JP3562460B2 - Electrophoresis device - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an electrophoresis apparatus used for electrophoresis for analyzing very small amounts of proteins, nucleic acids, drugs, and the like at high speed and high resolution, and more specifically, a chip for performing electrophoresis in a flow path formed inside a plate member The present invention relates to an electrophoresis apparatus using a device.
[0002]
[Prior art]
When analyzing very small amounts of proteins, nucleic acids, and the like, an electrophoresis apparatus has been conventionally used, and a typical example thereof is a capillary electrophoresis apparatus. A capillary electrophoresis apparatus is a device in which a glass capillary having an inner diameter of 100 μm or less is filled with an electrophoresis medium, a sample is introduced into one end, and a high voltage is applied between both ends to separate and develop an analyte in the capillary. It is. 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 a very small amount of sample such as DNA can be analyzed at high speed and with high resolution.
[0003]
Since the capillary has an outer diameter of about 100 to 500 μm and is easily broken, there is a problem that it is not easy for the user to handle the capillary when replacing it. Therefore, instead of a capillary that requires complicated handling, D.I. J. Harrison et al. / Anal. Chem. As shown in 1993, 283, 361-366, a microchip formed by bonding two substrates has been proposed. FIG. 4 shows an example of the microchip.
[0004]
The microchip 1 is composed of a pair of substrates 1a and 1b made of a transparent plate-like inorganic material (eg, glass, quartz, silicon, etc.) or plastic, and is formed on the surface of one substrate 1b by a semiconductor photolithography technique or a micromachining technique. Electrophoresis capillary grooves 3 and 5 that intersect each other are formed, and through holes are formed in the other substrate 1a at positions corresponding to the ends of the grooves 3 and 5, respectively, the anode reservoir 7a, the cathode reservoir 7c, the sample reservoir 7s, and the waste reservoir 7w. It is provided as. The microchip 1 is used in a state where both substrates 1a and 1b are overlapped and joined as shown in FIG. Such a microchip is also called a cross-channel micro-chip because two channels are formed to intersect.
[0005]
When electrophoresis is performed using this microchip 1, prior to analysis, for example, by pumping using a syringe, one of the reservoirs, for example, from the anode reservoir 7a to the inside of the grooves 3, 5 and the reservoirs 7a, 7c, 7s. , 7w is filled with the electrophoresis medium. Next, the electrophoresis medium filled in the reservoirs 7a, 7c, 7s, and 7w is removed, and a sample is injected into the sample reservoir 7s corresponding to one end of the shorter groove (sample injection flow path) 3; The buffer solution is injected into the reservoirs 7a, 7c, 7w.
[0006]
The microchip 1 into which the electrophoresis medium, the sample, and the buffer solution have been injected is mounted on an electrophoresis apparatus. A predetermined voltage is applied to each of the reservoirs 7a, 7c, 7s, and 7w to cause the sample to migrate into the groove 3 and to lead to the intersection 9 between the grooves 3,5. By switching the voltage applied to each of the reservoirs 7a, 7c, 7s, 7w, the sample existing at the intersection 9 is changed by the voltage between the reservoirs 7a, 7c at both ends of the longer groove (separation channel) 5. Inject into. After injecting the sample into the groove 5, the sample contained in the reservoir 7s is replaced with a buffer solution. Thereafter, a voltage for electrophoresis is applied to each of the reservoirs 7a, 7c, 7s, 7w, and the sample injected into the groove 5 is separated in the groove 5. By disposing a detector at an appropriate position in the groove 5, a sample separated by electrophoresis is detected. Detection is carried out by means such as an absorption spectrophotometry, a fluorescence photometry, an electrochemical or electric conductivity method.
[0007]
The analysis conditions such as the flow path design of the microchip 1 and the composition of the electrophoresis medium differ depending on the use and the sample. Microchips having other flow path designs include, for example, Ying Shi et al. / Anal. Chem. As shown in 1999, 71, 5354-5361, there is an electrophoresis microplate provided with a large number of separation channels radially. In recent years, a device having a size larger than a microchip, a device having a plurality of channels, and a device having a straight channel without an intersection of channels have been used. The chip device in the present invention includes all of them.
[0008]
[Problems to be solved by the invention]
Conventionally, loading the microchip with the electrophoretic medium, removing the electrophoretic medium in the reservoir, injecting the sample and buffer solution into the reservoir, removing the sample in the reservoir after injecting the sample into the separation channel, and removing the sample into the reservoir after removing the sample All buffer liquid injections were performed manually.
However, it is complicated for the operator to perform these operations manually using a syringe or the like.
Therefore, an object of the present invention is to provide an electrophoresis apparatus in which at least a part of these operations is automated.
[0009]
[Means for Solving the Problems]
In the electrophoresis apparatus of the present invention, a sample injection flow path and a separation flow path that intersect each other are formed inside the plate-like member, and reach the flow path at a position corresponding to the flow path on one surface of the plate-like member. An electrophoresis apparatus using a chip device in which a hole is formed as a reservoir, a chip holding mechanism for holding the chip device, and an electrophoresis device for filling a flow path and a reservoir with an electrophoresis medium via a reservoir of the chip device. A medium filling mechanism, a sample injection mechanism for injecting a sample into the reservoir, a voltage supply mechanism for supplying a voltage to each reservoir, a detection mechanism for detecting a sample separated in the flow path, And a control unit for controlling the mechanism to operate automatically.
[0010]
After the chip device is held by the chip holding mechanism, the electrophoresis medium filling mechanism is operated by the control unit to fill the flow path and the reservoir of the chip device with the electrophoresis medium. Activate the sample injection mechanism to inject the sample into the sample reservoir. The voltage supply mechanism is operated to supply a voltage to each reservoir to separate the sample in the channel. Activate the detection mechanism to detect the separated sample.
[0011]
The present invention provides an electrophoretic medium suction mechanism provided for each of the reservoirs for removing the electrophoretic medium filled in the reservoir, and simultaneously buffering the buffer solution in each of the reservoirs after the electrophoretic medium is removed. A buffer liquid injection mechanism provided for each of the reservoirs for injection is further provided, and the control unit controls the electrophoretic medium suction mechanism and the buffer liquid injection mechanism to operate automatically.
When using an electrophoretic medium that cannot be applied with a voltage by directly contacting the electrodes, the electrophoretic medium filling mechanism is activated to load the electrophoretic medium into the flow path of the chip device and the reservoir, and then the electrophoretic medium suction mechanism is activated. Then, the electrophoresis medium filled in the reservoir is removed. The buffer solution injection mechanism is operated to inject the buffer solution into the reservoir after the removal of the electrophoresis medium excluding the sample reservoir. Activate the sample injection mechanism to inject the sample into the sample reservoir after the electrophoresis medium has been removed. As a result, a voltage can be applied to the electrophoresis medium via the electrophoresis buffer. Thereafter, the voltage supply mechanism and the detection mechanism are operated to detect the separated sample.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
【Example】
FIG. 1 is a schematic perspective view showing one embodiment of the electrophoresis apparatus according to the present invention. The microchip 1 shown in FIG. 1 is the same as that of FIG.
A chip holding mechanism (not shown) for holding the microchip 1 as a chip device is provided, and the chip 1 is moved to positions A and B in the direction of arrow 11 in the figure by a moving mechanism provided in the chip holding mechanism. , C.
[0014]
Above the position A, a migration medium suction and buffer liquid injection port 13 is provided. The port 13 includes a nozzle fixing member 15 and four sets of suction nozzles 17 and discharge nozzles 19 fixed to the member 15 corresponding to the arrangement of the reservoirs 7a, 7c, 7s, and 7w when the chip 1 is positioned at the position A. It has. The suction nozzle 17 and the discharge nozzle 19 are respectively connected to independent syringes (not shown). Further, the port 13 is provided with an elevating mechanism (not shown) for elevating and lowering the member 15, and the member 15 is moved up and down in the direction of arrow 21 in the figure. When the tip 1 is located at the position A, the member 15 is lowered by the elevating mechanism so that the tips of the nozzles 17 and 19 enter the reservoirs 7a, 7c, 7s and 7w.
[0015]
The electrophoretic medium suction mechanism and the buffer liquid injection mechanism that constitute the present invention include the electrophoretic medium suction and buffer liquid injection port 13, a syringe, and an elevating mechanism.
As the nozzles 17 and 19, for example, a capillary made of resin can be used. However, the nozzles 17 and 19 are not limited to capillaries made of resin. For example, capillaries made of other materials such as glass capillaries can be used.
[0016]
Above position B, a sample loading syringe 23 constituting a sample injection mechanism and a loading medium loading port 25 constituting a loading medium loading mechanism are provided.
The sample injection mechanism includes a syringe 23 provided corresponding to the position of the sample reservoir 7s when the tip 1 is positioned at the position B, and a mechanism for moving the syringe 23 in a three-dimensional direction (see an arrow 27 in the figure). A moving mechanism (not shown) and a cylinder driving mechanism (not shown) for performing suction and discharge of the syringe 23 are provided. When the tip 1 is located at the position B, the suction and discharge port of the syringe 23 enters the sample reservoir 7s.
[0017]
The port 25 is connected to a syringe (not shown) for accommodating the electrophoresis medium, and has a nozzle 31 provided corresponding to the position of the anode reservoir 7a when the chip 1 is positioned at the position B. A sealing material 33 is provided at the tip of the nozzle 31. The electrophoretic medium filling mechanism includes a port 25, a syringe for pushing out the electrophoretic medium from the nozzle 31, and an elevating mechanism (not shown) for elevating the port 25 in the direction of arrow 35 in the figure. By the elevating mechanism, the port 25 is lowered when the chip 1 is located at the position B, and the tip of the nozzle 31 is brought into close contact with the anode port 7a by the sealing material 33.
[0018]
Above position C, an electrode port 37 is provided. The port 37 has an electrode fixing member 39 and four electrodes 41 fixed to the member 39 corresponding to the arrangement of the reservoirs 7a, 7c, 7s, 7w when the chip 1 is positioned at the position C. The electrode 41 is connected to a high voltage supply device (not shown). Further, the port 37 is provided with an elevating mechanism (not shown) for elevating and lowering the member 39, and the member 15 is moved up and down in the direction of arrow 43 in the figure. The voltage supply mechanism includes an electrode port 37, a high voltage supply device, and an elevating mechanism. The member 39 is lowered by the elevating mechanism so that the tip of the electrode 41 enters the reservoirs 7a, 7c, 7s, 7w when the chip 1 is at the position C.
[0019]
A detection optical system (detection mechanism) 45 is provided below the position C. When the chip 1 is located at the position C, the detection optical system 45 irradiates the detection light 47 to the detection position of the separation channel 5 between the intersection 9 and the anode reservoir 7a, and separates the detection light 47 by the ultraviolet absorption amount. This is for detecting a sample.
The chip holding mechanism, the electrophoresis medium suction and buffer liquid injection port 13, the sample injection mechanism, the electrophoresis medium filling mechanism, the voltage supply mechanism, and the detection optical system 45 are controlled by a control unit (not shown).
[0020]
FIG. 2 is a flowchart showing an operation example of this embodiment. The operation of this embodiment will be described with reference to FIGS. Here, an electrophoretic medium containing an organic polymer (hereinafter simply referred to as a polymer) was used.
The chip 1 is placed at the position B (step S1).
The loading medium loading port 25 is lowered, and the tip of the nozzle 31 is brought into close contact with the anode reservoir 7a of the chip 1 by the sealing material 33. The polymer is pressure-filled from the syringe containing the polymer into the channel of the chip 1 and the reservoirs 7c, 7s, 7w via the nozzle 31 and the anode reservoir 7a. When the polymer comes out of all of the reservoirs 7c, 7s, 7w, the filling is completed (step S2).
[0021]
The migration medium loading port 25 is returned upward, and the chip 1 is moved to the position A (step S3).
The electrophoretic medium suction and buffer liquid injection port 13 descends, causing the nozzles 17 and 19 to enter the polymer contained in the reservoirs 7a, 7c, 7s and 7w. The suction mechanism connected to the suction nozzle 17 operates, and the polymer contained in each of the reservoirs 7a, 7c, 7s, 7w is suctioned and removed via the suction nozzle 17 (step S4). After the removal of the polymer, the discharge mechanism connected to the discharge nozzle 19 corresponding to the reservoirs 7c, 7a, 7w operates, and the buffer liquid is injected from the discharge nozzle 19 into the reservoirs 7c, 7a, 7w excluding the sample reservoir 7s (step S5).
[0022]
The port 13 is returned upward, and the chip 1 is moved to the position B (step S6).
The syringe 23 moves, and the suction / discharge port of the syringe 23 enters the empty sample reservoir 7s. The syringe 23 performs a discharging operation, and injects a sample previously sucked into the syringe 23 from a sample port (not shown) into the sample reservoir 7s (step S7).
[0023]
The syringe 23 is returned upward, and the tip 1 is moved to the position C (step S8).
The electrode port 37 descends, bringing the electrode 41 into contact with the buffer solution or sample contained in the reservoirs 7a, 7c, 7s, 7w. After applying a predetermined voltage to the buffer solution or the sample contained in the reservoirs 7a, 7c, 7s, 7w via the electrode 41 to guide the sample to the intersection of the sample introduction channel and the separation channel, The voltage is switched to inject the sample into the separation channel (step S9).
[0024]
The port 37 is returned upward, and the chip 1 is moved to the position A (step S10).
The port 13 descends, causing the nozzles 17, 19 to enter the sample or buffer solution contained in the reservoirs 7a, 7c, 7s, 7w. The suction mechanism connected to the suction nozzle 17 corresponding to the sample reservoir 7s operates, and the excess sample remaining in the sample reservoir 7s is suctioned and removed via the suction nozzle 17 (step S11). After removing the sample, the ejection mechanism connected to the ejection nozzle 19 corresponding to the sample reservoir 7s operates to inject the buffer solution from the ejection nozzle 19 into the sample reservoir 7s (Step S12).
[0025]
The port 13 is returned upward, and the chip 1 is moved to the position C (step S13).
The electrode port 37 descends, bringing the electrode 41 into contact with the buffer solution contained in the reservoirs 7a, 7c, 7s, 7w. A predetermined voltage is applied to the reservoirs 7a, 7c, 7s, 7w via the electrodes 41 to perform electrophoretic separation of the sample injected into the separation channel, and the separated sample is detected by the detection optical system 45 (step). S14).
[0026]
As described above, in this embodiment, the microchip is filled with the polymer, the polymer is removed from the reservoir, the sample and the buffer solution are injected into the reservoir, the sample is removed from the reservoir after the sample is injected into the separation channel, and the sample after the sample is removed. Injection of the buffer solution into the reservoir, and separation and detection of the sample can all be performed automatically.
[0027]
FIG. 3 is a flowchart showing another operation example of this embodiment. The operation of this embodiment will be described with reference to FIGS. Here, an inorganic ionic buffer (hereinafter, referred to as an electrophoresis buffer) was used as an electrophoresis medium.
The chip 1 is placed at the position B (step S21).
The loading medium loading port 25 is lowered, and the tip of the nozzle 31 is brought into close contact with the anode reservoir 7a of the chip 1 by the sealing material 33. The migration buffer is filled from the syringe containing the migration buffer into the channel of the chip 1 and the reservoirs 7c, 7s, 7w via the nozzle 31 and the anode reservoir 7a. When the electrophoresis buffer comes out from all of the reservoirs 7c, 7s, 7w, the filling is completed (step S22). Here, the loading of the migration buffer is performed by the migration medium loading port 25. However, the chip 1 is moved to the position A, and a discharge mechanism connected to the migration medium suction and buffer liquid injection port 13, the discharge nozzle 19, and the discharge nozzle 19 is provided. It may be used to fill the electrophoresis buffer.
[0028]
After the migration medium loading port 25 returns to the top, the syringe 23 moves, and the suction and discharge port of the syringe 23 enters near the inlet of the sample introduction channel 3 in the sample reservoir 7s. The syringe 23 performs a discharging operation, and injects a sample previously sucked into the syringe 23 from a sample port (not shown) into the sample reservoir 7s (step S23).
[0029]
The syringe 23 is returned upward, and the tip 1 is moved to the position C (step S24).
The electrode port 37 descends, bringing the electrode 41 into contact with the electrophoresis buffer or sample contained in the reservoirs 7a, 7c, 7s, 7w. After applying a predetermined voltage to the electrophoresis buffer or the sample contained in the reservoirs 7a, 7c, 7s, 7w via the electrode 41 to guide the sample to the intersection of the sample introduction channel and the separation channel, The voltage is switched to inject the sample into the separation channel (step S25), and then the sample is subjected to electrophoretic separation, and the separated sample is detected by the detection optical system 45 (step S26).
Thus, in this embodiment, the filling of the electrophoresis buffer into the microchip, the injection of the sample into the reservoir, and the separation and detection of the sample can all be performed automatically.
[0030]
Here, the channel design of the chip 1 is arbitrary.
The electrophoresis medium is not particularly limited, and any electrophoresis medium such as an electrophoresis buffer such as an inorganic ionic buffer such as tris-boric acid, or an organic polymer such as hydroxymethylcellulose, hydroxyethylcellulose, or polyacrylamide. May be used.
The electrophoresis buffer and the buffer solution are not particularly limited, and tris-boric acid-EDTA (ethylenediaminetetraacetic acid) (TBE) or tris-TAPS (tetrapentylammonium 3- {tris (hydroxymethyl) methylamino} -1-) An appropriate material such as a propanesulfate-EDTA (TTE) system can be used depending on the electrophoresis medium, measurement conditions, and the like.
[0031]
In this embodiment, a syringe is used as a suction mechanism connected to the suction nozzle 17, but the present invention is not limited to this, and another suction mechanism such as a vacuum pump or an aspirator may be used. May be used.
Further, in this embodiment, the suction nozzles 17 are connected to independent syringes, respectively, but the present invention is not limited to this. At least the suction nozzles 17 for the sample reservoir 7s are connected to independent syringes. Alternatively, the suction nozzles 17 for the other reservoirs 7a, 7b, 7w may be connected to a common syringe. The same applies to a case where another suction mechanism is used in place of the syringe.
[0032]
In this embodiment, a syringe is used as a discharge mechanism connected to the discharge nozzle 19, but the present invention is not limited to this, and other discharge mechanisms, such as a peristaltic pump and a gas pressurizing mechanism, may be used. Other ejection mechanisms may be used.
Further, in this embodiment, the discharge nozzles 19 are connected to independent syringes, respectively, but the present invention is not limited to this. At least the discharge nozzle 19 for the sample reservoir 7s is connected to an independent syringe. Alternatively, the discharge nozzles 19 for the other reservoirs 7a, 7b, 7w may be connected to a common syringe. The same applies to a case where another ejection mechanism is used instead of the syringe.
In this embodiment, a syringe for the suction nozzle 17 and a syringe for the discharge nozzle 19 are provided. However, the present invention is not limited to this, and one common syringe and a common syringe are connected to the suction nozzle 17. A switching valve may be provided for switching to and connecting to the discharge nozzle 19, and the suction from the suction nozzle 17 and the discharge from the discharge nozzle 19 may be performed by switching the switching valve and operating the common syringe. This is the same when other suction and discharge mechanisms are used instead of the syringe.
[0033]
In this embodiment, a detection mechanism that detects a sample separated by ultraviolet absorption is used as a detection mechanism, but the present invention is not limited to this, and detection using one-color or multi-color fluorescence or detection light irradiated A detection mechanism using another detection principle, such as detection by scattering of light, may be used.
In this embodiment, a detection mechanism that detects a sample at a single detection position is used.However, the present invention is not limited to this, and a detection mechanism that detects an image in a predetermined range of a separation channel is used. May be used.
[0034]
In this embodiment, the microchip is moved between positions A, B, and C. However, the present invention is not limited to this, and the microchip is fixed to the chip holding mechanism, The medium suction mechanism, the buffer solution injection mechanism, the sample injection mechanism, the sample suction mechanism, the voltage supply mechanism or the detection mechanism, or a combination thereof may be moved above or below the fixed microchip.
[0035]
In this embodiment, a microchip having four reservoirs is used. However, the present invention is not limited to this, and can be applied to a microchip having five or more reservoirs. That is, the present invention can be applied to a microchip provided with a large number of separation channels.
Further, in this embodiment, a chip device in which a sample introduction channel and a separation channel that intersect with each other is used, but the chip device used in the present invention is not limited to this. For other chip devices, such as those with separation channels that do not have crossing channels, those with multiple channels intersecting with the separation channels, multi-channels, and large channels The present invention can also be applied. In these cases, it is preferable to change the nozzle configuration of the electrophoresis medium filling mechanism, electrophoresis medium suction mechanism, buffer solution injection mechanism, sample injection mechanism, and sample suction mechanism according to the arrangement of the reservoir.
[0036]
In this embodiment, a voltage supply mechanism provided with an electrode 41 for entering a buffer solution and a sample is provided. However, the present invention is not limited to this. For example, a chip-side electrode for supplying electricity to a reservoir on a microchip surface is provided. Is formed, a voltage supply mechanism having an electrode for connecting to the chip-side electrode can be used.
[0037]
【The invention's effect】
The electrophoresis apparatus of the present invention includes a chip holding mechanism, a migration medium filling mechanism, a sample injection mechanism, a voltage supply mechanism, a detection mechanism, and a control unit for controlling these mechanisms. Since these mechanisms are operated, the loading of the chip device with the electrophoresis medium, the injection of the sample into the reservoir, and the separation and detection of the sample can be performed automatically.
[0038]
An electrophoresis medium suction mechanism for removing the electrophoresis medium filled in the reservoir, and a buffer solution injection mechanism for injecting a buffer solution into the reservoir after the electrophoresis medium has been removed, the control unit includes: The electrophoretic medium suction mechanism and the buffer liquid injection mechanism are also controlled to operate automatically, the electrophoretic medium in the reservoir is removed by the electrophoretic medium suction mechanism, and the buffer liquid injection mechanism is inserted into the reservoir after the electrophoretic medium is removed. Thus, even when using an electrophoretic medium to which a voltage cannot be applied by directly contacting the electrodes, a voltage can be applied to the electrophoretic medium via the buffer solution.
In addition, by separately providing the sample injection mechanism, the electrophoretic medium suction mechanism, and the buffer solution injection mechanism, the analysis cycle time can be significantly reduced, such as the nozzle washing step can be omitted, resulting in higher throughput. Can be planned.
In addition, since each reservoir can be filled with the buffer solution at the same time, the influence of the head difference (head difference) can be reduced.
[Brief description of the drawings]
FIG. 1 is a schematic configuration perspective view showing one embodiment.
FIG. 2 is a flowchart illustrating an operation example of the embodiment.
FIG. 3 is a flowchart showing another operation example of the embodiment.
4A and 4B are diagrams illustrating an example of a microchip, in which FIG. 4A is a top view of one substrate, FIG. 4B is a top view of the other substrate, and FIG. FIG.
[Explanation of symbols]
Reference Signs List 1 Microchip 3 Sample injection channel 5 Separation channel 7a Anode reservoir 7c Cathode reservoir 7s Sample reservoir 7w Weight reservoir 13 Electrophoretic medium suction and buffer liquid injection port 15 Nozzle fixing member 17 Suction nozzle 19 Discharge nozzle 23 Sample loading syringe 25 Electrophoresis medium loading port 31 Nozzle 33 Seal material 37 Electrode port 39 Electrode fixing member 41 Electrode 45 Detection optical system

Claims (1)

In an electrophoresis apparatus using a chip device in which a flow path is formed inside the plate member and a hole reaching the flow path at a position corresponding to the flow path on one surface of the plate member is formed as a reservoir,
A chip holding mechanism for holding a chip device;
An electrophoresis medium filling mechanism for filling the electrophoresis medium into the flow path and the reservoir via the reservoir of the chip device,
A sample injection mechanism for injecting a sample into the reservoir,
A voltage supply mechanism for supplying a voltage to each of the reservoirs,
A detection mechanism for detecting the sample separated in the flow path,
For removing the electrophoresis medium filled in the reservoir, an electrophoresis medium suction mechanism provided corresponding to each of the reservoirs,
For simultaneously injecting buffer solution into each of the reservoirs after the electrophoresis medium has been removed, a buffer solution injection mechanism provided corresponding to each of the reservoirs,
An electrophoresis apparatus comprising: a control unit for controlling these mechanisms to operate automatically.

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