WO2009087753A1

WO2009087753A1 - Electrophoretic device and method for alignment

Info

Publication number: WO2009087753A1
Application number: PCT/JP2008/050023
Authority: WO
Inventors: Rintaro Yamamoto; Hiroyuki Matsumoto; Hikaru Shibata
Original assignee: Shimadzu Corporation
Priority date: 2008-01-07
Filing date: 2008-01-07
Publication date: 2009-07-16

Abstract

An electrophoretic device includes a transparent substrate (10) in which a flow path (12) for electrophoretic migration is provided, a detection optics system (30) which irradiates an excitation light onto the substrate (10) to detect a detection light excited by the excitation light, a movement unit (24) to move at least one of the substrate (10) and the detection optics system (30) in the direction perpendicular to the extending direction of the flow path (12), and a control unit (40) which determines an end position of the flow path (12) depending on a change in intensity of the detection light detected in association with movement to align the excitation light with the flow path (12).

Description

Electrophoresis apparatus and alignment method

The present invention relates to an electrophoresis apparatus capable of automatically adjusting the position of the optical axis of a detection optical system, and a method for aligning the optical system with respect to the flow path of the electrophoresis apparatus.

Electrophoresis devices have been developed that perform chemical analysis and chemical synthesis by forming microchannels on a substrate such as glass by microelectromechanical system (MEMS) technology. In chemical analysis by electrophoresis, molecules to be analyzed are labeled with a fluorescent dye for detection. The fluorescently labeled molecules are separated in the flow path by electrophoresis, and are identified by detecting the excited fluorescence with a detection optical system.

Electrophoresis in a minute channel can reduce the amount of sample and reagent used, and enables high-speed and highly efficient chemical reaction and chemical analysis. However, since the amount of analysis is very small, a highly sensitive detection method is required. Therefore, it is important to align the excitation light of the detection optical system.

When a surface-emitting light emitting device (LED) is used as the excitation light source of the fluorescent dye, the condensing diameter of the LED is as large as about 400 μm, assuming that the width of the electrophoresis channel is about 100 μm. What is necessary is just to make it a flow path fit in the condensing diameter of LED, when aligning excitation light to a flow path. Therefore, if the flow path is produced with a processing position accuracy within about ± 100 μm from the substrate end face of the electrophoresis apparatus, the flow path can be arranged within the condensing diameter of the LED.

In an electrophoresis apparatus used for a sequencer that determines the sequence of deoxyribonucleic acid (DNA) or the like, a very small amount of fluorescent dye is added to a sample, and thus a high-power excitation light source is required. It is required to use the output of excitation light effectively by using a laser diode (LD) as an excitation light source and condensing it. When the condensing diameter of the excitation light from the LD is about 30 μm to about 50 μm, it is necessary to irradiate the condensed excitation light into the channel having a width of about 100 μm. For this purpose, it is necessary to align the excitation light with a high positional accuracy of about ± 20 μm with respect to the flow path provided on the substrate of the electrophoresis apparatus. With the processing position accuracy of the flow path from the substrate end face of the electrophoresis apparatus, it is difficult to achieve such high-precision alignment. In addition, if fine particles such as dust are sandwiched between the alignment reference plane and the substrate end surface, accurate alignment becomes difficult.

In view of the above problems, the present invention provides an electrophoresis apparatus capable of aligning excitation light with high accuracy with respect to a flow path, and an optical system alignment method with respect to the flow path of the electrophoresis apparatus. For the purpose.

In order to achieve the above object, the first aspect of the present invention includes (a) a transparent substrate in which a flow path for electrophoresis is provided, and (b) irradiating the substrate with excitation light. A detection optical system that detects the detection light excited by the liquid crystal, (c) a moving unit that moves at least one of the substrate and the detection optical system in a direction perpendicular to the direction in which the flow path extends, and The gist of the present invention is an electrophoresis apparatus including a control unit that determines the position of the end of the flow path from the intensity change of the detected light and aligns the excitation light with the flow path.

According to the second aspect of the present invention, (a) excitation light is irradiated to a transparent substrate in which a flow path for electrophoresis is provided, and (b) the excitation light is perpendicular to the direction in which the flow path extends. (C) determine the position of the end of the flow path from the change in the intensity of the detected light, and (d) align the excitation light with the flow path. The gist is that it is a positioning method including.

FIG. 1 is a schematic diagram showing an example of the configuration of an electrophoresis apparatus according to an embodiment of the present invention. FIG. 2 is a schematic view showing a cross section taken along line AA of the cathode shown in FIG. FIG. 3 is a schematic view showing a cross section taken along line BB of the cathode shown in FIG. FIG. 4 is a schematic sectional view showing an example of the arrangement of the substrates of the electrophoresis plate according to the embodiment of the present invention. FIG. 5 is a diagram showing an example of the relationship between the detection light intensity from the substrate of the electrophoresis plate and the excitation light position according to the embodiment of the present invention. FIG. 6 is a diagram showing another example of the relationship between the detection light intensity from the substrate of the electrophoresis plate and the excitation light position according to the embodiment of the present invention. FIG. 7 is a flowchart showing an example of the alignment method according to the embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, it should be noted that the drawings are schematic and the configuration of the apparatus and system is different from the actual one. Therefore, a specific configuration should be determined in consideration of the following description. Moreover, it is a matter of course that portions having different structures and the like are included between the drawings.

The following embodiments of the present invention exemplify apparatuses and methods for embodying the technical idea of the present invention. The technical idea of the present invention is based on the material and shape of component parts. The structure, arrangement, etc. are not specified below. The technical idea of the present invention can be variously modified within the technical scope described in the claims.

(Electrophoresis device)
As shown in FIGS. 1 and 2, the electrophoresis apparatus according to the embodiment of the present invention includes an electrophoresis plate 1, a heating block 20, a moving unit 24, a detection optical system 30, a control unit 40, and the like. The electrophoresis plate 1 includes a substrate 10 and

reservoirs

16a and 16b. Inside the substrate 10, there is provided a flow path 12 for electrophoresis of a sample to be analyzed to which a fluorescent dye is added. Through

holes

14 a and 14 b that communicate from the surface of the substrate 10 to the flow path 12 are provided at both ends of the flow path 12 in the direction in which the flow path 12 extends. The

reservoirs

16a and 16b are installed on the surface of the substrate 10 so as to surround the through

holes

14a and 14b, respectively.

The electrophoresis plate 1 is installed on the upper surface of the heating block 20. A through hole 22 is provided in the heating block 20. The image of the flow path 12 projected on the cross section of the through hole 22 parallel to the upper surface of the heating block 20 extends from one end to the other end in the center of the cross section of the through hole 22. The heating block 20 is provided with a heater (not shown) for heating the electrophoresis plate 1.

The moving unit 24 is installed on the lower surface of the heating block 20. The moving unit 24 moves the heating unit block 20 in a direction orthogonal to the direction in which the flow path 12 extends.

The detection optical system 30 includes a light source 32, a filter 34, a lens 36, and a detector 38. The light source 32 emits excitation light that excites the fluorescent dye added to the sample. The filter 34 reflects the excitation light and transmits the fluorescence emitted from the excited fluorescent dye. The lens 36 irradiates the electrophoresis plate so that the excitation light reflected by the filter 34 is condensed on the flow path 12 through the through hole 22. The detector 38 detects the fluorescence emitted from the fluorescent dye. Here, a straight line passing through the lens 36, the filter 34, and the detector 38 is the optical axis Oa of the detection optical system 30.

The control unit 40 controls the moving unit 24 to move the electrophoresis plate 1 on the heating block 20 so that the optical axis Oa crosses the flow path 12. While moving the electrophoresis plate 1, the substrate 10 is irradiated with excitation light from the light source 32 of the detection optical system 30. The position of the end of the flow path 12 is determined from the change in the intensity of the detection light detected by the detector 38. The optical axis Oa is aligned with the center of the direction orthogonal to the extending direction of the flow path 12 obtained from the determined position of the end of the flow path 12.

As the substrate 10, a transparent substrate such as quartz glass, borosilicate glass, and resin is used. The width of the channel 12 is in the range of about 50 μm to about 100 μm. A metal such as aluminum (Al) is used as the heating block 20. As the moving unit 24, an eccentric cam of a motor-driven circular disk or the like is used. For example, if the circular disc is eccentric about 0.5 mm, an adjustment stroke of about 1 mm can be obtained.

As the light source 32 of the detection optical system 30, a laser light source such as a laser diode (LD) that emits excitation light having a wavelength of less than about 500 nm is used. As the filter 34, a filter such as a dichroic mirror, a long pass filter (LPF), or a combination of a dichroic mirror and an LPF is used. The filter 34 reflects excitation light having a wavelength of less than about 500 nm and transmits light having a wavelength of about 500 nm or more. The lens 36 collects excitation light in the flow path 12. As the detector 38, an optical amplification detector such as a photomultiplier tube (PMT) is used. The control unit 40 may be configured by dedicated hardware, or may have a substantially equivalent function by software using a central processing unit (CPU) of a normal computer system.

For example, as shown in FIG. 3, the substrate 10 includes a transparent first substrate 10a having a flow path 12 formed on the surface and a transparent second substrate 10b bonded to the surface of the first substrate 10a. The channel 12 is formed by photolithography and etching such as wet etching or dry etching. For example, the channel 12 has

curved end portions

2a and 2b having a convex curvature outward, and a flat bottom portion 4 between the

end portions

2a and 2b. In addition, the shape of the

edge parts

2a and 2b is not limited. Depending on the etching conditions, the end of the flow path 12 can be formed into a shape such as a substantially vertical cross section or a tapered cross section close to a straight line.

For example, the electrophoresis plate 1 is heated to about 50 ° C. to about 60 ° C. by the heating block 20 shown in FIG. The buffer solution is injected from the through hole 14a into the channel 12 that has been washed in advance. A sample to which a fluorescent dye is added is injected into the flow path 12 from the through hole 14b. Thereafter, the buffer solution is put into the

reservoirs

16a and 16b. An electrode (not shown) is inserted into each buffer solution of the

reservoirs

16a and 16b, and a sample is electrophoresed by applying a voltage. The sample reaches the vicinity of the optical axis Oa of the detection optical system 30 by electrophoresis. As shown in FIG. 3, the excitation light LB emitted from the light source 32 is condensed into the flow path 12 by the lens 36 through the filter 34. Fluorescence Fl is emitted from the fluorescent dye added to the sample by the excitation light. A part of the fluorescence Fl is detected by the detector 38 through the lens 36 and the filter 34.

In an electrophoresis apparatus used for a sequencer that determines the sequence of DNA or the like, a very small amount of fluorescent dye is added to a sample, and high-sensitivity detection is required. Therefore, it is necessary to align the optical axis Oa of the detection optical system 30 shown in FIG. For example, the width of the flow path 12 is about 100 μm, and the condensing diameter of the excitation light is about 30 μm to about 50 μm. It is required to align the optical axis Oa of the detection optical system 30 and the center of the flow path 12 with a high positional accuracy of about ± 20 μm in a cross section perpendicular to the direction perpendicular to the extending direction of the flow path 12. .

In the embodiment of the present invention, the optical axis of the detection optical system 30 is aligned with the flow path 12 using the detection optical system 30. As shown in FIG. 4, the direction orthogonal to the extending direction of the flow path 12 is taken as the x-axis. For example, with one end of the through hole 22 as the reference position O, the end 2a, the bottom 4 and the end 2b of the flow path 12 are respectively positioned between Xa and Xb, between Xb and Xc, and between Xc and Xd. Become.

2 controls the moving unit 24 and the detection optical system 30 to acquire the relationship between the position of the excitation light and the detection light intensity. Specifically, the electrophoresis plate 1 is moved so that the optical axis Oa traverses the flow path 12 while irradiating the substrate 10 with the excitation light emitted from the light source 32. As shown in FIG. 5, when excitation light is irradiated onto the substrate 10 at a position between the reference position and Xa, detection light with intensity Is due to autofluorescence of the substrate 10 is detected. When excitation light is irradiated between Xa and Xb or between Xc and Xd, detection light peaks Pl and Pr due to scattered light at the end surfaces of the

end portions

2a and 2b are detected. In addition, when excitation light is irradiated to a position between Xb and Xc, detection light having an intensity Ic is detected by autofluorescence such as a buffer solution injected into the flow path 12.

The alignment may be performed without injecting the solution into the flow path 12. In this case, as shown in FIG. 6, when the excitation light is irradiated to a position between Xb and Xc, the detection light by the autofluorescence of the substrate 10 decreases to the intensity Id according to the empty flow path 12.

Using the result of FIG. 5 or FIG. 6, the control unit 40 determines the center of the flow path 12. For example, the peak positions X1 and Xr of the detection light peaks Pl and Pr are obtained, and the center position of the flow path 12 is determined from (X1 + Xr) / 2. Alternatively, the region showing the substantially flat detection light intensity Ic or Id substantially corresponds to the position between Xb and Xc of the bottom 4 of the flow path 12. Therefore, the center position of the flow path 12 can be determined from (Xb + Xc) / 2. Similarly, the rising positions of the detection light peaks Pl and Pr from the detection light intensity Is substantially correspond to the positions Xa and Xd at both ends of the flow path 12. Therefore, the center position of the flow path 12 can be determined from (Xa + Xd) / 2. Furthermore, since the width of the flow path 12 is known, it is possible to determine the center position of the flow path 12 by determining at least one of the positions Xl, Xr, Xa, Xb, Xc, and Xd. is there.

In the embodiment of the present invention, the position of the flow path 12 is determined by irradiating the substrate 10 including the flow path 12 with excitation light using the detection optical system 30 provided in the electrophoresis apparatus. Therefore, the position of the flow path 12 is determined without depending on the processing accuracy of the flow path 12 and without being affected by fine particles such as dust which becomes a problem when the electrophoresis plate 1 is installed in the heating block 20. be able to. As described above, in the electrophoresis apparatus according to the embodiment of the present invention, the excitation light can be aligned with the flow path 12 with high accuracy.

(Positioning method)
Next, the alignment method according to the embodiment of the present invention will be described using the flowchart shown in FIG. In advance, a buffer solution is injected into the flow path 12 of the electrophoresis plate 1 shown in FIG. The control unit 40 controls the moving unit 24 to move the optical axis Oa of the detection optical system 30 to a predetermined reference position.

In step S100, the excitation light of the light source 32 is applied to the substrate 10 of the electrophoresis plate 1 through the filter 34 and the lens 36.

In step S101, the electrophoresis plate 1 is moved by the moving unit 24 in a direction orthogonal to the direction in which the flow path 12 extends.

In step S102, detection light emitted from the substrate 10 is detected by the detector 38 while moving the excitation light across the flow path 12.

In step S103, the position of the

end portions

2a and 2b of the flow path 12 is determined by the control unit 40 from the change in detected light intensity with respect to the position of the excitation light. The control unit 40 determines the center position of the flow path 12 from the positions of the

end portions

2 a and 2 b of the flow path 12. Thereafter, the optical axis Oa of the detection optical system 30 is aligned with the center position of the flow path 12 by the moving unit 24.

In the alignment method according to the embodiment of the present invention, the position of the flow path 12 is determined by irradiating the substrate 10 including the flow path 12 with excitation light using the detection optical system 30 provided in the electrophoresis apparatus. Therefore, the excitation light can be aligned with the flow path 12 with high accuracy.

(Other embodiments)
As described above, the present invention has been described according to the embodiment of the present invention. However, it should not be understood that the description and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples and operational techniques will be apparent to those skilled in the art.

In the embodiment of the present invention, the moving unit 24 is disposed on the lower surface of the heating block 20. However, the moving unit 24 may be disposed in the detection optical system 30. In this case, the electrophoresis plate 1 on the upper surface of the heating block 20 is fixed, and the detection optical system 30 is moved by the moving unit 24.

Thus, it goes without saying that the present invention includes various embodiments not described herein. Accordingly, the technical scope of the present invention is defined only by the invention specifying matters according to the scope of claims reasonable from the above description.

The present invention can be applied to an electrophoresis apparatus used for a sequencer for determining the sequence of DNA or the like.

Claims

A transparent substrate provided with a flow path for electrophoresis inside;
A detection optical system for irradiating the substrate with excitation light and detecting detection light excited by the excitation light;
A moving unit that moves at least one of the substrate and the detection optical system in a direction orthogonal to a direction in which the flow path extends;
A control unit that determines the position of the end of the flow path from the intensity change of the detection light detected with the movement and aligns the excitation light with the flow path. apparatus.
The electrophoresis apparatus according to claim 1, further comprising an electrophoresis plate on which the substrate is mounted.
The electrophoresis apparatus according to claim 2, wherein the moving unit includes a flat-core cam that drives the electrophoresis plate.
2. The electrophoresis apparatus according to claim 1, wherein the excitation light is laser light.
2. The electrophoresis apparatus according to claim 1, wherein the detection light is scattered light at an end of the flow path excited by the excitation light.
2. The electrophoresis apparatus according to claim 1, wherein the detection light is autofluorescence of the substrate excited by the excitation light.
Irradiate excitation light to a transparent substrate with a flow path for electrophoresis inside,
Detecting detection light excited by the excitation light while moving the excitation light in a direction perpendicular to the direction in which the flow path extends,
Determine the position of the end of the flow path from the change in intensity of the detection light,
Aligning the excitation light with the flow path.
The alignment method according to claim 7, wherein the excitation light is laser light.
The alignment method according to claim 7, wherein the detection light is scattered light at an end of the flow path.
10. The alignment method according to claim 9, wherein the position of the end of the flow path is determined from the peak position of the scattered light.
The alignment method according to claim 7, wherein the detection light is autofluorescence of the substrate excited by the excitation light.
12. The alignment method according to claim 11, wherein the position of the end of the flow path is determined from a flat part of autofluorescence of the substrate.