing the process in step S130 described with reference to FIG. The process in step S130 will be described with reference to FIGS.
- step S131 the signal generation unit 520 converts the intensity of the fluorescence FL into a signal value. Thereafter, step S133 is executed.
- step S133 the comparison unit 531 compares the signal value with the threshold value TH. Thereafter, step S135 is generated.
- Step S135 If the signal value is larger than the threshold value TH in step S135, the comparison unit 531 generates a pulse. Thereafter, step S137 is executed. In other cases, step S139 is executed.
- step S137 the determination unit 532 increases the passage amount by “1”. Thereafter, step S139 is executed.
- step S139 In step S139, if the detection work has been completed, the determination unit 532 outputs data PSD representing the passage amount. In other cases, step S131 is executed.
- FIG. 10 is a schematic diagram of the fluorescence detection apparatus 100B of the eighth embodiment. With reference to FIG. 10, the fluorescence detection apparatus 100B will be described.
- the code used in common between the third embodiment and the eighth embodiment means that the element to which the common code is attached has the same function as that of the third embodiment. Therefore, description of 3rd Embodiment is used for these elements.
- the fluorescence detection device 100B includes a light source 410, a measurement device 510, and a pump 600.
- the fluorescence detection device 100B further includes a microchip 120B.
- the microchip 120B includes an upper substrate 210, a lower substrate 320, and a light shielding film 300A.
- the microchip 120B further includes a filling material 311 filled in the slit 310A formed in the light shielding film 300A.
- the filling material 311 may be a transparent inorganic material such as glass or quartz, a transparent resin such as polydimethylsiloxane (PDMS), or another material transparent to the excitation light PL.
- PDMS polydimethylsiloxane
- the filling material 311 smoothes the boundary between the region where the slit 310A is formed and the region where the light shielding film 300A exists. Therefore, the channel 200A is disposed on a smoothed surface. As a result, the detection target DO can flow smoothly along the flow path 200A.
- FIG. 11 is a schematic diagram of the fluorescence detection apparatus 100C of the ninth embodiment. With reference to FIG. 11, the fluorescence detection apparatus 100C will be described.
- a symbol used in common between the eighth embodiment and the ninth embodiment means that an element to which the common symbol is attached has the same function as that of the eighth embodiment. Therefore, description of 8th Embodiment is used for these elements.
- the fluorescence detection device 100C includes a light source 410, a measurement device 510, and a pump 600.
- the fluorescence detection device 100C further includes a microchip 120C.
- the microchip 120C includes an upper substrate 210, a lower substrate 320, a light shielding film 300A, and a filling material 311.
- the microchip 120 ⁇ / b> C further includes a thin thin film 130 formed between the upper substrate 210 and the lower substrate 320. Since the thin film 130 covers the light shielding film 300A and the filling material 311, the liquid for flowing the detection target DO hardly corrodes the light shielding film 300A.
- the thickness of the thin film 130 is set so as not to cause excessive attenuation of the excitation light PL.
- the fluorescence detection apparatus may detect a plurality of types of detection targets.
- a technique for detecting a plurality of types of detection targets will be described.
- FIG. 12 is a schematic diagram of the fluorescence detection apparatus 100A described in relation to the third embodiment. A technique for detecting a plurality of types of detection objects will be described with reference to FIG.
- the pump 600 of the fluorescence detection apparatus 100A shown in FIG. 12 supplies the first detection target DO1 and the second detection target DO2 to the flow path 200A.
- the first detection target DO1 emits fluorescence FL stronger than the second detection target DO2 under irradiation of the excitation light PL.
- FIG. 13 is a timing chart of the detection signal DS.
- symbol used in common between 7th Embodiment and 10th Embodiment means that the element to which the said common code
- the comparison unit 531 compares the signal value represented by the detection signal DS with the first threshold value TH1 and the second threshold value TH2.
- the first threshold value TH1 is set to a value larger than the second threshold value TH2. If the signal value is larger than the first threshold value TH1, the comparison unit 531 generates data CRD representing the passage of the first detection target DO1. If the signal value is smaller than the first threshold value TH1 and larger than the second threshold value TH2, the comparison unit 531 generates data CRD representing the passage of the second detection target DO2.
- FIG. 14 is a schematic flowchart showing the process in step S130 described with reference to FIG. The processing in step S130 will be described with reference to FIGS. 7 and 12 to 14.
- Step S210 the signal generation unit 520 converts the intensity of the fluorescence FL into a signal value. Thereafter, step S220 is executed.
- step S220 the comparison unit 531 compares the signal value with threshold values (first threshold value TH1, second threshold value TH2). Thereafter, step S230 is generated.
- Step S230 If the signal value is larger than the second threshold value TH2 in step S230, step S240 is executed. In other cases, step S270 is executed.
- Step S240 If the signal value is larger than the first threshold value TH1 in step S240, the comparison unit 531 generates data CRD representing the passage of the first detection target DO1. Thereafter, step S250 is executed. In other cases, the comparison unit 531 generates data CRD representing the passage of the second detection target DO2. Thereafter, step S260 is executed.
- step S250 the determination unit 532 increases the passing amount of the first detection target DO1 by “1”. Thereafter, step S270 is executed.
- step S260 the determination unit 532 increases the passing amount of the second detection target DO2 by “1”. Thereafter, step S270 is executed.
- Step S270 In step S270, if the detection work has been completed, the determination unit 532 outputs data PSD representing the passage amounts of the first detection target DO1 and the second detection target DO2. In other cases, step S210 is executed.
- ⁇ Eleventh embodiment> The principle of the tenth embodiment makes it possible to identify the type of detection target based on the intensity of fluorescence. Alternatively, the type of detection target may be identified based on the wavelength of fluorescence. In the eleventh embodiment, a technique for identifying the type of detection target based on the wavelength of fluorescence is described.
- FIG. 15 is a schematic block diagram of the measurement unit 500.
- the structure of the measuring unit 500 will be described with reference to FIGS.
- symbol used in common between 1st Embodiment and 11th Embodiment means that the element to which the said common code
- the measurement unit 500 includes a signal generation unit 520D and an acquisition unit 530D.
- the signal generation unit 520D includes a first signal generation unit 521, a second signal generation unit 522, and a spectroscopic unit 523.
- Acquisition unit 530D includes a first comparison unit 541, a second comparison unit 542, and a determination unit 532D.
- 1st detection object DO1 and 2nd detection object DO2 are supplied to the flow path 200 as detection object DO.
- the user may use a plurality of types of miRNA processed using artificial nucleic acids as the first detection target DO1 and the second detection target DO2.
- the double strand formed by the artificial nucleic acid and the miRNA can emit fluorescence having different wavelengths (hue) depending on the type of the miRNA.
- the first detection target DO1 emits fluorescence FL1 having the wavelength ⁇ 1 under irradiation of the excitation light PL.
- the second detection object DO2 emits fluorescence FL2 having a wavelength ⁇ 2 under irradiation of the excitation light PL.
- the wavelength ⁇ 1 is longer than the wavelength ⁇ 2.
- the spectroscopic unit 523 may be a prism or a grating element. If the fluorescence FL1 is incident on the spectroscopic unit 523, the spectroscopic unit 523 emits the fluorescence FL1 to the first signal generation unit 521. If the fluorescence FL 2 is incident on the spectroscopic unit 523, the spectroscopic unit 523 emits the fluorescence FL 2 to the second signal generation unit 522.
- the first signal generation unit 521 generates the first detection signal DS1 representing the light amount of the fluorescence FL1 according to the principle described in relation to the seventh embodiment.
- the first detection signal DS1 is output from the first signal generation unit 521 to the first comparison unit 541.
- the second signal generation unit 522 generates the second detection signal DS2 representing the light amount of the fluorescence FL2 according to the principle described in relation to the seventh embodiment.
- the second detection signal DS2 is output from the second signal generation unit 522 to the second comparison unit 542.
- the first signal generation unit 521 and the second signal generation unit 522 may be line type image sensors.
- the first comparison unit 541 compares the first detection signal DS1 with a preset threshold value according to the principle described in relation to the seventh embodiment, and generates first data CRD1 representing the comparison result.
- the first data CRD1 is output from the first comparison unit 541 to the determination unit 532D.
- the second comparison unit 542 compares the second detection signal DS2 with a preset threshold according to the principle described in relation to the seventh embodiment, and generates second data CRD2 representing the comparison result.
- the second data CRD2 is output from the second comparison unit 542 to the determination unit 532D.
- the determination unit 532D refers to the first data CRD1, and determines the passage amount of the first detection target DO1 that has passed through the intersection 110.
- the determination unit 532D generates data PSD1 representing the passage amount of the first detection target DO1.
- the data PSD1 may be output to a computer (not shown) or other external device (not shown) connected to the fluorescence detection device 100 in a communicable manner.
- the determination unit 532D refers to the second data CRD2, and determines the passage amount of the second detection target DO2 that has passed through the intersection 110.
- the determination unit 532D generates data PSD2 representing the passage amount of the second detection target DO2.
- the data PSD2 may be output to a computer (not shown) or other external device (not shown) connected to the fluorescence detection device 100 in a communicable manner.
- ⁇ Twelfth embodiment> The principle of the eleventh embodiment uses a spectroscopic element to switch the propagation path of fluorescence according to the wavelength of fluorescence.
- the signal generation unit may have a function of identifying a wavelength (for example, a color image sensor).
- a detection signal generation technique by a signal generation unit having a function of identifying a wavelength will be described.
- FIG. 16 is a schematic flowchart showing a detection signal DS generation process by the signal generation unit 520. With reference to FIGS. 7 and 16, the generation process of the detection signal DS will be described.
- the reference numerals used in common among the seventh embodiment, the eleventh embodiment, and the twelfth embodiment are the same as those in the seventh embodiment or the eleventh embodiment. It means having. Therefore, description of 7th Embodiment or 11th Embodiment is used for these elements.
- Step S310 the signal generation unit 520 determines whether or not the wavelength of the fluorescence FL is the wavelength ⁇ 1 of the fluorescence from the first detection target DO1. If the wavelength of the fluorescence FL is the wavelength ⁇ 1 of the fluorescence from the first detection target DO1, step S320 is executed. In other cases, step S340 is executed.
- Step S320 the signal generation unit 520 generates the intensity of the fluorescence FL as a signal value according to the principle described in relation to the seventh embodiment. Thereafter, Step S330 is executed.
- Step S330 the signal generation unit 520 outputs a detection signal DS including information regarding the wavelength of the fluorescence FL and information regarding the intensity of the fluorescence FL.
- Step S340 the signal generation unit 520 determines whether or not the wavelength of the fluorescence FL is the wavelength ⁇ 2 of the fluorescence from the second detection target DO2. If the wavelength of the fluorescence FL is the wavelength ⁇ 2 of the fluorescence from the second detection target DO2, step S350 is executed. In other cases, step S360 is executed.
- Step S350 the signal generation unit 520 generates the intensity of the fluorescence FL as a signal value according to the principle described in relation to the seventh embodiment. Thereafter, Step S330 is executed.
- Step S360 the signal generation unit 520 processes the incidence of the fluorescence FL as noise.
- the principle of the eleventh embodiment and the twelfth embodiment is that the wavelength of the fluorescence emitted from the first detection target is different from the wavelength of the fluorescence emitted from the second detection target. If the excitation light emitted from the irradiation unit includes a light component suitable for fluorescence from the first detection target and fluorescence from the second detection target, the first detection target and the second detection target are under irradiation of the excitation light. Can emit strong fluorescence. In the thirteenth embodiment, a technique for achieving strong detection sensitivity by causing the first detection target and the second detection target to emit light strongly will be described.
- FIG. 17 is a conceptual diagram of the fluorescence detection apparatus 100E of the thirteenth embodiment.
- symbol used in common between 1st Embodiment and 13th Embodiment means that the element to which the said common code
- the fluorescence detection device 100E includes a flow path 200, a light shielding film 300, and a measurement unit 500.
- the fluorescence detection device 100E further includes an irradiation unit 400E.
- the irradiation unit 400E includes a first irradiation unit 411, a second irradiation unit 412, and a multiplexing unit 413.
- the first irradiation unit 411 emits the first excitation light PL1 to the multiplexing unit 413.
- the second irradiation unit 412 emits the second excitation light PL2 to the multiplexing unit 413.
- the multiplexing unit 413 multiplexes the first excitation light PL1 and the second excitation light PL2, and generates the excitation light PL.
- the excitation light PL propagates to the flow path 200 through the slit 310.
- the multiplexing unit 413 may be a dichroic mirror or another optical element that can multiplex the first excitation light PL1 and the second excitation light PL2.
- the present embodiment is not limited to a specific optical element used for the multiplexing unit 413.
- the first detection target DO1 and the second detection target DO2 flow along the flow path 200.
- the first detection target DO1 emits fluorescence more strongly under irradiation of the first excitation light PL1 than under irradiation of the second excitation light PL2.
- the second detection target DO2 emits fluorescence more strongly under irradiation of the second excitation light PL2 than under irradiation of the first excitation light PL1.
- the excitation light PL includes the optical component of the first excitation light PL1 and the optical component of the second excitation light PL2, the first detection target DO1 and the second detection target DO2 that pass through the intersection 110. Both can strongly emit fluorescence.
- the principle of the thirteenth embodiment is that a plurality of excitation lights different from each other in wavelength are emitted, and each of a plurality of types of detection targets is strongly fluorescently emitted.
- the irradiating unit may have a function of changing the wavelength.
- the fluorescence detection apparatus which has an irradiation part which has a wavelength change function is demonstrated.
- FIG. 18 is a conceptual diagram of the fluorescence detection device 100F of the fourteenth embodiment. With reference to FIG. 18, the fluorescence detection apparatus 100F will be described.
- a symbol used in common between the thirteenth embodiment and the fourteenth embodiment means that an element to which the common symbol is attached has the same function as that of the thirteenth embodiment. Therefore, the description of the thirteenth embodiment is applied to these elements.
- the fluorescence detection device 100F includes a flow path 200, a light shielding film 300, and a measurement unit 500.
- the fluorescence detection device 100F further includes an irradiation unit 400F.
- Irradiation unit 400 ⁇ / b> F includes a light source 410 and a wavelength changing unit 420.
- the light source 410 emits excitation light PL to the wavelength changing unit 420.
- the wavelength changing unit 420 changes the wavelength of the excitation light PL between the wavelength ⁇ 1 and the wavelength ⁇ 2. After the wavelength changing process, the excitation light PL propagates to the flow path 200 through the slit 310.
- the first detection target DO1 and the second detection target DO2 flow along the flow path 200.
- the wavelength of the excitation light PL is the wavelength ⁇ 1
- the first detection target DO1 emits fluorescence more strongly than the second detection target DO2.
- the wavelength of the excitation light PL is the wavelength ⁇ 2
- the second detection target DO2 emits fluorescence more strongly than the first detection target DO1. If the wavelength changing unit 420 changes the wavelength of the excitation light PL between the wavelength ⁇ 1 and the wavelength ⁇ 2 while each of the first detection target DO1 and the second detection target DO2 passes through the intersection 110, the first Both the first detection object DO1 and the second detection object DO2 can emit strong fluorescence.
- Microchips can be easily created based on the principle of the fluorescence detection apparatus described in connection with the first embodiment. Microchips may be produced in large quantities by MEMS (Micro Electro Mechanical Systems) creation technology or semiconductor manufacturing technology. Therefore, the manufacturing cost of the microchip is very low. In the fifteenth embodiment, various microchips are described.
- FIG. 19A is a schematic plan view of a microchip 121 according to the principle of the first embodiment.
- FIG. 19B is a schematic plan view of another microchip 122 according to the principle of the first embodiment.
- the microchips 121 and 122 will be described with reference to FIGS. 1, 19A, and 19B.
- symbol used in common between 1st Embodiment and 15th Embodiment means that the element to which the said common code
- a flow path 200, a first slit 331, and a second slit 332 are formed.
- Each of the first slit 331 and the second slit 332 corresponds to the slit 310 described with reference to FIG.
- the manufacturer of the microchip 121 prepares two substrates.
- the manufacturer forms the first slit 331 on one of the two substrates and the second slit 332 next to the first slit 331.
- the second slit 332 may be substantially parallel to the first slit 331.
- the manufacturer forms the flow path 200 on the other of the two substrates.
- the manufacturer superimposes the two substrates and causes the flow path 200 to intersect the first slit 331 and the second slit 332.
- an intersection IP1 defined by the first slit 331 and the flow path 200 and an intersection IP2 defined by the second slit 332 and the flow path 200 are formed.
- the first flow path is exemplified by the flow path 200.
- the first intersection is exemplified by the intersection IP1.
- the second intersection is exemplified by the intersection IP2.
- the measurement unit 500 described with reference to FIG. 1 may include a two-dimensional image sensor.
- the measurement unit 500 can identify the intersections IP1 and IP2 as different measurement points. Therefore, the user who uses the microchip 121 can simultaneously measure the passing amount of the detection target DO at the intersections IP1 and IP2.
- a slit 310, a first channel 201, and a second channel 202 are formed.
- Each of the first flow path 201 and the second flow path 202 corresponds to the flow path 200 described with reference to FIG.
- the manufacturer of the microchip 122 prepares two substrates.
- the manufacturer forms the first flow path 201 on one of the two substrates, and the second flow path 202 next to the first flow path 201.
- the second flow path 202 may be substantially parallel to the first flow path 201.
- the manufacturer forms the slit 310 on the other of the two substrates.
- the manufacturer superimposes the two substrates and causes the slit 310 to intersect the first flow path 201 and the second flow path 202.
- an intersection IP3 defined by the first flow path 201 and the slit 310 and an intersection IP4 defined by the second flow path 202 and the slit 310 are formed.
- the first slit is exemplified by the slit 310.
- the first intersection is exemplified by the intersection IP3.
- the third intersection is exemplified by the intersection IP4.
- the measurement unit 500 described with reference to FIG. 1 may include a two-dimensional image sensor.
- the measurement unit 500 can identify the intersections IP3 and IP4 as different measurement points. Therefore, the user who uses the microchip 122 can simultaneously measure the passing amount of the detection target DO at the intersections IP3 and IP4.
- FIG. 20 is a schematic flowchart showing a manufacturing process of the microchips 121 and 122. A manufacturing process of the microchips 121 and 122 will be described with reference to FIGS. 19A to 20.
- Step S410 the manufacturer prepares an upper substrate and a lower substrate. Thereafter, steps S420 and S430 are executed in parallel.
- step S420 the manufacturer forms a light shielding film on the lower substrate.
- the manufacturer may form the slit simultaneously with the formation of the light shielding film by using a masking technique.
- step S430 the manufacturer forms a flow path in the upper substrate.
- the formation of the flow path may depend on the etching technique.
- Step S440 the manufacturer places the upper substrate on the lower substrate.
- the intersection is easily formed by the intersection of the flow path and the slit. Therefore, unlike the prior art, the microchips 121 and 122 are produced without requiring a strict positioning operation. Therefore, the manufacturing cost of the microchips 121 and 122 is low.
- the detection target is detected without being immobilized in the microchips 121 and 122, the microchips 121 and 122 are easily reused.
- FIG. 21 is a schematic plan view of a microchip 123 having a large number of intersections.
- the microchip 123 will be described with reference to FIGS.
- symbol used in common between 1st Embodiment and 16th Embodiment means that the element to which the said common code
- the microchip 123 shown in FIG. 21 includes a first channel 201, a second channel 202, a third channel 203, a fourth channel 204, a first slit 331, a second slit 332, a third slit 333, and A fourth slit 334 is formed.
- the interval between the first channel 201 to the fourth channel 204 and the interval between the first slit 331 to the fourth slit 334 may be determined based on the resolution of the measurement unit 500.
- the distance between the first channel 201 to the fourth channel 204 and An interval between the first slit 331 to the fourth slit 334 is determined. If the resolution of the detector 514 is 0.5 ⁇ m, the distance between the first flow path 201 to the fourth flow path 204 and the distance between the first slit 331 to the fourth slit 334 are set to 0.8 ⁇ m. May be.
- the field of view defined by the objective lens 511, the optical filter 512, and the imaging lens 513 is 2.0 mm in diameter, and fluorescence detection is possible in 80% of the field of view, detection that flows through 2000 channels
- the target DO is detected at the same time. That is, the principle of the present embodiment can achieve a throughput 2000 times that of the prior art.
- the arithmetic unit 515 may perform an averaging process on the passing amount measured at each intersection.
- the arithmetic device 515 may improve the accuracy of the data related to the passage amount by using various arithmetic processes.
- the principle of the present embodiment is not limited to a specific calculation process executed by the calculation device 515.
- the principle of this embodiment is not limited to the number of flow paths, the specific number of slits, or the specific arrangement.
- the designer may arrange the flow paths radially and form arc-shaped slits.
- the techniques relating to the exemplary detection techniques described in connection with the various embodiments described above primarily comprise the following features.
- At least one flow path that guides a plurality of detection targets and at least one slit that is three-dimensionally intersected by the at least one flow path are formed.
- a light-shielding film, and an irradiation unit that irradiates excitation light to at least one intersecting portion where the at least one channel three-dimensionally intersects the at least one slit, and generates fluorescence from the plurality of detection targets;
- a measurement unit that measures the passage amounts of the plurality of detection targets that have passed through the at least one intersecting portion based on the intensity of fluorescence.
- the excitation light includes linearly polarized light along the extending direction of the at least one slit.
- the excitation light includes linearly polarized light along the extending direction of at least one slit
- the detection target can strongly emit fluorescence. Therefore, the fluorescence detection device can achieve high sensitivity.
- the at least one slit may have a width dimension smaller than the wavelength of the excitation light.
- the excitation light since at least one slit has a width dimension smaller than the wavelength of the excitation light, the excitation light does not unnecessarily propagate to the flow path. Therefore, the designer may not unnecessarily incorporate an optical element such as an optical filter into the fluorescence detection apparatus.
- the at least one channel may have a height dimension smaller than the width dimension of the at least one slit.
- At least one flow path has a height dimension smaller than the width dimension of at least one slit
- a plurality of detection targets can be separated and flow through the flow path. Therefore, the observer can observe a plurality of detection objects without performing the immobilization process.
- the excitation light that has passed through the at least one slit can propagate in the height direction of the flow path. Therefore, the fluorescence detection apparatus can efficiently detect each of the plurality of detection targets.
- the measurement unit may include a signal generation unit that generates a detection signal that represents the intensity of the fluorescence, and an acquisition unit that acquires information related to the passage amount from the detection signal.
- the fluorescence detection device can detect the amount of fluorescence and provide the observer with information regarding the amount of passage of the detection target.
- the detection signal may represent a signal value that changes according to the intensity of the fluorescence.
- the acquisition unit includes a comparison unit that compares the signal value and a threshold set for the signal value, and a determination unit that determines the passage amount based on a result of comparison by the comparison unit. But you can.
- the amount of passage is determined by the determination unit with little influence of noise.
- the fluorescence detection apparatus may further include a moving unit that moves the plurality of detection objects along the at least one flow path.
- the plurality of detection targets are appropriately moved by the moving unit in the flow path.
- the light shielding film may be opaque to the excitation light.
- the detection target is appropriately detected with almost no influence of the background light.
- the light shielding film may be a metal film.
- the light shielding film can appropriately shield the excitation light.
- the fluorescence detection apparatus may further include a filling material filled in the at least one slit.
- the filling material may be transparent to the excitation light.
- the filling material can smooth the area around the slit. Therefore, the plurality of detection targets can move smoothly in the flow path.
- the at least one slit may include a first slit and a second slit formed adjacent to the first slit.
- the at least one flow path may include a first flow path that three-dimensionally intersects the first slit and the second slit.
- the at least one intersection may include a first intersection defined by the first slit and the first flow path, and a second intersection defined by the second slit and the first flow path.
- the measurement unit may identify the first intersection as a measurement point different from the second intersection.
- the measurement unit identifies the first intersection as a measurement point different from the second intersection, so that the fluorescence detection apparatus can efficiently detect a plurality of detection targets.
- the at least one flow path may include a first flow path and a second flow path formed adjacent to the first flow path.
- the at least one slit may include a first slit that three-dimensionally intersects the first channel and the second channel.
- the at least one intersection includes a first intersection defined by the first slit and the first flow path, and a third intersection defined by the first slit and the second flow path. But you can.
- the measurement unit may identify the first intersection as a measurement point different from the third intersection.
- the measurement unit identifies the first intersection as a measurement point different from the third intersection, so that the fluorescence detection apparatus can efficiently detect a plurality of detection targets.
- the measurement unit may simultaneously measure the passage amounts of the plurality of detection targets that have passed through the first intersection and the passage amounts of the plurality of detection targets that have passed through the second intersection.
- the measurement unit simultaneously measures the passage amounts of the plurality of detection targets that have passed through the first intersection and the passage amounts of the plurality of detection targets that have passed through the second intersection. A plurality of detection targets can be efficiently detected.
- the measurement unit may simultaneously measure the passage amounts of the plurality of detection targets that have passed through the first intersection and the passage amounts of the plurality of detection targets that have passed through the third intersection.
- the measurement unit simultaneously measures the passage amounts of the plurality of detection targets that have passed the first intersection and the passage amounts of the plurality of detection targets that have passed the third intersection. A plurality of detection targets can be efficiently detected.
- the plurality of detection targets include a first detection target that emits a first fluorescence having a first wavelength, and a second detection target that emits a second fluorescence having a second wavelength different from the first wavelength; May be included.
- the detection signal may include a first detection signal that represents the intensity of the first fluorescence and a second detection signal that represents the intensity of the second fluorescence.
- the acquisition unit acquires, from the first detection signal, first information related to a passing amount of the first detection target that has passed through the at least one intersection, and from the second detection signal, the at least one You may acquire the 2nd information regarding the passage amount of the 2nd detection object which passed the intersection.
- the acquisition unit acquires, from the first detection signal, the first information related to the passage amount of the first detection target that has passed through at least one intersection, and from the second detection signal, at least one Since the second information related to the passage amount of the second detection target that has passed through the intersection is acquired, the fluorescence detection device can provide the observer with information regarding the passage amount of the first detection target and the second detection target.
- the irradiation unit may include a first irradiation unit that irradiates first excitation light as the excitation light, and a second irradiation unit that irradiates second excitation light as the excitation light.
- the first detection target may emit the first fluorescence more strongly under irradiation of the first excitation light than under irradiation of the second excitation light.
- the second detection target may emit the second fluorescence more strongly under the irradiation of the second excitation light than under the irradiation of the first excitation light.
- the irradiation unit includes the first irradiation unit that irradiates the first excitation light as the excitation light and the second irradiation unit that irradiates the second excitation light as the excitation light.
- the apparatus can detect the first detection target and the second detection target with high sensitivity.
- the excitation light may have a wavelength of 350 nm or more and 750 nm or less.
- the excitation light has a wavelength of 350 nm or more and 750 nm or less, fluorescence in the visible light region can be efficiently generated.
- the height dimension may be not less than 30 nm and not more than 150 nm.
- the excitation light can illuminate a plurality of detection targets before being excessively attenuated.
- the width dimension of the at least one flow path may be not less than 10 nm and not more than 500 nm.
- the fluorescence detection apparatus can be used to detect various detection targets.
- the fluorescence detection method includes a step of moving a plurality of detection targets in at least one flow path that sterically intersects at least one slit, and the at least one slit is at least the at least one slit. Irradiating excitation light including linearly polarized light along the extending direction of the at least one slit to at least one intersecting portion intersecting one flow path to generate fluorescence from the plurality of detection targets; Measuring the passage amounts of the plurality of detection targets that have passed through the at least one intersection.
- the detection target since the excitation light including linearly polarized light along the extending direction of the slit is irradiated, the detection target can strongly emit fluorescence. Therefore, the fluorescence detection method can achieve high sensitivity.
- the principle of the above-described embodiment can be suitably used for a technique for detecting a detection target.
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Abstract
The present application discloses a fluorescence detection device provided with at least one flow path for guiding a plurality of objects to be detected, a light shielding film on which at least one slit is formed that three-dimensionally intersects with the at least one flow path, at least one intersection portion where the at least one flow path three-dimensionally intersects with the at least one slit, an irradiation unit for irradiating excitation light and causing the plurality of objects to be detected to generate fluorescence, and a measurement unit for measuring the amount of the plurality of objects to be detected that have passed through the at least one intersection portion on the basis of the strength of the fluorescence. The excitation light includes linearly polarized light that follows the extension direction of the at least one slit.
Description
本発明は、蛍光法に基づいて蛍光発光する検出対象を検出するための検出技術に関する。
The present invention relates to a detection technique for detecting a detection target that emits fluorescence based on a fluorescence method.
バイオテクノロジの分野において、DNA(デオキシリボ核酸)、RNA(リボ核酸)や蛋白質といった検出対象を高い感度で検出する技術が要求されている。これらの検出対象を、検出するために利用される技術として、蛍光法が一般的に知られている。
In the field of biotechnology, a technology for detecting a detection target such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid) or protein with high sensitivity is required. As a technique used for detecting these detection targets, a fluorescence method is generally known.
観察者が、蛍光法を用いて、検出対象を観察するとき、検出対象の分子又は検出対象の分子と特異的に結合及び/又は反応する分子は、蛍光体によって標識化される。標識化された分子は、外部からの励起光の照射下で、蛍光発光する。観察者は、蛍光発光を検出し、検出対象の分子の生体特異反応の過程を観察することができる。あるいは、観察者は、蛍光発光を検出し、検出対象に対する定量化処理を行うこともできる。
When the observer observes the detection target using the fluorescence method, the molecule to be detected or the molecule that specifically binds and / or reacts with the molecule to be detected is labeled with the fluorescent substance. The labeled molecule emits fluorescence under irradiation of excitation light from the outside. The observer can detect fluorescence emission and observe the process of the biospecific reaction of the molecule to be detected. Alternatively, the observer can detect fluorescence emission and perform quantification processing on the detection target.
様々なRNAは、ウィルス、病原体や遺伝子の活動に関連する生体分子である。ウィルス、病原体や遺伝子の活動を研究するために、RNAの検出が利用される。RNAの検出技術として、RT-PCR(Reverse Transcription Polymerase Chain Reaction:逆転写ポリメラーゼ連鎖反応)法やマイクロアレイ法といった方法が例示される。これらの検出技術にも、上述の蛍光法が利用される。
Various RNAs are biomolecules related to the activity of viruses, pathogens and genes. RNA detection is used to study the activity of viruses, pathogens and genes. Examples of RNA detection techniques include RT-PCR (Reverse Transcription Polymerase Chain Reaction) method and microarray method. The above-described fluorescence method is also used for these detection techniques.
バイオテクノロジの分野において、PCR法と呼ばれるDNA増幅方法もよく利用される。PCR法は、DNAポリメラーゼと呼ばれる酵素の反応を利用する。PCR法は、DNA中の所望の領域のみを選択的に増幅することを可能にする。PCR法は、上述のRT-PCR法やマイクロアレイ法にも利用される。観察者が、PCR法を用いるならば、DNAを増幅させることができる。したがって、生体試料中のRNAの量が微量であり、蛍光検出が困難な条件の下においても、観察者は、DNAを増幅し、蛍光を検出することができる。
In the field of biotechnology, a DNA amplification method called a PCR method is often used. The PCR method utilizes an enzyme reaction called DNA polymerase. The PCR method makes it possible to selectively amplify only a desired region in DNA. The PCR method is also used for the above-mentioned RT-PCR method and microarray method. If the observer uses the PCR method, the DNA can be amplified. Therefore, even under conditions where the amount of RNA in the biological sample is very small and fluorescence detection is difficult, the observer can detect the fluorescence by amplifying the DNA.
上述の如く、蛍光法及びPCR法は、非常に有用である。しかしながら、PCR法は、複雑なプライマ設計、試薬の増加、厳密な温度管理や増幅バイアスといった課題を内包する。観察者が、使い捨てに適した微細なマイクロチップ構造の検出器(いわゆるμTAS(Micro Total Analysis Systems)を使用するならば、検出器の制限された空間に起因して、上述の課題は、顕著に現れる。
As described above, the fluorescence method and the PCR method are very useful. However, the PCR method has problems such as complicated primer design, increase in reagents, strict temperature control and amplification bias. If the observer uses a micro-microchip detector suitable for disposable use (so-called μTAS (Micro Total Analysis Systems)), the above-mentioned problem is notable due to the limited space of the detector. appear.
1つの分子から生体分子の活動を動的且つリアルタイムに観察することを可能にする蛍光観察装置(全反射照明蛍光顕微鏡)は、既に実用化されている。全反射照明蛍光顕微鏡は、落射照明型顕微鏡で用いられる励起光の入射技術とは異なり、高屈折率を有する材料(例えば、石英)から低屈折率を有する材料(例えば、水溶液)へ臨界角以上で入射した光の全反射によって生じたエバネッセント光を励起光として利用する。全反射が生じる界面からの距離が長くなると、エバネッセント光の強度は、指数関数的に減衰する。したがって、観察者が全反射照明蛍光顕微鏡を利用するならば、観察者が落射照明型顕微鏡を使用するときと比べて、励起光が照射される照射体積は非常に小さい。この結果、水のラマン散乱や様々な他の背景光は非常に低減される。このことは、大幅に向上された検出感度に帰結する。
A fluorescence observation apparatus (total reflection illumination fluorescence microscope) that enables dynamic and real-time observation of biomolecule activity from a single molecule has already been put into practical use. The total reflection illumination fluorescence microscope is different from the excitation light incident technique used in the epi-illumination microscope, and it exceeds the critical angle from a material having a high refractive index (for example, quartz) to a material having a low refractive index (for example, an aqueous solution). The evanescent light generated by the total reflection of the light incident on is used as excitation light. As the distance from the interface where total reflection occurs increases, the intensity of the evanescent light attenuates exponentially. Therefore, if the observer uses a total reflection illumination fluorescence microscope, the irradiation volume irradiated with the excitation light is very small compared to when the observer uses an epi-illumination microscope. As a result, water Raman scattering and various other background lights are greatly reduced. This results in a greatly improved detection sensitivity.
励起光の照射体積の低減を利用し、背景光を低減する技術は、特許文献1や非特許文献1において研究されている。特許文献1は、全反射エバネッセント光を利用した励起光よりも小さな照射体積を実現することに取り組む。特許文献1は、ナノ開口によって生じたエバネッセント光を励起光として利用し、蛍光検出の感度を向上させることを提案する。特許文献1によれば、励起光は、励起光の波長よりも短い直径約200nmのナノ開口が形成された薄膜に照射される。ナノ開口から漏れ出るエバネッセント光が、蛍光観察に利用される。ナノ開口から漏れ出るエバネッセント光の照射体積は、ナノ開口の大きさに依存する。したがって、特許文献1の技術は、全反射エバネッセント光の照射体積よりも遙かに小さい照射体積を達成することができる。
Techniques for reducing background light using reduction of the excitation light irradiation volume have been studied in Patent Document 1 and Non-Patent Document 1. Patent Document 1 addresses the realization of an irradiation volume smaller than that of excitation light using total reflection evanescent light. Patent Document 1 proposes to improve the sensitivity of fluorescence detection by using evanescent light generated by a nano-aperture as excitation light. According to Patent Document 1, excitation light is applied to a thin film in which nano openings having a diameter of about 200 nm shorter than the wavelength of excitation light are formed. Evanescent light leaking from the nano-aperture is used for fluorescence observation. The irradiation volume of the evanescent light leaking from the nano aperture depends on the size of the nano aperture. Therefore, the technique of Patent Document 1 can achieve an irradiation volume that is much smaller than the irradiation volume of total reflection evanescent light.
非特許文献2は、ナノ開口から漏れ出るエバネッセント光を利用した照明技術を開示する。非特許文献2によれば、ナノ開口内の底面部にDNAポリメラーゼが固定化される。蛍光体標識dCTPが、固定化されたDNAポリメラーゼに取り込まれる。
Non-Patent Document 2 discloses an illumination technique using evanescent light leaking from a nano-aperture. According to Non-Patent Document 2, DNA polymerase is immobilized on the bottom surface in the nano-opening. The phosphor-labeled dCTP is incorporated into the immobilized DNA polymerase.
特許文献2は、表面プラズモン共鳴によって生じた電場増強効果を利用し、蛍光強度を増加させることを提案する。特許文献3は、斜めに入射する励起光と、ナノ開口が形成された膜と基板との間に配置された低屈折率膜と、を利用することを提案する。特許文献3の技術は、励起光を効率的に入射させることを可能にする。
Patent Document 2 proposes to increase the fluorescence intensity by utilizing the electric field enhancement effect generated by surface plasmon resonance. Patent Document 3 proposes to use obliquely incident excitation light and a low-refractive index film disposed between a film in which a nano-aperture is formed and a substrate. The technique of Patent Document 3 enables excitation light to be incident efficiently.
ナノ開口から漏れ出るエバネッセント光は弱く、検出対象を十分に蛍光発光させないこともある。したがって、上述の技術は、検出感度の観点において、課題を内包している。
エ The evanescent light leaking from the nano aperture is weak, and the detection target may not be sufficiently fluorescent. Therefore, the above-described technique has a problem in terms of detection sensitivity.
本発明は、高い検出感度を達成することができる蛍光検出技術を提供することを目的とする。
An object of the present invention is to provide a fluorescence detection technique capable of achieving high detection sensitivity.
本発明の一の局面に係る蛍光検出装置は、複数の検出対象を案内する少なくとも1つの流路と、前記少なくとも1つの流路によって立体的に交差される少なくとも1つのスリットが形成された遮光膜と、前記少なくとも1つの流路が前記少なくとも1つのスリットに立体的に交差する少なくとも1つの交差部に、励起光を照射し、前記複数の検出対象から蛍光を生じさせる照射部と、前記蛍光の強度から、前記少なくとも1つの交差部を通過した前記複数の検出対象の通過量を計測する計測部と、を備える。前記励起光は、前記少なくとも1つのスリットの延設方向に沿う直線偏光を含む。
A fluorescence detection apparatus according to one aspect of the present invention includes a light shielding film in which at least one flow path for guiding a plurality of detection targets and at least one slit that is sterically intersected by the at least one flow path are formed. And at least one intersection where the at least one flow path sterically intersects the at least one slit is irradiated with excitation light to generate fluorescence from the plurality of detection targets, and the fluorescence And a measuring unit that measures the passage amounts of the plurality of detection targets that have passed through the at least one intersecting portion based on intensity. The excitation light includes linearly polarized light along the extending direction of the at least one slit.
本発明の他の局面に係る蛍光検出方法は、少なくとも1つのスリットに立体的に交差する少なくとも1つの流路内で複数の検出対象を移動させる段階と、前記少なくとも1つのスリットが前記少なくとも1つの流路に交差する少なくとも1つの交差部に、前記少なくとも1つのスリットの延設方向に沿う直線偏光を含む励起光を照射し、前記複数の検出対象から蛍光を生じさせる段階と、前記蛍光の光量から、前記少なくとも1つの交差部を通過した前記複数の検出対象の通過量を計測する段階と、を備える。
The fluorescence detection method according to another aspect of the present invention includes a step of moving a plurality of detection targets in at least one flow path that sterically intersects at least one slit, and the at least one slit is the at least one slit. Irradiating excitation light including linearly polarized light along the extending direction of the at least one slit to at least one intersecting portion intersecting the flow path to generate fluorescence from the plurality of detection targets; To measuring the passage amounts of the plurality of detection objects that have passed through the at least one intersection.
上述の蛍光検出技術は、高い検出感度を達成することができる。
The above-described fluorescence detection technology can achieve high detection sensitivity.
本発明の目的、特徴及び利点は、以下の詳細な説明と添付図面とによって、より明白となる。
The objects, features and advantages of the present invention will become more apparent from the following detailed description and the accompanying drawings.
例示的な蛍光検出技術の様々な実施形態が、図面を参照して説明される。尚、以下に説明される実施形態において、同一の構成要素に対して同一の符号が付されている。説明の明瞭化のため、重複する説明は省略される。図面に示される構成、配置或いは形状、図面に関連する記載並びに「上」、「下」、「右」、「左」といった方向を表す用語は、単に各実施形態の原理を容易に理解させることを目的とする。したがって、以下の実施形態の原理は、これらに何ら限定されない。
Various embodiments of exemplary fluorescence detection techniques are described with reference to the drawings. In the embodiments described below, the same components are denoted by the same reference numerals. For the sake of clarity of explanation, overlapping explanation is omitted. The configuration, arrangement or shape shown in the drawings, descriptions related to the drawings, and terms such as “up”, “down”, “right”, and “left” simply make it easy to understand the principle of each embodiment. With the goal. Therefore, the principle of the following embodiment is not limited to these.
<第1実施形態>
上述の特許文献1乃至4は、微小な開口部を通過した光を利用して、検出対象を蛍光発色させる。微小な開口部を通過した光が、検出対象の検出に選択的に利用されるので、背景光は、効果的に低減される。その一方で、検出対象の検出に用いられる光は、微弱となるので、検出対象は、強く発光しないこともある。このことは、低い検出感度に帰結する。第1実施形態において、検出対象を強く発光させ、高い検出感度を達成するための技術が説明される。 <First Embodiment>
In Patent Documents 1 to 4 described above, the detection target is fluorescently colored using light that has passed through a minute opening. Since the light that has passed through the minute opening is selectively used for detection of the detection target, the background light is effectively reduced. On the other hand, since the light used for detecting the detection target is weak, the detection target may not emit light strongly. This results in low detection sensitivity. In the first embodiment, a technique for causing a detection target to emit light strongly and achieving high detection sensitivity will be described.
上述の特許文献1乃至4は、微小な開口部を通過した光を利用して、検出対象を蛍光発色させる。微小な開口部を通過した光が、検出対象の検出に選択的に利用されるので、背景光は、効果的に低減される。その一方で、検出対象の検出に用いられる光は、微弱となるので、検出対象は、強く発光しないこともある。このことは、低い検出感度に帰結する。第1実施形態において、検出対象を強く発光させ、高い検出感度を達成するための技術が説明される。 <First Embodiment>
In Patent Documents 1 to 4 described above, the detection target is fluorescently colored using light that has passed through a minute opening. Since the light that has passed through the minute opening is selectively used for detection of the detection target, the background light is effectively reduced. On the other hand, since the light used for detecting the detection target is weak, the detection target may not emit light strongly. This results in low detection sensitivity. In the first embodiment, a technique for causing a detection target to emit light strongly and achieving high detection sensitivity will be described.
図1は、第1実施形態の蛍光検出装置100の概念図である。図1を参照して、蛍光検出装置100が説明される。
FIG. 1 is a conceptual diagram of the fluorescence detection apparatus 100 according to the first embodiment. With reference to FIG. 1, the fluorescence detection apparatus 100 will be described.
蛍光検出装置100は、流路200と、遮光膜300と、照射部400と、計測部500と、を備える。遮光膜300には、スリット310が形成される。流路200は、複数の検出対象DOを案内する。検出対象DOは、流路200に沿って移動する。検出対象DOは、DNA、RNAや蛋白質であってもよい。本実施形態の原理は、検出対象DOの特定の種類に限定されない。
The fluorescence detection apparatus 100 includes a flow path 200, a light shielding film 300, an irradiation unit 400, and a measurement unit 500. A slit 310 is formed in the light shielding film 300. The channel 200 guides a plurality of detection targets DO. The detection target DO moves along the flow path 200. The detection target DO may be DNA, RNA, or protein. The principle of this embodiment is not limited to a specific type of detection target DO.
スリット310は、流路200とは異なる方向に延びる。流路200は、スリット310に立体的に交差する。この結果、交差部110が規定される。流路200は、スリット310に対して直角であってもよい。代替的に、流路200は、スリット310に対して、他の交差角で交差してもよい。本実施形態の原理は、流路200とスリット310との間の特定の交差角に限定されない。
The slit 310 extends in a direction different from the flow path 200. The channel 200 intersects the slit 310 three-dimensionally. As a result, the intersection 110 is defined. The channel 200 may be perpendicular to the slit 310. Alternatively, the flow path 200 may intersect the slit 310 at other intersection angles. The principle of the present embodiment is not limited to a specific crossing angle between the flow path 200 and the slit 310.
照射部400は、交差部110に、励起光PLを照射する。励起光PLの一部は、スリット310及び流路200を順次通過する。この結果、励起光PLは、流路200に沿って流れる検出対象DOに到達する。励起光PLの他の一部は、遮光膜300によって遮蔽される。したがって、背景光は、十分に低減される。
The irradiation unit 400 irradiates the intersection 110 with excitation light PL. A part of the excitation light PL sequentially passes through the slit 310 and the flow path 200. As a result, the excitation light PL reaches the detection target DO that flows along the flow path 200. Another part of the excitation light PL is shielded by the light shielding film 300. Therefore, the background light is sufficiently reduced.
遮光膜300は、基板(図示せず)に蒸着された金属膜であってもよい。代替的に、遮光膜300は、照射部400から出射される励起光PLに対して不透明な他の材料から形成されてもよい。遮光膜300が、励起光PLに対して不透明な材料から形成されるならば、遮光膜300は、励起光PLを完全に遮蔽することができる。
The light shielding film 300 may be a metal film deposited on a substrate (not shown). Alternatively, the light shielding film 300 may be formed of another material that is opaque to the excitation light PL emitted from the irradiation unit 400. If the light shielding film 300 is formed of a material that is opaque to the excitation light PL, the light shielding film 300 can completely shield the excitation light PL.
遮光膜300が金属膜であるならば、遮光膜300は、高い消衰係数を有する。遮光膜300は、高い遮光特性を有することができるので、設計者は、遮光膜300の厚さを非常に小さな値に設定してもよい。アルミニウム、金、銀、クロム、白金、ゲルマニウムやタングステンといった様々な金属材料が、遮光膜300の形成に用いられてもよい。遮光膜300は、複数の種類の金属材料から形成された合金膜であってもよい。本実施形態の原理は、遮光膜300の特定の組成に限定されない。
If the light shielding film 300 is a metal film, the light shielding film 300 has a high extinction coefficient. Since the light shielding film 300 can have high light shielding characteristics, the designer may set the thickness of the light shielding film 300 to a very small value. Various metal materials such as aluminum, gold, silver, chromium, platinum, germanium, and tungsten may be used for forming the light shielding film 300. The light shielding film 300 may be an alloy film formed from a plurality of types of metal materials. The principle of the present embodiment is not limited to a specific composition of the light shielding film 300.
照射部400は、遮光膜300に対して略垂直に、励起光PLを入射させてもよい。代替的に、照射部400は、遮光膜300に対して励起光PLの他の入射角度を設定してもよい。本実施形態の原理は、遮光膜300に対する励起光PLの特定の入射角度に限定されない。
The irradiation unit 400 may make the excitation light PL enter substantially perpendicular to the light shielding film 300. Alternatively, the irradiation unit 400 may set another incident angle of the excitation light PL with respect to the light shielding film 300. The principle of the present embodiment is not limited to a specific incident angle of the excitation light PL with respect to the light shielding film 300.
照射部400は、イオンレーザ光源を含んでもよい。代替的に、照射部400は、LED光源といった広い帯域を有する光源であってもよい。更に代替的に、検出対象DOからの蛍光発光が得られるならば、照射部400は、白色光源を含んでもよい。本実施形態の原理は、照射部400の特定の構造に限定されない。
The irradiation unit 400 may include an ion laser light source. Alternatively, the irradiation unit 400 may be a light source having a wide band such as an LED light source. Further alternatively, the irradiation unit 400 may include a white light source if fluorescence emission from the detection target DO is obtained. The principle of this embodiment is not limited to a specific structure of the irradiation unit 400.
励起光PLの波長は、検出対象DOからの蛍光発光が得られるように、適切に設定される。励起光PLは、複数の波長の光成分を含んでもよい。
The wavelength of the excitation light PL is appropriately set so that fluorescence emission from the detection target DO can be obtained. The excitation light PL may include light components having a plurality of wavelengths.
励起光PLの最短波長は、350nm以上750nm以下の範囲に設定されてもよい。この場合、可視光域の蛍光FLが、効率的に検出される。本実施形態の原理は、励起光PLの特定の波長に限定されない。
The shortest wavelength of the excitation light PL may be set in a range from 350 nm to 750 nm. In this case, the fluorescence FL in the visible light range is detected efficiently. The principle of this embodiment is not limited to a specific wavelength of the excitation light PL.
検出対象DOは、蛍光体を用いて標識化されている。検出対象DOは、励起光PLを受け、蛍光FLを発することができる。蛍光FLは、計測部500へ伝搬する。計測部500は、蛍光FLを検出し、蛍光FLの光量から交差部110を通過した検出対象DOの量を計測する。したがって、検出対象DOの通過量は、固定化技術なしで適切に計測される。このことは、流路200及びスリット310が形成されたマイクロチップ(図示せず)の再利用を容易化する。検出対象DOの通過量は、検出対象DOの個数として表現されてもよい。代替的に、検出対象DOの通過量は、検出対象DOの濃度として表現されてもよい。本実施形態の原理は、検出対象DOの通過量の特定に表現に何ら限定されない。
The detection target DO is labeled with a phosphor. The detection target DO can receive the excitation light PL and emit fluorescence FL. The fluorescence FL propagates to the measurement unit 500. The measuring unit 500 detects the fluorescence FL and measures the amount of the detection target DO that has passed through the intersection 110 from the light amount of the fluorescence FL. Therefore, the passing amount of the detection target DO is appropriately measured without the immobilization technique. This facilitates the reuse of a microchip (not shown) in which the channel 200 and the slit 310 are formed. The passing amount of the detection target DO may be expressed as the number of detection target DOs. Alternatively, the passing amount of the detection target DO may be expressed as the concentration of the detection target DO. The principle of the present embodiment is not limited to the expression for specifying the passage amount of the detection target DO.
蛍光FLを検出するために、様々な光学的な技術が利用されてもよい。本実施形態の原理は、蛍光FLを検出するための特定の技術に限定されない。
Various optical techniques may be used to detect the fluorescence FL. The principle of this embodiment is not limited to a specific technique for detecting fluorescence FL.
励起光PLは、スリット310の延設方向に沿う直線偏光を含む。したがって、検出対象DOは、励起光PLによって強く照射される。励起光PLは、上述の直線偏光に加えて、他の光成分を含んでもよい。
The excitation light PL includes linearly polarized light along the extending direction of the slit 310. Therefore, the detection target DO is strongly irradiated with the excitation light PL. The excitation light PL may include other light components in addition to the above-described linearly polarized light.
照射部400は、直線偏光を出射する光源を有してもよい。代替的に、照射部400は、光源と、光源からの光を直線偏光にする光学素子(例えば、波長板や偏光フィルタ)を有してもよい。本実施形態の原理は、直線偏光を得るための特定の光学技術に限定されない。
The irradiation unit 400 may have a light source that emits linearly polarized light. Alternatively, the irradiation unit 400 may include a light source and an optical element (for example, a wavelength plate or a polarization filter) that linearly polarizes light from the light source. The principle of this embodiment is not limited to a specific optical technique for obtaining linearly polarized light.
励起光PLは、平行光であってもよい。代替的に、励起光PLは、収束光であってもよい。更に代替的に、励起光PLは、発散光であってもよい。
The excitation light PL may be parallel light. Alternatively, the excitation light PL may be convergent light. Further alternatively, the excitation light PL may be diverging light.
流路200は、矩形断面を有してもよい。代替的に、流路200は、他の断面形状(例えば、円形、楕円形や台形)を有してもよい。流路200の断面の角隅部は湾曲していてもよい。本実施形態の原理は、流路200の特定の形状に限定されない。
The flow path 200 may have a rectangular cross section. Alternatively, the channel 200 may have other cross-sectional shapes (eg, circular, oval or trapezoidal). The corners of the cross section of the channel 200 may be curved. The principle of this embodiment is not limited to a specific shape of the flow path 200.
スリット310は、矩形断面を有してもよい。代替的に、スリット310は、他の断面形状(例えば、円形、楕円形や台形)を有してもよい。スリット310の断面の角隅部は湾曲していてもよい。本実施形態の原理は、スリット310の特定の形状に限定されない。
The slit 310 may have a rectangular cross section. Alternatively, the slit 310 may have other cross-sectional shapes (eg, circular, oval or trapezoidal). The corners of the cross section of the slit 310 may be curved. The principle of this embodiment is not limited to a specific shape of the slit 310.
<第2実施形態>
第1実施形態の検出技術の原理に基づいて、検出対象は適切に検出される。第2実施形態において、蛍光検出方法が説明される。 Second Embodiment
Based on the principle of the detection technique of the first embodiment, the detection target is appropriately detected. In the second embodiment, a fluorescence detection method is described.
第1実施形態の検出技術の原理に基づいて、検出対象は適切に検出される。第2実施形態において、蛍光検出方法が説明される。 Second Embodiment
Based on the principle of the detection technique of the first embodiment, the detection target is appropriately detected. In the second embodiment, a fluorescence detection method is described.
図2は、第2実施形態の蛍光検出方法の概略的なフローチャートである。図1及び図2を参照して、蛍光検出方法が説明される。
FIG. 2 is a schematic flowchart of the fluorescence detection method of the second embodiment. With reference to FIGS. 1 and 2, the fluorescence detection method will be described.
(ステップS110)
ステップS110において、観察者は、標識化処理後の検出対象DOを流路200へ供給する。この結果、検出対象DOは、流路200に沿って移動する。観察者は、ポンプ(図示せず)を、流路200への検出対象DOの供給に利用してもよい。代替的に、観察者は、既知の電気泳動技術を利用して、検出対象DOを流路200内で移動させてもよい。本実施形態の原理は、検出対象DOを流路200内で移動させるための特定の技術に限定されない。流路200への検出対象DOの供給の後、ステップ120が実行される。 (Step S110)
In step S <b> 110, the observer supplies the detection target DO after the labeling process to theflow path 200. As a result, the detection target DO moves along the flow path 200. An observer may use a pump (not shown) for supplying the detection target DO to the flow path 200. Alternatively, the observer may move the detection target DO within the flow path 200 using a known electrophoresis technique. The principle of the present embodiment is not limited to a specific technique for moving the detection target DO in the flow path 200. Step 120 is executed after the supply of the detection target DO to the flow path 200.
ステップS110において、観察者は、標識化処理後の検出対象DOを流路200へ供給する。この結果、検出対象DOは、流路200に沿って移動する。観察者は、ポンプ(図示せず)を、流路200への検出対象DOの供給に利用してもよい。代替的に、観察者は、既知の電気泳動技術を利用して、検出対象DOを流路200内で移動させてもよい。本実施形態の原理は、検出対象DOを流路200内で移動させるための特定の技術に限定されない。流路200への検出対象DOの供給の後、ステップ120が実行される。 (Step S110)
In step S <b> 110, the observer supplies the detection target DO after the labeling process to the
(ステップS120)
ステップS120において、励起光PLが交差部110へ照射される。励起光PLの直線偏光成分は、スリット310から効率的に漏れ出るので、交差部110を通過する検出対象DOは、蛍光FLを強く発することができる。励起光PLの照射の後、ステップS130が実行される。 (Step S120)
In step S120, the excitation light PL is irradiated to theintersection 110. Since the linearly polarized light component of the excitation light PL efficiently leaks from the slit 310, the detection target DO that passes through the intersection 110 can emit fluorescence FL strongly. Step S130 is performed after irradiation of the excitation light PL.
ステップS120において、励起光PLが交差部110へ照射される。励起光PLの直線偏光成分は、スリット310から効率的に漏れ出るので、交差部110を通過する検出対象DOは、蛍光FLを強く発することができる。励起光PLの照射の後、ステップS130が実行される。 (Step S120)
In step S120, the excitation light PL is irradiated to the
(ステップS130)
ステップS130において、計測部500は、交差部110通過する検出対象DOの蛍光FLを検出し、検出対象DOの通過量を計測する。 (Step S130)
In step S <b> 130, themeasurement unit 500 detects the fluorescence FL of the detection target DO passing through the intersection 110 and measures the amount of passage of the detection target DO.
ステップS130において、計測部500は、交差部110通過する検出対象DOの蛍光FLを検出し、検出対象DOの通過量を計測する。 (Step S130)
In step S <b> 130, the
本実施形態の原理は、図2に示される各工程の順序によって何ら限定されない。ステップS120は、ステップS110と平行して行われてもよい。
The principle of this embodiment is not limited at all by the order of the steps shown in FIG. Step S120 may be performed in parallel with step S110.
<第3実施形態>
第1実施形態の検出技術の原理に基づいて、設計者は、様々な蛍光検出装置を設計することができる。第3実施形態において、例示的な蛍光検出装置が説明される。 <Third Embodiment>
Based on the principle of the detection technique of the first embodiment, the designer can design various fluorescence detection devices. In the third embodiment, an exemplary fluorescence detection device is described.
第1実施形態の検出技術の原理に基づいて、設計者は、様々な蛍光検出装置を設計することができる。第3実施形態において、例示的な蛍光検出装置が説明される。 <Third Embodiment>
Based on the principle of the detection technique of the first embodiment, the designer can design various fluorescence detection devices. In the third embodiment, an exemplary fluorescence detection device is described.
図3は、第3実施形態の蛍光検出装置100Aの概略図である。図1及び図3を参照して、蛍光検出装置100Aが説明される。
FIG. 3 is a schematic diagram of the fluorescence detection apparatus 100A of the third embodiment. The fluorescence detection apparatus 100A will be described with reference to FIGS.
蛍光検出装置100Aは、マイクロチップ120と、光源410と、計測装置510と、ポンプ600と、を備える。光源410は、図1を参照して説明された照射部400に対応する。計測装置510は、図1を参照して説明された計測部500に対応する。
The fluorescence detection device 100A includes a microchip 120, a light source 410, a measurement device 510, and a pump 600. The light source 410 corresponds to the irradiation unit 400 described with reference to FIG. The measuring device 510 corresponds to the measuring unit 500 described with reference to FIG.
マイクロチップ120は、上基板210と、下基板320と、遮光膜300Aと、を含む。遮光膜300Aは、上基板210と下基板320とによって挟まれる。遮光膜300Aは、図1を参照して説明された遮光膜300に対応する。
The microchip 120 includes an upper substrate 210, a lower substrate 320, and a light shielding film 300A. The light shielding film 300 </ b> A is sandwiched between the upper substrate 210 and the lower substrate 320. The light shielding film 300A corresponds to the light shielding film 300 described with reference to FIG.
下基板320は、ガラス製であってもよい。代替的に、下基板320は、励起光PLに対して透明な他の材料(例えば、石英といった無機材料やポリジメチルシロキサン(PDMS)といった透明樹脂)であってもよい。本実施形態の原理は、下基板320の特定の材質に限定されない。
The lower substrate 320 may be made of glass. Alternatively, the lower substrate 320 may be another material transparent to the excitation light PL (for example, an inorganic material such as quartz or a transparent resin such as polydimethylsiloxane (PDMS)). The principle of the present embodiment is not limited to a specific material for the lower substrate 320.
下基板320は、前面321と、前面321とは反対側の後面322と、前面321と後面322との間で延びる左面323と、左面323とは反対側の右面324と、を含む。遮光膜300Aには、前面321から後面322へ向けて延びるスリット310Aが形成される。スリット310Aは、図1を参照して説明されたスリット310に対応する。
The lower substrate 320 includes a front surface 321, a rear surface 322 opposite to the front surface 321, a left surface 323 extending between the front surface 321 and the rear surface 322, and a right surface 324 opposite to the left surface 323. A slit 310A extending from the front surface 321 to the rear surface 322 is formed in the light shielding film 300A. The slit 310A corresponds to the slit 310 described with reference to FIG.
下基板320は、励起光PLに対して透明である。したがって、励起光PLは、下基板320を通じて、スリット310Aへ伝搬する。スリット310Aの周囲の遮光膜300Aに到達した励起光PLは、遮光膜300Aによって遮られる一方で、スリット310Aに到達した励起光PLは、上基板210に向けて伝搬する。
The lower substrate 320 is transparent to the excitation light PL. Therefore, the excitation light PL propagates through the lower substrate 320 to the slit 310A. The excitation light PL that has reached the light shielding film 300A around the slit 310A is blocked by the light shielding film 300A, while the excitation light PL that has reached the slit 310A propagates toward the upper substrate 210.
上基板210は、ガラス製であってもよい。代替的に、上基板210は、蛍光FLに対して透明な他の材料(例えば、石英といった無機材料やポリジメチルシロキサン(PDMS)といった透明樹脂)であってもよい。上基板210は、下基板320と同一の材料から形成されてもよい。代替的に、上基板210は、下基板320とは異なる材料から形成されてもよい。本実施形態の原理は、上基板210の特定の材質に限定されない。
The upper substrate 210 may be made of glass. Alternatively, the upper substrate 210 may be another material transparent to the fluorescent FL (for example, an inorganic material such as quartz or a transparent resin such as polydimethylsiloxane (PDMS)). The upper substrate 210 may be formed from the same material as the lower substrate 320. Alternatively, the upper substrate 210 may be formed from a different material than the lower substrate 320. The principle of this embodiment is not limited to a specific material of the upper substrate 210.
上基板210は、前面211と、前面211とは反対側の後面212と、前面211と後面212との間で延びる左面213と、左面213とは反対側の右面214と、流路200Aを規定する流路壁215と、を含む。流路200Aは、左面213と右面214との間で延びる。したがって、流路200Aは、スリット310Aに立体的に交差する。流路200Aは、図1を参照して説明された流路200に対応する。
Upper substrate 210 defines front surface 211, rear surface 212 opposite to front surface 211, left surface 213 extending between front surface 211 and rear surface 212, right surface 214 opposite to left surface 213, and flow path 200A. And a flow path wall 215 that performs. The channel 200A extends between the left surface 213 and the right surface 214. Accordingly, the flow path 200A three-dimensionally intersects the slit 310A. The channel 200A corresponds to the channel 200 described with reference to FIG.
ポンプ600は、流路200Aへ複数の検出対象DOを供給し、検出対象DOを左面213から右面214へ流路200Aに沿って移動させる。本実施形態において、移動部は、ポンプ600によって例示される。
The pump 600 supplies a plurality of detection objects DO to the flow path 200A, and moves the detection objects DO from the left surface 213 to the right surface 214 along the flow path 200A. In the present embodiment, the moving unit is exemplified by the pump 600.
表面コート処理、疎水処理や親水処理といった様々な表面処理が、流路壁215に対して施与されてもよい。表面処理によって、検出対象DOは、流路壁215に付着しにくくなる。本実施形態の原理は、流路壁215に対する特定の表面処理に限定されない。
Various surface treatments such as surface coating treatment, hydrophobic treatment, and hydrophilic treatment may be applied to the flow path wall 215. The surface treatment makes it difficult for the detection object DO to adhere to the flow path wall 215. The principle of this embodiment is not limited to a specific surface treatment for the flow path wall 215.
検出対象DOは、miRNA(マイクロRNA:マイクロリボ核酸)であってもよい。miRNAは、細胞内に存在する生体分子である。miRNAは、約18~20の塩基を有する。miRNAは、は、特定のmRNA(メッセンジャRNA)を認識する。miRNAは、は、ポリA鎖の分解と翻訳の抑制を引き起こす。このため、miRNAは、ガンの発症や記憶の形成といった生命現象に関与していると考えられている。
The detection target DO may be miRNA (microRNA: microribonucleic acid). miRNAs are biomolecules that exist in cells. miRNA has about 18-20 bases. miRNA recognizes specific mRNA (messenger RNA). miRNAs cause poly A chain degradation and translational repression. For this reason, miRNA is considered to be involved in life phenomena such as cancer development and memory formation.
miRNAは、単独では、蛍光発光しない。したがって、検出対象DOとしてmiRNAが用いられるならば、miRNAと人工核酸とによって形成された二本鎖が利用可能である。人工核酸は、miRNAを認識し、二本鎖として結合する。人工核酸が二本鎖としてmiRNAに結合したときのみ、蛍光発光が得られる。WO2008-111485号公報は、miRNAの蛍光発光に利用可能な様々な人工核酸を開示する。
MiRNA alone does not emit fluorescence. Therefore, if miRNA is used as the detection target DO, a double strand formed by the miRNA and the artificial nucleic acid can be used. The artificial nucleic acid recognizes miRNA and binds as a double strand. Fluorescence emission is obtained only when the artificial nucleic acid is bound to miRNA as a double strand. WO 2008-111485 discloses various artificial nucleic acids that can be used for fluorescence emission of miRNA.
光源410は、514nmの波長の光を出射するArイオンレーザ光源であってもよい。Arイオンレーザ光源は、連続光として励起光PLを出射してもよい。代替的に、Arイオンレーザ光源は、パルス変調された光を励起光PLとして出射してもよい。励起光PLが、パルス変調された光であるならば、検出対象DOが照明される期間は短くなる。したがって、検出対象DOの蛍光の退色は生じにくくなる。本実施形態の原理は、励起光PLの特定の出射パターンに限定されない。
The light source 410 may be an Ar ion laser light source that emits light having a wavelength of 514 nm. The Ar ion laser light source may emit the excitation light PL as continuous light. Alternatively, the Ar ion laser light source may emit pulse-modulated light as excitation light PL. If the excitation light PL is pulse-modulated light, the period during which the detection target DO is illuminated is shortened. Therefore, the fading of the fluorescence of the detection target DO is less likely to occur. The principle of this embodiment is not limited to a specific emission pattern of the excitation light PL.
上述の如く、スリット310Aに到達した励起光PLは、上基板210に向けて伝搬する。この結果、流路200Aを流れる検出対象DOは、励起光PLによって照明される。励起光PLに照らされた検出対象DOは、蛍光FLを発する。光源410が、514nmの波長の光を励起光PLとして出射するならば、蛍光FLは、約540nmの波長を有してもよい。蛍光FLは、計測装置510に向けて伝搬する。計測装置510は、蛍光FLを検出する。計測装置510は、蛍光FLの強度を表す検出信号を生成する。
As described above, the excitation light PL that has reached the slit 310A propagates toward the upper substrate 210. As a result, the detection target DO flowing through the flow path 200A is illuminated by the excitation light PL. The detection target DO illuminated by the excitation light PL emits fluorescence FL. If the light source 410 emits light having a wavelength of 514 nm as excitation light PL, the fluorescence FL may have a wavelength of about 540 nm. The fluorescence FL propagates toward the measuring device 510. Measuring device 510 detects fluorescence FL. The measuring device 510 generates a detection signal representing the intensity of the fluorescence FL.
計測装置510は、対物レンズ511と、光学フィルタ512と、結像レンズ513と、ディテクタ514と、演算装置515と、を含む。対物レンズ511は、蛍光FL及び対物レンズ511に到達した励起光PLを集光する。光学フィルタ512は、蛍光FLの通過を許容する一方で、励起光PLを遮光する。結像レンズ513は、ディテクタ514に蛍光FLを結像する。ディテクタ514は、蛍光FLの強度を表す検出信号を生成する。検出信号は、ディテクタ514から演算装置515に出力される。演算装置515は、検出信号から検出対象DOの通過量に関する情報(例えば、流路200Aとスリット310Aとの交差部を通過した検出対象DOの個数や検出対象DOの濃度)を取得する。本実施形態において、信号生成部は、ディテクタ514によって例示される。取得部は、演算装置515によって例示される。
The measuring device 510 includes an objective lens 511, an optical filter 512, an imaging lens 513, a detector 514, and an arithmetic device 515. The objective lens 511 condenses the fluorescence FL and the excitation light PL that has reached the objective lens 511. The optical filter 512 blocks the excitation light PL while allowing the fluorescence FL to pass therethrough. The imaging lens 513 images the fluorescence FL on the detector 514. The detector 514 generates a detection signal indicating the intensity of the fluorescence FL. The detection signal is output from the detector 514 to the arithmetic device 515. The arithmetic device 515 acquires information on the amount of passage of the detection target DO (for example, the number of detection target DOs passing through the intersection of the flow path 200A and the slit 310A and the concentration of the detection target DO) from the detection signal. In the present embodiment, the signal generation unit is exemplified by the detector 514. The acquisition unit is exemplified by the arithmetic device 515.
遮光膜300Aが、高い遮光特性を有するならば、設計者は、光学フィルタ512を除去してもよい。代替的に、設計者は、光学フィルタ512に代えて、分光技術を用いて、蛍光FLを抽出してもよい。本実施形態の原理は、計測装置510の特定の構造に限定されない。
The designer may remove the optical filter 512 if the light shielding film 300A has high light shielding characteristics. Alternatively, the designer may extract the fluorescence FL using a spectroscopic technique instead of the optical filter 512. The principle of this embodiment is not limited to a specific structure of the measuring device 510.
<第4実施形態>
スリットの寸法が適切に設定されるならば、励起光は、スリットの近傍に留まることができる。この結果、設計者は、励起光を遮光するための光学フィルタを組み込むことなく、計測部を設計することができる。第4実施形態において、スリットの設計原理が説明される。 <Fourth embodiment>
If the slit dimensions are set appropriately, the excitation light can remain in the vicinity of the slit. As a result, the designer can design the measurement unit without incorporating an optical filter for shielding the excitation light. In the fourth embodiment, the design principle of the slit will be described.
スリットの寸法が適切に設定されるならば、励起光は、スリットの近傍に留まることができる。この結果、設計者は、励起光を遮光するための光学フィルタを組み込むことなく、計測部を設計することができる。第4実施形態において、スリットの設計原理が説明される。 <Fourth embodiment>
If the slit dimensions are set appropriately, the excitation light can remain in the vicinity of the slit. As a result, the designer can design the measurement unit without incorporating an optical filter for shielding the excitation light. In the fourth embodiment, the design principle of the slit will be described.
図4は、スリット310の概略的な断面図である。図1及び図4を参照して、スリット310の設計原理が説明される。
FIG. 4 is a schematic sectional view of the slit 310. The design principle of the slit 310 will be described with reference to FIGS. 1 and 4.
スリット310の深さSDは、遮光膜300の厚さとして定義されてもよい。スリット310の幅SWは、スリット310の厚さ方向に対して直交する方向の寸法として定義されてもよい。尚、スリット310の断面寸法に関するこれらの定義は、本実施形態の原理を何ら限定しない。
The depth SD of the slit 310 may be defined as the thickness of the light shielding film 300. The width SW of the slit 310 may be defined as a dimension in a direction orthogonal to the thickness direction of the slit 310. In addition, these definitions regarding the cross-sectional dimension of the slit 310 do not limit the principle of this embodiment at all.
図4は、励起光PLの最短波長WLを示す。スリット310の幅SWは、最短波長WLよりも小さな値に設定される。励起光PLの最短波長WLが、350nm以上750nm以下の範囲に設定されるならば、スリット310の幅SWは、200nmに設定されてもよい。この場合、励起光PLは、遮光膜300を完全に通過することなく、スリット310の近傍において、局所的に存在することができる。本実施形態の原理は、スリット310の幅SWの特定の値に限定されない。
FIG. 4 shows the shortest wavelength WL of the excitation light PL. The width SW of the slit 310 is set to a value smaller than the shortest wavelength WL. If the shortest wavelength WL of the excitation light PL is set in the range of 350 nm or more and 750 nm or less, the width SW of the slit 310 may be set to 200 nm. In this case, the excitation light PL can locally exist in the vicinity of the slit 310 without completely passing through the light shielding film 300. The principle of the present embodiment is not limited to a specific value of the width SW of the slit 310.
スリット310の幅SWの値が、励起光PLの最短波長WLの値に近似しているならば、複数の検出対象DOが励起光PLに同時に曝されやすくなる。このことは、検出精度の低下を意味する。スリット310の幅SWが過度に小さな値に設定されるならば、スリット310の近傍に局在する励起光PLが、流路200の断面を十分に覆うことができないこともある。このことは、交差部110において蛍光発光することなく通過する検出対象DOの増加を意味する。したがって、設計者は、流路200の高さ寸法を更に考慮して、スリット310の幅SWを設定してもよい。
If the value of the width SW of the slit 310 approximates the value of the shortest wavelength WL of the excitation light PL, a plurality of detection objects DO are easily exposed to the excitation light PL simultaneously. This means a decrease in detection accuracy. If the width SW of the slit 310 is set to an excessively small value, the excitation light PL localized in the vicinity of the slit 310 may not sufficiently cover the cross section of the channel 200. This means an increase in the detection target DO that passes through the intersection 110 without emitting fluorescence. Therefore, the designer may set the width SW of the slit 310 in consideration of the height dimension of the flow path 200.
スリット310の深さSDは、遮光膜300の遮光特性を考慮して、適切に設定されてもよい。遮光膜300が、過度に薄いならば、遮光膜300は、励起光PLを十分に遮光することはできない。このことは、交差部110以外の領域において蛍光発光を引き起こす。交差部110以外の領域での蛍光発光は、計測部500が生成する検出信号に含まれるノイズを増加させる。遮光膜300が、過度に厚いならば、スリット310の近傍に局在する励起光PLは、流路200に十分に到達しない。遮光膜300の厚さは、20nm以上200nm以下の範囲に設定されてもよい。遮光膜300が、アルミニウムから形成されるならば、設計者は、遮光膜300の厚さを60nmに設定してもよい。
The depth SD of the slit 310 may be appropriately set in consideration of the light shielding characteristics of the light shielding film 300. If the light shielding film 300 is excessively thin, the light shielding film 300 cannot sufficiently shield the excitation light PL. This causes fluorescence emission in a region other than the intersection 110. Fluorescence emission in a region other than the intersection 110 increases noise included in the detection signal generated by the measurement unit 500. If the light shielding film 300 is excessively thick, the excitation light PL localized in the vicinity of the slit 310 does not reach the flow path 200 sufficiently. The thickness of the light shielding film 300 may be set in a range of 20 nm to 200 nm. If the light shielding film 300 is made of aluminum, the designer may set the thickness of the light shielding film 300 to 60 nm.
<第5実施形態>
特許文献1の従来技術は、微小開口が形成された開口膜上に、検出対象としての生体分子を含む溶液層を形成する。微小開口から漏れ出るエバネッセント場が、生体分子の蛍光検出に利用される。励起光は、微小開口の近傍のみを照らすので、生体分子の観測確率は、不安定である。このことは、生体分子の低下や検出時間の増大を引き起こす。したがって、従来技術は、生体分子の定量的な評価のために、検量線を必要とする。検量線を用いた定量的な評価では、生体分子の個体数を高い精度で検出することは困難である。 <Fifth Embodiment>
In the prior art of Patent Document 1, a solution layer containing a biomolecule as a detection target is formed on an opening film in which a minute opening is formed. The evanescent field leaking from the minute aperture is used for fluorescence detection of biomolecules. Since the excitation light illuminates only the vicinity of the minute aperture, the observation probability of the biomolecule is unstable. This causes a decrease in biomolecules and an increase in detection time. Therefore, the prior art requires a calibration curve for quantitative evaluation of biomolecules. In quantitative evaluation using a calibration curve, it is difficult to detect the number of biomolecules with high accuracy.
特許文献1の従来技術は、微小開口が形成された開口膜上に、検出対象としての生体分子を含む溶液層を形成する。微小開口から漏れ出るエバネッセント場が、生体分子の蛍光検出に利用される。励起光は、微小開口の近傍のみを照らすので、生体分子の観測確率は、不安定である。このことは、生体分子の低下や検出時間の増大を引き起こす。したがって、従来技術は、生体分子の定量的な評価のために、検量線を必要とする。検量線を用いた定量的な評価では、生体分子の個体数を高い精度で検出することは困難である。 <Fifth Embodiment>
In the prior art of Patent Document 1, a solution layer containing a biomolecule as a detection target is formed on an opening film in which a minute opening is formed. The evanescent field leaking from the minute aperture is used for fluorescence detection of biomolecules. Since the excitation light illuminates only the vicinity of the minute aperture, the observation probability of the biomolecule is unstable. This causes a decrease in biomolecules and an increase in detection time. Therefore, the prior art requires a calibration curve for quantitative evaluation of biomolecules. In quantitative evaluation using a calibration curve, it is difficult to detect the number of biomolecules with high accuracy.
溶液層は、開口膜とカバーガラスとの間に形成される。開口膜上の溶液層の領域には、複数の生体分子が存在することもある。したがって、背景光は、十分に低減されないこともある。
The solution layer is formed between the opening film and the cover glass. There may be a plurality of biomolecules in the region of the solution layer on the opening membrane. Therefore, the background light may not be sufficiently reduced.
特許文献2及び3は、微小開口中での検出対象の固定化技術を開示する。特許文献2及び3の従来技術は、微小開口の近傍に検出対象と特異的に反応する反応物質を予め固定化することを必要とする。このことは、蛍光検出装置の製造コストを増大させる。加えて、蛍光検出装置の再利用は困難になる。
Patent Documents 2 and 3 disclose a technique for immobilizing a detection target in a minute opening. The prior arts of Patent Documents 2 and 3 require that a reactive substance that specifically reacts with the detection target be immobilized in the vicinity of the minute opening. This increases the manufacturing cost of the fluorescence detection device. In addition, it becomes difficult to reuse the fluorescence detection apparatus.
特許文献4の技術は、ラマン散乱光を用いて、照射体積を低減させることを可能にする。特許文献4の技術は、微小開口を更に用いて、背景光を低減させる。しかしながら、特許文献4の技術は、微小開口と、電場増強に用いられる突起構造を高い精度(数nmレベル)で位置合わせすることを要求する。突起構造自体も、高い精度(数nmレベル)で形成される必要がある。このことは、蛍光検出装置の製造コスト及び製造の困難性を増大させる。
The technique of Patent Document 4 makes it possible to reduce the irradiation volume using Raman scattered light. The technique of Patent Document 4 further reduces the background light by further using a minute aperture. However, the technique of Patent Document 4 requires that the minute aperture and the protrusion structure used for electric field enhancement be aligned with high accuracy (several nm level). The protrusion structure itself needs to be formed with high accuracy (several nm level). This increases the manufacturing cost and manufacturing difficulty of the fluorescence detection device.
第1実施形態に関連して説明された流路が、適切に設計されるならば、複数の検出対象は、1つずつ流れることができる。計測部は、流路を流れる検出対象を1つずつ検出し、検量線を要することなく、検出対象の通過量を精度よく検出することができる。加えて、検出対象は、固定化されなくともよい。したがって、検出対象の検出は、短時間しかかからない。加えて、検出対象の検出に要する準備も簡素化される。第5実施形態において、流路の設計原理が説明される。
If the flow path described in relation to the first embodiment is appropriately designed, a plurality of detection targets can flow one by one. The measurement unit detects the detection target flowing through the flow channel one by one, and can accurately detect the passage amount of the detection target without requiring a calibration curve. In addition, the detection target may not be fixed. Therefore, the detection of the detection target takes only a short time. In addition, the preparation required for detecting the detection target is simplified. In the fifth embodiment, the design principle of the flow path will be described.
図5は、流路200の概略的な断面図である。図1及び図5を参照して、流路200の設計原理が説明される。
FIG. 5 is a schematic cross-sectional view of the flow path 200. The design principle of the flow path 200 will be described with reference to FIGS.
流路200の高さCHは、励起光PLの伝搬方向におけるスリット310の寸法として定義されてもよい。流路200の幅CWは、流路200の高さ方向に対して直交する方向の寸法として定義されてもよい。尚、流路200の断面寸法に関するこれらの定義は、本実施形態の原理を何ら限定しない。
The height CH of the flow path 200 may be defined as the dimension of the slit 310 in the propagation direction of the excitation light PL. The width CW of the flow channel 200 may be defined as a dimension in a direction orthogonal to the height direction of the flow channel 200. In addition, these definitions regarding the cross-sectional dimension of the flow path 200 do not limit the principle of this embodiment at all.
流路200の高さCHは、スリット310の幅SWよりも小さな値に設定されてもよい。この結果、スリット310の近傍で局在する励起光PLは、流路200の断面を覆うことができる。本実施形態において、高さ寸法は、高さCHによって例示される。
The height CH of the channel 200 may be set to a value smaller than the width SW of the slit 310. As a result, the excitation light PL localized near the slit 310 can cover the cross section of the flow path 200. In the present embodiment, the height dimension is exemplified by the height CH.
スリット310からの距離が長くなるにつれて、スリット310の近傍で局在する励起光PLの強度は、指数関数的に低減する。したがって、流路200の高さCHに大きな値が設定されるならば、流路200を通過する検出対象DOの蛍光発光は弱くなる。したがって、流路200の高さCHは、30nm以上150nm以下の範囲に設定されてもよい。励起光PLとして、350nm以上750nm以下の最低波長を有する直線偏光が用いられるならば、高さCHは、100nmに設定されてもよい。
As the distance from the slit 310 increases, the intensity of the excitation light PL localized near the slit 310 decreases exponentially. Therefore, if a large value is set for the height CH of the flow path 200, the fluorescence emission of the detection target DO passing through the flow path 200 becomes weak. Therefore, the height CH of the channel 200 may be set in a range of 30 nm or more and 150 nm or less. If linearly polarized light having a minimum wavelength of 350 nm or more and 750 nm or less is used as the excitation light PL, the height CH may be set to 100 nm.
流路200の幅CWは、検出対象DOの大きさに基づいて設定されてもよい。流路200の幅CWの値が、10nm以上500nm以下の範囲に設定されるならば、使用者は、検出対象DOとして、様々な生体細胞を流路200に供給することができる。
The width CW of the flow path 200 may be set based on the size of the detection target DO. If the value of the width CW of the flow path 200 is set in the range of 10 nm to 500 nm, the user can supply various biological cells to the flow path 200 as the detection target DO.
<第6実施形態>
本発明者等は、FDTD法(Poynting:富士通株式会社製)を用いて、スリットの延設方向に沿う直線偏光を照射することの光学的な効果を検証した。第6実施形態において、本発明者等が行ったシミュレーションが説明される。 <Sixth Embodiment>
The present inventors verified the optical effect of irradiating linearly polarized light along the extending direction of the slit, using the FDTD method (Poying: manufactured by Fujitsu Limited). In the sixth embodiment, a simulation performed by the present inventors will be described.
本発明者等は、FDTD法(Poynting:富士通株式会社製)を用いて、スリットの延設方向に沿う直線偏光を照射することの光学的な効果を検証した。第6実施形態において、本発明者等が行ったシミュレーションが説明される。 <Sixth Embodiment>
The present inventors verified the optical effect of irradiating linearly polarized light along the extending direction of the slit, using the FDTD method (Poying: manufactured by Fujitsu Limited). In the sixth embodiment, a simulation performed by the present inventors will be described.
図6A及び図6Bは、第3実施形態に関連して説明されたマイクロチップ120の概略的な断面図である。図6A及び図6Bは、流路200Aの中心線上のマイクロチップ120の断面を表す。図6Aに示される励起光PLは、スリット310Aの延設方向に方向付けられた直線偏光である。図6Bに示される励起光PLは、スリット310Aの延設方向に対して直角に方向付けられた直線偏光である。図6A及び図6Bに示される複数の曲線それぞれは、同一の光強度を表す等高線である。図3、図6A及び図6Bを参照して、本発明者等が行ったシミュレーションが説明される。
6A and 6B are schematic cross-sectional views of the microchip 120 described in relation to the third embodiment. 6A and 6B represent a cross section of the microchip 120 on the center line of the flow path 200A. The excitation light PL shown in FIG. 6A is linearly polarized light oriented in the extending direction of the slit 310A. The excitation light PL shown in FIG. 6B is linearly polarized light oriented at right angles to the extending direction of the slit 310A. Each of the plurality of curves shown in FIGS. 6A and 6B is a contour line representing the same light intensity. A simulation performed by the present inventors will be described with reference to FIGS. 3, 6A, and 6B.
本発明者等は、第4実施形態に関連して説明された設計原理に従って、スリット310Aの幅を200nmに設定した。加えて、本発明者等は、遮光膜300Aの厚さを60nmに設定した。
The inventors set the width of the slit 310A to 200 nm in accordance with the design principle described in relation to the fourth embodiment. In addition, the present inventors set the thickness of the light shielding film 300A to 60 nm.
本発明者等は、第5実施形態に関連して説明された設計原理に従って、流路200Aの高さ及び幅を、100nmに設定した。
The present inventors set the height and width of the flow path 200A to 100 nm in accordance with the design principle described in relation to the fifth embodiment.
図6A及び図6Bに示される如く、遮光膜300Aは、励起光PLを適切に遮っている。励起光PLの一部は、スリット310Aに入り込む。図6A及び図6Bにおいて、スリット310Aの近傍に局在する励起光PLは、等高線によって表されている。図6及び図6Bのスリット310Aの近傍に局在する励起光PLから、流路200Aと遮光膜300Aに形成されたスリット310Aとの立体交差構造は、励起光PLの照射体積を低減することが分かる。したがって、上述の実施形態の原理は、高いSN比の下で検出対象DOを検出することを可能にする。
6A and 6B, the light shielding film 300A appropriately shields the excitation light PL. A part of the excitation light PL enters the slit 310A. 6A and 6B, the excitation light PL localized in the vicinity of the slit 310A is represented by contour lines. From the excitation light PL localized near the slit 310A in FIGS. 6 and 6B, the three-dimensional intersection structure of the flow path 200A and the slit 310A formed in the light shielding film 300A can reduce the irradiation volume of the excitation light PL. I understand. Therefore, the principle of the above-described embodiment makes it possible to detect the detection target DO under a high S / N ratio.
図6Aと図6Bとを比較すると、図6Aは、図6Bよりもスリット310Aから流路200Aに入り込む多くの等高線を示す。このことは、直線偏光の向きが、スリット310Aの延設方向に沿うならば、励起光PLが、流路200Aを強く照らすことができることを意味する。したがって、設計者は、励起光PLの直線偏光の向きを、スリット310Aの延設方向に略平行に設定してもよい。
6A is compared with FIG. 6B, FIG. 6A shows more contour lines entering the flow path 200A from the slit 310A than in FIG. 6B. This means that the excitation light PL can strongly illuminate the flow path 200A if the direction of linearly polarized light is along the extending direction of the slit 310A. Therefore, the designer may set the direction of the linearly polarized light of the excitation light PL to be substantially parallel to the extending direction of the slit 310A.
第5実施形態に関連して説明された設計原理に従って形成された流路200Aの断面積は非常に小さい。したがって、流路200Aを流れる検出対象DOも非常に小さい。小さな検出対象DOに、図6Bに示されるような微弱な励起光が照射されても、蛍光検出に不十分な蛍光しか得られない。図6Aに示されるような強い励起光が小さな検出対象DOに照射されるならば、蛍光検出に十分な強い蛍光発光が得られる。したがって、計測装置510は、流路200Aとスリット310Aとの交差部を通過した検出対象DOを精度よくカウントすることができる。
The cross-sectional area of the flow path 200A formed according to the design principle described in relation to the fifth embodiment is very small. Therefore, the detection target DO flowing through the flow path 200A is also very small. Even if a small detection object DO is irradiated with weak excitation light as shown in FIG. 6B, only fluorescence insufficient for fluorescence detection can be obtained. If strong excitation light as shown in FIG. 6A is irradiated to a small detection object DO, strong fluorescence emission sufficient for fluorescence detection can be obtained. Therefore, the measuring device 510 can accurately count the detection target DO that has passed through the intersection between the flow path 200A and the slit 310A.
<第7実施形態>
蛍光検出装置の計測部は、蛍光の強度から検出対象の通過量を見出すための様々なデータ処理構造を有してもよい。第7実施形態において、計測部の例示的な構造が説明される。 <Seventh embodiment>
The measurement unit of the fluorescence detection apparatus may have various data processing structures for finding the passage amount of the detection target from the fluorescence intensity. In the seventh embodiment, an exemplary structure of the measurement unit will be described.
蛍光検出装置の計測部は、蛍光の強度から検出対象の通過量を見出すための様々なデータ処理構造を有してもよい。第7実施形態において、計測部の例示的な構造が説明される。 <Seventh embodiment>
The measurement unit of the fluorescence detection apparatus may have various data processing structures for finding the passage amount of the detection target from the fluorescence intensity. In the seventh embodiment, an exemplary structure of the measurement unit will be described.
図7は、計測部500の概略的なブロック図である。図1、図3及び図7を参照して、計測部500の構造が説明される。
FIG. 7 is a schematic block diagram of the measurement unit 500. The structure of the measuring unit 500 will be described with reference to FIGS.
計測部500は、信号生成部520と、取得部530と、を含む。蛍光FLは、信号生成部520に入射する。信号生成部520は、蛍光FLの入射に応じて、蛍光FLの強度を表す検出信号DSを生成する。検出信号DSは、信号生成部520から取得部530へ出力される。信号生成部520は、図3を参照して説明されたディテクタ514に対応する。
The measurement unit 500 includes a signal generation unit 520 and an acquisition unit 530. The fluorescence FL is incident on the signal generation unit 520. The signal generation unit 520 generates a detection signal DS representing the intensity of the fluorescence FL in response to the incidence of the fluorescence FL. The detection signal DS is output from the signal generation unit 520 to the acquisition unit 530. The signal generation unit 520 corresponds to the detector 514 described with reference to FIG.
取得部530は、比較部531と、判定部532と、を含む。比較部531は、検出信号DSを予め設定された閾値と比較し、比較結果を表すデータCRDを生成する。データCRDは、比較部531から判定部532へ出力される。判定部532は、データCRDを参照し、交差部110を通過した検出対象DOの通過量を判定する。判定部532によって判定された通過量を表すデータPSDは、蛍光検出装置100に通信可能に接続されたコンピュータ(図示せず)や他の外部装置(図示せず)へ出力されてもよい。取得部530は、図3を参照して説明された演算装置515に対応する。
The acquisition unit 530 includes a comparison unit 531 and a determination unit 532. The comparison unit 531 compares the detection signal DS with a preset threshold value, and generates data CRD representing the comparison result. The data CRD is output from the comparison unit 531 to the determination unit 532. The determination unit 532 determines the passage amount of the detection target DO that has passed through the intersection 110 by referring to the data CRD. The data PSD representing the passage amount determined by the determination unit 532 may be output to a computer (not shown) or other external device (not shown) connected to the fluorescence detection device 100 so as to be communicable. The acquisition unit 530 corresponds to the arithmetic device 515 described with reference to FIG.
図8は、検出信号DSのタイミングチャートである。図1、図7及び図8を参照して、計測部500が実行する演算処理が説明される。
FIG. 8 is a timing chart of the detection signal DS. With reference to FIGS. 1, 7, and 8, calculation processing executed by the measurement unit 500 will be described.
蛍光FLの強度が高いならば、検出信号DSは、高い信号値を有してもよい。蛍光FLの強度が低いならば、検出信号DSは、低い信号値を有してもよい。信号値は、信号電圧の大きさであってもよい。代替的に、信号値は、信号電流の大きさであってもよい。
If the intensity of the fluorescence FL is high, the detection signal DS may have a high signal value. If the intensity of the fluorescence FL is low, the detection signal DS may have a low signal value. The signal value may be the magnitude of the signal voltage. Alternatively, the signal value may be the magnitude of the signal current.
比較部531は、信号値が閾値THを超えている間に立ち上がるパルスを有するパルス信号をデータCRDとして生成してもよい。判定部532は、パルス信号中のパルスの数をカウントする。図8に示される如く、時刻T1と時刻T2との間に、3つのパルスが存在するならば、判定部532は、時刻T1から時刻T2までの期間に3つの検出対象DOが交差部110を通過したと判定してもよい。
The comparison unit 531 may generate a pulse signal having a pulse that rises while the signal value exceeds the threshold value TH as the data CRD. The determination unit 532 counts the number of pulses in the pulse signal. As shown in FIG. 8, if there are three pulses between time T1 and time T2, the determination unit 532 determines that the three detection target DOs cross the intersection 110 during the period from time T1 to time T2. You may determine that it has passed.
図9は、図2を参照して説明されたステップS130中の処理を表す概略的なフローチャートである。図7乃至図9を参照して、ステップS130中の処理が説明される。
FIG. 9 is a schematic flowchart showing the process in step S130 described with reference to FIG. The process in step S130 will be described with reference to FIGS.
(ステップS131)
ステップS131において、信号生成部520は、蛍光FLの強度を信号値に変換する。その後、ステップS133が実行される。 (Step S131)
In step S131, thesignal generation unit 520 converts the intensity of the fluorescence FL into a signal value. Thereafter, step S133 is executed.
ステップS131において、信号生成部520は、蛍光FLの強度を信号値に変換する。その後、ステップS133が実行される。 (Step S131)
In step S131, the
(ステップS133)
ステップS133において、比較部531は、信号値を閾値THと比較する。その後、ステップS135が生成される。 (Step S133)
In step S133, thecomparison unit 531 compares the signal value with the threshold value TH. Thereafter, step S135 is generated.
ステップS133において、比較部531は、信号値を閾値THと比較する。その後、ステップS135が生成される。 (Step S133)
In step S133, the
(ステップS135)
ステップS135において、信号値が閾値THよりも大きいならば、比較部531は、パルスを生成する。その後、ステップS137が実行される。他の場合には、ステップS139が実行される。 (Step S135)
If the signal value is larger than the threshold value TH in step S135, thecomparison unit 531 generates a pulse. Thereafter, step S137 is executed. In other cases, step S139 is executed.
ステップS135において、信号値が閾値THよりも大きいならば、比較部531は、パルスを生成する。その後、ステップS137が実行される。他の場合には、ステップS139が実行される。 (Step S135)
If the signal value is larger than the threshold value TH in step S135, the
(ステップS137)
ステップS137において、判定部532は、通過量を「1」だけ増加させる。その後、ステップS139が実行される。 (Step S137)
In step S137, thedetermination unit 532 increases the passage amount by “1”. Thereafter, step S139 is executed.
ステップS137において、判定部532は、通過量を「1」だけ増加させる。その後、ステップS139が実行される。 (Step S137)
In step S137, the
(ステップS139)
ステップS139において、検出作業が終了されているならば、判定部532は、通過量を表すデータPSDを出力する。他の場合には、ステップS131が実行される。 (Step S139)
In step S139, if the detection work has been completed, thedetermination unit 532 outputs data PSD representing the passage amount. In other cases, step S131 is executed.
ステップS139において、検出作業が終了されているならば、判定部532は、通過量を表すデータPSDを出力する。他の場合には、ステップS131が実行される。 (Step S139)
In step S139, if the detection work has been completed, the
<第8実施形態>
流路が、第5実施形態に関連して説明された設計原理に基づいて設計されるならば、流路は非常に狭くなる。したがって、設計者は、検出対象が流路に沿って円滑に流れるための技術を蛍光検出装置に組み込んでもよい。第8実施形態において、検出対象の円滑な流動を得るための技術が説明される。 <Eighth Embodiment>
If the flow path is designed based on the design principle described in connection with the fifth embodiment, the flow path becomes very narrow. Therefore, the designer may incorporate a technique for allowing the detection target to smoothly flow along the flow path into the fluorescence detection apparatus. In the eighth embodiment, a technique for obtaining a smooth flow of a detection target will be described.
流路が、第5実施形態に関連して説明された設計原理に基づいて設計されるならば、流路は非常に狭くなる。したがって、設計者は、検出対象が流路に沿って円滑に流れるための技術を蛍光検出装置に組み込んでもよい。第8実施形態において、検出対象の円滑な流動を得るための技術が説明される。 <Eighth Embodiment>
If the flow path is designed based on the design principle described in connection with the fifth embodiment, the flow path becomes very narrow. Therefore, the designer may incorporate a technique for allowing the detection target to smoothly flow along the flow path into the fluorescence detection apparatus. In the eighth embodiment, a technique for obtaining a smooth flow of a detection target will be described.
図10は、第8実施形態の蛍光検出装置100Bの概略図である。図10を参照して、蛍光検出装置100Bが説明される。第3実施形態及び第8実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第3実施形態と同一の機能を有することを意味する。したがって、第3実施形態の説明は、これらの要素に援用される。
FIG. 10 is a schematic diagram of the fluorescence detection apparatus 100B of the eighth embodiment. With reference to FIG. 10, the fluorescence detection apparatus 100B will be described. The code used in common between the third embodiment and the eighth embodiment means that the element to which the common code is attached has the same function as that of the third embodiment. Therefore, description of 3rd Embodiment is used for these elements.
第3実施形態と同様に、蛍光検出装置100Bは、光源410と、計測装置510と、ポンプ600と、を備える。蛍光検出装置100Bは、マイクロチップ120Bを更に備える。
As in the third embodiment, the fluorescence detection device 100B includes a light source 410, a measurement device 510, and a pump 600. The fluorescence detection device 100B further includes a microchip 120B.
第3実施形態と同様に、マイクロチップ120Bは、上基板210と、下基板320と、遮光膜300Aと、を含む。マイクロチップ120Bは、遮光膜300Aに形成されたスリット310Aに充填された充填材料311を更に含む。充填材料311は、ガラスや石英といった透明な無機材料、ポリジメチルシロキサン(PDMS)といった透明な樹脂や励起光PLに対して透明な他の材料であってもよい。
As in the third embodiment, the microchip 120B includes an upper substrate 210, a lower substrate 320, and a light shielding film 300A. The microchip 120B further includes a filling material 311 filled in the slit 310A formed in the light shielding film 300A. The filling material 311 may be a transparent inorganic material such as glass or quartz, a transparent resin such as polydimethylsiloxane (PDMS), or another material transparent to the excitation light PL.
充填材料311は、スリット310Aが形成された領域と遮光膜300Aが存在する領域との間の境界を平滑化する。したがって、流路200Aは、平滑化された面上に配置されることとなる。この結果、検出対象DOは、流路200Aに沿って円滑に流れることができる。
The filling material 311 smoothes the boundary between the region where the slit 310A is formed and the region where the light shielding film 300A exists. Therefore, the channel 200A is disposed on a smoothed surface. As a result, the detection target DO can flow smoothly along the flow path 200A.
<第9実施形態>
遮光膜の腐食は、遮光膜の遮光特性を劣化させる。第9実施形態において、遮光膜の腐食を防止するための技術が説明される。 <Ninth Embodiment>
The corrosion of the light shielding film deteriorates the light shielding characteristics of the light shielding film. In the ninth embodiment, a technique for preventing corrosion of the light shielding film will be described.
遮光膜の腐食は、遮光膜の遮光特性を劣化させる。第9実施形態において、遮光膜の腐食を防止するための技術が説明される。 <Ninth Embodiment>
The corrosion of the light shielding film deteriorates the light shielding characteristics of the light shielding film. In the ninth embodiment, a technique for preventing corrosion of the light shielding film will be described.
図11は、第9実施形態の蛍光検出装置100Cの概略図である。図11を参照して、蛍光検出装置100Cが説明される。第8実施形態及び第9実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第8実施形態と同一の機能を有することを意味する。したがって、第8実施形態の説明は、これらの要素に援用される。
FIG. 11 is a schematic diagram of the fluorescence detection apparatus 100C of the ninth embodiment. With reference to FIG. 11, the fluorescence detection apparatus 100C will be described. A symbol used in common between the eighth embodiment and the ninth embodiment means that an element to which the common symbol is attached has the same function as that of the eighth embodiment. Therefore, description of 8th Embodiment is used for these elements.
第8実施形態と同様に、蛍光検出装置100Cは、光源410と、計測装置510と、ポンプ600と、を備える。蛍光検出装置100Cは、マイクロチップ120Cを更に備える。
As in the eighth embodiment, the fluorescence detection device 100C includes a light source 410, a measurement device 510, and a pump 600. The fluorescence detection device 100C further includes a microchip 120C.
第8実施形態と同様に、マイクロチップ120Cは、上基板210と、下基板320と、遮光膜300Aと、充填材料311と、を含む。マイクロチップ120Cは、上基板210と下基板320との間に形成された薄い薄膜130を更に含む。薄膜130は、遮光膜300A及び充填材料311を覆うので、検出対象DOを流動させるための液体は、遮光膜300Aを腐食させにくくなる。薄膜130の厚さは、励起光PLの過度の減衰を引き起こさないように設定される。
Similarly to the eighth embodiment, the microchip 120C includes an upper substrate 210, a lower substrate 320, a light shielding film 300A, and a filling material 311. The microchip 120 </ b> C further includes a thin thin film 130 formed between the upper substrate 210 and the lower substrate 320. Since the thin film 130 covers the light shielding film 300A and the filling material 311, the liquid for flowing the detection target DO hardly corrodes the light shielding film 300A. The thickness of the thin film 130 is set so as not to cause excessive attenuation of the excitation light PL.
<第10実施形態>
蛍光検出装置は、複数の種類の検出対象を検出してもよい。第10実施形態において、複数の種類の検出対象を検出するための技術が説明される。 <Tenth Embodiment>
The fluorescence detection apparatus may detect a plurality of types of detection targets. In the tenth embodiment, a technique for detecting a plurality of types of detection targets will be described.
蛍光検出装置は、複数の種類の検出対象を検出してもよい。第10実施形態において、複数の種類の検出対象を検出するための技術が説明される。 <Tenth Embodiment>
The fluorescence detection apparatus may detect a plurality of types of detection targets. In the tenth embodiment, a technique for detecting a plurality of types of detection targets will be described.
図12は、第3実施形態に関連して説明された蛍光検出装置100Aの概略図である。図12を参照して、複数の種類の検出対象を検出するための技術が説明される。
FIG. 12 is a schematic diagram of the fluorescence detection apparatus 100A described in relation to the third embodiment. A technique for detecting a plurality of types of detection objects will be described with reference to FIG.
図12に示される蛍光検出装置100Aのポンプ600は、第1検出対象DO1と第2検出対象DO2とを流路200Aに供給する。第1検出対象DO1は、励起光PLの照射下で、第2検出対象DO2よりも強い蛍光FLを発する。
The pump 600 of the fluorescence detection apparatus 100A shown in FIG. 12 supplies the first detection target DO1 and the second detection target DO2 to the flow path 200A. The first detection target DO1 emits fluorescence FL stronger than the second detection target DO2 under irradiation of the excitation light PL.
図13は、検出信号DSのタイミングチャートである。図7及び図13を参照して、第1検出対象DO1及び第2検出対象DO2の通過量に関する情報を得るために計測部500が実行する演算処理が説明される。第7実施形態及び第10実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第7実施形態と同一の機能を有することを意味する。したがって、第7実施形態の説明は、これらの要素に援用される。
FIG. 13 is a timing chart of the detection signal DS. With reference to FIG.7 and FIG.13, the calculation process which the measurement part 500 performs in order to obtain the information regarding the passage amount of the 1st detection object DO1 and the 2nd detection object DO2 is demonstrated. The code | symbol used in common between 7th Embodiment and 10th Embodiment means that the element to which the said common code | symbol was attached | subjected has the same function as 7th Embodiment. Therefore, description of 7th Embodiment is used for these elements.
比較部531は、検出信号DSが表す信号値を、第1閾値TH1と第2閾値TH2とに比較する。第1閾値TH1は、第2閾値TH2よりも大きな値に設定されている。信号値が、第1閾値TH1よりも大きいならば、比較部531は、第1検出対象DO1の通過を表すデータCRDを生成する。信号値が、第1閾値TH1よりも小さく、且つ、第2閾値TH2よりも大きいならば、比較部531は、第2検出対象DO2の通過を表すデータCRDを生成する。
The comparison unit 531 compares the signal value represented by the detection signal DS with the first threshold value TH1 and the second threshold value TH2. The first threshold value TH1 is set to a value larger than the second threshold value TH2. If the signal value is larger than the first threshold value TH1, the comparison unit 531 generates data CRD representing the passage of the first detection target DO1. If the signal value is smaller than the first threshold value TH1 and larger than the second threshold value TH2, the comparison unit 531 generates data CRD representing the passage of the second detection target DO2.
図14は、図2を参照して説明されたステップS130中の処理を表す概略的なフローチャートである。図7、図12乃至図14を参照して、ステップS130中の処理が説明される。
FIG. 14 is a schematic flowchart showing the process in step S130 described with reference to FIG. The processing in step S130 will be described with reference to FIGS. 7 and 12 to 14.
(ステップS210)
ステップS210において、信号生成部520は、蛍光FLの強度を信号値に変換する。その後、ステップS220が実行される。 (Step S210)
In step S210, thesignal generation unit 520 converts the intensity of the fluorescence FL into a signal value. Thereafter, step S220 is executed.
ステップS210において、信号生成部520は、蛍光FLの強度を信号値に変換する。その後、ステップS220が実行される。 (Step S210)
In step S210, the
(ステップS220)
ステップS220において、比較部531は、信号値を閾値(第1閾値TH1,第2閾値TH2)と比較する。その後、ステップS230が生成される。 (Step S220)
In step S220, thecomparison unit 531 compares the signal value with threshold values (first threshold value TH1, second threshold value TH2). Thereafter, step S230 is generated.
ステップS220において、比較部531は、信号値を閾値(第1閾値TH1,第2閾値TH2)と比較する。その後、ステップS230が生成される。 (Step S220)
In step S220, the
(ステップS230)
ステップS230において、信号値が第2閾値TH2よりも大きいならば、ステップS240が実行される。他の場合には、ステップS270が実行される。 (Step S230)
If the signal value is larger than the second threshold value TH2 in step S230, step S240 is executed. In other cases, step S270 is executed.
ステップS230において、信号値が第2閾値TH2よりも大きいならば、ステップS240が実行される。他の場合には、ステップS270が実行される。 (Step S230)
If the signal value is larger than the second threshold value TH2 in step S230, step S240 is executed. In other cases, step S270 is executed.
(ステップS240)
ステップS240において、信号値が第1閾値TH1よりも大きいならば、比較部531は、第1検出対象DO1の通過を表すデータCRDを生成する。その後、ステップS250が実行される。他の場合には、比較部531は、第2検出対象DO2の通過を表すデータCRDを生成する。その後、ステップS260が実行される。 (Step S240)
If the signal value is larger than the first threshold value TH1 in step S240, thecomparison unit 531 generates data CRD representing the passage of the first detection target DO1. Thereafter, step S250 is executed. In other cases, the comparison unit 531 generates data CRD representing the passage of the second detection target DO2. Thereafter, step S260 is executed.
ステップS240において、信号値が第1閾値TH1よりも大きいならば、比較部531は、第1検出対象DO1の通過を表すデータCRDを生成する。その後、ステップS250が実行される。他の場合には、比較部531は、第2検出対象DO2の通過を表すデータCRDを生成する。その後、ステップS260が実行される。 (Step S240)
If the signal value is larger than the first threshold value TH1 in step S240, the
(ステップS250)
ステップS250において、判定部532は、第1検出対象DO1の通過量を「1」だけ増加させる。その後、ステップS270が実行される。 (Step S250)
In step S250, thedetermination unit 532 increases the passing amount of the first detection target DO1 by “1”. Thereafter, step S270 is executed.
ステップS250において、判定部532は、第1検出対象DO1の通過量を「1」だけ増加させる。その後、ステップS270が実行される。 (Step S250)
In step S250, the
(ステップS260)
ステップS260において、判定部532は、第2検出対象DO2の通過量を「1」だけ増加させる。その後、ステップS270が実行される。 (Step S260)
In step S260, thedetermination unit 532 increases the passing amount of the second detection target DO2 by “1”. Thereafter, step S270 is executed.
ステップS260において、判定部532は、第2検出対象DO2の通過量を「1」だけ増加させる。その後、ステップS270が実行される。 (Step S260)
In step S260, the
(ステップS270)
ステップS270において、検出作業が終了されているならば、判定部532は、第1検出対象DO1及び第2検出対象DO2の通過量を表すデータPSDを出力する。他の場合には、ステップS210が実行される。 (Step S270)
In step S270, if the detection work has been completed, thedetermination unit 532 outputs data PSD representing the passage amounts of the first detection target DO1 and the second detection target DO2. In other cases, step S210 is executed.
ステップS270において、検出作業が終了されているならば、判定部532は、第1検出対象DO1及び第2検出対象DO2の通過量を表すデータPSDを出力する。他の場合には、ステップS210が実行される。 (Step S270)
In step S270, if the detection work has been completed, the
<第11実施形態>
第10実施形態の原理は、蛍光の強度に基づき、検出対象の種類を識別することを可能にする。代替的に、検出対象の種類は、蛍光の波長に基づき、識別されてもよい。第11実施形態において、蛍光の波長に基づき、検出対象の種類を識別する技術が説明される。 <Eleventh embodiment>
The principle of the tenth embodiment makes it possible to identify the type of detection target based on the intensity of fluorescence. Alternatively, the type of detection target may be identified based on the wavelength of fluorescence. In the eleventh embodiment, a technique for identifying the type of detection target based on the wavelength of fluorescence is described.
第10実施形態の原理は、蛍光の強度に基づき、検出対象の種類を識別することを可能にする。代替的に、検出対象の種類は、蛍光の波長に基づき、識別されてもよい。第11実施形態において、蛍光の波長に基づき、検出対象の種類を識別する技術が説明される。 <Eleventh embodiment>
The principle of the tenth embodiment makes it possible to identify the type of detection target based on the intensity of fluorescence. Alternatively, the type of detection target may be identified based on the wavelength of fluorescence. In the eleventh embodiment, a technique for identifying the type of detection target based on the wavelength of fluorescence is described.
図15は、計測部500の概略的なブロック図である。図1、図12及び図15を参照して、計測部500の構造が説明される。第1実施形態及び第11実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第1実施形態と同一の機能を有することを意味する。したがって、第1実施形態の説明は、これらの要素に援用される。
FIG. 15 is a schematic block diagram of the measurement unit 500. The structure of the measuring unit 500 will be described with reference to FIGS. The code | symbol used in common between 1st Embodiment and 11th Embodiment means that the element to which the said common code | symbol was attached | subjected has the same function as 1st Embodiment. Therefore, description of 1st Embodiment is used for these elements.
計測部500は、信号生成部520Dと、取得部530Dと、を含む。信号生成部520Dは、第1信号生成部521と、第2信号生成部522と、分光部523と、を含む。取得部530Dは、第1比較部541と、第2比較部542と、判定部532Dと、を含む。
The measurement unit 500 includes a signal generation unit 520D and an acquisition unit 530D. The signal generation unit 520D includes a first signal generation unit 521, a second signal generation unit 522, and a spectroscopic unit 523. Acquisition unit 530D includes a first comparison unit 541, a second comparison unit 542, and a determination unit 532D.
第1検出対象DO1及び第2検出対象DO2(図12を参照)は、検出対象DOとして、流路200に供給される。使用者は、第1検出対象DO1と第2検出対象DO2として、人工核酸を用いて処理された複数の種類のmiRNAを用いてもよい。人工核酸とmiRNAとによって形成された二本鎖は、miRNAの種類に応じて、異なる波長(色相)の蛍光を発することができる。
1st detection object DO1 and 2nd detection object DO2 (refer FIG. 12) are supplied to the flow path 200 as detection object DO. The user may use a plurality of types of miRNA processed using artificial nucleic acids as the first detection target DO1 and the second detection target DO2. The double strand formed by the artificial nucleic acid and the miRNA can emit fluorescence having different wavelengths (hue) depending on the type of the miRNA.
第1検出対象DO1は、励起光PLの照射下で、波長λ1を有する蛍光FL1を発する。第2検出対象DO2は、励起光PLの照射下で、波長λ2を有する蛍光FL2を発する。波長λ1は、波長λ2よりも長い。
The first detection target DO1 emits fluorescence FL1 having the wavelength λ1 under irradiation of the excitation light PL. The second detection object DO2 emits fluorescence FL2 having a wavelength λ2 under irradiation of the excitation light PL. The wavelength λ1 is longer than the wavelength λ2.
蛍光FL1,FL2は、分光部523に入射する。分光部523は、プリズムやグレーティング素子であってもよい。蛍光FL1が分光部523に入射するならば、分光部523は、第1信号生成部521へ、蛍光FL1を出射する。蛍光FL2が分光部523に入射するならば、分光部523は、第2信号生成部522へ、蛍光FL2を出射する。
Fluorescence FL1 and FL2 enter the spectroscopic unit 523. The spectroscopic unit 523 may be a prism or a grating element. If the fluorescence FL1 is incident on the spectroscopic unit 523, the spectroscopic unit 523 emits the fluorescence FL1 to the first signal generation unit 521. If the fluorescence FL 2 is incident on the spectroscopic unit 523, the spectroscopic unit 523 emits the fluorescence FL 2 to the second signal generation unit 522.
第1信号生成部521は、第7実施形態に関連して説明された原理に従って、蛍光FL1の光量を表す第1検出信号DS1を生成する。第1検出信号DS1は、第1信号生成部521から第1比較部541へ出力される。第2信号生成部522は、第7実施形態に関連して説明された原理に従って、蛍光FL2の光量を表す第2検出信号DS2を生成する。第2検出信号DS2は、第2信号生成部522から第2比較部542へ出力される。第1信号生成部521及び第2信号生成部522は、ライン型のイメージセンサであってもよい。
The first signal generation unit 521 generates the first detection signal DS1 representing the light amount of the fluorescence FL1 according to the principle described in relation to the seventh embodiment. The first detection signal DS1 is output from the first signal generation unit 521 to the first comparison unit 541. The second signal generation unit 522 generates the second detection signal DS2 representing the light amount of the fluorescence FL2 according to the principle described in relation to the seventh embodiment. The second detection signal DS2 is output from the second signal generation unit 522 to the second comparison unit 542. The first signal generation unit 521 and the second signal generation unit 522 may be line type image sensors.
第1比較部541は、第7実施形態に関連して説明された原理に従って、第1検出信号DS1を予め設定された閾値と比較し、比較結果を表す第1データCRD1を生成する。第1データCRD1は、第1比較部541から判定部532Dへ出力される。
The first comparison unit 541 compares the first detection signal DS1 with a preset threshold value according to the principle described in relation to the seventh embodiment, and generates first data CRD1 representing the comparison result. The first data CRD1 is output from the first comparison unit 541 to the determination unit 532D.
第2比較部542は、第7実施形態に関連して説明された原理に従って、第2検出信号DS2を予め設定された閾値と比較し、比較結果を表す第2データCRD2を生成する。第2データCRD2は、第2比較部542から判定部532Dへ出力される。
The second comparison unit 542 compares the second detection signal DS2 with a preset threshold according to the principle described in relation to the seventh embodiment, and generates second data CRD2 representing the comparison result. The second data CRD2 is output from the second comparison unit 542 to the determination unit 532D.
判定部532Dは、第1データCRD1を参照し、交差部110を通過した第1検出対象DO1の通過量を判定する。判定部532Dは、第1検出対象DO1の通過量を表すデータPSD1を生成する。データPSD1は、蛍光検出装置100に通信可能に接続されたコンピュータ(図示せず)や他の外部装置(図示せず)へ出力されてもよい。
The determination unit 532D refers to the first data CRD1, and determines the passage amount of the first detection target DO1 that has passed through the intersection 110. The determination unit 532D generates data PSD1 representing the passage amount of the first detection target DO1. The data PSD1 may be output to a computer (not shown) or other external device (not shown) connected to the fluorescence detection device 100 in a communicable manner.
判定部532Dは、第2データCRD2を参照し、交差部110を通過した第2検出対象DO2の通過量を判定する。判定部532Dは、第2検出対象DO2の通過量を表すデータPSD2を生成する。データPSD2は、蛍光検出装置100に通信可能に接続されたコンピュータ(図示せず)や他の外部装置(図示せず)へ出力されてもよい。
The determination unit 532D refers to the second data CRD2, and determines the passage amount of the second detection target DO2 that has passed through the intersection 110. The determination unit 532D generates data PSD2 representing the passage amount of the second detection target DO2. The data PSD2 may be output to a computer (not shown) or other external device (not shown) connected to the fluorescence detection device 100 in a communicable manner.
<第12実施形態>
第11実施形態の原理は、分光素子を利用して、蛍光の波長に応じて、蛍光の伝搬経路を切り替える。代替的に、信号生成部は、波長を識別する機能を有してもよい(例えば、カラーイメージセンサ)。第12実施形態において、波長を識別する機能を有する信号生成部による検出信号の生成技術が説明される。 <Twelfth embodiment>
The principle of the eleventh embodiment uses a spectroscopic element to switch the propagation path of fluorescence according to the wavelength of fluorescence. Alternatively, the signal generation unit may have a function of identifying a wavelength (for example, a color image sensor). In the twelfth embodiment, a detection signal generation technique by a signal generation unit having a function of identifying a wavelength will be described.
第11実施形態の原理は、分光素子を利用して、蛍光の波長に応じて、蛍光の伝搬経路を切り替える。代替的に、信号生成部は、波長を識別する機能を有してもよい(例えば、カラーイメージセンサ)。第12実施形態において、波長を識別する機能を有する信号生成部による検出信号の生成技術が説明される。 <Twelfth embodiment>
The principle of the eleventh embodiment uses a spectroscopic element to switch the propagation path of fluorescence according to the wavelength of fluorescence. Alternatively, the signal generation unit may have a function of identifying a wavelength (for example, a color image sensor). In the twelfth embodiment, a detection signal generation technique by a signal generation unit having a function of identifying a wavelength will be described.
図16は、信号生成部520による検出信号DSの生成工程を表す概略的なフローチャートである。図7及び図16を参照して、検出信号DSの生成工程が説明される。第7実施形態、第11実施形態及び第12実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第7実施形態又は第11実施形態と同一の機能を有することを意味する。したがって、第7実施形態又は第11実施形態の説明は、これらの要素に援用される。
FIG. 16 is a schematic flowchart showing a detection signal DS generation process by the signal generation unit 520. With reference to FIGS. 7 and 16, the generation process of the detection signal DS will be described. The reference numerals used in common among the seventh embodiment, the eleventh embodiment, and the twelfth embodiment are the same as those in the seventh embodiment or the eleventh embodiment. It means having. Therefore, description of 7th Embodiment or 11th Embodiment is used for these elements.
(ステップS310)
ステップS310において、信号生成部520は、蛍光FLの波長が、第1検出対象DO1からの蛍光の波長λ1であるか否かを判定する。蛍光FLの波長が、第1検出対象DO1からの蛍光の波長λ1であるならば、ステップS320が実行される。他の場合には、ステップS340が実行される。 (Step S310)
In step S310, thesignal generation unit 520 determines whether or not the wavelength of the fluorescence FL is the wavelength λ1 of the fluorescence from the first detection target DO1. If the wavelength of the fluorescence FL is the wavelength λ1 of the fluorescence from the first detection target DO1, step S320 is executed. In other cases, step S340 is executed.
ステップS310において、信号生成部520は、蛍光FLの波長が、第1検出対象DO1からの蛍光の波長λ1であるか否かを判定する。蛍光FLの波長が、第1検出対象DO1からの蛍光の波長λ1であるならば、ステップS320が実行される。他の場合には、ステップS340が実行される。 (Step S310)
In step S310, the
(ステップS320)
ステップS320において、信号生成部520は、第7実施形態に関連して説明された原理に従って、蛍光FLの強度を信号値に生成する。その後、ステップS330が実行される。 (Step S320)
In step S320, thesignal generation unit 520 generates the intensity of the fluorescence FL as a signal value according to the principle described in relation to the seventh embodiment. Thereafter, Step S330 is executed.
ステップS320において、信号生成部520は、第7実施形態に関連して説明された原理に従って、蛍光FLの強度を信号値に生成する。その後、ステップS330が実行される。 (Step S320)
In step S320, the
(ステップS330)
ステップS330において、信号生成部520は、蛍光FLの波長に関する情報と蛍光FLの強度に関する情報とを含む検出信号DSを出力する。 (Step S330)
In step S330, thesignal generation unit 520 outputs a detection signal DS including information regarding the wavelength of the fluorescence FL and information regarding the intensity of the fluorescence FL.
ステップS330において、信号生成部520は、蛍光FLの波長に関する情報と蛍光FLの強度に関する情報とを含む検出信号DSを出力する。 (Step S330)
In step S330, the
(ステップS340)
ステップS340において、信号生成部520は、蛍光FLの波長が、第2検出対象DO2からの蛍光の波長λ2であるか否かを判定する。蛍光FLの波長が、第2検出対象DO2からの蛍光の波長λ2であるならば、ステップS350が実行される。他の場合には、ステップS360が実行される。 (Step S340)
In step S340, thesignal generation unit 520 determines whether or not the wavelength of the fluorescence FL is the wavelength λ2 of the fluorescence from the second detection target DO2. If the wavelength of the fluorescence FL is the wavelength λ2 of the fluorescence from the second detection target DO2, step S350 is executed. In other cases, step S360 is executed.
ステップS340において、信号生成部520は、蛍光FLの波長が、第2検出対象DO2からの蛍光の波長λ2であるか否かを判定する。蛍光FLの波長が、第2検出対象DO2からの蛍光の波長λ2であるならば、ステップS350が実行される。他の場合には、ステップS360が実行される。 (Step S340)
In step S340, the
(ステップS350)
ステップS350において、信号生成部520は、第7実施形態に関連して説明された原理に従って、蛍光FLの強度を信号値に生成する。その後、ステップS330が実行される。 (Step S350)
In step S350, thesignal generation unit 520 generates the intensity of the fluorescence FL as a signal value according to the principle described in relation to the seventh embodiment. Thereafter, Step S330 is executed.
ステップS350において、信号生成部520は、第7実施形態に関連して説明された原理に従って、蛍光FLの強度を信号値に生成する。その後、ステップS330が実行される。 (Step S350)
In step S350, the
(ステップS360)
ステップS360において、信号生成部520は、ノイズとして蛍光FLの入射を処理する。 (Step S360)
In step S360, thesignal generation unit 520 processes the incidence of the fluorescence FL as noise.
ステップS360において、信号生成部520は、ノイズとして蛍光FLの入射を処理する。 (Step S360)
In step S360, the
<第13実施形態>
第11実施形態及び第12実施形態の原理は、第1検出対象から発せられる蛍光の波長を第2検出対象から発せられる蛍光の波長から相違させる。照射部から発せられる励起光が、第1検出対象からの蛍光及び第2検出対象からの蛍光に適した光成分を含むならば、第1検出対象及び第2検出対象は、励起光の照射下で強く蛍光発光することができる。第13実施形態において、第1検出対象及び第2検出対象を強く発光させ、高い検出感度を達成するための技術が説明される。 <13th Embodiment>
The principle of the eleventh embodiment and the twelfth embodiment is that the wavelength of the fluorescence emitted from the first detection target is different from the wavelength of the fluorescence emitted from the second detection target. If the excitation light emitted from the irradiation unit includes a light component suitable for fluorescence from the first detection target and fluorescence from the second detection target, the first detection target and the second detection target are under irradiation of the excitation light. Can emit strong fluorescence. In the thirteenth embodiment, a technique for achieving strong detection sensitivity by causing the first detection target and the second detection target to emit light strongly will be described.
第11実施形態及び第12実施形態の原理は、第1検出対象から発せられる蛍光の波長を第2検出対象から発せられる蛍光の波長から相違させる。照射部から発せられる励起光が、第1検出対象からの蛍光及び第2検出対象からの蛍光に適した光成分を含むならば、第1検出対象及び第2検出対象は、励起光の照射下で強く蛍光発光することができる。第13実施形態において、第1検出対象及び第2検出対象を強く発光させ、高い検出感度を達成するための技術が説明される。 <13th Embodiment>
The principle of the eleventh embodiment and the twelfth embodiment is that the wavelength of the fluorescence emitted from the first detection target is different from the wavelength of the fluorescence emitted from the second detection target. If the excitation light emitted from the irradiation unit includes a light component suitable for fluorescence from the first detection target and fluorescence from the second detection target, the first detection target and the second detection target are under irradiation of the excitation light. Can emit strong fluorescence. In the thirteenth embodiment, a technique for achieving strong detection sensitivity by causing the first detection target and the second detection target to emit light strongly will be described.
図17は、第13実施形態の蛍光検出装置100Eの概念図である。図17を参照して、蛍光検出装置100Eが説明される。第1実施形態及び第13実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第1実施形態と同一の機能を有することを意味する。したがって、第1実施形態の説明は、これらの要素に援用される。
FIG. 17 is a conceptual diagram of the fluorescence detection apparatus 100E of the thirteenth embodiment. With reference to FIG. 17, the fluorescence detection apparatus 100E will be described. The code | symbol used in common between 1st Embodiment and 13th Embodiment means that the element to which the said common code | symbol was attached | subjected has the same function as 1st Embodiment. Therefore, description of 1st Embodiment is used for these elements.
第1実施形態と同様に、蛍光検出装置100Eは、流路200と、遮光膜300と、計測部500と、を備える。蛍光検出装置100Eは、照射部400Eを更に備える。照射部400Eは、第1照射部411と、第2照射部412と、合波部413と、を含む。
As in the first embodiment, the fluorescence detection device 100E includes a flow path 200, a light shielding film 300, and a measurement unit 500. The fluorescence detection device 100E further includes an irradiation unit 400E. The irradiation unit 400E includes a first irradiation unit 411, a second irradiation unit 412, and a multiplexing unit 413.
第1照射部411は、第1励起光PL1を合波部413へ出射する。第2照射部412は、第2励起光PL2を合波部413へ出射する。合波部413は、第1励起光PL1と第2励起光PL2とを合波し、励起光PLを生成する。励起光PLは、スリット310を通じて、流路200へ伝搬する。合波部413は、ダイクロイックミラーや、第1励起光PL1と第2励起光PL2とを合波することができる他の光学素子であってもよい。本実施形態は、合波部413に用いられる特定の光学素子に限定されない。
The first irradiation unit 411 emits the first excitation light PL1 to the multiplexing unit 413. The second irradiation unit 412 emits the second excitation light PL2 to the multiplexing unit 413. The multiplexing unit 413 multiplexes the first excitation light PL1 and the second excitation light PL2, and generates the excitation light PL. The excitation light PL propagates to the flow path 200 through the slit 310. The multiplexing unit 413 may be a dichroic mirror or another optical element that can multiplex the first excitation light PL1 and the second excitation light PL2. The present embodiment is not limited to a specific optical element used for the multiplexing unit 413.
第1検出対象DO1及び第2検出対象DO2は、流路200に沿って流れる。第1検出対象DO1は、第2励起光PL2の照射下よりも、第1励起光PL1の照射下において、強く蛍光発光する。第2検出対象DO2は、第1励起光PL1の照射下よりも、第2励起光PL2の照射下において、強く蛍光発光する。上述の如く、励起光PLは、第1励起光PL1の光成分と、第2励起光PL2の光成分と、を含むので、交差部110を通過する第1検出対象DO1及び第2検出対象DO2はともに強く蛍光発光することができる。
The first detection target DO1 and the second detection target DO2 flow along the flow path 200. The first detection target DO1 emits fluorescence more strongly under irradiation of the first excitation light PL1 than under irradiation of the second excitation light PL2. The second detection target DO2 emits fluorescence more strongly under irradiation of the second excitation light PL2 than under irradiation of the first excitation light PL1. As described above, since the excitation light PL includes the optical component of the first excitation light PL1 and the optical component of the second excitation light PL2, the first detection target DO1 and the second detection target DO2 that pass through the intersection 110. Both can strongly emit fluorescence.
<第14実施形態>
第13実施形態の原理は、波長において互いに相違する複数の励起光を出射し、複数の種類の検出対象それぞれを強く蛍光発光させる。代替的に、照射部は、波長を変更する機能を有してもよい。第14実施形態において、波長変更機能を有する照射部を有する蛍光検出装置が説明される。 <Fourteenth embodiment>
The principle of the thirteenth embodiment is that a plurality of excitation lights different from each other in wavelength are emitted, and each of a plurality of types of detection targets is strongly fluorescently emitted. Alternatively, the irradiating unit may have a function of changing the wavelength. In 14th Embodiment, the fluorescence detection apparatus which has an irradiation part which has a wavelength change function is demonstrated.
第13実施形態の原理は、波長において互いに相違する複数の励起光を出射し、複数の種類の検出対象それぞれを強く蛍光発光させる。代替的に、照射部は、波長を変更する機能を有してもよい。第14実施形態において、波長変更機能を有する照射部を有する蛍光検出装置が説明される。 <Fourteenth embodiment>
The principle of the thirteenth embodiment is that a plurality of excitation lights different from each other in wavelength are emitted, and each of a plurality of types of detection targets is strongly fluorescently emitted. Alternatively, the irradiating unit may have a function of changing the wavelength. In 14th Embodiment, the fluorescence detection apparatus which has an irradiation part which has a wavelength change function is demonstrated.
図18は、第14実施形態の蛍光検出装置100Fの概念図である。図18を参照して、蛍光検出装置100Fが説明される。第13実施形態及び第14実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第13実施形態と同一の機能を有することを意味する。したがって、第13実施形態の説明は、これらの要素に援用される。
FIG. 18 is a conceptual diagram of the fluorescence detection device 100F of the fourteenth embodiment. With reference to FIG. 18, the fluorescence detection apparatus 100F will be described. A symbol used in common between the thirteenth embodiment and the fourteenth embodiment means that an element to which the common symbol is attached has the same function as that of the thirteenth embodiment. Therefore, the description of the thirteenth embodiment is applied to these elements.
第13実施形態と同様に、蛍光検出装置100Fは、流路200と、遮光膜300と、計測部500と、を備える。蛍光検出装置100Fは、照射部400Fを更に備える。照射部400Fは、光源410と、波長変更部420と、を含む。
As in the thirteenth embodiment, the fluorescence detection device 100F includes a flow path 200, a light shielding film 300, and a measurement unit 500. The fluorescence detection device 100F further includes an irradiation unit 400F. Irradiation unit 400 </ b> F includes a light source 410 and a wavelength changing unit 420.
光源410は、励起光PLを波長変更部420へ出射する。波長変更部420は、励起光PLの波長を、波長λ1と波長λ2との間で変更する。波長変更処理の後、励起光PLは、スリット310を通じて、流路200へ伝搬する。
The light source 410 emits excitation light PL to the wavelength changing unit 420. The wavelength changing unit 420 changes the wavelength of the excitation light PL between the wavelength λ1 and the wavelength λ2. After the wavelength changing process, the excitation light PL propagates to the flow path 200 through the slit 310.
第1検出対象DO1及び第2検出対象DO2は、流路200に沿って流れる。励起光PLの波長が、波長λ1であるとき、第1検出対象DO1は、第2検出対象DO2よりも強く蛍光発光する。励起光PLの波長が、波長λ2であるとき、第2検出対象DO2は、第1検出対象DO1よりも強く蛍光発光する。第1検出対象DO1及び第2検出対象DO2それぞれが、交差部110を通過する間に、波長変更部420が、波長λ1と波長λ2との間で励起光PLの波長を変更するならば、第1検出対象DO1及び第2検出対象DO2はともに強く蛍光発光することができる。
The first detection target DO1 and the second detection target DO2 flow along the flow path 200. When the wavelength of the excitation light PL is the wavelength λ1, the first detection target DO1 emits fluorescence more strongly than the second detection target DO2. When the wavelength of the excitation light PL is the wavelength λ2, the second detection target DO2 emits fluorescence more strongly than the first detection target DO1. If the wavelength changing unit 420 changes the wavelength of the excitation light PL between the wavelength λ1 and the wavelength λ2 while each of the first detection target DO1 and the second detection target DO2 passes through the intersection 110, the first Both the first detection object DO1 and the second detection object DO2 can emit strong fluorescence.
<第15実施形態>
第1実施形態に関連して説明された蛍光検出装置の原理に基づいて、様々なマイクロチップが容易に作成可能である。マイクロチップは、MEMS(Micro Electro Mechanical Systems)作成技術や半導体製造技術によって、大量に生産されてもよい。したがって、マイクロチップの製造コストは、非常に低廉である。第15実施形態において、様々なマイクロチップが説明される。 <Fifteenth embodiment>
Various microchips can be easily created based on the principle of the fluorescence detection apparatus described in connection with the first embodiment. Microchips may be produced in large quantities by MEMS (Micro Electro Mechanical Systems) creation technology or semiconductor manufacturing technology. Therefore, the manufacturing cost of the microchip is very low. In the fifteenth embodiment, various microchips are described.
第1実施形態に関連して説明された蛍光検出装置の原理に基づいて、様々なマイクロチップが容易に作成可能である。マイクロチップは、MEMS(Micro Electro Mechanical Systems)作成技術や半導体製造技術によって、大量に生産されてもよい。したがって、マイクロチップの製造コストは、非常に低廉である。第15実施形態において、様々なマイクロチップが説明される。 <Fifteenth embodiment>
Various microchips can be easily created based on the principle of the fluorescence detection apparatus described in connection with the first embodiment. Microchips may be produced in large quantities by MEMS (Micro Electro Mechanical Systems) creation technology or semiconductor manufacturing technology. Therefore, the manufacturing cost of the microchip is very low. In the fifteenth embodiment, various microchips are described.
図19Aは、第1実施形態の原理に従うマイクロチップ121の概略的な平面図である。図19Bは、第1実施形態の原理に従う他のマイクロチップ122の概略的な平面図である。図1、図19A及び図19Bを参照して、マイクロチップ121,122が説明される。第1実施形態及び第15実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第1実施形態と同一の機能を有することを意味する。したがって、第1実施形態の説明は、これらの要素に援用される。
FIG. 19A is a schematic plan view of a microchip 121 according to the principle of the first embodiment. FIG. 19B is a schematic plan view of another microchip 122 according to the principle of the first embodiment. The microchips 121 and 122 will be described with reference to FIGS. 1, 19A, and 19B. The code | symbol used in common between 1st Embodiment and 15th Embodiment means that the element to which the said common code | symbol was attached | subjected has the same function as 1st Embodiment. Therefore, description of 1st Embodiment is used for these elements.
図19Aに示されるマイクロチップ121には、流路200、第1スリット331及び第2スリット332が形成される。第1スリット331及び第2スリット332それぞれは、図1を参照して説明されたスリット310に対応する。
In the microchip 121 shown in FIG. 19A, a flow path 200, a first slit 331, and a second slit 332 are formed. Each of the first slit 331 and the second slit 332 corresponds to the slit 310 described with reference to FIG.
マイクロチップ121の製造者は、2つの基板を用意する。製造者は、2つの基板のうち一方に、第1スリット331と、第1スリット331の隣に第2スリット332と、を形成する。第2スリット332は、第1スリット331に略平行であってもよい。製造者は、2つの基板のうち他方に、流路200を形成する。製造者は、2つの基板を重ね合わせ、流路200を、第1スリット331と第2スリット332とに交差させる。この結果、第1スリット331と流路200とによって規定される交差点IP1及び第2スリット332と流路200とによって規定される交差点IP2が形成される。本実施形態において、第1流路は、流路200によって例示される。第1交差点は、交差点IP1によって例示される。第2交差点は、交差点IP2によって例示される。
The manufacturer of the microchip 121 prepares two substrates. The manufacturer forms the first slit 331 on one of the two substrates and the second slit 332 next to the first slit 331. The second slit 332 may be substantially parallel to the first slit 331. The manufacturer forms the flow path 200 on the other of the two substrates. The manufacturer superimposes the two substrates and causes the flow path 200 to intersect the first slit 331 and the second slit 332. As a result, an intersection IP1 defined by the first slit 331 and the flow path 200 and an intersection IP2 defined by the second slit 332 and the flow path 200 are formed. In the present embodiment, the first flow path is exemplified by the flow path 200. The first intersection is exemplified by the intersection IP1. The second intersection is exemplified by the intersection IP2.
図1を参照して説明された計測部500は、2次元イメージセンサを含んでもよい。この場合、計測部500は、交差点IP1,IP2を異なる計測点として識別することができる。したがって、マイクロチップ121を使用する使用者は、交差点IP1,IP2における検出対象DOの通過量を同時に計測することができる。
The measurement unit 500 described with reference to FIG. 1 may include a two-dimensional image sensor. In this case, the measurement unit 500 can identify the intersections IP1 and IP2 as different measurement points. Therefore, the user who uses the microchip 121 can simultaneously measure the passing amount of the detection target DO at the intersections IP1 and IP2.
図19Bに示されるマイクロチップ122には、スリット310、第1流路201及び第2流路202が形成される。第1流路201及び第2流路202それぞれは、図1を参照して説明された流路200に対応する。
In the microchip 122 shown in FIG. 19B, a slit 310, a first channel 201, and a second channel 202 are formed. Each of the first flow path 201 and the second flow path 202 corresponds to the flow path 200 described with reference to FIG.
マイクロチップ122の製造者は、2つの基板を用意する。製造者は、2つの基板のうち一方に、第1流路201と、第1流路201の隣に第2流路202と、を形成する。第2流路202は、第1流路201に略平行であってもよい。製造者は、2つの基板のうち他方に、スリット310を形成する。製造者は、2つの基板を重ね合わせ、スリット310を、第1流路201と第2流路202とに交差させる。この結果、第1流路201とスリット310とによって規定される交差点IP3及び第2流路202とスリット310とによって規定される交差点IP4が形成される。本実施形態において、第1スリットは、スリット310によって例示される。第1交差点は、交差点IP3によって例示される。第3交差点は、交差点IP4によって例示される。
The manufacturer of the microchip 122 prepares two substrates. The manufacturer forms the first flow path 201 on one of the two substrates, and the second flow path 202 next to the first flow path 201. The second flow path 202 may be substantially parallel to the first flow path 201. The manufacturer forms the slit 310 on the other of the two substrates. The manufacturer superimposes the two substrates and causes the slit 310 to intersect the first flow path 201 and the second flow path 202. As a result, an intersection IP3 defined by the first flow path 201 and the slit 310 and an intersection IP4 defined by the second flow path 202 and the slit 310 are formed. In the present embodiment, the first slit is exemplified by the slit 310. The first intersection is exemplified by the intersection IP3. The third intersection is exemplified by the intersection IP4.
図1を参照して説明された計測部500は、2次元イメージセンサを含んでもよい。この場合、計測部500は、交差点IP3,IP4を異なる計測点として識別することができる。したがって、マイクロチップ122を使用する使用者は、交差点IP3,IP4における検出対象DOの通過量を同時に計測することができる。
The measurement unit 500 described with reference to FIG. 1 may include a two-dimensional image sensor. In this case, the measurement unit 500 can identify the intersections IP3 and IP4 as different measurement points. Therefore, the user who uses the microchip 122 can simultaneously measure the passing amount of the detection target DO at the intersections IP3 and IP4.
図20は、マイクロチップ121,122の製造工程を表す概略的なフローチャートである。図19A乃至図20を参照して、マイクロチップ121,122の製造工程が説明される。
FIG. 20 is a schematic flowchart showing a manufacturing process of the microchips 121 and 122. A manufacturing process of the microchips 121 and 122 will be described with reference to FIGS. 19A to 20.
(ステップS410)
ステップS410において、製造者は、上基板と下基板とを用意する。その後、ステップS420,S430が平行して実行される。 (Step S410)
In step S410, the manufacturer prepares an upper substrate and a lower substrate. Thereafter, steps S420 and S430 are executed in parallel.
ステップS410において、製造者は、上基板と下基板とを用意する。その後、ステップS420,S430が平行して実行される。 (Step S410)
In step S410, the manufacturer prepares an upper substrate and a lower substrate. Thereafter, steps S420 and S430 are executed in parallel.
(ステップS420)
ステップS420において、製造者は、下基板に遮光膜を形成する。製造者は、マスキング技術を用いて、遮光膜の形成と同時に、スリットを形成してもよい。 (Step S420)
In step S420, the manufacturer forms a light shielding film on the lower substrate. The manufacturer may form the slit simultaneously with the formation of the light shielding film by using a masking technique.
ステップS420において、製造者は、下基板に遮光膜を形成する。製造者は、マスキング技術を用いて、遮光膜の形成と同時に、スリットを形成してもよい。 (Step S420)
In step S420, the manufacturer forms a light shielding film on the lower substrate. The manufacturer may form the slit simultaneously with the formation of the light shielding film by using a masking technique.
(ステップS430)
ステップS430において、製造者は、上基板に流路を形成する。流路の形成は、エッチング技術に依存してもよい。 (Step S430)
In step S430, the manufacturer forms a flow path in the upper substrate. The formation of the flow path may depend on the etching technique.
ステップS430において、製造者は、上基板に流路を形成する。流路の形成は、エッチング技術に依存してもよい。 (Step S430)
In step S430, the manufacturer forms a flow path in the upper substrate. The formation of the flow path may depend on the etching technique.
(ステップS440)
ステップS440において、製造者は、上基板を下基板に重ねる。流路とスリットとの交差によって、交差点は容易に形成される。したがって、従来技術とは異なり、マイクロチップ121,122は、厳密な位置決め作業を要することなく作成される。したがって、マイクロチップ121,122の製造コストは低廉である。加えて、検出対象は、マイクロチップ121,122内で固定化されることなく検出されるので、マイクロチップ121,122は、容易に再利用される。 (Step S440)
In step S440, the manufacturer places the upper substrate on the lower substrate. The intersection is easily formed by the intersection of the flow path and the slit. Therefore, unlike the prior art, the microchips 121 and 122 are produced without requiring a strict positioning operation. Therefore, the manufacturing cost of the microchips 121 and 122 is low. In addition, since the detection target is detected without being immobilized in the microchips 121 and 122, the microchips 121 and 122 are easily reused.
ステップS440において、製造者は、上基板を下基板に重ねる。流路とスリットとの交差によって、交差点は容易に形成される。したがって、従来技術とは異なり、マイクロチップ121,122は、厳密な位置決め作業を要することなく作成される。したがって、マイクロチップ121,122の製造コストは低廉である。加えて、検出対象は、マイクロチップ121,122内で固定化されることなく検出されるので、マイクロチップ121,122は、容易に再利用される。 (Step S440)
In step S440, the manufacturer places the upper substrate on the lower substrate. The intersection is easily formed by the intersection of the flow path and the slit. Therefore, unlike the prior art, the
<第16実施形態>
第15実施形態に関連して説明された原理に従って、製造者は、多数の交差点を有するマイクロチップを容易に形成することができる。第16実施形態において、多数の交差点を有するマイクロチップが説明される。 <Sixteenth Embodiment>
According to the principle described in connection with the fifteenth embodiment, the manufacturer can easily form a microchip having a large number of intersections. In the sixteenth embodiment, a microchip having multiple intersections is described.
第15実施形態に関連して説明された原理に従って、製造者は、多数の交差点を有するマイクロチップを容易に形成することができる。第16実施形態において、多数の交差点を有するマイクロチップが説明される。 <Sixteenth Embodiment>
According to the principle described in connection with the fifteenth embodiment, the manufacturer can easily form a microchip having a large number of intersections. In the sixteenth embodiment, a microchip having multiple intersections is described.
図21は、多数の交差点を有するマイクロチップ123の概略的な平面図である。図3及び図21を参照して、マイクロチップ123が説明される。第1実施形態及び第16実施形態の間で共通して用いられる符号は、当該共通の符号が付された要素が、第1実施形態と同一の機能を有することを意味する。したがって、第1実施形態の説明は、これらの要素に援用される。
FIG. 21 is a schematic plan view of a microchip 123 having a large number of intersections. The microchip 123 will be described with reference to FIGS. The code | symbol used in common between 1st Embodiment and 16th Embodiment means that the element to which the said common code | symbol was attached | subjected has the same function as 1st Embodiment. Therefore, description of 1st Embodiment is used for these elements.
図21に示されるマイクロチップ123には、第1流路201、第2流路202、第3流路203、第4流路204、第1スリット331、第2スリット332、第3スリット333及び第4スリット334が形成される。第1流路201乃至第4流路204の間の間隔並びに第1スリット331乃至第4スリット334の間の間隔は、計測部500の分解能に基づいて決定されてもよい。
The microchip 123 shown in FIG. 21 includes a first channel 201, a second channel 202, a third channel 203, a fourth channel 204, a first slit 331, a second slit 332, a third slit 333, and A fourth slit 334 is formed. The interval between the first channel 201 to the fourth channel 204 and the interval between the first slit 331 to the fourth slit 334 may be determined based on the resolution of the measurement unit 500.
マイクロチップ123が、図3を参照して説明されたマイクロチップ120に代えて利用されるならば、ディテクタ514の分解能に基づいて、第1流路201乃至第4流路204の間の間隔並びに第1スリット331乃至第4スリット334の間の間隔が決定される。ディテクタ514の分解能が、0.5μmであるならば、第1流路201乃至第4流路204の間の間隔並びに第1スリット331乃至第4スリット334の間の間隔は、0.8μmに設定されてもよい。
If the microchip 123 is used instead of the microchip 120 described with reference to FIG. 3, based on the resolution of the detector 514, the distance between the first channel 201 to the fourth channel 204 and An interval between the first slit 331 to the fourth slit 334 is determined. If the resolution of the detector 514 is 0.5 μm, the distance between the first flow path 201 to the fourth flow path 204 and the distance between the first slit 331 to the fourth slit 334 are set to 0.8 μm. May be.
対物レンズ511、光学フィルタ512及び結像レンズ513によって規定される視野が、直径2.0mmであり、且つ、視野の8割で蛍光検出が可能であるならば、2000本の流路を流れる検出対象DOが同時に検出される。すなわち、本実施形態の原理は、従来技術よりも2000倍のスループットを達成することができる。
If the field of view defined by the objective lens 511, the optical filter 512, and the imaging lens 513 is 2.0 mm in diameter, and fluorescence detection is possible in 80% of the field of view, detection that flows through 2000 channels The target DO is detected at the same time. That is, the principle of the present embodiment can achieve a throughput 2000 times that of the prior art.
演算装置515は、各交差点で計測された通過量に対して平均化処理を施与してもよい。演算装置515は、様々な演算処理を用いて、通過量に関するデータの精度を向上してもよい。本実施形態の原理は、演算装置515が実行する特定の演算処理に限定されない。
The arithmetic unit 515 may perform an averaging process on the passing amount measured at each intersection. The arithmetic device 515 may improve the accuracy of the data related to the passage amount by using various arithmetic processes. The principle of the present embodiment is not limited to a specific calculation process executed by the calculation device 515.
本実施形態の原理は、流路の本数及びスリットの特定の本数や特定の配置に限定されない。設計者は、流路を放射状に配置し、且つ、円弧状のスリットを形成してもよい。
The principle of this embodiment is not limited to the number of flow paths, the specific number of slits, or the specific arrangement. The designer may arrange the flow paths radially and form arc-shaped slits.
上述の様々な実施形態の原理は、蛍光検出技術の用途に応じて、適切に組み合わされてもよい。
The principles of the various embodiments described above may be combined appropriately depending on the application of the fluorescence detection technique.
上述の様々な実施形態に関連して説明された例示的な検出技術に関する技術は、以下の特徴を主に備える。
The techniques relating to the exemplary detection techniques described in connection with the various embodiments described above primarily comprise the following features.
上述の実施形態の一の局面に係る蛍光検出装置は、複数の検出対象を案内する少なくとも1つの流路と、前記少なくとも1つの流路によって立体的に交差される少なくとも1つのスリットが形成された遮光膜と、前記少なくとも1つの流路が前記少なくとも1つのスリットに立体的に交差する少なくとも1つの交差部に、励起光を照射し、前記複数の検出対象から蛍光を生じさせる照射部と、前記蛍光の強度から、前記少なくとも1つの交差部を通過した前記複数の検出対象の通過量を計測する計測部と、を備える。前記励起光は、前記少なくとも1つのスリットの延設方向に沿う直線偏光を含む。
In the fluorescence detection device according to one aspect of the above-described embodiment, at least one flow path that guides a plurality of detection targets and at least one slit that is three-dimensionally intersected by the at least one flow path are formed. A light-shielding film, and an irradiation unit that irradiates excitation light to at least one intersecting portion where the at least one channel three-dimensionally intersects the at least one slit, and generates fluorescence from the plurality of detection targets; And a measurement unit that measures the passage amounts of the plurality of detection targets that have passed through the at least one intersecting portion based on the intensity of fluorescence. The excitation light includes linearly polarized light along the extending direction of the at least one slit.
上記構成によれば、励起光は、少なくとも1つのスリットの延設方向に沿う直線偏光を含むので、検出対象は、強く蛍光発光することができる。したがって、蛍光検出装置は、高い感度を達成することができる。
According to the above configuration, since the excitation light includes linearly polarized light along the extending direction of at least one slit, the detection target can strongly emit fluorescence. Therefore, the fluorescence detection device can achieve high sensitivity.
上記構成において、前記少なくとも1つのスリットは、前記励起光の波長よりも小さな幅寸法を有してもよい。
In the above configuration, the at least one slit may have a width dimension smaller than the wavelength of the excitation light.
上記構成によれば、少なくとも1つのスリットは、励起光の波長よりも小さな幅寸法を有するので、励起光は、流路へ不必要に伝搬しない。したがって、設計者は、光学フィルタといった光学素子を蛍光検出装置へ不必要に組み込まなくてもよい。
According to the above configuration, since at least one slit has a width dimension smaller than the wavelength of the excitation light, the excitation light does not unnecessarily propagate to the flow path. Therefore, the designer may not unnecessarily incorporate an optical element such as an optical filter into the fluorescence detection apparatus.
上記構成において、前記少なくとも1つの流路は、前記少なくとも1つのスリットの前記幅寸法よりも小さな高さ寸法を有してもよい。
In the above configuration, the at least one channel may have a height dimension smaller than the width dimension of the at least one slit.
上記構成によれば、少なくとも1つの流路は、少なくとも1つのスリットの幅寸法よりも小さな高さ寸法を有するので、複数の検出対象それぞれは分離して、流路を流れることができる。したがって、観察者は、固定化処理を施与することなく、複数の検出対象を観察することができる。加えて、少なくとも1つのスリットを通過した励起光は、流路の高さ方向に伝搬することができる。したがって、蛍光検出装置は、複数の検出対象それぞれを効率的に検出することができる。
According to the above configuration, since at least one flow path has a height dimension smaller than the width dimension of at least one slit, a plurality of detection targets can be separated and flow through the flow path. Therefore, the observer can observe a plurality of detection objects without performing the immobilization process. In addition, the excitation light that has passed through the at least one slit can propagate in the height direction of the flow path. Therefore, the fluorescence detection apparatus can efficiently detect each of the plurality of detection targets.
上記構成において、前記計測部は、前記蛍光の前記強度を表す検出信号を生成する信号生成部と、前記検出信号から前記通過量に関する情報を取得する取得部と、を含んでもよい。
In the above-described configuration, the measurement unit may include a signal generation unit that generates a detection signal that represents the intensity of the fluorescence, and an acquisition unit that acquires information related to the passage amount from the detection signal.
上記構成によれば、蛍光検出装置は、蛍光の光量を検出し、検出対象の通過量に関する情報を観察者に提供することができる。
According to the above configuration, the fluorescence detection device can detect the amount of fluorescence and provide the observer with information regarding the amount of passage of the detection target.
上記構成において、前記検出信号は、前記蛍光の前記強度に応じて変化する信号値を表してもよい。前記取得部は、前記信号値と前記信号値に対して設定された閾値とを比較する比較部と、前記比較部による比較の結果に基づいて、前記通過量を判定する判定部と、を含んでもよい。
In the above configuration, the detection signal may represent a signal value that changes according to the intensity of the fluorescence. The acquisition unit includes a comparison unit that compares the signal value and a threshold set for the signal value, and a determination unit that determines the passage amount based on a result of comparison by the comparison unit. But you can.
上記構成によれば、検出信号の信号値は、閾値と比較されるので、通過量は、ノイズの影響をほとんど受けることなく、判定部によって判定される。
According to the above configuration, since the signal value of the detection signal is compared with the threshold value, the amount of passage is determined by the determination unit with little influence of noise.
上記構成において、蛍光検出装置は、前記少なくとも1つの流路に沿って前記複数の検出対象を移動させる移動部を更に備えてもよい。
In the above configuration, the fluorescence detection apparatus may further include a moving unit that moves the plurality of detection objects along the at least one flow path.
上記構成によれば、複数の検出対象は、流路中で、移動部によって適切に移動される。
According to the above configuration, the plurality of detection targets are appropriately moved by the moving unit in the flow path.
上記構成において、前記遮光膜は、前記励起光に対して不透明であってもよい。
In the above configuration, the light shielding film may be opaque to the excitation light.
上記構成によれば、遮光膜は、励起光に対して不透明であるので、検出対象は、背景光の影響をほとんど受けることなく、適切に検出される。
According to the above configuration, since the light-shielding film is opaque to the excitation light, the detection target is appropriately detected with almost no influence of the background light.
上記構成において、前記遮光膜は、金属膜であってもよい。
In the above configuration, the light shielding film may be a metal film.
上記構成によれば、遮光膜は、励起光を適切に遮蔽することができる。
According to the above configuration, the light shielding film can appropriately shield the excitation light.
上記構成において、蛍光検出装置は、前記少なくとも1つのスリットに充填された充填材料を更に備えてもよい。前記充填材料は、前記励起光に対して透明であってもよい。
In the above configuration, the fluorescence detection apparatus may further include a filling material filled in the at least one slit. The filling material may be transparent to the excitation light.
上記構成によれば、充填材料は、スリットの周囲の領域を平滑化することができる。したがって、複数の検出対象は、流路内を円滑に移動することができる。
According to the above configuration, the filling material can smooth the area around the slit. Therefore, the plurality of detection targets can move smoothly in the flow path.
上記構成において、前記少なくとも1つのスリットは、第1スリットと、前記第1スリットの隣に形成された第2スリットと、を含んでもよい。前記少なくとも1つの流路は、前記第1スリットと前記第2スリットとに立体的に交差する第1流路を含んでもよい。前記少なくとも1つの交差部は、前記第1スリットと前記第1流路とによって規定される第1交差点と、前記第2スリットと前記第1流路によって規定される第2交差点と、を含んでもよい。前記計測部は、前記第1交差点を前記第2交差点とは異なる計測点として識別してもよい。
In the above configuration, the at least one slit may include a first slit and a second slit formed adjacent to the first slit. The at least one flow path may include a first flow path that three-dimensionally intersects the first slit and the second slit. The at least one intersection may include a first intersection defined by the first slit and the first flow path, and a second intersection defined by the second slit and the first flow path. Good. The measurement unit may identify the first intersection as a measurement point different from the second intersection.
上記構成によれば、計測部は、第1交差点を第2交差点とは異なる計測点として識別するので、蛍光検出装置は、複数の検出対象を効率的に検出することができる。
According to the above configuration, the measurement unit identifies the first intersection as a measurement point different from the second intersection, so that the fluorescence detection apparatus can efficiently detect a plurality of detection targets.
上記構成において、前記少なくとも1つの流路は、第1流路と、前記第1流路の隣に形成された第2流路と、を含んでもよい。前記少なくとも1つのスリットは、前記第1流路と前記第2流路とに立体的に交差する第1スリットを含んでもよい。前記少なくとも1つの交差部は、前記第1スリットと前記第1流路とによって規定される第1交差点と、前記第1スリットと前記第2流路とによって規定される第3交差点と、を含んでもよい。前記計測部は、前記第1交差点を前記第3交差点とは異なる計測点として識別してもよい。
In the above configuration, the at least one flow path may include a first flow path and a second flow path formed adjacent to the first flow path. The at least one slit may include a first slit that three-dimensionally intersects the first channel and the second channel. The at least one intersection includes a first intersection defined by the first slit and the first flow path, and a third intersection defined by the first slit and the second flow path. But you can. The measurement unit may identify the first intersection as a measurement point different from the third intersection.
上記構成によれば、計測部は、第1交差点を第3交差点とは異なる計測点として識別するので、蛍光検出装置は、複数の検出対象を効率的に検出することができる。
According to the above configuration, the measurement unit identifies the first intersection as a measurement point different from the third intersection, so that the fluorescence detection apparatus can efficiently detect a plurality of detection targets.
上記構成において、前記計測部は、前記第1交差点を通過した前記複数の検出対象の通過量と、前記第2交差点を通過した前記複数の検出対象の通過量と、を同時に計測してもよい。
In the above configuration, the measurement unit may simultaneously measure the passage amounts of the plurality of detection targets that have passed through the first intersection and the passage amounts of the plurality of detection targets that have passed through the second intersection. .
上記構成によれば、計測部は、第1交差点を通過した複数の検出対象の通過量と、第2交差点を通過した複数の検出対象の通過量と、を同時に計測するので、蛍光検出装置は、複数の検出対象を効率的に検出することができる。
According to the above configuration, the measurement unit simultaneously measures the passage amounts of the plurality of detection targets that have passed through the first intersection and the passage amounts of the plurality of detection targets that have passed through the second intersection. A plurality of detection targets can be efficiently detected.
上記構成において、前記計測部は、前記第1交差点を通過した前記複数の検出対象の通過量と、前記第3交差点を通過した前記複数の検出対象の通過量と、を同時に計測してもよい。
In the above configuration, the measurement unit may simultaneously measure the passage amounts of the plurality of detection targets that have passed through the first intersection and the passage amounts of the plurality of detection targets that have passed through the third intersection. .
上記構成によれば、計測部は、第1交差点を通過した複数の検出対象の通過量と、第3交差点を通過した複数の検出対象の通過量と、を同時に計測するので、蛍光検出装置は、複数の検出対象を効率的に検出することができる。
According to the above configuration, the measurement unit simultaneously measures the passage amounts of the plurality of detection targets that have passed the first intersection and the passage amounts of the plurality of detection targets that have passed the third intersection. A plurality of detection targets can be efficiently detected.
上記構成において、前記複数の検出対象は、第1波長を有する第1蛍光を発する第1検出対象と、前記第1波長とは異なる第2波長を有する第2蛍光を発する第2検出対象と、を含んでもよい。前記検出信号は、前記第1蛍光の強度を表す第1検出信号と、前記第2蛍光の強度を表す第2検出信号と、を含んでもよい。前記取得部は、前記第1検出信号から、前記少なくとも1つの交差部を通過した前記第1検出対象の通過量に関する第1情報を取得し、且つ、前記第2検出信号から、前記少なくとも1つの交差部を通過した前記第2検出対象の通過量に関する第2情報を取得してもよい。
In the above configuration, the plurality of detection targets include a first detection target that emits a first fluorescence having a first wavelength, and a second detection target that emits a second fluorescence having a second wavelength different from the first wavelength; May be included. The detection signal may include a first detection signal that represents the intensity of the first fluorescence and a second detection signal that represents the intensity of the second fluorescence. The acquisition unit acquires, from the first detection signal, first information related to a passing amount of the first detection target that has passed through the at least one intersection, and from the second detection signal, the at least one You may acquire the 2nd information regarding the passage amount of the 2nd detection object which passed the intersection.
上記構成によれば、取得部は、第1検出信号から、少なくとも1つの交差部を通過した第1検出対象の通過量に関する第1情報を取得し、且つ、第2検出信号から、少なくとも1つの交差部を通過した第2検出対象の通過量に関する第2情報を取得するので、蛍光検出装置は、第1検出対象及び第2検出対象の通過量に関する情報を観察者に提供することができる。
According to the above configuration, the acquisition unit acquires, from the first detection signal, the first information related to the passage amount of the first detection target that has passed through at least one intersection, and from the second detection signal, at least one Since the second information related to the passage amount of the second detection target that has passed through the intersection is acquired, the fluorescence detection device can provide the observer with information regarding the passage amount of the first detection target and the second detection target.
上記構成において、前記照射部は、前記励起光として、第1励起光を照射する第1照射部と、前記励起光として、第2励起光を照射する第2照射部と、を含んでもよい。前記第1検出対象は、前記第2励起光の照射下よりも前記第1励起光の照射下において前記第1蛍光を強く発してもよい。前記第2検出対象は、前記第1励起光の前記照射下よりも前記第2励起光の前記照射下において前記第2蛍光を強く発してもよい。
In the above-described configuration, the irradiation unit may include a first irradiation unit that irradiates first excitation light as the excitation light, and a second irradiation unit that irradiates second excitation light as the excitation light. The first detection target may emit the first fluorescence more strongly under irradiation of the first excitation light than under irradiation of the second excitation light. The second detection target may emit the second fluorescence more strongly under the irradiation of the second excitation light than under the irradiation of the first excitation light.
上記構成によれば、照射部は、励起光として、第1励起光を照射する第1照射部と、励起光として、第2励起光を照射する第2照射部と、を含むので、蛍光検出装置は、第1検出対象及び第2検出対象を高い感度で検出することができる。
According to the above configuration, the irradiation unit includes the first irradiation unit that irradiates the first excitation light as the excitation light and the second irradiation unit that irradiates the second excitation light as the excitation light. The apparatus can detect the first detection target and the second detection target with high sensitivity.
上記構成において、前記励起光は、350nm以上750nm以下の波長を有してもよい。
In the above configuration, the excitation light may have a wavelength of 350 nm or more and 750 nm or less.
上記構成によれば、励起光は、350nm以上750nm以下の波長を有するので、可視光域の蛍光を効率的に発生させることができる。
According to the above configuration, since the excitation light has a wavelength of 350 nm or more and 750 nm or less, fluorescence in the visible light region can be efficiently generated.
上記構成において、前記高さ寸法は、30nm以上150nm以下であってもよい。
In the above configuration, the height dimension may be not less than 30 nm and not more than 150 nm.
上記構成によれば、高さ寸法は、30nm以上150nm以下であるので、励起光は、過度に減衰する前に、複数の検出対象を照明することができる。
According to the above configuration, since the height dimension is not less than 30 nm and not more than 150 nm, the excitation light can illuminate a plurality of detection targets before being excessively attenuated.
上記構成において、前記少なくとも1つの流路の幅寸法は、10nm以上500nm以下であってもよい。
In the above configuration, the width dimension of the at least one flow path may be not less than 10 nm and not more than 500 nm.
上記構成によれば、少なくとも1つの流路の幅寸法は、10nm以上500nm以下であるので、蛍光検出装置は、様々な検出対象を検出するために利用可能である。
According to the above configuration, since the width dimension of at least one flow path is 10 nm or more and 500 nm or less, the fluorescence detection apparatus can be used to detect various detection targets.
上述の実施形態の他の局面に係る蛍光検出方法は、少なくとも1つのスリットに立体的に交差する少なくとも1つの流路内で複数の検出対象を移動させる段階と、前記少なくとも1つのスリットが前記少なくとも1つの流路に交差する少なくとも1つの交差部に、前記少なくとも1つのスリットの延設方向に沿う直線偏光を含む励起光を照射し、前記複数の検出対象から蛍光を生じさせる段階と、前記蛍光の光量から、前記少なくとも1つの交差部を通過した前記複数の検出対象の通過量を計測する段階と、を備える。
The fluorescence detection method according to another aspect of the above-described embodiment includes a step of moving a plurality of detection targets in at least one flow path that sterically intersects at least one slit, and the at least one slit is at least the at least one slit. Irradiating excitation light including linearly polarized light along the extending direction of the at least one slit to at least one intersecting portion intersecting one flow path to generate fluorescence from the plurality of detection targets; Measuring the passage amounts of the plurality of detection targets that have passed through the at least one intersection.
上記構成によれば、スリットの延設方向に沿う直線偏光を含む励起光が照射されるので、検出対象は、強く蛍光発光することができる。したがって、蛍光検出方法は、高い感度を達成することができる。
According to the above configuration, since the excitation light including linearly polarized light along the extending direction of the slit is irradiated, the detection target can strongly emit fluorescence. Therefore, the fluorescence detection method can achieve high sensitivity.
上述の実施形態の原理は、検出対象を検出するための技術に好適に利用可能である。
The principle of the above-described embodiment can be suitably used for a technique for detecting a detection target.
Claims (19)
- 複数の検出対象を案内する少なくとも1つの流路と、
前記少なくとも1つの流路によって立体的に交差される少なくとも1つのスリットが形成された遮光膜と、
前記少なくとも1つの流路が前記少なくとも1つのスリットに立体的に交差する少なくとも1つの交差部に、励起光を照射し、前記複数の検出対象から蛍光を生じさせる照射部と、
前記蛍光の強度から、前記少なくとも1つの交差部を通過した前記複数の検出対象の通過量を計測する計測部と、を備え、
前記励起光は、前記少なくとも1つのスリットの延設方向に沿う直線偏光を含むことを特徴とする蛍光検出装置。 At least one flow path for guiding a plurality of detection targets;
A light-shielding film in which at least one slit that is three-dimensionally intersected by the at least one flow path is formed;
An irradiation unit that emits excitation light to at least one intersecting portion in which the at least one channel three-dimensionally intersects the at least one slit to generate fluorescence from the plurality of detection targets;
A measurement unit that measures the amount of passage of the plurality of detection objects that have passed through the at least one intersection from the intensity of the fluorescence, and
The fluorescence detection apparatus, wherein the excitation light includes linearly polarized light along an extending direction of the at least one slit. - 前記少なくとも1つのスリットは、前記励起光の波長よりも小さな幅寸法を有することを特徴とする請求項1に記載の蛍光検出装置。 The fluorescence detection device according to claim 1, wherein the at least one slit has a width dimension smaller than a wavelength of the excitation light.
- 前記少なくとも1つの流路は、前記少なくとも1つのスリットの前記幅寸法よりも小さな高さ寸法を有することを特徴とする請求項2に記載の蛍光検出装置。 The fluorescence detection device according to claim 2, wherein the at least one flow path has a height dimension smaller than the width dimension of the at least one slit.
- 前記計測部は、前記蛍光の前記強度を表す検出信号を生成する信号生成部と、前記検出信号から前記通過量に関する情報を取得する取得部と、を含むことを特徴とする請求項1乃至3のいずれか1項に記載の蛍光検出装置。 The said measurement part contains the signal generation part which produces | generates the detection signal showing the said intensity | strength of the said fluorescence, and the acquisition part which acquires the information regarding the said passage amount from the said detection signal, The Claim 1 thru | or 3 characterized by the above-mentioned. The fluorescence detection device according to any one of the above.
- 前記検出信号は、前記蛍光の前記強度に応じて変化する信号値を表し、
前記取得部は、前記信号値と前記信号値に対して設定された閾値とを比較する比較部と、前記比較部による比較の結果に基づいて、前記通過量を判定する判定部と、を含むことを特徴とする請求項4に記載の蛍光検出装置。 The detection signal represents a signal value that varies according to the intensity of the fluorescence,
The acquisition unit includes a comparison unit that compares the signal value with a threshold set for the signal value, and a determination unit that determines the passage amount based on a result of comparison by the comparison unit. The fluorescence detection apparatus according to claim 4. - 前記少なくとも1つの流路に沿って前記複数の検出対象を移動させる移動部を更に備えることを特徴とする請求項1乃至5のいずれか1項に記載の蛍光検出装置。 6. The fluorescence detection apparatus according to claim 1, further comprising a moving unit that moves the plurality of detection targets along the at least one flow path.
- 前記遮光膜は、前記励起光に対して不透明であることを特徴とする請求項1乃至6のいずれか1項に記載の蛍光検出装置。 The fluorescence detection apparatus according to any one of claims 1 to 6, wherein the light shielding film is opaque to the excitation light.
- 前記遮光膜は、金属膜であることを特徴とする請求項1乃至7のいずれか1項に記載の蛍光検出装置。 The fluorescence detection apparatus according to any one of claims 1 to 7, wherein the light shielding film is a metal film.
- 前記少なくとも1つのスリットに充填された充填材料を更に備え、
前記充填材料は、前記励起光に対して透明であることを特徴とする請求項1乃至8のいずれか1項に記載の蛍光検出装置。 A filling material filling the at least one slit;
The fluorescence detection apparatus according to claim 1, wherein the filling material is transparent to the excitation light. - 前記少なくとも1つのスリットは、第1スリットと、前記第1スリットの隣に形成された第2スリットと、を含み、
前記少なくとも1つの流路は、前記第1スリットと前記第2スリットとに立体的に交差する第1流路を含み、
前記少なくとも1つの交差部は、前記第1スリットと前記第1流路とによって規定される第1交差点と、前記第2スリットと前記第1流路によって規定される第2交差点と、を含み、
前記計測部は、前記第1交差点を前記第2交差点とは異なる計測点として識別することを特徴とする請求項1乃至9のいずれか1項に記載の蛍光検出装置。 The at least one slit includes a first slit and a second slit formed adjacent to the first slit,
The at least one flow path includes a first flow path that three-dimensionally intersects the first slit and the second slit,
The at least one intersection includes a first intersection defined by the first slit and the first flow path, and a second intersection defined by the second slit and the first flow path,
10. The fluorescence detection device according to claim 1, wherein the measurement unit identifies the first intersection as a measurement point different from the second intersection. 11. - 前記少なくとも1つの流路は、第1流路と、前記第1流路の隣に形成された第2流路と、を含み、
前記少なくとも1つのスリットは、前記第1流路と前記第2流路とに立体的に交差する第1スリットを含み、
前記少なくとも1つの交差部は、前記第1スリットと前記第1流路とによって規定される第1交差点と、前記第1スリットと前記第2流路とによって規定される第3交差点と、を含み、
前記計測部は、前記第1交差点を前記第3交差点とは異なる計測点として識別することを特徴とする請求項1乃至9のいずれか1項に記載の蛍光検出装置。 The at least one flow path includes a first flow path and a second flow path formed adjacent to the first flow path,
The at least one slit includes a first slit that three-dimensionally intersects the first channel and the second channel,
The at least one intersection includes a first intersection defined by the first slit and the first flow path, and a third intersection defined by the first slit and the second flow path. ,
10. The fluorescence detection device according to claim 1, wherein the measurement unit identifies the first intersection as a measurement point different from the third intersection. 11. - 前記計測部は、前記第1交差点を通過した前記複数の検出対象の通過量と、前記第2交差点を通過した前記複数の検出対象の通過量と、を同時に計測することを特徴とする請求項10に記載の蛍光検出装置。 The said measurement part measures simultaneously the passage amount of these detection objects which passed the said 1st intersection, and the passage amount of these detection objects which passed the said 2nd intersection. 10. The fluorescence detection apparatus according to 10.
- 前記計測部は、前記第1交差点を通過した前記複数の検出対象の通過量と、前記第3交差点を通過した前記複数の検出対象の通過量と、を同時に計測することを特徴とする請求項11に記載の蛍光検出装置。 The said measurement part measures simultaneously the passage amount of these detection objects which passed the said 1st intersection, and the passage amount of these detection objects which passed the said 3rd intersection. 11. The fluorescence detection apparatus according to 11.
- 前記複数の検出対象は、第1波長を有する第1蛍光を発する第1検出対象と、前記第1波長とは異なる第2波長を有する第2蛍光を発する第2検出対象と、を含み、
前記検出信号は、前記第1蛍光の強度を表す第1検出信号と、前記第2蛍光の強度を表す第2検出信号と、を含み、
前記取得部は、前記第1検出信号から、前記少なくとも1つの交差部を通過した前記第1検出対象の通過量に関する第1情報を取得し、且つ、前記第2検出信号から、前記少なくとも1つの交差部を通過した前記第2検出対象の通過量に関する第2情報を取得することを特徴とする請求項5に記載の蛍光検出装置。 The plurality of detection objects include a first detection object that emits a first fluorescence having a first wavelength, and a second detection object that emits a second fluorescence having a second wavelength different from the first wavelength,
The detection signal includes a first detection signal representing the intensity of the first fluorescence, and a second detection signal representing the intensity of the second fluorescence,
The acquisition unit acquires, from the first detection signal, first information related to a passing amount of the first detection target that has passed through the at least one intersection, and from the second detection signal, the at least one The fluorescence detection apparatus according to claim 5, wherein second information related to a passing amount of the second detection target that has passed through the intersection is acquired. - 前記照射部は、前記励起光として、第1励起光を照射する第1照射部と、前記励起光として、第2励起光を照射する第2照射部と、を含み、
前記第1検出対象は、前記第2励起光の照射下よりも前記第1励起光の照射下において前記第1蛍光を強く発し、
前記第2検出対象は、前記第1励起光の照射下よりも前記第2励起光の照射下において前記第2蛍光を強く発することを特徴とする請求項14に記載の蛍光検出装置。 The irradiation unit includes a first irradiation unit that irradiates first excitation light as the excitation light, and a second irradiation unit that irradiates second excitation light as the excitation light,
The first detection object emits the first fluorescence more strongly under irradiation of the first excitation light than under irradiation of the second excitation light,
The fluorescence detection device according to claim 14, wherein the second detection target emits the second fluorescence more strongly under irradiation of the second excitation light than under irradiation of the first excitation light. - 前記励起光は、350nm以上750nm以下の波長を有することを特徴とする請求項1乃至14のいずれか1項に記載の蛍光検出装置。 The fluorescence detection apparatus according to any one of claims 1 to 14, wherein the excitation light has a wavelength of 350 nm or more and 750 nm or less.
- 前記高さ寸法は、30nm以上150nm以下であることを特徴とする請求項3に記載の蛍光検出装置。 4. The fluorescence detection apparatus according to claim 3, wherein the height dimension is 30 nm or more and 150 nm or less.
- 前記少なくとも1つの流路の幅寸法は、10nm以上500nm以下であることを特徴とする請求項1乃至17に記載の蛍光検出装置。 18. The fluorescence detection device according to claim 1, wherein a width dimension of the at least one flow path is 10 nm or more and 500 nm or less.
- 少なくとも1つのスリットに立体的に交差する少なくとも1つの流路内で複数の検出対象を移動させる段階と、
前記少なくとも1つのスリットが前記少なくとも1つの流路に交差する少なくとも1つの交差部に、前記少なくとも1つのスリットの延設方向に沿う直線偏光を含む励起光を照射し、前記複数の検出対象から蛍光を生じさせる段階と、
前記蛍光の光量から、前記少なくとも1つの交差部を通過した前記複数の検出対象の通過量を計測する段階と、を備えることを特徴とする蛍光検出方法。 Moving a plurality of detection targets in at least one flow path that sterically intersects at least one slit;
The at least one intersection where the at least one slit intersects the at least one flow path is irradiated with excitation light including linearly polarized light along the extending direction of the at least one slit, and fluorescence is emitted from the plurality of detection targets. A stage of generating
Measuring the amount of passage of the plurality of detection targets that have passed through the at least one intersecting portion from the amount of the fluorescence, and a method for detecting fluorescence.
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