JPH10148628A - Microchip electrophoresis device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To automate the operation of electrophoresis of a microchip. SOLUTION: In a microchip 1 in a dry state, a sample injecting opening S1 and an electrophoresis liquid reservoir B1 are positioned at a location P, where a rod 13 is lowered and at a location I, where a needle 12 is lowered respectively by the movement of a tray 10 in Y directions. After the needle 12 and rod 13 are each lowered to the locations I and P on the microchip 1 to fill a separating channel 2-1 and sample introducing channel 3-1 with a electrophoresis liquid, A sample is injected into the sample injection opening S1 from a syringe 22. Then the needle 12 and rod 13 are raised, and the tray 10 on which the microchip 1 is fixed is again moved in Y directions to match the detection point Ml of the channels to the location of a part to be detected and to perform a regular analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for analyzing very small amounts of proteins and nucleic acids at high speed and with high resolution. More specifically, the present invention provides a pair of transparent plate-like members, A groove through which the liquid flows is formed on the surface,
The other plate-shaped agent is provided with a through-hole at a position corresponding to the groove, and these plate-shaped members are opposed to each other with the groove inside, and the separation channel and the sample introduction channel cross each other by the groove. The separation channel is filled with the electrophoresis liquid using the microchip on which the sample is formed, the sample is introduced from the sample introduction channel into the separation channel, and the sample is separated by applying an electrophoresis voltage between both ends of the separation channel. The present invention relates to a microchip electrophoresis apparatus for performing electrophoretic separation on a path.

[0002]

2. Description of the Related Art Electrophoresis apparatuses have been used to analyze very small amounts of proteins and nucleic acids.
A typical example is a capillary electrophoresis apparatus. The capillary electrophoresis device is a glass capillary with an inner diameter of about 50 μm or less filled with an electrophoresis buffer, a sample is introduced at one end, and a high voltage is applied between both ends to expand an analyte in the capillary. It is to let. The surface area is large relative to the volume inside the capillary,
That is, since the cooling efficiency is high, 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.

A capillary has an outer shape of several tens μm to 1 μm.
Since it is as thin as about 00 μm and easily broken, there is a problem that it is not easy for the user to handle the capillary when replacing it. Therefore, DJ Harrison et al./Anal. Chi
m. As indicated in Acta 283 (1993) 361-366, two
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 glass substrates 51, 52, and electrophoresis capillary grooves 54, crossing each other by etching on the surface of one substrate 52.
The other substrate 51 is provided with through holes 53 at positions corresponding to the ends of the grooves 54 and 55.

When this microchip is used, both substrates 51 and 52 are overlapped as shown in FIG. 1C, and the electrophoresis running liquid is injected into the grooves 54 and 55 from one of the through holes 53. Thereafter, a sample is injected into the through hole 53 at one end of the shorter groove 54, an electrode is inserted between the through holes 53 at both ends of the groove 54, and a high voltage is applied for a predetermined time. As a result, the sample is dispersed in the groove 54.

Next, the through holes 5 at both ends of the longer groove 55
Electrodes are inserted into 3, 53, and a migration voltage is applied. Thus, the sample existing at the intersection 56 between the two grooves 54 and 55 electrophoreses in the groove 55. The separation component is detected by disposing a detector such as an ultraviolet-visible spectrophotometer, a fluorometer, and an electrochemical detector at an appropriate position in the groove 55. Electrophoresis using such a microchip is known to have features such as high-speed operation, ultra-trace analysis, and small size.
It has the potential to be an unprecedented unique analyzer.

In the conventional technology using a microchip, filling of the electrophoresis liquid into the flow paths 54 and 55 from the electrophoresis liquid through hole 53, which is indispensable before analysis, and filling the electrophoresis liquid into the through hole 53 for sample injection. All sample injections are manual. The through-hole for the electrophoresis running liquid serves as a reservoir for the electrophoresis running fluid, and the through-hole for injecting the sample corresponds to a sample container. As an operation before analysis, the electrophoresis running liquid is manually sent from one of the through holes 53 using a syringe or the like, and the sample is injected from another through hole 53 at one end of the sample introduction groove 54 with another syringe.

[0007]

The operation of filling the electrophoretic solution and injecting the sample is not only complicated, but also results in a longer overall analysis time including the injection of the electrophoretic solution and the sample. Does not take advantage of the characteristics of microchip electrophoresis that is possible. Accordingly, an object of the present invention is to automate and facilitate the operation of microchip electrophoresis.

[0008]

In the microchip electrophoresis apparatus of the present invention, a moving mechanism for moving the microchip in parallel with the above-mentioned parallel line in order to automate the filling of the microchip with the electrophoresis liquid and the injection of the sample. And a sample injection mechanism for moving the sample supply needle up and down on the movement trajectory of the sample injection hole of the microchip to inject the sample into the sample injection hole, and an electrophoresis solution supply port for supplying the electrophoresis solution to the microchip. An electrophoretic liquid injection mechanism that moves up and down on the movement trajectory of the electrophoretic liquid injection hole and injects the electrophoretic liquid into the electrophoretic liquid injection hole, the sample injection hole is located just below the needle of the sample injection mechanism, and the electrophoretic liquid injection hole is Control of the movement of the microchip by the movement mechanism so as to position the position just below the electrophoresis liquid supply port of the injection mechanism and the detection point at the position of the detection unit, respectively, and the sample injection mechanism and the electrophoresis liquid injector And a control unit for controlling the operation.

When the microchip is mounted on the moving mechanism in a dry state where the flow path is not filled with the electrophoretic liquid and the operation is started, the moving mechanism moves the sample injection hole and the electrophoretic liquid injection hole of the microchip respectively. It is positioned just below the sample supply needle and just below the electrophoresis liquid supply port. The sample supply needle and the electrophoresis liquid supply port are lowered to the microchip, and the electrophoresis liquid is filled and the sample is injected.

Thereafter, the sample supply needle and the electrophoretic solution supply port are both raised, the microchip is moved by the moving mechanism, the detection point is positioned at the position of the detection section, and the analysis is performed. After the analysis, the microchip is further moved by the moving mechanism, and the filling of the electrophoresis liquid, the sample injection, and the analysis are similarly repeated using the unused flow path.

[0011]

FIG. 2 shows a microchip used in one embodiment. As shown in FIG. 1, the microchip 1 has a set of capillary grooves 2-1 and 3-1 intersecting each other on one surface of a pair of transparent glass substrates by etching, and 2-2 and 3-2. Are formed on the other substrate, and the through holes B1, D1, S1,
W1, B2, D2, S2, and W2 are provided. The capillary groove 2-1 is a separation channel, and the electrophoresis solution reservoirs B1 and D1 are disposed at both ends thereof, respectively.
Reference numeral -1 denotes a sample introduction channel, in which a sample injection port S1 is arranged at one end and a sample waste liquid reservoir W1 is arranged at the other end. The set of the two flow paths 2-1 and 3-1 is set as a first flow path set, and a sample having a volume at the intersection of the flow paths 2-1 and 3-1 is analyzed. Similarly, the capillary groove 2-2 is a separation channel, and electrophoresis liquid reservoirs B2 and D2 are respectively disposed at both ends thereof. The capillary groove 3-2 is a sample introduction channel and a sample inlet S2 is provided at one end and a sample waste liquid is provided at the other end. The reservoir W2 is arranged.
The set of the two flow paths 2-2 and 3-2 is used as a second flow path set, and a sample having a volume at the intersection of the flow paths 2-2 and 3-2 is analyzed.

In the microchip 1, two sets of flow paths are formed, and the sample injection ports S1 and S2, the electrophoresis solution reservoirs D1 and D2, and the detection points M1 and M2 of each set are arranged on the same straight line. And the straight lines are parallel to each other. The direction of those straight lines is defined as the Y direction. The microchip 1 is moved in its linear direction (Y direction) by a moving mechanism described later.

FIGS. 3A and 3B show respective through holes B1, D1, D1 and D2.
FIG. 3 shows an electrode pattern 4 for applying a voltage to S1, W1, B2, D2, S2, and W2. (A) is a plan view, and (B) is a cross-sectional view taken along the line AA '.
The electrode patterns 4 connected to the through holes are gathered in one connector section 5 and operability is improved by connecting the connector section 5 to a power supply device. The electrode pattern 4 is formed on the surface of the microchip 1 by a method such as vapor deposition.

In the example of FIG. 3, since the reservoirs having the same function are conducting, a voltage is simultaneously applied to the reservoirs having the same function. Therefore, after injecting the sample into one channel set, the electrophoresis of the channel set is performed,
After that, even if the sample is injected into the next channel set, the sample injection cannot be performed collectively even in the channel set of the same microchip.

FIG. 4 shows an example in which another electrode is provided for each channel set. In this example, since an independent terminal is used in each flow channel set, a sample can be injected into both flow channel sets of the microchip, and electrophoresis can be performed by sequentially applying a voltage. The number of flow channel sets provided in one microchip is not limited to two. One set may be provided as shown in FIG. 5A, or three or more sets may be provided as shown in FIG.

FIG. 6 and FIG. 7 show an embodiment. Reference numeral 10 denotes a microchip loading tray on which the microchip 1 can be mounted and fixed. Tray 10
Are supported so as to be movable in the Y direction. Tray 1
The gear 10a is formed on the side of the gear 10a.
Are engaged with the gear 11b driven to rotate by the motor M1, so that the tray 10 moves in the Y direction. The sample injection ports S1 and S2 of the microchip 1 are moved to the position I where the sample injection needle 12 is lowered by the movement of the tray 10, and the electrophoretic solution reservoirs B1 and B2 are positioned at the position P where the electrophoretic solution pressurized liquid sending rod 13 is lowered. The detection points M1 and M2 are respectively positioned at the positions of the detection units.

The rod 13 having the electrophoresis liquid supply port is provided with a seal member 14 at the tip thereof for keeping airtight by contacting the surface of the microchip 1. The needle 12 and the rod 13 are fixed to one block 15, and the block 1
Reference numeral 5 denotes an X mount 16 that moves in the X direction and an arm of a Z mount 17 that moves in the vertical direction, and is supported so as to be able to move horizontally in the X direction and up and down in the vertical direction.

A spring 18 is provided between the rod 13 and the block 15 to press the sealing member 14 at the tip against the surface of the microchip 1 with an appropriate pressure. A weak spring 19 is provided for bringing the tip of the needle 12 into contact with the bottom of the sample inlets S1 and S2 of the microchip 1.

The proximal end of the rod 13 is connected via a valve 21 to a syringe 20 which sucks the electrophoresis running liquid.
When the valve 21 is opened and the syringe 20 is pushed in, the electrophoresis running liquid is discharged from the tip of the rod 13. Rod 13, seal member 14, valve 21, and syringe 20
Constitutes an electrophoresis liquid injection mechanism. The base end of the needle 12 is connected to a syringe 20 so that the syringe 20 can suck and discharge a sample.

A washing bottle 23 and a sample container 25 arranged on a sample tray 24 are arranged on the side of the tray 10, and the rod 13 and the needle 12 move in the washing bottle 23 to be immersed and washed. Then, the needle 12 moves and is immersed in the sample container 25 to suck the sample. The sample container 25 contains a standard sample or an analysis sample, and about 1 μl of the sample is sucked into the needle.

The operation of the motor M1, the X mount 16, the Z mount 17, the syringes 20, 22 and the valve 21 is performed by a drive circuit 26, which is controlled by a CPU corresponding to a control unit. In FIG. 6, two sets of the needle 12 and the rod 13 are illustrated, but only one set is actually provided, and the figure shows a state of movement.

Next, the operation of this embodiment will be described. When the dry microchip 1 is mounted on the tray 10 and the operation is started, the tray 10 is driven by the motor M1.
Is moved in the Y direction, and the sample inlet S1 and the electrophoresis solution reservoir B1 of the microchip 1 are positioned at the position P where the rod 13 descends and the position I where the needle 12 descends, respectively. On the other hand, after the needle 12 sucks the sample from the sample container 25, the needle 12 and the rod 13 move to positions I and P, respectively. The needle 12 and the rod 13 descend on the microchip 1, and the rod 13 is
As a result, the sealing member at the tip is pressed against the surface of the microchip 1, and the tip of the needle 12 is brought into contact with the bottom of the sample inlet S1 by the spring 19. In this state, the electrophoresis running liquid is sent by the syringe 20 and the separation channel 2-1 and the sample introduction channel 3-1 of the microchip 1 are filled with the electrophoresis running solution. It is injected into the inlet S1.

Thereafter, the needle 1 is moved by the Z mount 17.
2 and the rod 13 are raised, the tray 10 on which the microchip 1 is fixed moves again in the Y direction, and the detection point M1 of the flow path is adjusted to the position of the detection unit. As in the conventional analysis, a voltage is applied to the sample inlet S1 and the sample waste liquid reservoir W1 for a predetermined period of time, and then the voltage is switched to the electrophoresis solution reservoirs B1 and D1, so that the intersection of the two flow paths 2-1 and 3-1 can be analyzed. The sample is introduced in the form of a plug in the direction of the separation channel 2-1 and is separated and detected.

The needle 12 and the rod 13 move in the direction of the washing bottle 23. First, after the needle 12 is washed by the washing bottle 23, the rod 13 is inserted into the washing bottle 23 and the needle 12 is inserted into the sample container 25, respectively. The cleaning of the rod 13 and the suction of the next sample into the needle 12 are performed.

The tray 10 is moved again in the Y direction by the motor M1, and the sample inlet S2 and the electrophoresis solution reservoir B2 of the microchip 1 are positioned at the position P where the rod 13 descends and the position I where the needle 12 descends, respectively. ,
Filling the electrophoresis liquid into the first set of flow paths, filling the electrophoresis liquid into the second set of flow paths,
Sample injection and analysis are performed.

The present invention includes the following as another aspect. The microchip has at least two sets of a separation channel and a sample introduction channel that intersect each other, and the electrophoretic solution injection hole, the sample injection hole of the sample introduction channel, and the detection point of those sets are parallel to each other. They are arranged so as to be located on a line. Furthermore, in an actual analysis, it is necessary to analyze at least two times of the standard sample and the actual sample. Therefore, in a microchip provided with only one set of the sample introduction channel and the separation channel, it is necessary to wash the channel once during the analysis. The manual operation results in a longer time.

When two or more channel sets are provided in the microchip, the standard sample is analyzed in the first channel set, and the unknown sample is analyzed in the other channel sets. For example, a plurality of samples can be sequentially and continuously analyzed using the same microchip. After the electrophoresis liquid is filled into the flow path of the microchip and the sample is injected, the time required for analysis is extremely short, so when analyzing the standard sample and the unknown sample in separate flow paths of one microchip However, the overall analysis time is short.

[0028]

According to the present invention, there are provided a mechanism for moving the microchip, a mechanism for automatically supplying the electrophoretic solution to the flow path of the microchip, and a mechanism for automatically injecting the sample into the flow path of the microchip. Therefore, the electrophoresis operation using the microchip can be easily performed. In addition, by moving the microchip in a direction perpendicular to the separation channel, it can also serve as a detection point positioning mechanism.

[Brief description of the drawings]

FIG. 1 is a view showing a conventional microchip, and FIG.
(B) is a plan view showing a transparent plate member constituting the microchip, and (C) is a front view.

FIG. 2 is a plan view showing a microchip used in one embodiment.

3A and 3B are diagrams showing an example of an electrode pattern for applying a voltage to each through hole of the microchip of FIG. 2; FIG. 3A is a plan view, and FIG. FIG.

FIG. 4 is a plan view showing another example of an electrode pattern for applying a voltage to each through hole of the microchip of FIG. 2;

FIGS. 5A and 5B are plan views showing another example of a microchip provided with an electrode for applying a voltage to each through hole.

FIG. 6 is a perspective view showing one embodiment.

FIG. 7 is a schematic configuration diagram showing a control system of the embodiment.

[Description of Signs] 1 Microchip 2-1, 2-2 Separation flow path 3-1, 3-2 Sample introduction flow path 4 Electrode pattern 5 Connector section 10 Microchip loading tray 12 Sample injection needle 13 Electrophoresis liquid addition Rod for pumping liquid 16 X mount 17 Z mount 20, 22 Syringe 26 Drive circuit B1, B2 Electrophoresis liquid reservoir S1, S2 Sample injection port

Claims (1)

[Claims] 1. A pair of transparent plate members are provided, a groove through which a liquid flows is formed on at least one of the plate members, and a through hole is provided in a position corresponding to the groove in the other plate member. Using a microchip in which a separation channel and a sample introduction channel are formed in which these plate-shaped members are faced with the groove inside and cross each other by the groove, the separation channel is filled with the electrophoresis liquid, In a microchip electrophoresis apparatus for introducing a sample from a sample introduction channel to a separation channel and applying an electrophoresis voltage between both ends of the separation channel to electrophoretically separate the sample in the separation channel, A moving mechanism for moving the sample in parallel with the line, a sample injection mechanism for moving a needle for supplying the sample up and down on the movement trajectory of the sample injection hole to inject the sample into the sample injection hole, and an electrophoretic solution for supplying the electrophoretic solution Supply port is the electrophoresis An electrophoretic liquid injection mechanism that moves up and down on the movement trajectory of the liquid injection hole to inject the electrophoretic liquid into the electrophoretic liquid injection hole; and a position where the sample injection hole is located immediately below the needle of the sample injection mechanism and the electrophoretic liquid injection hole. Control of the movement of the microchip by the moving mechanism so as to position the position immediately below the electrophoretic liquid supply port of the electrophoretic liquid injection mechanism and the detection point at the position of the detection unit, respectively, and the sample injection mechanism and the electrophoretic liquid injection mechanism And a control unit for controlling the operation of the microchip electrophoresis apparatus.