WO2014167792A1 - Fluorescence detection device and fluorescence detection method - Google Patents

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

Fluorescence detection apparatus and fluorescence detection method

The present invention relates to a detection technique for detecting a detection target that emits fluorescence based on a fluorescence method.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

The above-described fluorescence detection technology can achieve high detection sensitivity.

The objects, features and advantages of the present invention will become more apparent from the following detailed description and the accompanying drawings.

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). 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. It is a schematic block diagram of the measurement part of the fluorescence detection apparatus shown by FIG. 1 (11th Embodiment). It is a schematic flowchart showing the detection signal production | generation process by the signal production | generation part of the measurement part shown by FIG. 7 (12th Embodiment). It is a conceptual diagram of the fluorescence detection apparatus of 13th Embodiment. It is a conceptual diagram of the fluorescence detection apparatus of 14th Embodiment. It is a schematic plan view of a microchip (15th Embodiment). It is a schematic plan view of a microchip (15th Embodiment). 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).

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.

<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.

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. 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.

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.

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.

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.

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. 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.

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.

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.

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. 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.

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. 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.

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.

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.

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. 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.

(Step S120)
In 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.

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.

<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.

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. 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.

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. In the present embodiment, 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 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. 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.

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.

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.

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.

<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. In addition, 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.

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.

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.

<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.

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.

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.

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.

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. In addition, 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. 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.

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.

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.

<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.

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.

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.

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.

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.

<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.

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.

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.

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)
In 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)
In 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)
In 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.

<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.

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.

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.

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.

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.

<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.

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.

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.

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.

<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.

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. 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.

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)
In 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)
In 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)
In 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)
In 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. 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.

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 (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.

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.

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.

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. 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.

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)
In step S310, the signal 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.

(Step S320)
In 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)
In 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)
In step S340, the signal 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.

(Step S350)
In 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)
In step S360, the signal generation unit 520 processes the incidence of the fluorescence FL as noise.

<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.

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.

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.

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. 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.

<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.

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.

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.

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.

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.

<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.

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.

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.

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.

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.

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.

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.

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.

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)
In step S410, the manufacturer prepares an upper substrate and a lower substrate. Thereafter, steps S420 and S430 are executed in parallel.

(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.

(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.

(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.

<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.

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.

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.

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.

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.

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 principles of the various embodiments described above may be combined appropriately depending on the application of the fluorescence detection technique.

The techniques relating to the exemplary detection techniques described in connection with the various embodiments described above primarily comprise the following features.

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.

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.

In the above configuration, the at least one slit may have a width dimension smaller than the wavelength of the excitation light.

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.

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.

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.

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.

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.

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.

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.

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.

According to the above configuration, the plurality of detection targets are appropriately moved by the moving unit in the flow path.

In the above configuration, the light shielding film may be opaque to the excitation light.

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.

In the above configuration, the light shielding film may be a metal film.

According to the above configuration, the light shielding film can appropriately shield the excitation light.

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.

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.

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.

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.

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.

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.

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. .

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.

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. .

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.

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.

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.

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.

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.

In the above configuration, the excitation light may have a wavelength of 350 nm or more and 750 nm or less.

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.

In the above configuration, the height dimension may be not less than 30 nm and not more than 150 nm.

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.

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.

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.

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.

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.

The principle of the above-described embodiment can be suitably used for a technique for detecting a detection target.

Claims (19)

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. The fluorescence detection apparatus according to any one of claims 1 to 6, wherein the light shielding film is opaque to the excitation light.
8. The fluorescence detection apparatus according to any one of claims 1 to 7, wherein the light shielding film is a metal film.
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
14. 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.
15. 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.
16. 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.
17. 4. The fluorescence detection apparatus according to claim 3, wherein the height dimension is 30 nm or more and 150 nm or less.
18. 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.
19. 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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