JP5488511B2 - Electrophoresis device and electrophoretic analyzer - Google Patents

Description

  The present invention includes a separation channel and a sample reservoir provided at one end of the separation channel, and performs electrophoretic separation of a sample injected into the sample reservoir by applying a voltage to both ends of the separation channel. And an electrophoretic analyzer using the electrophoretic device.

FIG. 5 shows an example of a conventional microchip type electrophoresis device.
This electrophoretic device is configured by overlapping two transparent substrates 52 and 54, and a separation channel 56 is formed therein. A sample well 56a is provided at one end of the separation channel 56, and a sample reservoir 58 is provided above the sample well 56a. In addition, a waste liquid well 56b is provided at the other end of the separation channel 56, and a waste liquid reservoir 60 is provided above the well.

An example of the electrophoresis process using the electrophoresis device of FIG. 5 will be described with reference to FIG.
First, the separation medium is discharged into the sample well 56a from the nozzle that discharges the separation medium (step S11). After moving the separation medium discharge nozzle, a mechanism that can supply air toward the sample well 56a while sealing the upper surface of the sample well 56a is used, and the sample well is sealed by the pressure of air while sealing the upper surface of the sample well 56a. The separation medium 56a is fed into the separation channel 56 (steps S12 and S13). At this time, the separation medium is pumped until the separation medium comes out to the side of the waste liquid well 56b opposite to the sample reservoir 58, and the inside of the separation channel 56 is filled with the separation medium. The separation channel 56 can be filled with the separation medium from the waste liquid well 56b side.

  The separation medium in the wells 56a and 56b is sucked by the suction nozzle, and the separation medium is left only in the separation channel 56 (step S14). The waste liquid reservoir 60 is filled with the buffer solution, and the sample is dispensed into the sample well 56a (steps S15 and S16). Samples are introduced by inserting electrodes into the liquids of the wells 56a and 56b and applying a voltage across the separation channel 56 (step S17). Thereafter, the sample in the sample well 56a is removed (step S18), and a buffer is injected into the sample reservoir 58 (step S19). Electrophoresis is performed by inserting electrodes into the reservoirs 58 and 60 and applying a voltage across the separation channel 56 (step S20). When electrophoresis of a new sample is performed using the same microchip after the completion of electrophoresis, the process returns to step S11 and the operations up to step S20 are repeated.

US Pat. No. 5,653,050

  In the electrophoresis device shown in FIG. 5, as a separation medium filling mechanism for filling the separation channel 56 with the separation medium, the upper surface of the well 56 a or 56 b is sealed in addition to the nozzle for discharging the separation medium. A special mechanism capable of supplying air is required, and the configuration of the apparatus is complicated. In addition, when filling the separation channel into the separation channel, it is necessary to go through two steps: a step of discharging the separation medium from the nozzle and a step of sealing the upper surface of the well and supplying air. However, since the upper surface of the well has a large area, it is not easy to completely seal it. If the sealing of the upper surface of the well is not complete when the separation channel 56 is filled, the separation medium to the separation channel 56 The reproducibility of the analysis results deteriorates due to incomplete filling of the liquid or bubbles in the separation medium.

  Furthermore, when electrophoresis of a plurality of samples is performed continuously, it is necessary to go through two steps: a step of discharging a separation medium from a nozzle and a step of sealing the upper surface of the well and supplying air for each sample. There is a further risk of problems such as poor filling of the separation medium and mixing of bubbles into the separation medium. Therefore, in order to reduce the risk of such a problem, the structure of the separation medium filling mechanism in the separation flow path is simplified and the upper surface of the well does not have to be sealed via the reservoir when filling the separation medium. It is desirable to make it.

  On the other hand, an electrophoretic device that is not a microchip type has been proposed that does not require a sealing step on the upper surface of the reservoir when filling the separation channel into the separation channel (see Patent Document 1). In the proposed apparatus, a valve is provided between the separation medium filling mechanism and the separation flow path. When the separation flow path is filled with the separation medium, the valve is opened, and thereafter, when electrophoresis is performed, the valve is closed. In this configuration, there is no risk of air bubbles being mixed into the separation medium because there is no operation of sealing the upper surface of the reservoir when filling the separation channel into the separation channel. However, in this configuration, since the valve exists on the flow path, there is a problem that the dead volume increases and the amount of the separation medium used increases.

  Accordingly, an object of the present invention is to simplify the configuration and processing necessary for filling a separation channel into a separation channel without increasing the dead volume.

  The present invention provides a separation channel for performing electrophoretic separation of a sample introduced to one end by applying a voltage to both ends, and one end of the separation channel on the sample introduction side. And a separation medium injecting unit for injecting a separation medium into the other end side flow path to the other end portion at the same time. According to this configuration, since the separation medium injection part is connected in the middle of the separation flow path, the separation medium filling mechanism can be connected to the separation medium injection part even when the analysis is repeated a plurality of times. By connecting the separation medium filling mechanism every time the separation medium is injected, the possibility of air being mixed into the separation medium in the analysis channel is suppressed. Further, an operation of sealing the upper surface of the well every time the separation medium is filled becomes unnecessary.

  An electrophoretic analyzer of the present invention uses the electrophoretic device of the present invention, and includes a separation medium filling mechanism that fills a separation channel from a separation medium injection portion of the electrophoretic device, and an electrophoretic device. At least a voltage application unit that applies a voltage for electrophoresis between both ends of the separation channel, and a detector that detects a sample component electrophoretically separated in the separation channel of the electrophoresis device. A series of operations including filling the separation medium into the channel, electrophoretic separation of the sample in the separation flow path, and detection of the electrophoretic separated sample components, and the separation medium filling mechanism as the separation medium injection part of the electrophoresis device Repeated multiple times in a connected state.

  In the electrophoretic device of the present invention, when a detection position for detecting a separation component is provided on the one-end flow path, the separation medium in the one-end flow path is replaced during the repeated electrophoretic analysis. Is preferably carried out reliably. This is because if the separation medium or sample component used in the previous electrophoresis remains in the one end side flow path, it greatly affects the next sample measurement result. On the other hand, even if such a residue is present in the flow path on the other end side of the separation flow path, the influence on the measurement result is small compared to the case where it remains in the flow path on the one end side. Therefore, when a detection position for detecting a separation component is provided on the one end side flow path, the flow path resistance of the other end side flow path of the separation flow path is set equal to or higher than that of the one end side flow path. It is preferable to do. Then, when the separation medium is injected from the separation medium injection section, the flow rate of the separation medium flowing in each of the one end-side flow path and the other end-side flow path is the same or larger in the one end-side flow path. The flow rate of the separation medium in the channel can be secured, and the presence of the residue in the one end side channel can be suppressed.

  In the electrophoretic device of the present invention, the separation medium injected from the separation medium injection section is simultaneously injected into the one end side flow path and the other end side flow path of the separation flow path, so that the separation medium is completely in the one end side flow path. If the separation medium is completely filled in the other end-side flow path before being filled, the separation medium is wasted from the other end-side flow path until the one end-side flow path is completely filled with the separation medium. It will be discharged. Therefore, the separation medium injected from the separation medium injection section is filled in both the flow paths at the same time, or the one end side flow path and the other end side of the separation flow path so that the one end side flow path completes the filling first. It is preferable that the length of the flow path and the flow path resistance are set. By doing so, the waste of the separation medium can be reduced.

  A reservoir for storing the liquid discharged from the other end is provided at the other end of the separation channel, and the reservoir has a capacity larger than that of the sample-side reservoir on one end side, and is used for the separation medium to be drained. It is preferable that the size is set such that a plurality of times can be stored. Then, it is possible to perform the next electrophoresis without collecting the separation medium waste from the other end of the separation channel after electrophoresis. As a result, when electrophoresis is repeatedly performed for a plurality of samples, the separation medium recovery operation can be reduced, and the operating rate is improved.

  According to the present invention, since the separation medium injection portion is provided in the middle of the separation flow path, a step of sealing the upper surface of the well is not necessary when filling the separation medium, and the structure of the separation medium filling mechanism is more than conventional. It can be simplified. Even when the separation medium filling mechanism is connected to the separation medium injection section, it is possible to apply a voltage to both ends of the separation flow path, and thus electrophoresis can be performed while the separation medium filling mechanism is connected. As a result, it becomes possible to continuously and repeatedly perform electrophoresis of a plurality of samples while the separation medium filling mechanism is connected to the separation medium injection section, and the separation medium filling accuracy in the separation flow path is improved. This reduces the risk of air bubbles being mixed in, improves the reproducibility of analysis results by electrophoresis, and improves the operating rate. In addition, since the separation medium injected from the separation medium injection section is configured to be injected simultaneously to one end side and the other end side of the separation flow path, the separation medium injection destination is switched between the one end side and the other end side. Therefore, a mechanism such as a valve is unnecessary, and the dead volume can be reduced.

It is a flow path lineblock diagram showing roughly composition of one example of an electrophoretic analysis device using an electrophoresis device of one example. It is a figure which shows an example of a microchip type electrophoresis device, (A) is a top view, (B) is sectional drawing when cut along the one end side flow path of a separation flow path, (C) is a separation medium It is sectional drawing when it cut | disconnects along the one end side flow path of the separation flow path which shows the state which connected the discharge syringe. It is a figure which shows the other example of a microchip type electrophoresis device, (A) is a top view, (B) is sectional drawing when cut | disconnecting along a separation flow path, (C) is a separation-medium discharge syringe. It is sectional drawing when cut | disconnecting along the separation flow path which shows the connected state. 3 is a flowchart for explaining an example of electrophoresis processing using the electrophoresis device of FIG. 2. It is a figure which shows an example of the microchip part of the electrophoresis device using the conventional microchip, (A) is a top view, (B) is sectional drawing when cut | disconnecting along a separation flow path. 6 is a flowchart for explaining an example of electrophoresis processing using the electrophoresis device of FIG. 5.

A schematic configuration of one embodiment of the electrophoretic analyzer will be described with reference to FIG.
An electrophoretic analyzer uses an electrophoretic device, and only an essential part of the electrophoretic device is schematically shown in FIG. The separation flow path 2 of the electrophoresis device is subjected to electrophoretic separation of the sample introduced into the one end 2a by applying a voltage between the one end 2a and the other end 2b. A separation medium injection unit 10 for supplying a separation medium is provided in the middle of the separation channel 2, and the separation medium is injected from the separation medium injection unit 10 into the separation channel 2. A part of the separation channel 2 is provided with a detection position 12 where a sample component separated by electrophoresis is detected.

  The electrophoretic analyzer includes a separation medium filling mechanism 16 that fills the separation channel 2 from the separation medium injection unit 10 of the electrophoresis device, and one end 2a and the other end 2b of the separation channel 2 of the electrophoresis device. At least a voltage application unit 18 for applying a voltage for sample introduction and electrophoresis in between, and a detector 14 for detecting a sample component electrophoretically separated in the separation channel 2 of the electrophoresis device at a detection position 12 are provided. ing.

  The electrophoretic analyzer separates a series of operations including filling of the separation medium into the separation channel 2, electrophoretic separation of the sample in the separation channel 2, and detection of the sample components separated by electrophoresis. The medium filling mechanism 16 is repeated a plurality of times while being connected to the separation medium injection section 12 of the electrophoresis device. In order to perform this series of operations, a control device (not shown) that controls the operations can be provided, and the series of operations can be automatically performed. Such a control device can be realized by a computer dedicated to the electrophoretic analyzer or a general-purpose computer such as a personal computer connected to the outside.

  In addition, a part of this series of operations can be performed manually.

  The separation flow path 2 of the electrophoresis device is a flow path for performing electrophoretic separation of the sample introduced into the one end 2a by applying a voltage between the one end 2a and the other end 2b by the voltage applying unit 18. A separation medium injection unit 10 for supplying a separation medium is provided in the middle of the separation flow path 2. By connecting the separation medium filling mechanism 16 to the separation medium injection section 10, the separation medium is separated into the separation flow path 2 at one end side flow path 4 to the one end 2a on the sample introduction side and the other end side flow path to the other end 2b. 6 can be injected simultaneously. Detection of sample components separated by electrophoresis is configured to be detected by a detector 14 at a detection position 12. In FIG. 1, the detection position 12 is provided on the one end side flow path 4, but may be provided on the other end side flow path 6.

  When the detection position 12 is provided on the one end side flow path 4, it is important to reduce the risk of remaining of the previous separation medium or the like in the one end side flow path 4 rather than the other end side flow path 6. Therefore, in order to obtain a sufficient flow rate of the separation medium injected into the one end side channel 4, the one end side channel 4 and the other end side channel 6 of the separation channel 2 have the same or other channel resistance. It is preferable that the end channel is set to be higher. The channel resistances of the one end side channel 4 and the other end side channel 6 can be controlled by adjusting the length and inner diameter of each channel. One end side channel in the case where the sample is repeatedly and continuously electrophoresed by ensuring the flow rate of the separation medium at a certain flow rate or more in the one end side channel 4 by adjusting the channel resistance of both channels 4 and 6. The replacement efficiency of the separation medium in 4 is improved.

  Further, the lengths and inner diameters of both flow paths 4 and 6 are set so that the filling of the separation medium injected from the separation medium injection section 10 is completed simultaneously in the one end side flow path 4 and the other end side flow path 6. It is preferable. The fact that the filling of the separation medium is completed simultaneously in the one end side flow path 4 and the other end side flow path 6 means that the leading ends of the separation medium injected into both the flow paths 4 and 6 are simultaneously the one end 2a and the other end 2b of the separation flow path 2. Means to reach. Since the one end side flow path 4 must be completely filled with a new separation medium for the next sample, the filling of the separation medium in the other end side flow path 6 is completed first, Until the filling of the separation medium in the one end side flow path 4 is completed, the separation medium continues to be discharged from the other end side flow path 6, so that the separation medium is wasted. If it is set so that the filling of the separation medium in both flow paths 4 and 6 is completed at the same time, or if it is set so that the filling of the separation medium in at least one end-side flow path 4 is completed first, such It is possible to eliminate unnecessary consumption of the separation medium.

Next, an example of a microchip type electrophoresis device will be described with reference to FIG.
This electrophoretic device is constructed by laminating two transparent substrates 26 and 28, and a V-shaped separation channel 30 is formed inside as viewed from above. The separation channel 30 is configured by a groove formed in the substrate 26. A sample well 30a for sample introduction is provided at one end of the separation channel 30, and a waste liquid well 30b is provided at the other end. A sample reservoir 34 is provided at a position above the sample well 30a on the upper surface of the transparent substrate 26, and a waste liquid reservoir 36 is provided at a position above the waste liquid well 30b. In this example, the electrophoretic device is composed of two substrates 26 and 28, and a channel groove is formed on one of the substrates, but PDMS (polydimethylsiloxane) is used between the two substrates. The flow path forming sheet may be sandwiched, and the flow path may be formed as a through groove in the flow path forming sheet.

  A separation medium injection portion 38 formed of a through hole is provided at the position of the V-shaped apex portion of the separation flow path 30 of the substrate 26. Separation is achieved by sealing the upper surface of the separation medium injection unit 38 and the lower surface of the nozzle of the separation medium discharge syringe 39 which is a separation medium filling mechanism in close contact with the peripheral surface of the edge of the through hole constituting the separation medium injection unit 38. The medium discharge syringe 39 can be connected to the V-shaped apex portion of the separation channel 30. Since the separation medium injection part 38 is constituted by a through-hole having a small inner diameter, it is easy to seal the connection part between the separation medium injection part 38 and the separation medium discharge syringe 39 and prevent air bubbles from entering the separation flow path. can do. In order to improve the adhesion between the lower surface of the nozzle of the separation medium discharge syringe 39 and the peripheral surface of the edge of the through hole constituting the separation medium injection portion 38, an O-ring 39a is provided as a seal member on the lower surface of the nozzle.

  The flow path 32 from the V-shaped apex portion where the separation medium injection part 38 is provided in the separation flow path 30 to the one end 30a corresponds to the one end side flow path 4 in FIG. 1, and the other end 30b from the V-shaped apex portion. The flow path 33 up to this corresponds to the flow path 6 on the other end side. In this example, the channel 32 and the channel 33 are set to have the same channel length and the same inner diameter so as to have the same channel resistance. Since the flow resistance and the length of both the flow paths 32 and 33 are the same, the separation medium injected from the separation medium injection portion 38 at the V-shaped apex portion of the separation flow path 30 by the separation medium filling mechanism 39 is separated from the separation flow path. The both ends 30a and 30b of the 30 are reached almost simultaneously, and wasteful consumption of the separation medium is suppressed.

  The waste liquid reservoir 36 has a larger capacity than the sample reservoir 34. The waste liquid reservoir 36 can store a larger amount of liquid than the amount of buffer liquid necessary for electrophoresis, and is pushed out from the end 30b of the flow path 33 by a new separation medium injected by the separation medium discharge syringe 39. It has the capacity to store multiple batches of old separation media.

  If there is no significant difference between the composition of the separation medium and the buffer solution, the electrode can be inserted into the sample reservoir 34 and the waste fluid reservoir 36 at the time of electrophoresis at a constant position. Even if an old separation medium is mixed, the resistance value between both ends 30a and 30b of the separation flow path 30 hardly changes, and therefore the separation medium mixed with the buffer solution in the waste liquid reservoir 36 hardly affects the measurement result of electrophoresis. . That is, as in this example, if the capacity of the waste liquid reservoir 36 is increased so that a plurality of old separation media can be stored, it is possible to continuously remove the waste liquid in the waste liquid reservoir 36 without performing the operation of removing the separation medium. Electrophoresis can be performed repeatedly.

  Further, the separation channel 30 is V-shaped, and the positions of the sample reservoir 34 and the waste liquid reservoir 36 are provided close to each other, so that a nozzle for performing processing such as supply of buffer liquid and suction of liquid and electrodes for applying voltage are provided. Can be driven on a single axis and at a short distance, there is an effect that the configuration of the apparatus for processing this device can be simplified.

  Note that the separation channel may not be V-shaped, and may be linear as shown in FIG. In the example of FIG. 3, the reservoirs 34 and 36 have a cylindrical shape, but the reservoirs 34 and 36 may have any shape. In this example, the detection position is provided on the other end side flow path 33, but may be provided on the one end side flow path 32 when a migration distance sufficient for sample separation can be secured.

  Next, an example of electrophoresis processing using the electrophoresis device of FIG. 2 will be described using the flowchart of FIG.

  In this electrophoresis, for example, LPA (linear polyacrylamide) is used as a separation medium, and TTE (tetrapentylammonium 3- {tris (hydroxymethyl) methylamino} -1-propanesulfate) -EDTA) is used as a buffer solution. And not limited to them.

  First, in order to supply the separation medium, a separation medium discharge syringe 39 having an O-ring on the lower surface of the syringe nozzle is used. The separation medium discharge syringe 39 is connected to the separation flow path 30 by pressing the O-ring on the lower surface of the nozzle of the syringe 39 against the surface around the edge of the through hole constituting the separation medium injection portion 38 (step S1). . The separation medium discharge syringe 39 is driven to discharge so as to discharge a predetermined amount of the separation medium, and the separation medium is filled in both the flow paths 32 and 33 of the separation flow path 30 (step S2). The discharge amount of the separation medium from the separation medium discharge syringe 39 is set to an amount larger than the amount by which the separation medium can be filled in both the flow paths 32 and 33, and both ends 30a, A small amount of separation medium overflows from 30b.

  Even if the nozzle of the separation medium discharge syringe 39 is connected to the separation medium injection section 38, the resistance value between the both ends 30a and 30b of the separation flow path 30 is not affected. It remains connected to section 38.

  After the separation medium overflowing the sample well 30a at one end is removed by suction with a suction nozzle (step S3), a predetermined amount of sample is supplied to the sample well 30a (step S4). Regarding the filling of the buffer liquid into the waste liquid reservoir 36, since the buffer liquid is not filled in the waste liquid reservoir 36 in the first electrophoresis, the buffer liquid is supplied to the waste liquid reservoir 36, and in the second and subsequent electrophoresis, the waste liquid reservoir is filled. Since the buffer liquid already exists in 36, the buffer liquid is not filled in the waste liquid reservoir 36 again, and the existing buffer liquid is used (steps S5 and S6).

  Next, an electrode is inserted into the sample in the sample well 30a and the buffer solution in the waste liquid reservoir 36 to apply a voltage, and the sample is introduced into one end of the separation channel 32 (step S7). After the sample is introduced into the separation medium at one end of the separation channel 30, the sample remaining in the sample well 30a is sucked and removed by the suction nozzle (step S8). Thereafter, a predetermined amount of buffer solution is supplied to the sample reservoir 34 (step S9).

  Thereafter, electrodes are inserted into the buffer liquid in the sample reservoir 34 and the buffer liquid in the waste liquid reservoir 36 to apply a voltage and electrophoresis is performed (step S10). The sample components separated by electrophoresis are detected by placing a detector at the detection position. As a detection method, for example, optical fluorescence detection at a detection position, UV (ultraviolet) absorption detection, chemiluminescence detection, electrochemical detection provided with a working electrode and a detection electrode, or an electrospray method from the end of the separation channel There are methods such as ionization and detection with a mass spectrometer.

  When electrophoresis of the next sample is performed continuously after the electrophoresis is completed, the operation from step S2 is repeatedly performed as the second and subsequent electrophoresis processes. That is, the electrodes for electrophoresis are temporarily moved from the respective reservoirs 34 and 36, and a predetermined amount of the separation medium is discharged from the separation medium discharge syringe 39 to separate the separation medium in the one end side flow path 32 and the other end side flow path 33. Is replaced (step S2). The old separation medium used in the previous electrophoresis is pushed out from both flow paths 32 and 33 and discharged to the respective reservoirs 34 and 36.

  The buffer solution and separation medium in the sample well 30a are removed by suction with a suction nozzle (step S3), and a predetermined amount of sample is supplied to the sample well 30a (step S4). The waste liquid reservoir 36 contains the old separation medium and sample pushed out from the other end side flow path 33 in addition to the buffer liquid, but the amount is small relative to the amount of the buffer liquid. Since there is no significant difference in composition, the buffer solution in the waste solution reservoir 36 is not replaced. Thereafter, as in the first electrophoresis operation, in this state, electrodes are inserted into the liquids of the sample well 30a and the waste liquid well 30b to apply a voltage, and the sample is placed in the separation medium at one end of the separation channel 30. After introduction (step S7) and removal of the residual sample in the sample well 30a by suction with a suction nozzle (step S8), a buffer solution is supplied to the sample reservoir 34 (step S9), and the sample reservoir 34 and waste liquid reservoir An electrode is inserted into the buffer solution 36 to apply a voltage, and the sample is electrophoresed (step S10).

2,30 Separation channel 2a, 30a Separation channel one end 2b, 30b Separation channel other end 4,32 One end side channel 6,33 Other end side channel 10,38 Separation medium injection part 12 Detection position 14 Detector 16 , 39 Separation medium filling mechanism 18 Voltage application unit 26, 28 Transparent substrate 34 Sample reservoir 36 Waste liquid reservoir

Claims (4)

A separation channel for performing electrophoretic separation of a sample introduced at one end by applying a voltage to both ends;
Separation for simultaneously injecting the separation medium into the one end side channel to the one end portion on the sample introduction side and the other end side channel to the other end portion provided in the middle of the separation channel. A medium injection part;
A sample injection reservoir provided at the one end;
A reservoir for storing the liquid provided at the other end and discharged from the other end ;
The reservoir provided at the other end has a larger capacity than the sample injection reservoir, and is set to a size having a capacity that is at least twice as large as the amount of separation medium to be drained per time. . A detection position for detecting a separation component is provided on the one end side flow path,
The electrophoretic device according to claim 1, wherein a flow path resistance of the other end side flow path is set to be equal to or higher than that of the one end side flow path. The one end side flow path and the other end side flow path have the same length and inner diameter, and are set so that the separation medium injected from the separation medium injection section is filled in both flow paths simultaneously. 2. The electrophoresis device according to 2. The electrophoresis device according to any one of claims 1 to 3 ,
A separation medium filling mechanism for filling the separation flow path from the separation medium injection portion of the electrophoresis device to the separation flow path;
A voltage application unit for applying a voltage for electrophoresis between both ends of the separation flow path of the electrophoresis device;
Using an electrophoretic analyzer comprising at least a detector for detecting sample components electrophoretically separated in a separation channel of the electrophoresis device,
A series of steps including filling the separation channel into the separation channel by the separation medium filling mechanism , electrophoretic separation of the sample in the separation channel by the voltage application unit , and detection of the sample component electrophoretically separated by the detector An electrophoresis analysis method in which the operation is repeated a plurality of times in a state where the separation medium filling mechanism is connected to a separation medium injection section of the electrophoresis device.

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