JP3661320B2 - Microchip electrophoresis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for analyzing trace amounts of proteins, nucleic acids, and the like at high speed and with high resolution. More specifically, the present invention includes a pair of transparent plate-like members, and a groove through which liquid flows on the surface of at least one plate-like member. The other plate-shaped member is formed with a through hole at a position corresponding to the groove, and the plate-like member is stretched with the groove inside and the separation channel and the sample intersect each other by the groove. Using a microchip in which an introduction channel is formed, the separation channel is filled with the electrophoresis solution, the sample is introduced from the sample introduction channel to the separation channel, and an electrophoresis voltage is applied between both ends of the separation channel. The present invention relates to a microchip electrophoretic apparatus for performing electrophoretic separation on a separation channel.
[0002]
[Prior art]
In the case of analyzing a very small amount of protein or nucleic acid, an electrophoresis apparatus has been conventionally used, and a typical example is a capillary electrophoresis apparatus. A capillary electrophoresis device is a glass capillary with an inner diameter of about 50 μm or less, filled with an electrophoresis buffer, and after introducing a sample to one end, a high voltage is applied between the two ends to develop the analyte in the capillary. It is something to be made. Since the capillary has a large surface area relative to the volume, that is, the cooling efficiency is high, it is possible to apply a high voltage, and it is possible to analyze a trace amount sample such as DNA with high speed and high resolution.
[0003]
Since the capillaries are thin and easily broken to a few tens of μm to 100 μm, the capillaries have a problem that they are not easy to handle when replacing the capillary. Therefore, as shown in DJ Harrison et al./ Anal. Chim. Acta 283 (1993) 361-366, a capillary electrophoresis chip (called a microchip) formed by joining two substrates is proposed. Has been. An example of the microchip is shown in FIG. A pair of transparent glass substrates 51 and 52 are formed, and electrophoresis capillary grooves 54 and 55 that cross each other are formed on the surface of one substrate 52 by etching, and the other substrate 51 corresponds to the ends of the grooves 54 and 55. A through hole 53 is provided at a position.
[0004]
When this microchip is used, both the substrates 51 and 52 are overlapped as shown in (C), and the electrophoretic solution 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, 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.
[0005]
Next, an electrode is inserted into the through holes 53 at both ends of the longer groove 55, and an electrophoresis voltage is applied. As a result, the sample present at the intersecting portion 56 of both grooves 54 and 55 is electrophoresed in the groove 55. By disposing a detector such as an ultraviolet-visible spectrophotometer, a fluorophotometer, or an electrochemical detector at an appropriate position in the groove 55, the separation component is detected.
Electrophoresis using such a microchip is known to have features such as high-speed operation, ultra-trace analysis, and compactness. -It has the potential to become a reliable analyzer.
[0006]
In the conventional technology using a microchip, filling of the electrophoretic liquid from the through hole 53 for the electrophoretic liquid, which is indispensable before the analysis, into the flow paths 54 and 55, and injection of the sample into the through hole 53 for injecting the sample Are all manually operated. The through hole for the electrophoretic liquid serves as a reservoir for the electrophoretic liquid, and the through hole into which the sample is injected corresponds to the sample container. As an operation before the analysis, the electrophoresis solution is manually fed from any one of the through holes 53 by a syringe or the like, and the sample is injected from another through hole 53 at one end of the sample introduction groove 54 by another syringe.
[0007]
[Problems to be solved by the invention]
Electrophoresis filling and sample injection operations are not only complicated, but also result in longer overall analysis time including injection of the electrophoresis solution and sample, and the characteristics of microchip electrophoresis that can be separated in seconds. I will not make use of it.
Therefore, the present invention aims to automate and facilitate the operation of microchip electrophoresis.
[0008]
[Means for Solving the Problems]
In the microchip electrophoresis apparatus of the present invention, in order to automate the filling of the electrophoresis solution into the microchip and the sample injection, the moving mechanism for moving the microchip and the needle for supplying the sample move the sample injection hole of the microchip. The sample injection mechanism that moves up and down on the locus and injects the sample into the sample injection hole, and the electrophoresis solution supply port that supplies the electrophoresis solution moves up and down on the movement locus of the electrophoresis solution injection hole of the microchip. Electrophoretic liquid injection mechanism for injecting the electrophoretic liquid into the injection hole, the sample injection hole at the position immediately below the needle of the sample injection mechanism, the electrophoretic liquid injection hole at the position immediately below the electrophoretic liquid supply port of the electrophoretic liquid injection mechanism, and the separation channel A control unit for controlling the movement of the microchip by the moving mechanism so as to position the detection point at the position of the detecting unit, and for controlling the operation of the sample injection mechanism and the electrophoresis solution injection mechanism, respectively. There.
[0009]
When the microchip is mounted on the moving mechanism and the operation is started in a dry state where the electrophoresis solution is not filled in the flow path, the sample injection hole and the electrophoresis solution injection hole of the microchip are used for sample supply by the moving mechanism. Positioned just below the needle and just below the electrophoresis solution supply port. The sample supply needle and the electrophoresis solution supply port descend to the microchip, and the electrophoresis solution is filled and the sample is injected.
[0010]
Thereafter, both the sample supply needle and the electrophoresis solution supply port rise, the microchip is moved by the moving mechanism, the detection point is positioned at the position of the detection unit, and the analysis is performed.
After the analysis is completed, the microchip is further moved by a moving mechanism, and the filling of the electrophoretic solution, the sample injection, and the analysis are repeated using the unused channel.
[0011]
【Example】
FIG. 2 shows a microchip used in one embodiment. In the same manner as shown in FIG. 1, the microchip 1 has a pair of capillary grooves 2-1 and 3-1 that intersect each other by etching on one surface of a pair of transparent glass substrates, and 2-2 and 3-2. The other substrate is provided with through holes B1, D1, S1, W1, B2, D2, S2, and W2 at positions corresponding to the ends of the grooves. The capillary groove 2-1 is a separation channel, and electrophoresis solution reservoirs B1 and D1 are arranged at both ends thereof. The capillary groove 3-1 is a sample introduction channel, and has a sample inlet S1 at one end and a sample waste liquid reservoir W1 at the other end. Is arranged. A set of the two flow paths 2-1 and 3-1 is used as a first flow path set, and a sample of the 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 solution reservoirs B2 and D2 are respectively arranged at both ends thereof. The capillary groove 3-2 is a sample introduction channel and has a sample inlet S2 at one end and a sample waste liquid at the other end. A reservoir W2 is arranged. The set of the two flow paths 2-2 and 3-2 is set as a second flow path set, and a sample of the volume at the intersection of the flow paths 2-2 and 3-2 is analyzed.
[0012]
In the microchip 1, two sets of flow paths are formed, and the sample inlets S1 and S2, the electrophoresis solution reservoirs D1 and D2, and the detection points M1 and M2 are arranged on the same straight line. The straight lines are parallel to each other. The direction of these straight lines is defined as the Y direction. The microchip 1 is moved in the linear direction (Y direction) by a moving mechanism described later.
[0013]
3A and 3B show an electrode pattern 4 for applying a voltage to each of the through holes B1, D1, 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 pattern 4 connected to each through hole is concentrated in one connector portion 5, and the operability is improved by connecting the connector portion 5 to the power supply device. The electrode pattern 4 is formed on the surface of the microchip 1 by a method such as vapor deposition.
[0014]
In the example of FIG. 3, since the reservoirs having the same function are electrically connected, a voltage is simultaneously applied to the reservoirs having the same function. Therefore, even if the same microchip channel set is used, after the sample is injected into one channel set, the channel set is migrated and then the sample is injected into the next channel set. Sample injection cannot be performed collectively.
[0015]
On the other hand, FIG. 4 is an example in which another electrode is provided for each channel set. In this example, since an independent terminal is used in each channel set, the sample can be injected into both 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 used as shown in FIG. 5A, or three or more sets may be used as shown in FIG.
[0016]
One embodiment is shown in FIG. 6 and FIG.
Reference numeral 10 denotes a microchip loading tray on which the microchip 1 can be mounted and fixed. The tray 10 is supported so as to be movable in the Y direction. A gear 10a is formed on the side of the tray 10, and the gear 10a meshes with a gear 10b whose rotation is driven by the motor 11, whereby 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, and the electrophoresis solution reservoirs B1 and B2 are moved to the position P where the electrophoresis solution pressurizing and feeding rod 13 is lowered. The detection points M1 and M2 are respectively positioned at the positions of the detection units.
[0017]
The rod 13 having the electrophoresis solution supply port is provided with a seal member 14 at the tip thereof for contacting the surface of the microchip 1 and maintaining airtightness. The needle 12 and the rod 13 are fixed to one block 15, and the block 15 is moved horizontally in the X direction and vertically moved in the vertical direction by the arm of the X mount 16 that moves in the X direction and the Z mount 17 that moves in the vertical direction. It is supported movably.
[0018]
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 into contact with the bottoms of the sample inlets S1 and S2 of the microchip 1.
[0019]
The proximal end portion of the rod 13 is connected to a syringe 20 that sucks the electrophoresis solution through a valve 21. When the valve 21 is opened and the syringe 20 is pushed, the electrophoresis solution is discharged from the tip of the rod 13. The rod 13, the seal member 14, the valve 21, and the syringe 20 constitute an electrophoresis solution injection mechanism.
The proximal end portion of the needle 12 is connected to a syringe 20 so that the sample can be sucked and discharged by the syringe 20.
[0020]
On the side of the tray 10, a cleaning container 23 and a sample container 25 disposed on the sample tray 24 are disposed. The cleaning bottle 23 is moved by being immersed in the rod 13 and the needle 12, respectively, and cleaned. In 25, the needle 12 moves and is immersed, and the sample is sucked. A standard sample or an analysis sample is accommodated in the sample container 25, and about 1 μl of the sample is sucked into the needle.
[0021]
The operations of the motor M1, the X mount 16, the Z mount 17, the syringes 20 and 22, and the valve 21 are performed by a drive circuit 26, and the drive circuit 26 is controlled by a CPU corresponding to a control unit.
In FIG. 6, the needle 12 and the rod 13 are depicted as having two pairs, but only one pair is actually provided, and the figure shows the state of movement.
[0022]
Next, the operation of this embodiment will be described.
When the microchip 1 in the dry state is mounted on the tray 10 and the operation is started, the tray 10 is moved in the Y direction by the motor M1, and the rod 13 is connected to the sample inlet S1 of the microchip 1 and the electrophoresis solution reservoir B1. The position P is lowered and the position I is lowered by the needle 12. 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 are lowered onto the microchip 1, and the tip of the rod 13 is pressed against the surface of the microchip 1 by the spring 18, and the tip of the needle 12 is brought to the bottom of the sample inlet S 1 by the spring 19. Contacted. In this state, the electrophoresis solution is fed 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 solution. It is injected into the inlet S1.
[0023]
Thereafter, the needle 12 and the rod 13 are raised by the Z mount 17, the tray 10 to which the microchip 1 is fixed moves again in the Y direction, and the detection point M1 of the flow path is aligned with the position of the detection unit.
As in the conventional analysis, a voltage is applied to the sample inlet S1 and the sample waste reservoir W1 for a predetermined time, and then the voltage is switched to the electrophoresis solution reservoirs B1 and D1, so that The sample is introduced in the form of a plug in the direction of the separation channel 2-1, separated and detected.
[0024]
The needle 12 and the rod 13 move in the direction of the cleaning bottle 23. After the needle 12 is first cleaned with the cleaning bottle 23, the rod 13 is inserted into the cleaning bottle 23 and the needle 12 is inserted into the sample container 25. Washing and inhalation of the next sample into the needle 12 are performed.
[0025]
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 is lowered and the position I where the needle 12 is lowered, respectively. In the same manner as the filling of the electrophoretic solution into the eye channel set, the sample injection, and the analysis, the filling of the electrophoretic solution into the second channel set, the sample injection, and the analysis are performed.
[0026]
The present invention includes the following as other aspects. The microchip includes at least two pairs of separation channels and sample introduction channels that intersect each other, and the electrophoresis solution injection holes, the sample injection holes of the sample introduction channels, and the detection points of these groups are parallel to each other. It is arranged so as to be located on the line.
Furthermore, in actual analysis, it is necessary to analyze the standard sample and the actual sample at least twice. Therefore, in the case of a microchip having only one set of sample introduction channel and separation channel, the channel must be washed once during the analysis. As a result, manual operation results in more time.
[0027]
Therefore, when two or more channel sets are provided on the microchip, the standard sample is analyzed with the first channel set, and the unknown sample is analyzed with the other channel set. For example, a plurality of samples can be sequentially analyzed with the same microchip. After the microchip flow path is filled with the electrophoresis solution and the sample is injected, the time required for the analysis is extremely short, so the standard sample and the unknown sample are analyzed in separate flow paths of one microchip. However, the overall analysis time is short.
[0028]
【The invention's effect】
In the present invention, a mechanism for moving the microchip, a mechanism for automatically supplying the electrophoresis solution to the flow path of the microchip, and a mechanism for automatically injecting the sample into the flow path of the microchip are provided. The used electrophoresis operation can be easily performed.
Further, by moving the microchip in a direction orthogonal to the separation channel, it can also serve as a detection point positioning mechanism.
[Brief description of the drawings]
1A and 1B are diagrams showing a conventional microchip, in which FIGS. 1A and 1B are plan views showing a transparent plate member constituting the microchip, and FIG. 1C is a front view;
FIG. 2 is a plan view showing a microchip used in one embodiment.
3 is a diagram showing an example of an electrode pattern for applying a voltage to each through hole of the microchip of FIG. 2, (A) is a plan view, and (B) is a position along the line AA ′ of (A). 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; FIG.
FIGS. 5A and 5B are plan views showing other examples of microchips each provided with an electrode for applying a voltage to each through hole. FIGS.
FIG. 6 is a perspective view showing an embodiment.
FIG. 7 is a schematic configuration diagram showing a control system of the same embodiment;
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Microchip 2-1, 2-2 Separation flow path 3-1, 3-2 Sample introduction flow path 4 Electrode pattern 5 Connector part 10 Microchip loading tray 12 Sample injection needle 13 Electrophoresis liquid pressurizing and feeding rod 16 X mount 17 Z mount 20, 22 Syringe 26 Drive circuit B1, B2 Electrophoresis liquid reservoir S1, S2 Sample inlet

Claims (1)

A pair of transparent plate-like members is provided, and a groove through which liquid flows is formed on the surface of at least one plate-like member, and the other plate-like member is provided with a through hole at a position corresponding to the groove. Using a microchip in which a separation channel and a sample introduction channel that are crossed with each other are formed with the groove inside, and the separation channel and the sample introduction channel are filled with the electrophoresis solution, and the sample introduction In a microchip electrophoresis apparatus that introduces a sample from a flow channel into a separation flow channel, applies an electrophoresis voltage between both ends of the separation flow channel, and electrophoretic separates the sample in the separation flow channel,
A moving mechanism for moving the microchip;
A sample injection mechanism for injecting the sample into the sample injection hole by moving the needle for supplying the sample up and down on the movement locus of the sample injection hole which is one of the through holes;
An electrophoretic liquid injection mechanism for injecting an electrophoretic liquid into the electrophoretic liquid injection hole by moving the electrophoretic liquid supply port for supplying the electrophoretic liquid up and down on the movement trajectory of the electrophoretic liquid injection hole which is another one of the through holes;
Position the sample injection hole directly below the needle of the sample injection mechanism, position the electrophoresis solution injection hole directly below the electrophoresis solution supply port of the electrophoresis solution injection mechanism, and the detection point on the separation channel at the position of the detection unit. A microchip electrophoresis apparatus comprising: a control unit that controls the movement of the microchip by the movement mechanism and the operation of the sample injection mechanism and the electrophoresis solution injection mechanism.

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