JP2002148236A - Electrophoretic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an electrophoretic apparatus whose detection accuracy is enhanced by a method wherein the flow direction of an electroosmotic flow in a part of an analytical flow channel on the side opposite to a detector is reversed regarding a flow-channel crossing part. SOLUTION: The flow direction of the electroosmotic flow in an analytical supply flow channel 31 as a part of the analytical flow channel 3 is reversed. Thereby, the width of a sample region inside the flow channel 3 is narrowed, and the flow into the flow channel 3 of a sample as an object, to be analyzed, to become a detection noise is prevented. Since the width is made narrow at a point of time when the sample is introduced to the flow channel 3, a region in which adjacent sample zones are overlapped is reduced, the noise is reduced, and the detection accuracy of the electrophoretic apparatus is enhanced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoresis apparatus used for analyzing proteins, peptides, amino acids, neurotransmitters, hormones, nucleic acids and the like in living organisms and trace substances contained in environment, food, medicine and the like. .

[0002]

2. Description of the Related Art In recent years, a sample flow path and an analysis flow path which are composed of a pair of transparent substrates and cross each other are formed on the surface of one substrate, and the other substrate corresponds to the ends of the sample flow path and the analysis flow path. An electrophoresis apparatus having a reservoir as a through hole at a position where the electrophoresis is performed has been actively developed. A high voltage is applied to the electrodes inserted into each reservoir, and the sample existing at the intersection of the flow paths is electrophoresed in the analysis flow path. In a sample that is a mixed solution composed of multiple components, the speed of electrophoresis is different for each component, and therefore, each sample is separated in the analysis channel. This technique is generally called an electrophoresis chip, and enables a very small amount of an object to be analyzed.

[0003] Such a conventional electrophoresis apparatus is disclosed in Japanese Patent Application Laid-Open No. H11-326274. In this apparatus, a sheath formation forming channel for forming a sheath flow at an intersection with the analysis channel is provided. At the time of sample introduction, a sheath flow is formed at the intersection of the sample channel and the analysis channel. The sample is introduced into the analysis channel as a thin layer having a uniform thickness.

[0004] Further, there is one described in Japanese Patent Application Laid-Open No. 10-10088. In this apparatus, an electroosmotic flow pump is connected to flow a buffer in a direction opposite to the flow of the electroosmotic flow, thereby effectively increasing the flow path length.

[0005]

However, in the electrophoresis chip, when the sample is introduced from the sample introduction channel into the analysis channel at the intersection of the channels before the start of the analysis, molecular diffusion, electric field, etc. Is introduced into the analysis flow channel while expanding from the flow channel intersection. Japanese Patent Application Laid-Open No. H11-326274 discloses that the influence of spreading a sample on a flow path for analysis when a sample is applied to a flow path for sample introduction by applying a voltage to the flow path intersection is controlled. When switching from both ends of the introduction flow channel to both ends of the analysis flow channel, the effect of spreading from the crossing of the flow channel to the analysis flow channel is not considered, and there is a problem in that the analysis target amount increases and the detection accuracy decreases. In the apparatus disclosed in Japanese Patent Application Laid-Open No. H10-10088, it is necessary to separately prepare a pump flow path and a power supply therefor, which complicates the apparatus.

In a conventional electrophoresis chip, even after a sample is cut from the sample introduction channel to the analysis channel, the sample is moved from the introduction channel by the electric field generated near the intersection of the channels. The sample continues to flow into the flow path, and the sample larger than the amount to be analyzed at the intersection of the flow path flows into the flow path for analysis. There was a problem to do.

[0007] It is an object of the present invention to provide an electrophoresis apparatus in which a part of a flow channel for analysis is provided with a part in which the flow direction of an electroosmotic flow is reversed in order to increase detection accuracy.

[0008]

The above object is attained by coating the wall of the analysis flow channel at a portion opposite to the detection side with respect to the flow channel intersection with the sample introduction flow channel.

[0009]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a top view of the configuration of an embodiment of an electrophoresis apparatus realized by the present invention, wherein FIG.
(B) is an enlarged view near the intersection of the flow paths.

In FIG. 1A, an electrophoresis substrate 11
Is an electrophoresis chip provided with a sample introduction channel 2 and an analysis channel 3. The two flow paths intersect at a flow path intersection 9. At both ends of each of the sample introduction channel 2 and the analysis channel 3, a migration buffer reservoir 4a, a migration buffer waste reservoir 4b, a sample reservoir 4c, and a sample waste reservoir 4d, which are solution reservoirs, are provided. The detector 8 is installed on the side of the electrophoresis buffer waste reservoir 4b in the drawing of the analysis channel 3. Channel power supply 1
0a and 10b are connected to the solution storage electrodes 5a to 5d.

In FIG. 1B, the flow direction of the electroosmotic flow is indicated by solid arrows. The sample supply flow path 21 of the sample introduction flow path 2, the sample supply flow path 21, the sample waste storage side of the sample waste flow path 22, the buffer storage side of the sample supply flow path 31, and the sample supply flow path 21. The detector side of the flow path 3 is referred to as an analysis waste flow path 32. An electric field is applied so that the electrode 5b becomes a positive electrode and the electrode 5a becomes a negative electrode.

Here, the electroosmotic flow is generally generated as follows. The channel wall surface is negatively charged when it comes into contact with the solution, and the negative charge attracts a positive charge in the solution to the channel wall surface and localizes it. When an electric field is applied to the flow path, the localized positive charges move toward the negative electrode, and at that time, the surrounding solution is dragged, so that the entire solution flows toward the negative electrode. In the present invention, the flow path wall of the analysis supply flow path 31 is coated to flow from the negative electrode to the positive electrode. Therefore, the electroosmotic flow flows to the detector side in the analysis supply channel 31 and
In the analysis waste flow channel 32, it flows to the side opposite to the detector side.

FIG. 2 is a top view of an operation example of an analysis process by an electrophoresis chip provided with a sample introduction channel and an analysis channel. The region 20 where the sample exists in the flow path is indicated by hatching.

In FIG. 2A, two flow paths, a sample introduction flow path 2 and an analysis flow path 3, are filled with a buffer solution, and the sample reservoir 4c is a mixed solution containing a multi-component. Inject. Solution storage electrodes 5c and 5d
The sample is subjected to electrophoresis by applying a high voltage from the flow channel power supply 10a to the sample collection channel 4d in the direction from the sample reservoir 4c to the sample waste reservoir 4d.
Introduce to the extent that exceeds.

Next, in FIG. 2 (B), by applying a high voltage from the flow channel power supply 10b to the solution storage electrode 5a and the solution storage electrode 5b, the sample cut out from the flow path intersection 9 is used for analysis. Electrophoresis is performed in the passage 3, and the analysis is started. Here, a sample separated for each component is preferably detected by fluorescence emission of the sample by a generally optical detector 8 installed at at least one point on the analysis channel 3. .

FIGS. 3 and 4 show flow paths in the analysis process shown in FIG. 2 from the time immediately after the sample is introduced into the flow path intersection 9 to the time immediately after the start of the analysis in which the electrophoresis is performed in the flow path 3 for analysis. It is the top view which expanded the vicinity of the intersection part 9. FIG. 3 shows the case of the conventional example, and FIG. 4 shows the case of the present invention, and shows (A1) to (A3) with time. The flow of the electroosmotic flow is indicated by a solid arrow, the flow of the buffer itself generated by the electroosmotic flow or generated by being pushed by the electroosmotic flow is indicated by a white arrow, and the migration of the sample is indicated by a thick broken arrow. Show. The sample to be analyzed is negatively charged.

When a sample is introduced from the sample introduction channel 2 into the analysis channel 3 near the channel intersection 9, it is affected by molecular diffusion, an electric field, and the like, and the sample is introduced from the channel intersection 9. It is introduced while spreading to 3. At this time, as shown in FIG. 3 (A1), the sample spreads from the channel intersection 9 to the analysis channel 3 side in a trapezoidal shape.

In FIG. 3 (A1), in the conventional example, when the direction of the analysis waste channel 32 is the positive electrode and the direction of the analysis supply channel 31 is the negative electrode with respect to the flow path intersection 9, the electroosmotic flow is From the analysis supply channel 31. The buffer is pushed out of the analysis waste flow channel 32 by the electroosmotic flow generated in the analysis waste flow channel 32, and the flow passage intersection 9
And flows into the sample supply channel 21 and the sample discard channel 22 in two directions.

In FIG. 3 (A2), the electric field is formed in the analysis flow channel 3 in a direction parallel to the longitudinal direction of the flow channel.
In the vicinity of the flow path intersection 9, the sample supply flow path 21 and the sample waste flow path 22 are formed in a swelling shape with the flow path intersection 9 interposed therebetween. Negatively charged sample 201
Out of 203, the sample 201 receives the force from the electric field formed in the sample supply flow path 21 and the sample waste flow path 22 while receiving the resistance from the flow of the buffer, in the direction of the flow path intersection 9. And flows into the analysis waste channel 32. The sample 202 migrates while receiving resistance from the flow of the buffer in the direction opposite to the migration direction, and flows into the flow channel intersection 9. The sample 203 migrates in the analysis waste channel 32 while receiving the resistance in the reverse direction from the buffer similarly to the sample 202.

In FIG. 3 (A3), the sample 20 existing in the sample supply channel 21 and the sample
Continues flowing into the analysis waste channel 32 due to the electric field formed in a bulging shape from the channel intersection 9, so that a sample area surrounded by an ellipse 90 is generated near the wall surface of the analysis waste channel 32. The analysis target to be electrophoresed in the analysis waste channel 32 is a sample in an area 91 surrounded by an ellipse, and the detector 8 detects the fluorescence emission intensity and the like generated in the area 91. At this time, the detector 8 also detects the light emission in the sample area surrounded by the ellipse 90, and the area 9
For 1 it is noise.

The sample 202 is supplied to the analysis supply channel 31.
Since electrophoresis is performed while receiving the resistance of the buffer flowing through the inside due to the electroosmotic flow, the length surrounded by the ellipse 91, which is the length of the sample area to be analyzed, becomes longer, and the sample areas for each component are easily overlapped, and detection is performed. It becomes difficult to do.

In FIG. 4 (A1), the flow direction of the electroosmotic flow in the analysis supply flow path 31 of the present invention is generated in a direction opposite to that of the conventional example. At this time, the flow of the buffer flowing from the analysis flow path 3 into the sample supply flow path 21 and the sample waste flow path 22 includes the flow from the analysis supply flow path 31 in addition to the flow from the analysis waste flow path 32 of the conventional example. As a result, the flow rate increases and the flow velocity also increases.

Therefore, in FIG. 4 (A2), the resistance of the negatively charged sample 201 from the buffer becomes larger than that in the conventional example of FIG. 3 (A2). Electrophoresis is performed in the direction of the analysis waste channel 32 by receiving the force from the electric field formed in the sample supply channel 21 and the sample waste channel 22 in a bulging shape from the analysis channel 3, but the analysis waste is performed. The amount that continues to flow into the flow path 32 is smaller than in the conventional example. Therefore, FIG.
The ellipse 9 near the wall of the analysis waste channel 32 in (A3)
The sample area surrounded by 0 is smaller than that in FIG. 3A3, the noise is reduced, and the detection accuracy is improved.

On the other hand, in FIG. 4 (A2), the sample 202 in the analysis supply channel 31 has the same electrophoresis direction and the buffer flow direction. The speed of flowing into 9 increases. The sample 203 migrates in the sample waste channel 32 while receiving the resistance in the reverse direction from the buffer. Therefore, in FIG. 4A3, the length of the ellipse 91, which is the length of the analysis target sample region, is shorter than that in FIG.

The effect of the sample area length on detection is as follows.

FIG. 5 shows an electrophoresis pattern when a mixed solution of two or more components is analyzed by the electrophoresis apparatus realized by the present invention. The flow path 3 for analysis is detected by the detector 8.
The case where the luminescence intensity of the fluorescence exhibited by the sample developed in the figure is recorded as waveform data with respect to the time axis is shown. The broken line indicates the conventional method and the solid line indicates the present invention.

FIG. 5A shows waveform data obtained when the detector 8 is installed in the vicinity of the flow path intersection 9.
With respect to the time axis, the leading position of the sample in the analysis waste channel 32 was set to the same position in the conventional example and the present invention. Compared to the conventional example, in the embodiment of the present invention, the sample migrates at a high migration speed in the analysis supply channel 31, so that the trailing end of the sample area is located forward in the time axis direction to have a narrow waveform, and the length of the ellipse 91 is longer. Becomes shorter.

FIG. 5B shows that two components in the sample solution were electrophoresed in the analysis channel 3 when the detector 8 was set apart from the channel intersection 9 in the analysis channel 3. It is an example of waveform data sometimes obtained. Regarding the comparison between the conventional example and the present invention example, the influence of diffusion or the like when developed in the analysis flow channel 3 is small, and therefore can be ignored. In the example of the present invention, since the electrophoresis is performed in the analysis channel in a sample area shorter than that of the conventional example, overlapping portions of the developed waveform data of two adjacent samples are reduced. Further, since the area on the graph obtained from the waveform data is the same in the present invention and that in the conventional example, the peak value of the waveform data, that is, the emission intensity increases as the length of the waveform data decreases. Therefore, since it is easy to determine the peaks of the waveforms of two adjacent samples from the waveform data, the detection accuracy is improved, and the detector 8 is simplified by not using an expensive and sensitive detection system. Becomes possible. Further, if the peak can be easily distinguished, the separation can be performed in a short flow path, so that the effect of miniaturizing the whole apparatus and increasing the speed can be obtained.

FIG. 6 shows an example of a method for manufacturing an electrophoresis chip according to the present invention.

In FIG. 6A, a sample introduction flow path is formed on a substrate 1a made of glass, such as quartz or Pyrex (registered trademark), a semiconductor material such as silicon, or a polymer such as PDMS (polydimethylsiloxane). 2 and an analysis channel 3 are provided. The processing method is preferably a general photo fabrication technology.

In FIG. 6B, the mask 1b is
a. The mask 1b is provided with a hole 31b through which only the analysis supply flow path 31 exits. By spraying a spray-like surface coating agent from above the mask 1b, only the analysis supply channel 31 is coated.

In the present invention, since the electroosmotic flow in the analysis supply channel 31 is generated so as to flow in the positive direction, the channel wall surface when the electroosmotic flow is generated is generally negatively charged. Contrary to the state, the flow path wall surface of the analysis supply flow path 31 needs to be positively charged. Therefore, when a glass material is used for the substrate 1a, for example, an acidic coating agent such as polyvinyl alcohol having a pH of about 2 or less may be used by spraying. After coating, the effect of the coating can be improved by heating and drying the substrate.

In FIG. 5 (C), a colorless and transparent substrate 1c, preferably made of the same material as the substrate 1a and having a through hole as each of the solution reservoirs 4a to 4d, is joined to the substrate 1a. An electrophoresis substrate 11 is obtained. Optical bonding is preferred as a joining method, and electric discharge machining is preferred as a processing method of a through hole.

The present invention has the following effects by adopting the structure of the above embodiment. Analysis channel 3
The flow direction of the electroosmotic flow is reversed in the analysis supply channel 31 and the analysis waste channel 32 so that the length of the sample area for electrophoresis in the analysis channel 3 is reduced. Since it becomes shorter in the road direction, the detection accuracy is improved and the detector 8
And the effect of downsizing the electrophoresis apparatus by shortening the analysis flow path 3 and increasing the speed of the apparatus.

[0036]

According to the electrophoresis apparatus of the present invention, the flow direction of the electroosmotic flow at the portion of the flow path for analysis opposite to the detector side with respect to the flow path intersection is reversed, so that the flow path in the flow path for analysis is reduced. In this case, the sample area of the sample is narrowed, and the flow of the sample, which is a detection noise or more than the analysis target, into the analysis channel is prevented. Therefore, there is less overlap between the two adjacent sample areas that have been developed, and noise is further reduced. This improves detection accuracy and simplifies the detector system by eliminating the need for expensive detection systems. The effect that can be performed. For the same reason, the size and speed of the electrophoresis apparatus can be reduced.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of an embodiment of an electrophoresis apparatus realized by the present invention.

FIG. 2 is a top view showing an operation example of an analysis process by the electrophoresis apparatus realized by the present invention.

FIG. 3 is a top view showing an operation example of an analysis process by a conventional electrophoresis apparatus.

FIG. 4 is a top view showing an operation example of an analysis process by the electrophoresis apparatus realized in the example of the present invention.

FIG. 5 is a schematic diagram showing an example of an electrophoresis pattern by electrophoresis realized in the present invention.

FIG. 6 is a perspective view schematically showing a process of another example of the production process of the present invention.

[Explanation of symbols]

1a ... substrate, 1b ... substrate, 2 ... sample introduction channel, 3
... Analysis flow path, 4a ... Solution reservoir, 4b ... Solution reservoir, 4c
... Solution reservoir, 4d ... Solution reservoir, 5a ... Solution reservoir electrode, 5
b: solution storage electrode, 5c: solution storage electrode, 5d: solution storage electrode, 8: detector, 9: flow path intersection, 10a: flow path power supply, 10b: flow path power supply, 11: substrate for electrophoresis, 21 …
Sample supply channel, 22: sample waste channel, 31: analysis supply channel, 32: analysis waste channel.

Claims (1)

[Claims] 1. An electrophoresis apparatus provided with a sample introduction channel, an analysis channel, a sample introduction electrode, and an analysis electrode, wherein an intersection between the sample introduction channel and the analysis channel is provided. By coating the flow path surface of the analysis flow path on the upstream side in the sample migration direction at the time of analysis from the intersection, the electricity flowing in the analysis flow path in the forward direction from the intersection in the analysis flow path An electrophoresis apparatus characterized by generating a permeation flow.