JP2012242128A - Microchip electrophoretic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve dispersion of separation performance of respective microchips in a microchip electrophoretic apparatus loaded with a plurality of microchips and capable of repeatedly performing electrophoretic analysis by respective microchips.SOLUTION: A control part 38 includes a sample solution dispensation volume setting part 102, a separation medium dispensation volume setting part 104 and a sample solution/separation medium dispensation volume setting part 106. The sample solution dispensation volume setting part 102 individually sets sample solution dispensation volumes to respective microchips 5-1 to 5-4 so as to respectively obtain optimum separation performance in electrophoretic analysis of four microchips 5-1 to 5-4 (Fig.2).

Description

  The present invention relates to a microchip electrophoresis method and a microchip electrophoresis apparatus for executing the microchip electrophoresis method.

In microchip electrophoresis, a sample such as DNA, RNA, or protein introduced into one end of a separation main channel is electrophoresed in the direction of the other end of the channel by a voltage applied across the channel. Separate and detect.
In microchip electrophoresis, an apparatus has been developed that automatically uses a microchip to repeatedly fill a buffer solution, dispense a sample, perform electrophoresis, and detect a separated sample component.

  In order to increase the operating rate of analysis, there is a microchip electrophoresis apparatus that is equipped with a plurality of microchips and that can repeatedly perform electrophoretic analysis on these microchips (see Patent Document 1). A microchip having a reservoir opened on the surface at each end of the flow path is used. When the microchip is mounted on the microchip processing apparatus, the microchip is held so that the reservoir is on the upper surface. The separation buffer liquid is filled into the flow path by supplying the separation buffer liquid to one reservoir by the separation buffer liquid filling and discharging unit, and pressing the air supply port onto the reservoir to supply air. This is done by pushing into the channel. After filling the flow path with the separation buffer solution, the separation buffer solution overflowing from the other reservoir is sucked by the suction nozzle. Thereafter, the sample is dispensed into a sample supply reservoir.

U.S. Pat. No. 7,678,254

  In such an apparatus, conventionally, the apparatus has been configured on the assumption that the same separation accuracy can be obtained each time in each microchip. However, in practice, it has been found that the separation performance of each microchip varies.

  Accordingly, an object of the present invention is to improve the dispersion of separation performance in a microchip in a microchip electrophoresis apparatus in which a plurality of microchips are mounted and electrophoresis analysis can be repeatedly performed on each microchip. Is.

  A microchip electrophoresis apparatus targeted by the present invention includes a plurality of microchips provided with a flow path including at least a separation flow path inside a plate-like member, and provided with reservoirs opened at the ends of the flow paths. A chip holding unit that holds the reservoir fixedly on the upper surface, a buffer liquid filling mechanism that fills the microchip flow path with a separation buffer liquid that serves as a separation medium, and a predetermined microchip chip that sucks a sample solution A dispensing probe for injecting into the reservoir, a probe moving mechanism for moving the dispensing probe between the inhalation position of the sample solution to be dispensed and the dispensing position on the microchip, and the liquid in the reservoir are aspirated A microphone by performing electrophoretic separation of a new sample after replacing the used separation medium with a new separation medium in each of the suction nozzle and the microchip Each electrophoresis separation buffer solution filling mechanism to repeat the chip, the dispensing probe, and a control unit for controlling the probe moving mechanism and operation of the suction nozzle, those having at least.

  There may be a variation in separation accuracy between the microchips mounted on the microchip electrophoresis apparatus. One of the causes is individual difference of each microchip. In other words, since there is an individual difference in each microchip due to processing accuracy and the like, it is difficult to position the dispensing nozzle of the automatic sample injection mechanism (autosampler) at exactly the same position for all the microchips. Therefore, there is a variation in the dispensing position of the liquid into the reservoir of each microchip, and the variation causes a variation in the state of the sample in the reservoir of each microchip, which causes a variation in separation accuracy in each microchip. I found out. And it turned out that this appears more noticeably as the amount of sample dispensed becomes smaller.

  Therefore, in the first microchip electrophoresis apparatus according to the present invention, the control unit sets the sample solution dispensing amount set in advance for each microchip, which is obtained in advance for obtaining the optimum separation performance. The control unit controls the dispensing operation of the sample solution by the dispensing probe for each microchip based on the sample solution dispensing amount set in the sample solution dispensing amount setting unit. As a result, the sample solution is dispensed under conditions that provide optimum separation performance for each microchip.

  Further, the control unit further includes a separation medium dispensing amount setting unit in which a separation medium dispensing amount obtained in advance for obtaining optimum separation performance is set for each microchip, and the control unit dispenses the separation medium dispensing It is preferable to control the separation medium filling operation by the buffer liquid filling mechanism for each microchip based on the separation medium dispensing amount set in the amount setting unit. Then, the buffer solution as the separation medium can be dispensed under the condition that the optimum separation performance can be obtained for each microchip, and the separation performance due to the dispersion accuracy of the separation medium between the microchips can be reduced. Variations can be eliminated.

  In addition, when performing electrophoretic analysis using different buffer solutions as separation media, the physical properties such as surface tension and viscosity differ depending on the type of buffer solution. In some cases, the dispensing accuracy may decrease. If the amount of the separation medium filled in the electrophoresis channel is changed by changing the dispensed amount of the buffer solution, the result of the electrophoretic analysis is affected, and the reproducibility of the analysis result is deteriorated. However, the conventional apparatus has not been configured in consideration of the influence of the difference in the characteristics of the buffer solution on the dispensing accuracy.

  Therefore, in the second microchip electrophoresis apparatus according to the present invention, the control unit determines the amount of sample solution dispensed and / or the amount of separation medium dispensed in advance for obtaining optimum separation performance as the type of separation medium. A sample solution / separation medium dispensing amount setting unit set for each, and the control unit sets the sample solution dispensing amount and / or separation medium dispensing amount set in the sample solution / separation medium dispensing amount setting unit. Based on the separation medium type, the dispensing operation of the sample solution by the dispensing probe and / or the filling operation of the separation medium by the buffer liquid filling mechanism is controlled. Thereby, the dispersion | variation in the separation performance by the dispersion | variation in the dispensing precision produced by the characteristic of a separation medium can be improved.

  According to the first microchip electrophoresis apparatus of the present invention, the control unit sets the sample solution dispensing amount set in advance for each microchip to obtain the sample solution dispensing amount obtained in advance for obtaining the optimum separation performance. And the control unit controls the dispensing operation of the sample solution by the dispensing probe for each microchip based on the sample solution dispensing amount set in the sample solution dispensing amount setting unit. This can improve the variation in separation performance caused by the variation in dispensing accuracy, and enhance the reproducibility of analysis results.

  In the second microchip electrophoresis apparatus, the control unit sets the sample solution dispensing amount and / or separation medium dispensing amount obtained in advance for obtaining the optimum separation performance for each type of separation medium. A separation medium dispensing amount setting unit, and the control unit determines the type of separation medium based on the sample solution dispensing amount and / or the separation medium dispensing amount set in the sample solution / separation medium dispensing amount setting unit. Since the dispensing operation of the sample solution by the dispensing probe and / or the separation medium filling operation by the buffer liquid filling mechanism is controlled every time, the variation in separation performance due to the variation in dispensing accuracy caused by the characteristics of the separation medium is improved, Highly reproducible analysis results can be obtained regardless of the separation medium specified.

It is a block diagram which shows roughly the part regarding the control part in the microchip electrophoresis apparatus of one Example. It is a perspective view which shows roughly the principal part of the microchip electrophoresis apparatus of the Example. It is a figure which shows an example of a microchip, (A) and (B) are top views which show the transparent plate-shaped member which comprises a microchip, (C) is a front view of a microchip. It is a top view which shows a specific example of the microchip. It is sectional drawing which shows roughly the connection state of the air supply port and microchip at the time of filling with a separation medium in the microchip electrophoresis apparatus. It is a perspective view which shows operation | movement of one Example in order of a process. It is a perspective view which shows operation | movement of the Example in order of a subsequent process. It is a perspective view which shows operation | movement of the Example further in order of a subsequent process. It is a perspective view which shows operation | movement of the Example further in order of a subsequent process. It is a flowchart which shows the process sequence in operation | movement of the Example. It is a graph which shows the relationship between the theoretical plate number of the analysis result obtained by each microchip, and the setting value of the dispensing amount of a sample solution, (A) is the actual dispensing amount and theoretical plate number of the sample solution to a microchip. (B) is experimental data showing the relationship between the dispensed amount after correction so that the optimum dispensed amount of the sample solution to the microchip matches in each microchip and the number of theoretical plates. .

FIG. 1 is a block diagram schematically showing a part related to a control unit in an embodiment in which the present invention is applied to a microchip electrophoresis apparatus 1.
The dispensing unit 2 includes a dispensing probe driving mechanism provided with a dispensing probe. The dispensing probe of the dispensing unit 2 sucks the separation buffer solution or sample by the syringe pump 4 and injects it into one end of the electrophoresis channel of the microchip, and is provided in common for the plurality of electrophoresis channels. ing.

  The separation buffer solution filling / discharging unit 16 fills the electrophoresis channel with the separation buffer solution injected into one end of the electrophoresis channel by air pressure, and discharges unnecessary separation buffer solution by the suction pump unit 23. The separation buffer liquid filling / discharging unit 16 is also provided in common for a plurality of electrophoresis channels to be processed. The electrophoresis high-voltage power supply unit 26 applies the electrophoresis voltage to each of the electrophoresis channels independently. The fluorescence measurement unit 31 is an example of a detection unit that detects a sample component separated in the electrophoresis channel.

  When the separation buffer liquid filling and sample injection into one electrophoresis channel are completed, the control unit 38 operates the dispensing unit 2 so as to shift to the separation buffer liquid filling and sample injection into the next electrophoresis channel. The operation of the high voltage power supply unit 26 for electrophoresis is controlled so as to cause electrophoresis by applying an electrophoresis voltage in the electrophoresis channel after the sample injection is completed, and the detection operation by the fluorescence measuring unit 31 is controlled. It is. The personal computer 40 is an external control device for instructing the operation of the microchip electrophoretic device and for taking in and processing data obtained by the fluorescence measuring unit 31. The control unit 38 includes a sample solution dispensing amount setting unit 102, a separation medium dispensing amount setting unit 104, and a sample solution / separation medium dispensing amount setting unit 106. These setting units 102, 104 and 106 will be described later.

  FIG. 2 schematically shows a main part of a microchip electrophoresis apparatus according to an embodiment. Four microchips 5-1 to 5-4 are fixedly held by the holding portion 7. As will be described in detail later, each of the microchips 5-1 to 5-4 is formed with one electrophoresis channel for processing one sample. The microchips 5-1 to 5-4 are fixed to the holding unit 7 during the analysis operation.

  In order to dispense the separation buffer solution and the sample into the microchips 5-1 to 5-4, the dispensing unit 2 includes a syringe pump 4 that performs suction and discharge, and a dispensing probe 8 that includes a dispensing nozzle. And a container 10 for the cleaning liquid, and the dispensing probe 8 and the container 10 for the cleaning liquid are connected to the syringe pump 4 via the three-way electromagnetic valve 6. The separation buffer solution and the sample are respectively accommodated in holes on the microtiter plate 12 and are dispensed into the microchips 5-1 to 5-4 by the dispensing unit 2. The separation buffer solution may be stored in a dedicated container and placed near the microtiter plate 12. Reference numeral 14 denotes a cleaning unit for cleaning the dispensing probe 8, and the cleaning liquid overflows.

  The dispensing unit 2 connects the three-way electromagnetic valve 6 in the direction in which the dispensing probe 8 and the syringe pump 4 are connected, and sucks the separation buffer solution or the sample into the dispensing probe 8, and the dispensing probe 8 is connected to the microchip 5. It moves to -1-5-4, and it discharges to the electrophoresis flow path in any one of microchips 5-1 to 5-4 with the syringe pump 4. When the dispensing probe 8 is washed, the three-way solenoid valve 6 is switched to a direction in which the syringe pump 4 and the washing liquid container 10 are connected, the washing liquid is sucked into the syringe pump 4, and then the dispensing probe 8 is washed in the washing section 14. Then, the three-way solenoid valve 6 is switched to the side where the syringe pump 4 and the dispensing probe 8 are connected, and washing is performed by discharging the washing liquid from the inside of the dispensing probe 8.

  Separation buffers for the four microchips 5-1 to 5-4 are used to fill the flow path with the separation buffer solution dispensed in the reservoir at one end of the electrophoresis flow paths of the microchips 5-1 to 5-4. A liquid filling / discharging unit 16 is provided in common. The separation buffer liquid filling / discharging unit 16 moves onto the microchips 5-1 to 5-4, and the air supply port 18 is placed on the reservoir at one end of the electrophoresis channel of any one of the microchips 5-1 to 5-4. The suction nozzle 22 is inserted into another reservoir, air is blown from the air supply port 18 to push the separation buffer solution into the electrophoresis channel, and the separation buffer solution overflowing from the other reservoir is pushed into the nozzle. The air is sucked from 22 with a suction pump and discharged to the outside.

  In order to apply the voltage for electrophoresis independently to the electrophoresis channel of each of the microchips 5-1 to 5-4, the high-voltage power supply for electrophoresis 26 (26 -1 to 26-4) are provided.

  A fluorescence measuring unit 31 for detecting the sample components electrophoretically separated in the separation channels 55 of the microchips 5-1 to 5-4 is provided for each of the microchips 5-1 to 5-4. The LEDs (light emitting diodes) 30-1 to 30-4 that irradiate a part of the electrophoresis channel and the sample components that move through the electrophoresis channel are excited by the excitation light from the LEDs 30-1 to 30-4. Optical fibers 32-1 to 32-4 that receive the generated fluorescence, and a filter 34-1 that removes the excitation light component from the fluorescence from the optical fibers 32-1 to 32-4 and transmits only the fluorescence component. , 34-2 and a photomultiplier tube 36 for receiving fluorescence. Here, since the filters 34-1 and 34-2 that transmit different fluorescence are used, the microchips 51-1 and 51-2 and 51-3 and 51-4 can detect different fluorescence. it can. However, when the same fluorescence is detected by the four microchips 51-1 to 51-4, one filter can be used in common. By causing the LEDs 30-1 to 30-4 to emit light at different times, the fluorescence from the four microchips 5-1 to 5-4 can be identified and detected by one photomultiplier tube 36. The excitation light source is not limited to the LED, and an LD (laser diode) may be used.

  3 and 4 show an example of the microchip in this embodiment. The microchip in the present invention refers to such an electrophoresis apparatus in which an electrophoresis channel is formed in a substrate, and is not necessarily limited to a small size.

  As shown in FIG. 3, the microchip 5 is composed of a pair of transparent substrates (quartz glass or other glass substrate or resin substrate) 51, 52, and on the surface of one substrate 52, as shown in (B). The capillary grooves 54 and 55 for electrophoresis intersecting each other are formed, and the reservoir 53 is provided as a through hole in the other substrate 51 at a position corresponding to the end of the grooves 54 and 55 as shown in FIG. Both substrates 51 and 52 are overlapped and joined as shown in FIG. 4C, and capillary grooves 54 and 55 are separated into a separation channel 55 for electrophoresis separation of the sample and a sample for introducing the sample into the separation channel. Let it be an introduction channel 54.

  The microchip 5 is basically the one shown in FIG. 3, but in order to facilitate handling, the electrode terminal for applying a voltage is previously formed on the chip as shown in FIG. Is used. FIG. 4 is a plan view of the microchip 5. The four reservoirs 53 are also ports for applying a voltage to the flow paths 54 and 55. Ports # 1 and # 2 are ports located at both ends of the sample introduction channel 54, and ports # 3 and # 4 are ports located at both ends of the separation channel 55. In order to apply a voltage to each port, electrode patterns 61 to 64 formed on the surface of the chip 5 are formed so as to extend from the respective ports to the side end portions of the microchip 5. 26-1 to 26-4.

  FIG. 5 schematically shows a connection state between the air supply port 18 and the microchip 5 in the buffer filling / discharging unit 16. An O-ring 20 is provided at the tip of the air supply port 18, and the air supply port 18 is pressed against one reservoir of the microchip 5 to press the air supply port with respect to the electrophoresis channel of the microchip 5. 18 can be attached in a secret manner, and air can be pressurized from the air supply port 18 and sent out into the flow path. A nozzle 22 is inserted into the other reservoir, and an unnecessary separation buffer solution overflowing from the flow path is sucked and discharged.

Here, returning to FIG. 1, the sample solution dispensing amount setting unit 102, the separation medium dispensing amount setting unit 104, and the sample solution / separation medium dispensing amount setting unit 106 of the control unit 38 will be described.
The sample solution dispensing amount setting unit 102 is provided with the microchips 5-1 to 5-4 so that optimum separation performance can be obtained by the electrophoretic analysis in the four microchips 5-1 to 5-4 (FIG. 2). The sample solution dispensing amount for each of the above is configured individually.

  Even if the set values of the sample solution dispensing amounts for the microchips 5-1 to 5-4 are all the same, the microchips 5-1 to 5-4 are caused by individual differences of the microchips 5-1 to 5-4. Variations occur in the dispensing accuracy of the sample solution to the sample, and due to the variations, the separation performance of the electrophoretic analysis in each microchip 5-1 to 5-4 varies. Therefore, if the same value is uniformly set as the amount of the sample solution dispensed to each microchip 5-1 to 5-4, and electrophoretic analysis is performed, only results with different separation performance between the microchips can be obtained. , The reproducibility of the analysis results will deteriorate. Therefore, the sample solution dispensing amount setting unit 102 of the control unit 38 sets the dispensing amount of the sample solution for obtaining the optimum separation performance in each of the microchips 5-1 to 5-4, and the set amount of the sample solution. The dispensing unit 2 is controlled so as to dispense.

  The amount of sample container dispensed to each of the microchips 5-1 to 5-4 is set based on experimental values obtained in advance through experiments. The experimental value can be obtained from the experimental data as shown in FIG. 11A, and such an experimental value may be held by the sample solution dispensing amount setting unit 102 or stored in the PC 40. It may be stored in the memory.

In the experimental data shown in FIG. 11A, the same value is set as the amount of sample solution dispensed to the microchips 5-1 to 5-4 (Chip # 1 to Chip # 4), and the electrophoretic analysis is performed. It is experimental data of the relationship between the theoretical plate number of the analysis result obtained by each microchip 5-1 to 5-4, and the setting value of the amount of sample solution dispensed. In this experiment, DNA-1000 kit (product of Shimadzu Corporation) was used as a separation buffer solution, and φ × 174-HaeIII marker (Promega) was used as a sample solution from TE buffer (Tris and ethylenediaminetetraacetic acid (EDTA)). A buffer solution (Nacalai Tesque) diluted 100 times. In this data, the horizontal axis is the set value (μL) of the sample solution dispensing amount, and the vertical axis is the theoretical plate number. The theoretical plate number N was calculated by the half width method of the following equation. In addition, t _r is the retention time, W _0.5h is a full width at half maximum.
_{N = 5.54 (t r / W} 0.5h) 2

  As shown in FIG. 11 (A), when a plurality of dispensing volume conditions are set and measured in each microchip, the dispensing volume (hereinafter referred to as the theoretical plate number) becomes the maximum among the set dispensing volumes. , Referred to as the optimum dispensing amount), but the optimum dispensing amount is different for each of the microchips 5-1 to 5-4. Therefore, based on this data, the optimum dispensing amount of each of the microchips 5-1 to 5-4 is stored, and the sample solution dispensing amount setting unit 102 determines the optimum dispensing amount as a dispensing amount setting value. Are individually set in the microchips 5-1 to 5-4.

When the amount of the sample solution dispensed to all the microchips 5-1 to 5-4 is uniformly set to a specified value (2.75 μL) as in the past, the number of theoretical plates of the microchips 5-1 to 5-4 As follows, the separation performance varies among microchips.
Microchip 5-1: 151975
Microchip 5-2: 117365
Microchip 5-3: 104421
Microchip 5-4: 111240

On the other hand, by correcting the set amount of the sample solution to each of the microchips 5-1 to 5-4 to the optimum dispensing amount, as shown in FIG. -1 to 5-4 can be corrected so that the optimal dispensing volume becomes the specified value (2.75 μL), and as shown below, the difference in the number of theoretical plates between microchips is reduced, and the variation in separation performance is improved. In any microchip, high separation performance can be obtained. Here, the correction means that when a prescribed value of 2.75 μL is set as a dispensing volume, the dispensing volume setting value for each microchip is corrected to be an optimal dispensing volume, and the sample solution with the optimal dispensing volume is corrected. Is dispensed.
Microchip 5-1: 151975
Microchip 5-2: 135663
Microchip 5-3: 155572
Microchip 5-4: 161934

  Similarly to the sample solution, the dispensing accuracy of the separation buffer solution dispensed as a separation medium to the microchips 5-1 to 5-4 varies depending on individual differences of the microchips 5-1 to 5-4. is there. Therefore, the separation medium injection amount setting unit 104 individually sets the separation medium dispensing amount to each of the microchips 5-1 to 5-4. The separation medium dispensing amount is set based on experimental values, and such experimental values are obtained from experimental data measured in advance for the dispensing amount with the maximum number of theoretical plates as in the sample solution.

  The dispensing accuracy also changes depending on the type of separation buffer solution. Therefore, when the sample solution / separation medium dispensing amount setting unit 106 inputs the type of separation medium used by the analyst, the optimal amount of sample solution and separation medium is dispensed according to the type of separation medium. Set. The optimal amount of sample solution and separation medium to be dispensed is based on experimental values obtained as the dispensing amount that maximizes the number of theoretical plates from the relational data between the number of theoretical plates and the amount dispensed in advance using each separation medium. Is set.

  In this microchip processing apparatus, the microchip is repeatedly used in a state where it is fixed to the holding unit 7 without moving. The processing procedure in this case is shown in FIGS. 6 to 9 and described with reference to the flowchart of FIG. To do. Reference numerals (A to U) in the flowchart of FIG. 10 mean the reference numerals of the steps in FIGS. In this process, the microchip used in the previous analysis is washed, the flow path is filled with the separation buffer solution, and the current flows normally through the flow channel with each reservoir filled with the separation buffer solution. This is a series of steps in which an electrophoresis test is performed and then the sample is dispensed to start electrophoresis, and the dispensing probe and the suction nozzle are washed.

  (A) shows one microchip 5. The microchip 5 is the one shown in FIGS. 3 and 4, and is provided so that the separation channel 55 and the sample introduction channel 54 intersect each other, and a reservoir 53 is formed at the end of each channel 54, 55. It is a thing. The reservoirs No. 1 to No. 4 in FIG. 4 are denoted by reference numerals 53-1 to 53-4 here.

  (B) The analysis of the previous sample is completed, the separation buffer solution remains in the flow path and each reservoir, and the separated sample also remains in the separation buffer solution.

  (C) First, in order to wash the sample supply reservoir 53-1, only the suction nozzle 22-1 is inserted into the reservoir 53-1. The suction nozzle 22-2 and the suction nozzle 22-3 also move up and down simultaneously with the suction nozzle 22-1, but the length of the suction nozzle 22-1 is longer than the other suction nozzles 22-2 and 22-3. Therefore, only the suction nozzle 22-1 is inserted into the reservoir 53-1, and is pressed against the bottom of the reservoir 53-1, but the other suction nozzles 22-2 and 22-3 are respectively Are not inserted into the corresponding reservoirs 53-2 and 53-3. By being sucked from the suction nozzle 22-1 in this state, the separation buffer solution in the reservoir 53-1 is sucked and removed.

(D) Wash water is supplied from the dispensing probe 8 to the reservoir 53-1.
(E) The suction nozzle 22-1 is again inserted into the reservoir 53-1, and the washing water is sucked and discharged.
(F) Washing water is supplied again from the dispensing probe 8 to the reservoir 53-1.

  (G) Next, the suction nozzles 22-1 to 22-3 are inserted into the reservoirs 53-1 to 53-3, respectively. At this time, the three suction nozzles 22-1 to 22-3 are inserted into the respective reservoirs 53-1 to 53-3. The suction nozzles 22-1 to 22-3 can be expanded and contracted by a spring mechanism, and the suction nozzle 22-1 is the longest when the tip of the suction nozzles 22-1 to 22-3 is not in contact with anything. It is so attached. The tips of the suction nozzles 22-1 to 22-3 are pressed against the bottoms of the reservoirs 53-1 to 53-3, so that the suction nozzles 22-1 to 22-3 become the bottoms of the respective reservoirs 53-1 to 53-3. Abut. The liquid is simultaneously sucked and removed from the three suction nozzles 22-1 to 22-3. The dispensing probe 8 is inserted into the rinse port 100 to discharge the entire amount of washing water in the dispensing probe 8 and the inside and outside of the dispensing probe 8 are washed.

  (H) The fourth suction nozzle 22-4 is inserted into the other reservoir 53-4. The suction nozzle 22-4 (not shown in FIG. 2) is provided separately from the three suction nozzles 22-1 to 22-3, and is close to the air supply port cylinder 18 shown in FIG. Is arranged. The suction nozzle 22-4 is also pressed against the bottom of the reservoir 53-4. The separation buffer solution in the reservoir 53-4 is sucked and removed from the suction nozzle 22-4. The dispensing probe 8 aspirates the separation buffer solution from the reagent container 91 containing the buffer solution.

(I) The dispensing probe 8 is moved to the reservoir 53-4, and the separation buffer solution is dispensed as a separation medium. The dispensing amount of the separation buffer liquid is an amount set for each microchip by the separation medium dispensing amount setting unit 104 (FIG. 1).
(J) The air supply port 18 is pressed onto the reservoir 53-4 while maintaining airtightness, and air is supplied from the reservoir 53-4 to the flow path. Suction nozzles 22-1 to 22-3 are inserted into the other reservoirs 53-1 to 53-3, respectively, and the separation buffer solution overflowing from the flow paths to the respective reservoirs 53-1 to 53-3 is sucked. Removed.

(K) The suction nozzle 22-4 is inserted into the reservoir 53-4, and the separation buffer solution in the reservoir 53-4 is sucked and removed. As a result, the separation buffer solution remains only in the flow path.
(L) to (O) The separation buffer solution is sequentially dispensed to the reservoirs 53-1 to 53-4 by the dispensing probe 8.

(P) An electrode is inserted into each reservoir and a migration test is performed. Here, it is confirmed whether dust or bubbles are mixed in the flow path by detecting the current value between the electrodes. Here, the voltage applied to the flow path may be the same as the electrophoresis voltage for separating the sample, but may be lower than that.
The dispensing probe 8 into which the separation buffer solution has been dispensed is inserted into the rinse port 100, and the entire amount of the separation buffer solution in the dispensing probe 8 is discharged and the inside and outside of the dispensing probe 8 are washed.

  If it is determined in this migration test step that the separation buffer is normally filled into the flow path, the process proceeds to the next step (Q) to inject the sample for analysis. If it is not determined that the buffer is normally filled, the process returns to step (B) to refill the flow path with the separation buffer solution.

  The number of times (N) that allows the refilling of the separation buffer liquid into the flow path is set in advance, and the separation buffer is normally filled into the flow path even if the separation buffer liquid is refilled by that number of times. If it is not determined that the process has been performed, the microchip is removed from the holding unit 7 and replaced with another microchip, and the process returns to step (B). The number N of times that the refilling of the separation buffer solution is allowed is not particularly limited, but 2 or 3 is suitable, for example.

(Q) The suction nozzle 22-1 is inserted only into the sample supply reservoir 53-1, and the separation buffer solution in the reservoir 53-1 is sucked and removed.
(R) The cleaning liquid is supplied from the dispensing nozzle 8 to the sample supply reservoir 53-1. The cleaning liquid is pure water, for example.
(S) The suction nozzle 22-1 is inserted into the sample supply reservoir 53-1, and the cleaning liquid is sucked and removed.

(T) A predetermined amount of sample solution is dispensed from the dispensing probe 8 into the sample supply reservoir 53-1. The dispensing amount of the sample solution is an amount set for each microchip by the sample solution dispensing amount setting unit 102 (FIG. 1).
(U) An electrode is inserted into each of the reservoirs 53-1 to 53-4, a voltage for supplying a sample is applied, and the sample is guided to the intersection of the flow paths 54 and 55.

(V) The applied voltage is switched to the voltage for electrophoretic separation, and the sample is electrophoresed in the direction of the reservoir 53-4 in the separation channel 55 and separated.
(W) After completion of the analysis, each of the suction nozzles 22-1 to 22-4 is inserted into the phosphorus spool 102, the cleaning liquid is sucked, the inside and outside of the nozzle are cleaned, and the probe 8 is inserted into the rinse port 100 so Washed.
In the embodiment, the control unit 38 includes all of the sample solution dispensing amount setting unit 102, the separation medium dispensing amount setting unit 104, and the sample solution / separation medium dispensing amount setting unit 106. One or two may be provided.

2 Dispensing unit 4 Syringe pump 5, 5-1 to 5-4 Microchip 8 Dispensing probe 8b Groove 16 Separation buffer solution filling / discharging unit 22-1 to 22-4 Suction nozzle 26 (26-1 to 26-4 ) Electrophoresis high voltage power supply unit 38 Control unit 102 Sample solution dispensing amount setting unit 104 Separation medium dispensing amount setting unit 106 Sample solution / separation medium dispensing amount setting unit

Claims (3)

A plurality of microchips provided with a flow path including at least a separation flow path inside the plate-like member and provided with reservoirs opened at the ends of the flow paths are fixed and held so that the reservoir is on the upper surface. A chip holder to
A buffer liquid filling mechanism that fills the flow path of the microchip with a separation buffer liquid as a separation medium;
A dispensing probe for inhaling and injecting a sample solution into a predetermined reservoir of the microchip;
A probe moving mechanism for moving the dispensing probe between a suction position of a sample solution to be dispensed and a dispensing position on the microchip;
A suction nozzle for sucking the liquid in the reservoir;
The buffer solution filling mechanism to repeat the electrophoretic separation in each of the microchips by performing electrophoretic separation of a new sample after exchanging the used separation medium with a new separation medium in each of the microchips, In a microchip electrophoresis apparatus comprising at least a dispensing probe, a probe moving mechanism, and a control unit that controls the operation of the suction nozzle,
The control unit includes a sample solution dispensing amount setting unit in which a sample solution dispensing amount obtained in advance for obtaining an optimum separation performance is set for each microchip, and the control unit includes the sample solution dispensing amount. A microchip electrophoresis apparatus, wherein a dispensing operation of a sample solution by the dispensing probe is controlled for each microchip based on a sample solution dispensing amount set in a dispensing amount setting unit. The control unit further includes a separation medium dispensing amount setting unit in which a separation medium dispensing amount obtained in advance for obtaining optimum separation performance is set for each microchip, and the control unit includes the separation medium. The microchip electrophoresis apparatus according to claim 1, wherein a separation medium filling operation by the buffer liquid filling mechanism is controlled for each microchip based on a separation medium dispensing amount set in a dispensing amount setting unit. Chip holding that has a flow path including at least a separation flow path inside the plate-like member, and holds a plurality of microchips provided with reservoirs opened at the ends of the flow paths so that the reservoir is on the upper surface And
A buffer liquid filling mechanism that fills the flow path of the microchip with a separation buffer liquid as a separation medium;
A dispensing probe for inhaling and injecting a sample solution into a predetermined reservoir of the microchip;
A probe moving mechanism for moving the dispensing probe between a suction position of a sample solution to be dispensed and a dispensing position on the microchip;
A suction nozzle for sucking the liquid in the reservoir;
The buffer solution filling mechanism to repeat the electrophoretic separation in each of the microchips by performing electrophoretic separation of a new sample after exchanging the used separation medium with a new separation medium in each of the microchips, In a microchip electrophoresis apparatus comprising at least a dispensing probe, a probe moving mechanism, and a control unit that controls the operation of the suction nozzle,
The control unit includes a sample solution / separation medium dispensing amount setting unit in which a sample solution dispensing amount and / or a separation medium dispensing amount obtained in advance for obtaining an optimum separation performance is set for each type of separation medium. And the control unit uses the dispensing probe for each type of separation medium based on the sample solution dispensing amount and / or the separation medium dispensing amount set in the sample solution / separation medium dispensing amount setting unit. A microchip electrophoresis apparatus characterized by controlling a dispensing operation of a sample solution and / or a filling operation of a separation medium by the buffer solution filling mechanism.

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