JPH11160278A - Microchip electrophoretic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve detecting accuracy and operability of a microchip electrophoretic apparatus. SOLUTION: Incident light from an optical system is illuminated to a slit 13. The slit 13 is formed on a surface of a microchip 9 and has an opening only on a separate channel 11. The light passed through the slit 13 is illuminated only to the channel 11. As a result, its detecting accuracy can be enhanced. And, when the microchip 9 is fixed to a predetermined position of the electrophoretic apparatus, even if it is not considerably severely positioned, the light can be illuminated only to the channel 11 via the slit 13. As a consequence, in the case of frequently replacing the chip, it can be efficiently replaced, and hence its operability can be improved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoresis apparatus for analyzing a very small amount of sample at ultra-high speed and high separation, and more particularly, to a separation flow formed on the inside by bonding two transparent plate members. The present invention relates to a microchip electrophoresis apparatus using a capillary electrophoresis chip for performing electrophoresis on a road.

[0002]

2. Description of the Related Art Electrophoresis has been conventionally used for analyzing very small amounts of proteins, nucleic acids, and the like, and a capillary electrophoresis apparatus is an example of a technique for realizing the apparatus. A capillary electrophoresis device is a device in which a glass capillary having an inner diameter of 100 μm or less is filled with an electrophoresis buffer, a sample is introduced into one end, and a high voltage is applied between both ends to separate and develop an analyte in the capillary. It is. Since the inside of the capillary has a large surface area with respect to the volume, that is, high cooling efficiency, a high voltage can be applied, and a very small amount of sample such as DNA can be analyzed at high speed and with high resolution.

[0003] In recent years, the use of fused silica
DJ Harrison et al./Ana is a form in which faster analysis and smaller equipment can be expected in place of capillaries.
As shown in l. Chim. Acta 283 (1993) 361-366, a capillary electrophoresis chip (referred to as a microchip) formed by joining two substrates has been proposed. FIG. 1 shows an example of the microchip. A pair of transparent substrates (generally, glass, quartz, resin, etc.)
Electrophoresis capillary grooves 54 and 55 that partially cross each other are formed on the surface of one substrate 52 by etching or the like, and the other substrate 51 has reservoirs 53 at positions corresponding to the ends of the grooves 54 and 55. It is provided as a through hole. The volume of the reservoir 53 is determined by the hole diameter and the thickness of the substrate 51.

When this microchip is used, both substrates 51 and 52 are overlapped as shown in FIG. 1C, and the electrophoresis running solution is injected into the grooves 54 and 55 from one of the reservoirs 53.
Thereafter, the sample is injected into the reservoir 53 at one end of the shorter groove 54, and a high voltage is applied between the reservoirs 53 at both ends of the groove 54 for a predetermined time. As a result, the sample is dispersed in the groove 54. Next, an electrophoresis voltage is applied to the reservoirs 53 at both ends of the longer groove 55. This allows
The sample existing at the intersection 56 between the two grooves 54 and 55 is
Electrophoresis inside. Since the volume of the sample developed in the groove 55 is extremely small, for example, a laser detector is used for detection. A detector is arranged at an appropriate position in the groove 55, and a laser beam is focused on one point on the groove 55 corresponding to the position, thereby sequentially detecting separated samples carried in order by electrophoresis. I do. The focus of the laser light is adjusted to an appropriate position by moving the laser light or adjusting a positioning device to which the microchip is fixed.

In an electrophoresis apparatus using a capillary, a UV (ultraviolet) absorption detector is widely used. Since the capillary has a circular cross section, it can be easily fixed to a fixing member having a V-shaped fixing position. By forming a slit having a width equal to or smaller than the inner diameter of the capillary in the fixing member, light can be applied only to the flow path through which the sample passes.

An electrophoresis apparatus using a microchip has a U
When applying V-absorption detection, a slit is placed between the incident optical system and the microchip to block stray light components that pass through other than the flow path in order to converge light from the light source only on the flow path. There must be. 2A and 2B show an example of a case where light is applied to the entire separation channel and light transmitted through the channel is detected by a linear array photodetector. FIG. 2A is a perspective view showing the vicinity of a detection unit, and FIG. Is a cross-sectional view in a direction perpendicular to the separation channel of the microchip, and (C) is a separation channel 5 in a circle in (B).
It is an expanded sectional view of.

In order to irradiate incident light from an optical system (not shown) to only the separation channel of the microchip 3, the slit 1 is provided on the light incident side of the microchip 3 with a rectangular opening. On the opposite side of the microchip 3, there is provided a PDA (photodiode array) 7 in which a plurality of photodetectors for detecting light transmitted through the separation channel are arranged. A separation channel 5 is formed in the microchip 3. For example, the slit 1 in which the width of the opening is smaller than 50 μm
When light is incident on the chip 3, the light passing through the slit hits the separation channel 5 (for example, 50 μm in width) on the chip 3, and the refractive index effect of the electrophoresis solution in the separation channel 5 and the chip 3 and the separation channel 5
The light transmitted through the separation channel 5 spreads due to the etching shape described above, and impinges on the PDA 7 (B). Then, by analyzing the detection data from the PDA 7, the separated components of the sample are confirmed.

[0008]

In the example as shown in FIG. 2, in addition to the distance between the slit 1 and the microchip 3,
Due to the thickness of the microchip 3 itself, the distance from the light passing through the slit 1 to the separation channel 5 becomes longer than the width of the separation channel 5, so that the optical axis adjustment becomes difficult. In particular, when it is assumed that the microchip 3 is frequently replaced, it takes time and effort to adjust the focal point of the light to the separation channel 5, and a large problem remains in operability. In addition, if the slit 1 is formed using a metal material that is easy to process by etching or the like, there is a possibility that an electrode (not shown) provided on the microchip 3 may discharge in order to apply a migration voltage. If the distance between the slit 1 and the microchip 3 is increased, the above problem becomes more serious.

Therefore, an object of the present invention is to improve the detection accuracy and operability of a microchip electrophoresis apparatus using a microchip by simply and accurately focusing light on a separation channel. is there.

[0010]

A microchip electrophoresis apparatus according to the present invention includes a pair of transparent plate members, wherein at least one of the plate members has a surface on which a groove for flowing a liquid is formed, and the other plate member. The plate is provided with a through hole at a position corresponding to the groove, these plate-shaped members are bonded with the groove inside, forming a separation channel and a sample introduction channel partially intersecting with each other by the groove. In a microchip electrophoresis apparatus for measuring light absorption or fluorescence of a part or the whole of a separation channel as a detection means, using a capillary electrophoresis chip as the capillary electrophoresis chip,
A plate-like member having a slit having an opening only on the separation channel is formed on the outer surface on the light incident side of the plate member.
A slit is formed directly on the surface of the plate-shaped member so that the distance between the slit and the separation channel is minimized, and light having a width equal to or less than the channel width is passed. Since the slit is formed so as to open only on the separation channel, light is applied only to the separation channel.

[0011]

3A and 3B are views showing an embodiment, in which FIG. 3A is a perspective view, and FIG. 3B is a cross-sectional view in a direction perpendicular to the separation channel of the microchip. A slit 13 having an opening only on the separation channel 11 and made of, for example, an organic thin film material is formed on the surface of the microchip 9 on the side where light is irradiated. Examples of the organic thin film include a polyimide-based material (e.g., polyimide isoindoloximezolinedione) which has excellent insulating properties and can be hardened and formed flat on the microchip surface only by heating, but is not limited thereto. .
On the opposite side of the microchip 9, a PDA 15 in which a plurality of photodetectors for detecting light transmitted through the separation channel 11 are arranged.
Is provided.

The slit 13 is irradiated with incident light from an optical system (not shown). Since the slit 13 is formed on the surface of the microchip 9 and has an opening only on the separation channel 11, the incident light passing through the slit 13 is applied only to the separation channel 11. As a result, detection accuracy can be improved. Further, when the microchip 9 is fixed at a predetermined position of the electrophoresis apparatus, the slit 13 can irradiate only the separation channel 11 with incident light, even if the microchip 9 is not precisely positioned. As a result, when the chip is frequently replaced, the chip can be replaced easily, so that the operability can be improved.

The sample components are separated by electrophoresis from the separation channel 11.
While moving through the inside, the outputs from each light receiving element of the PDA 15 are collectively captured at regular time intervals, and then the moving speed of each component is calculated and corrected, so that the migration time is equal to the number of light receiving elements. Get response information. After the analysis, the integrated averaging process of each component is performed. Also, after the time for separating the target component has elapsed, the application of the electrophoresis voltage is stopped,
The electrophoresis may be stopped, and during that time, the data may be repeatedly taken and averaged.

Next, an example of a method for manufacturing the slit 13 made of, for example, polyimide will be described. For example, a solution-like prepolymer is dropped on the chip 9, the chip 9 is rotated at a high speed to perform spin coating, and the thickness of the prepolymer is made uniform. Perform formation. Next, using a laser whose output is sufficiently controlled (for example, YAG or excimer),
An opening of about 50 μm is formed on the separation channel 11. When spin coating the prepolymer, the separation channel 11
Paste a slit-shaped film on top and mask it,
After removing the mask after spin coating, sintering may be performed. By using an organic insulating material for the slit 13,
A short circuit with an electrode for applying a high voltage to the separation channel during electrophoresis can be avoided.

FIG. 4 is a diagram showing the result of examining the spectral transmittance of the polyimide film. The sample was Kapton 200F
001, Kapton 200F919, Kapton 300F0
21, Kapton black polyimide (the above are manufactured by Toray DuPont), Upilex 25R 25 μm, 50R 50 μ
m, 25S 25 μm, 50S 50 μm (the above are manufactured by Ube Industries). As shown in FIG. 4, these samples can sufficiently block the light in the ultraviolet region and can be used as slits. When black polyimide is used, it can be used as a slit even in the visible region.

FIG. 5 is a diagram showing another embodiment.
(A) is a top view, and (B) is a cross-sectional view at the AA ′ position in (A). Cover plate 17a and base plate 1
A separation channel 19a and a sample channel 19b formed by, for example, etching are formed on a base plate 17b of the microchip 17 composed of 7b. The cover plate 17a has a reservoir 21 having a through hole at a position corresponding to the separation channel 19a and the sample channel 19b.
Are formed respectively. The periphery of the reservoir 21 is made of, for example, an Au film.
A for applying a voltage to the sample channel 9a and the sample channel 19b
The u electrode 23 is formed. Also, cover plate 1
An Au pattern 25 serving as a slit having an opening on the separation channel 19a is formed on 7a. An insulating film 2 such as a silicon oxide film so as to cover the Au pattern 25;
7 are formed.

When the Au electrode 23 is formed by, for example, photolithography and etching, an Au pattern 25 serving as a slit is formed at the same time, and then an insulating film 27 is formed so that at least the Au pattern 25 is completely covered. , Can create slits. When the insulating film 27 is formed using, for example, a silicon oxide film, a monosilane gas (SiH ₄ ) is thermally oxidized in an oxidizing atmosphere and deposited on the Au pattern 25 by a CVD (Chemical Vaper Deposition) method, and is deposited by a sputtering method. ,
The silicon oxide film is beaten by bombarding the quartz glass with Ar ions and deposited on the Au pattern 25. By forming the insulating film 27 on the Au pattern 25, a short circuit between the Au electrode 23 and the Au pattern 25 can be prevented. Although a silicon oxide film was used as the insulating film, the present invention is not limited to this.

[0018]

The microchip electrophoresis apparatus according to the present invention uses a microchip in which a slit having an opening only on the separation channel is formed on the outer surface of the microchip on the side irradiated with incident light. It is possible to irradiate the separation channel with light while minimizing stray light without strictly adjusting the separation channel and the optical axis formed on the separation channel. As a result, light can be easily and accurately focused on the separation flow path, frequent replacement of microchips is possible, and detection accuracy and operability of the microchip electrophoresis apparatus can be improved.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating an example of a conventional microchip.

FIG. 2 is an example of a case in which light is applied to the entire separation channel and light transmitted through the channel is detected by a linear array photodetector;
(A) is a perspective view, (B) is a cross-sectional view in a direction perpendicular to the separation flow path of the microchip, and (C) is an enlarged cross-sectional view of the separation flow path 5 in a circle in (B).

FIGS. 3A and 3B are diagrams illustrating an embodiment, in which FIG.
(B) is a cross-sectional view in a direction perpendicular to the separation channel of the microchip.

FIG. 4 is a diagram showing a result of examining a spectral transmittance of a polyimide film.

FIG. 5 is a diagram showing another embodiment, in which (A) is a top view,
(B) is a cross-sectional view at AA 'position in (A).

[Explanation of symbols]

 9 Microchip 11 Separation channel 13 Slit 15 PDA

Claims (1)

[Claims] 1. A pair of transparent plate-shaped members, wherein a groove through which a liquid flows is formed on a surface of at least one of the plate-shaped members, and a hole penetrating the other plate-shaped member at a position corresponding to the groove. A capillary electrophoresis chip is used in which these plate-shaped members are bonded together with their grooves inside, and a separation channel and a sample introduction channel partially intersecting with each other by the grooves are used. In the microchip electrophoresis apparatus for measuring light absorption or fluorescence of a part or the whole of the plate, the capillary electrophoresis chip has a slit having an opening only on the separation channel on the outer surface of the plate member on the light incident side. A microchip electrophoresis apparatus, characterized in that a microchip is formed.