JPH08304339A - Capillary electrophoretic device - Google Patents

Abstract

PURPOSE: To facilitate the injection of a precise quantity of a sample, and perform a component detection with high concentration sensitivity. CONSTITUTION: A sample injecting tip 13 and a detecting tip 14 to be connected to both ends of a capillary 11 housed in a cartridge 12 are formed. These are formed by laminating two glass plates, and an electrophoretic groove is formed on the laminated surfaces by micro-machining. In the sample injecting chip 13, a sample drawing groove is formed, and only a sample in the common part between this and the electrophoretic groove is sent to the capillary 11.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a capillary electrophoresis device.

[0002]

2. Description of the Related Art A method for accurately and quickly analyzing a small amount of biological components is always required in a wide range of fields related to biotechnology, such as researches such as molecular structure analysis, clinical fields and pharmaceutical fields. Is. High Performance Liquid Chromatograph (HPLC) is currently the most commonly used separation and analysis method.
Separation of complex biopolymers such as NA is often difficult,
Moreover, a large amount of sample is required for analysis. On the other hand, capillary electrophoresis (CE) using fused silica tubes with an inner diameter of 100 μm or less has a higher resolution than HPLC and often requires a short analysis time. It is suitable for separation between similar components and has been widely studied. Since CE generally uses a fused silica capillary with an inner diameter of 25 to 100 μm, heat can be dissipated relatively easily even when electrophoresing at ~ 500 V / cm.
It is possible to obtain as many column efficiencies as possible. Moreover, unlike conventional electrophoresis, online detection can be performed with a part of the capillary, and it has a theoretical advantage that the sample consumption is extremely small (several nanoliters) as compared with HPLC.

Further, in CE, the separation selectivity can be easily changed only by exchanging the buffer solution used for the electrophoresis while leaving the capillary column as it is, and therefore the examination of the separation conditions is extremely difficult as compared with the conventional separation analysis method. Easy to. HP
If you try to change the separation mode by exchanging the column with LC, in addition to exchanging the mobile phase, it is necessary to thoroughly replace and wash the pump, piping, mixer and other flow paths, so it is usually a day. The separation mode is not changed in the analysis of.

However, the characteristics of capillaries having the above-mentioned advantages are conversely limited, and the concentration sensitivity is about 10 to 100 times lower than that of HPLC due to the extremely small amount of sample, and generally 10 ⁻⁶ M for capillaries of up to 50 μm. The degree is the detection limit.

[0005]

The conventional capillary electrophoresis has the following problems on the sample injection side. Conventionally, the injection of a predetermined amount of sample into the capillary has been performed by injecting the sample from the end of the capillary by a length corresponding to the amount. At that time, the sample injection method is roughly classified into a method of injecting by applying a differential pressure to both ends of the capillary and a method of injecting by electrophoresis. However, in the case of the method using the differential pressure, in order to accurately inject a small amount of sample of several nanoliters, it is necessary to apply an extremely weak pressure difference to both ends of the capillary and inject it for a sufficient time. Further, in the case of the electrophoretic injection method, the migration speed varies depending on the component, so that the application is limited, such as injection when using a gel-filled capillary.

Further, when the end portion of the capillary 60 is obliquely cut (FIG. 6A) or when the polyimide protective film 61 on the surface of the capillary 60 is peeled off (FIG. 6B), the end portion of the capillary 60 is removed. The attachment of the sample 62 to the surface becomes irregular, and the sample injection length may vary in any method. There is also a problem that the amount of actually injected sample can only be indirectly known.

On the other hand, the detection side has the following problems. In order to apply a migration voltage, both ends of the capillary must be immersed in a migration solution tank with electrodes inserted.
Therefore, the detection of the components separated by electrophoresis must be performed while the sample is inside the capillary. In order to optically detect the light, the light must be emitted in the direction crossing the capillary, but in this case, the light passes through the sample only at a very short distance corresponding to the inner diameter of the capillary, It was disadvantageous in terms of density sensitivity.

The present invention has been made to solve these problems, and its purpose is to easily inject a precise amount of sample and to detect a component with high concentration sensitivity. Another object of the present invention is to provide a capillary electrophoresis apparatus capable of performing the above.

[0009]

A capillary electrophoresis device according to the present invention made to solve the above-mentioned problems comprises a)
A capillary, b) sample injection tip, c) detection tip,
It is characterized by having. Here, the sample injection chip b) is formed by laminating two flat plates, and b1) a sample stretching groove and b2) a migration groove (first migration groove) are formed between both flat plates. The first migration groove b2) shares some grooves with the sample stretching groove b1). In addition, at one end of the first migration groove b2),
A connection port (first connection port) for connecting to one end of the capillary is provided. The detection chip c) is also made by laminating two flat plates, and a migration groove (second migration groove) between both plates
Are formed. The second migration groove is provided with a detection unit and a connection port (second connection port) at one end for connecting to the other end of the capillary.

[0010]

When performing the capillary electrophoresis analysis, one end of the capillary is connected to the first connection port of the sample injection chip and the other end is connected to the second connection port of the detection chip. When injecting a sample, first, the sample is injected into one end of the sample extending groove of the sample injecting chip, and the sample is diffused over the entire sample extending groove by electrophoresis or differential pressure (pressing). Next, a migration voltage is applied between the migration groove (first migration groove) of the sample injection chip and the migration groove (second migration groove) of the detection chip. As a result, the sample in the common portion of the sample stretching groove and the first migration groove migrates in the order of the first migration groove → capillary → second migration groove, and the components are separated therebetween. The separated components are detected in the detection section of the second migration groove.

[0011]

In the sample injection chip of the capillary electrophoresis apparatus according to the present invention, only the sample existing in the common portion of the sample stretching groove and the migration groove migrates, and the volume of the common portion can be accurately known. Therefore, accurate quantitative analysis can be performed.

In the detection chip, it is possible to form a detection part of any shape in the chip. Therefore, for example, when performing absorption detection, a Z-shaped portion is formed in the migration groove to increase the optical path length. It is possible to improve the density sensitivity.

In the capillary electrophoresis apparatus according to the present invention, the sample injection portion and the detection portion are each made into a chip, so that it is easy to handle. In addition, since the chips can be manufactured with accurate dimensions by the recent micromachining technology, it is possible to carry out an analysis with little variation and good reproducibility.

[0014]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A capillary electrophoresis apparatus which is an embodiment of the present invention will be described with reference to FIGS. As shown in FIG. 1A, the capillary electrophoresis apparatus of the embodiment is composed of a cartridge 12 accommodating a capillary 11, a sample injection chip 13 and a detection chip 14 connected to both ends of the capillary. The cartridge 12 includes a main body 12a and a lid 12b that can be opened and closed with respect to the main body 12a.
2a is provided with a capillary storage groove 12c for winding and storing the capillary 11. Capillary storage groove 12
The main body 12a and the lid 12b in the portion c are provided with a large number of ventilation holes 12d penetrating them so that the heat generated during the migration can be dissipated by the fan 15 (FIG. 1).
(B)). It should be noted that the cartridge 12 may be provided with a barcode or the like for identifying the capillary 11 to be used, the electrophoretic liquid, or the like.

When the capillaries 11 are housed in the cartridge 12, both ends of the capillaries 11 are made to project a little from both side surfaces of the cartridge 12, and at that portion,
The sample injection chip 13 and the detection chip 14 are connected. An engaging tool for fixing the sample injection chip 13 and the detection chip 14 to the cartridge 12 may be provided in the cartridge 12 or each of the chips 13 and 14.

Sample injection chip 13 and detection chip 14
Is composed of two glass plates laminated together,
For convenience of handling, the entire size is a rectangle with sides of about 2 to 5 cm. Note that a semiconductor material such as silicon can be used to facilitate the etching process of the groove and the like described later, but in this case, it is necessary to form an insulating film such as a thermal oxide film on the surface of the groove. As shown in FIG.
The sample injection tip 13 is constituted by laminating two glass plates 21 and 22, and the surface of one of the glass plates 21 is subjected to micromachining such as etching,
Sample stretching groove 24 having a common part 23 and migration groove 2
And 5 are provided. The cross-sectional area of these grooves is the same as the cross-sectional area of the capillary 11 to be used (for example, 100 to 70).
0 μm ² ). Of course, the grooves 24, 2 of the same pattern are formed on the bonding surface of the other glass plate 22.
5 may be formed. Further, both glass plates 21 and 22 are
After processing of grooves, holes, electrodes, etc., which will be described later, they are bonded and fixed by thermal bonding or anodic bonding.

A capillary 1 is provided at one end of the migration groove 25.
A connection port 26 for receiving 1 is provided, and at the other end, a migration liquid suction port 27 that penetrates the lower glass plate 21 and opens downward is provided. In addition, the sample stretching groove 2
Reservoir portions (reservoirs) 28 and 29 are provided at both ends of 4. These storage portions 28 and 29 are formed as holes penetrating the upper glass plate 22 at positions corresponding to the ends of the sample stretching grooves 24 formed in the lower glass plate 21. These electrophoretic liquid inlet 27 and reservoirs 28, 29
Are provided with electrodes 30, 31, 32 for applying a voltage to the electrophoretic liquid present therein. Electrode 3
0, 31, 32 are made of a conductive thin film formed by photolithography on the surface of one glass plate 21, and are formed so as to reach the end surface of the glass plate 21 continuously (see FIG. 2B). ).

As shown in FIG. 3, a migration groove 35 is formed in the detection chip 14 in the same manner as in the case of the sample injection chip 13, a capillary connection port 36 is provided at one end thereof, and a drainage liquid is provided at the other end thereof. A mouth 38 is provided. Also, the drain port 38
An electrode 39 for applying an electrophoretic voltage is provided in the.
The migration groove 35 is bent in the middle and has a slightly long linear detection unit 3.
7 are provided. The light emitting side fiber 40 is provided at both end portions of the detection portion 37 of both glass plates constituting the detection chip 14.
And a hole for inserting the light receiving side fiber 41.

The method of using the capillary electrophoresis apparatus of this embodiment having the above construction is as follows. Capillary connection ports 26 and 36 of the sample injection tip 13 and the detection tip 14 are inserted into both ends of the capillary 11 protruding from both side surfaces of the cartridge 12 so that both tips 13 and 14 are inserted.
Is fixed to the cartridge 12. At this time, it is desirable to seal the periphery of the capillary connection ports 26, 36 with a sealing material. Next, the migration liquid injection tube 33 is inserted into the migration liquid suction port 27, and the tip thereof is immersed in the migration liquid tank 34. In this state, pressure is applied to the liquid surface of the electrophoretic liquid tank 34 to fill the grooves 24, 25, 35 and the capillary 11 with the electrophoretic liquid. Next, the migration liquid inlet 27 and the reservoir 2
Electrodes 30, 31, 3 corresponding to 8, 29 and the drainage port 38
Conductors are connected to 2, 39 to form a circuit as shown in FIG.

First, a small amount of the sample is injected into the reservoir 28 and the switches S2 and S3 are closed to store the sample in the reservoir 2.
The sample is made to migrate from 8 to the storage section 29, and the sample is drawn into the sample drawing groove 24. Next, the switches S2 and S3 are opened and the switches S1 and S4 are closed. As a result, a high voltage source (HV) 46 is provided at both ends of the migration groove 25-capillary 11-migration groove 35.
Is applied with the migration voltage from the sample stretching groove 24 and the migration groove 2
Only the sample existing in the common portion 23 with 5 migrates from the migration groove 25 to the capillary 11, and the components are separated in the capillary 11.

The components separated by the capillary 11 enter the migration groove 35 of the detection chip 14, and the detection section 37 detects the components by UV absorption, fluorescence detection or the like. In the detection unit 37 of the detection chip 14 of the present embodiment, the optical path length is sufficiently long as shown in FIG. 3, so that highly sensitive detection can be performed.

After the analysis, the sample stretching groove 24, the migration grooves 25 and 35, and the capillaries can be washed by pressurizing the liquid surface of the migration liquid tank 34. Also, the electrode 31 →
It is also possible to wash by electrophoresis by applying a voltage between the electrode 30 and the electrode 31 → the electrode 32.

Another example of the sample injection tip 13 is shown in FIG. In this example, when a voltage is applied between the electrodes T1 and T2 when the sample is stretched, the sample enters the shorter common portion 53a, and when a voltage is applied between the electrodes T1 and T3, the longer common portion 53a is applied. The sample enters the portion 53b. After that, by applying an electrophoretic voltage between the electrode T4 and the electrode 39 of the detection chip, electrophoretic analysis of the sample can be performed with two kinds of amounts.

Also, in the detection chip 14, in addition to lengthening the detection section 37 as shown in FIG. 4, for example, multiple reflection surfaces are formed above and below the migration groove 35 to multiple-analyze the analysis light. The density sensitivity can also be increased by.

As described above, in the capillary electrophoresis analyzer of the present embodiment, the samples to be analyzed are only those existing in the common parts 23, 53a, 53b, and the volumes of these common parts 23, 53a, 53b. Can be accurately known (may be actually measured, but if the machining accuracy of micromachining is high, the design volume can be used as it is), so that highly accurate analysis can be performed. Further, in the detection chip 14, the detection unit 3
Since 7 can be set in various shapes, detection can be performed with high density sensitivity. Further, in the method of using the capillary itself as a detection cell as in the past, alignment of the capillary with respect to the detection part was troublesome, but in the apparatus of this embodiment, if a standard detection chip is placed at a predetermined position,
It is possible to freely change the analysis conditions by exchanging only the capillary cartridge while keeping the detection unit in the optimum state.

[Brief description of drawings]

FIG. 1 is a plan view (a) and a side view (b) of a capillary electrophoresis device which is an embodiment of the present invention.

FIG. 2 is a plan view (a) and a cross-sectional view (b) of a sample injection tip of an example.

FIG. 3 is a plan view of a detection chip.

FIG. 4 is a configuration diagram of an electric circuit.

FIG. 5 is a plan view of another sample injection tip.

FIG. 6 is a cross-sectional view illustrating a problem when a sample is injected into a conventional capillary.

[Explanation of symbols]

 11 ... Capillary 12 ... Capillary cartridge 12a ... Main body 12b ... Lid 12c ... Capillary storage groove 12d ... Vent hole 13 ... Sample injection chip 14 ... Detection chip 15 ... Fans 21, 22 ... Glass plate 23 ... Common part 24 ... Sample stretching groove 25, 35 ... Electrophoresis groove 26, 36 ... Capillary connection port 27 ... Electrophoresis liquid suction port 28, 29 ... Reservoir 30, 31, 32, 39 ... Electrode 33 ... Electrophoresis liquid injection pipe 34 ... Electrophoresis liquid tank 37 ... Detection unit 38 ... Drainage port 40 ... Light emitting side fiber 41 ... Light receiving side fiber 53a, 53b ... Common groove

Claims (1)

[Claims] 1. A) a capillary and b) two flat plates bonded together, and between the flat plates, b1) a sample stretch groove and b2) a groove which shares a part with the sample stretch groove. And a sample injection chip having a migration groove provided at one end with a connection port for connecting to one end of the capillary, and c) two transparent flat plates are stuck together, and between the transparent flat plates. A capillary electrophoresis device, comprising: a detection unit; and a detection chip having a migration groove formed at one end thereof and a connection port for connecting to the other end of the capillary.