JPH11326274A - Member for electrophoresis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a member which enhances the separating capability of the electrophoresis of a microchip. SOLUTION: In order to guide a sample to a crossing part 12 on the surface of a baseplate 4 at a microchip 2, a voltage of 1.2 kV is applied to reservoirs 18a to 18c, 18f, a voltage of 1.0 kV is applied to a reservoir 18e, and a reservoir 18d is grounded. At this time, the potential of the crossing part 12 becomes about 0.87 V. A sheath flow which is shown in (C), (D) is formed in a sample flow passage 10 by an electroosmotic flow. The sample is guided to the crossing part 12 along a slanted line part (the central part of the sheath flow). A voltage of 1.0 kV is applied to the reservoir 18d so that the sample which is guided to the crossing part 12 is guided to an analytical flow passage 8, and the reservoir 18f is grounded. In order to prevent the sample in an excessive amount from flowing into from the sample flow passage 10, a voltage of 0.5 kV is applied to the reservoirs 18a to 18c, 18d. In this manner, a thin sample plug can be introduced into the analytical flow passage 8 as shown in (E).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a member used in an electrophoresis apparatus for analyzing a very small amount of sample at high speed and high separation. The present invention relates to an electrophoretic member having a passage formed therein.

[0002]

2. Description of the Related Art In the case of analyzing a very small amount of protein, nucleic acid, or the like, an electrophoresis method has been conventionally used, 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 at one end side, and a high voltage is applied between both ends to develop an analyte in the capillary. is there. 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 D
A very small amount of sample such as NA can be analyzed at high speed and with high resolution.

In recent years, DJ Harrison et al./Science 261 (19) has been proposed as a form that can be expected to increase the speed of analysis and reduce the size of the apparatus in place of a glass capillary that is complicated to handle.
93) 895-897 and Anal. Chim. Acta 283 (1993) 361-366.
As shown in (1), a chip (referred to as a microchip) used for capillary electrophoresis 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.) 51 and 52 are formed. A sample flow path 54 and an analysis flow path 55 are formed on the surface of one substrate 52 so as to intersect each other. Channel 54 and analysis channel 5
The reservoir 53 is provided as a through hole at a position corresponding to the end of the fifth.

When this microchip is used, both substrates 51 and 52 are overlapped as shown in FIG. 1C, and an electrophoresis running liquid is injected from one of the reservoirs 53 into a sample channel 54 and an analysis channel 55. Thereafter, a sample is injected into the reservoir 53 at one end of the sample flow path 54, electrodes are inserted into the respective reservoirs 53, and a predetermined high voltage is applied only for a predetermined time. As a result, the sample flows into the sample channel 54 and the analysis channel 55.
Is moved to the intersection 56 of. Next, a predetermined voltage for electrophoresis is applied to each reservoir 53. Thus, the sample existing at the intersection 56 electrophoreses in the analysis channel 55.
By arranging a detector at an appropriate position in the analysis channel 55, a separated component is detected.

[0005]

When the sample is guided to the intersection 56, the shape of the sample (also called a sample plug) at the intersection 56 depends on the gradient of the potential, and the shape becomes a wedge or trapezoid. . When an electrophoresis voltage is applied to separate the sample, the sample is guided to the analysis channel 55 while keeping the wedge or trapezoid. Therefore, the length of the sample plug differs depending on the position in the width direction of the analysis channel 55. This is thought to degrade the resolution and is not desirable.

Accordingly, an object of the present invention is to improve the separation performance of microchip electrophoresis by making the shape of a sample plug in an analysis channel a thin layer having a uniform thickness.

[0007]

The electrophoretic member according to the present invention comprises a pair of transparent plate members, and at least one of the plate members has a surface on which a groove for flowing liquid is formed. Is provided with a through hole at a position corresponding to the groove, and these plate-like members are bonded together with the groove inside to form a flow path by the groove, and the groove separates the sample. An analysis flow path, a sample flow path that intersects with the analysis flow path and guides the sample to the intersection, a third flow path that intersects with the sample injection side end of the sample flow path and the intersection, It is provided with.

[0008] The analysis flow path, the sample flow path, and the third flow path are filled with an electrophoresis buffer, and a sample is injected into one end of the sample flow path. When a predetermined voltage for sample introduction is applied to each end of the analysis channel, the sample channel, and the third channel, one end where the sample of the sample channel is injected from the end of each channel is the other end. A flow is created by the electro-osmotic flow towards the side. Thereby, the sheath flow from the intersection of the sample flow path and the third flow path to the other end side of the sample flow path through the intersection of the sample flow path and the analysis flow path to the other end side. Is formed. Since the sample is guided to the intersection of the sample channel and the analysis channel along the center of the sheath flow, the shape of the sample at the intersection is a uniform layer. Thereafter, when a predetermined voltage for electrophoresis is applied to each end of each flow channel, the sample is led to the analysis flow channel as a layer having a uniform thickness.

[0009]

FIG. 2 shows an embodiment, in which (A) is a front view and (B) is an embodiment.
Is a plan view, (C) and (D) are enlarged schematic diagrams showing the flow of the sample at the intersection of the flow paths in (B) when the sample is introduced, and (E) is the migration of the sample during electrophoresis in (D). FIG. The microchip 2 includes a base plate 4 made of quartz and a cover plate 6. On the surface of the base plate 4, grooves intersecting each other are formed as an analysis channel 8 and a sample channel 10, forming an intersection 12. A third channel that intersects between the sample injection side end of the sample channel and the intersection 12 is formed as a sheath-forming channel 14, and the intersection 1
6 are formed.

In the cover plate 6, the reservoirs 18a, 18b, 18c, 18d, 18e, and 1 are located at positions corresponding to the ends of the analysis channel 8, the sample channel 10, and the sheath-forming channel 14.
8f is provided as a through hole. The length of the analysis channel 8 is 28 mm, and intersects with the sample channel 10 at a position 7 mm from the end on the reservoir 18e side to form an intersection 12. The length of the sample channel 10 is 14 mm, and the reservoir 1
At a position 3.5 mm from the end on the side of 8a,
4 intersects with the analysis channel 8 at a position of 7 mm. The length between the intersections 12, 16 is 3.5 mm. The analysis channel 8, the sample channel 10, and the sheath forming channel 14 have a width of 50 μm and a depth of 20 μm.

FIG. 3 is a schematic perspective view showing one embodiment of a microchip electrophoresis apparatus to which this embodiment is applied. FIG.
In order to irradiate a predetermined range of the analysis flow channel 8 of the microchip 2 with light, light from a light source 20 linearly extending along the analysis flow channel 8 is converted into parallel light by a cylindrical lens 22, and a band-pass filter 24. And the light having a predetermined wavelength transmitted through the band-pass filter 24 is condensed by the cylindrical lens 26 into the separation channel of the microchip 2 and is incident. On the opposite side of the microchip 2, a cylindrical lens 28 is provided for condensing the light transmitted through the separation channel, and the light condensed by the cylindrical lens 28 is incident on a photocell array 30 of a photodetector and detected. Is done. The cylindrical lenses 22, 26, and 28, the bandpass filter 24, and the photocell array 30 are shorter than the portion of the analysis channel 8 where the sample migrates, but have substantially the same length as the portion of the analysis channel 8 where the sample migrates. doing.
The photocell array 30 includes a plurality of photodiodes arranged as detection elements on a straight line in the length direction of the analysis channel.

The operation of this embodiment will be described with reference to FIGS. For example, calcein having a concentration of 5 μM is injected as a sample into the reservoir 18a, and a tris-borate buffer (p
H8.2) was injected as an electrophoresis buffer to fill the flow path with the electrophoresis buffer. At this time, each of the reservoirs 18a to 18f
Make the liquid level equal.

Each of the reservoirs 18a to 18f has an electrode pattern formed thereon, or otherwise has an electrode (not shown).
Insert In order to guide the sample to the intersection 12, 1.2 kV is applied to the reservoirs 18a to 18c, 18f and the reservoir 18e.
, A voltage of 1.0 kV is applied, and the reservoir 18d is grounded. At this time, the potential of the intersection 12 is about 0.78V. Electroosmotic flow occurs from the reservoirs 18a to 18c, 18e, 18f toward the reservoir 18d, and the sample flow path 10
, A sheath flow as shown in FIGS. 2C and 2D is formed. The sample is guided to the intersection 12 by migrating the hatched portions in FIGS. The shape of the sample at the intersection 12 is a thin layer along the oblique line (the center of the sheath flow).

Next, a voltage of 1.0 kV is applied to the reservoir 18e and the reservoir 18f is grounded in order to guide the sample led to the intersection 12 to the analysis channel 8. In order to prevent an excessive amount of the sample from flowing from the sample channel 10, a voltage of 0.5 kV is applied to the reservoirs 18a to 18c and 18d. In this way, a thin sample plug can be introduced into the analysis channel as shown in FIG.

In the embodiment shown in FIG. 3, the light from the light source 20 is converted into parallel light by the cylindrical lens 22, passes through the band-pass filter 24, becomes light of a specific wavelength only, and is analyzed by the cylindrical lens 26 on the microchip 2. Light is collected on the path 8. The light that has passed through the analysis channel 8 is converted into parallel light again by the cylindrical lens 28 and is irradiated on the photocell array 30. In order to detect the separated sample, scanning is repeatedly performed within a range not exceeding the saturation charge amount of the junction capacitance of the photodiode of the photocell array 30, and the obtained data is subjected to integration and averaging processing, and the band of the absorbed sample component is peaked. As an imaging output. By temporarily stopping the migration of the sample during scanning, the S / N ratio can be improved in proportion to the square root of the number of scans. Since the sample plug introduced into the analysis channel 8 is a thin layer having a uniform thickness in the width direction of the separation channel 8,
The separation state of the sample can be improved.

In this embodiment, a microchip electrophoresis apparatus which performs light detection over a wide range of the analysis flow path is used, but a microchip electrophoresis apparatus which performs light detection at a specific position in the analysis flow path may be used. The microchip according to the present invention is a device that can apply a desired voltage to each reservoir.
It can be applied to any microchip electrophoresis apparatus. Further, in this embodiment, the sheath flow is formed by supplying the liquid by applying a voltage, but the sheath flow may be formed by using pressure.

[0017]

As described above, the electrophoretic member according to the present invention comprises an analysis channel for separating a sample, a sample channel that intersects the analysis channel and guides the sample to the intersection, and an end of the sample channel on the sample injection side. Intersecting between the section and the intersection, and a sheath forming flow path for forming a sheath flow at the intersection with the analysis flow path. Since the sample is introduced into the analysis channel as a thin layer having a uniform thickness by forming a sheath flow, the resolution of electrophoresis can be improved.

[Brief description of the drawings]

FIGS. 1A and 1B are diagrams showing a microchip, and FIGS. 1A and 1B are plan views showing a transparent plate member constituting the microchip; FIGS.
(C) is a front view of the microchip.

2 (A) is a front view, FIG. 2 (B) is a plan view, and FIGS. 2 (C) and 2 (D) show the flow of the sample at the intersection of the flow paths in FIG. 2 (B). (E) is a schematic diagram showing the migration of the sample during electrophoresis in (D).

FIG. 3 is a schematic perspective view illustrating an embodiment of a microchip electrophoresis apparatus to which the embodiment is applied.

[Explanation of symbols]

2 Microchip 4 Base plate 6 Cover plate 8 Analysis flow path 10 Sample flow path 12, 16 Intersection 14 Sheath formation flow path 18a, 18b, 18c, 18d, 18e, 18f
Reservoir

Claims (1)

[Claims] 1. A plate comprising a pair of transparent plate members, a groove through which a liquid flows is formed on a surface of at least one of the plate members, and a through hole is provided at a position corresponding to the groove in one of the plate members. In an electrophoresis member in which these plate-shaped members are bonded together with the groove inside and the groove forms a flow path, the groove includes an analysis flow path for separating a sample, and the analysis flow path. A sample flow path that intersects and guides the sample to the intersection, and a third flow path that intersects between the sample injection side end of the sample flow path and the intersection. Electrophoresis components.