JP5477341B2 - Microchip electrophoresis method and apparatus - Google Patents

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 having an electrophoresis flow path to automatically fill a separation medium, dispense a sample, perform electrophoresis, and detect a separated sample component.

  A microchip having a reservoir opened at the substrate surface at the end of the flow path is used, and when the microchip is mounted on the microchip electrophoresis apparatus, the microchip is held so that the reservoir is on the upper surface. For example, when the separation buffer solution is used also as the separation medium, the separation medium is filled into the flow path by supplying the separation buffer solution to one reservoir by the separation buffer solution filling and discharging unit and pressing the air supply port on the reservoir. The separation buffer solution is pushed into the flow path by supplying air. 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 the reservoir for sample supply, the separation buffer solution is dispensed into all the reservoirs, and then a migration voltage is applied between the reservoirs.

  In order to increase the operation rate of analysis, an electrophoretic analyzer has been developed in which a plurality of microchips are fixedly arranged and each microchip is repeatedly used (see Patent Document 1).

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

  In the case of repeatedly using a microchip, it was considered that the influence of the sample of the previous analysis and the electrophoresis medium is removed by exchanging the electrophoresis medium after the previous analysis is completed. However, when the microchip is repeatedly used, the analysis performance and analysis reproducibility such as the number of theoretical plates and detection time may be lowered.

  In view of the above, the present invention has an object to suppress a decrease in analysis performance and analysis reproducibility when a microchip is repeatedly used.

  When the microchip is used repeatedly, the components contained in the previous analysis sample solution and the components contained in the separation medium are adsorbed on the surface of the microchip flow path, and these adsorbed components are used for the performance of the electrophoresis apparatus, especially the theory. It was found that analysis performance such as the number of plates and detection time, and performance such as analysis reproducibility may be affected.

  The present invention provides a microchip comprising a flow path comprising a fine tube including at least a separation flow path inside a plate-shaped member, and a reservoir opened at the surface of the plate-shaped member at an end of the flow path. In the microchip electrophoresis method in which the reservoir is arranged so that the flow path is in the horizontal direction and the analysis process by electrophoretic separation in each microchip is repeated, the start of multiple analysis processes in the microchip Before, after every analysis process or after completion of a plurality of analysis processes, a cleaning process including the following processes (1) to (3) is performed.

(1) supplying a fixed amount of cleaning liquid to one reservoir in a state where the flow path and the reservoir are empty;
(2) Thereafter, a process of holding the cleaning liquid for a certain time and introducing the cleaning liquid into the flow path by capillary action; and (3) supplying pressurized gas from one reservoir and sucking the cleaning liquid from the other reservoir. Removing the cleaning liquid of the flow path and the reservoir.

  In the cleaning process, as a method of introducing the cleaning liquid into the flow path, it is possible to supply the cleaning liquid to one reservoir and then pressurize with air pressure to introduce the cleaning liquid into the flow path. Such a method is adopted. In such a case, the cleaning liquid may leak from other reservoirs, resulting in a decrease in the number of theoretical plates in the subsequent electrophoresis, an abnormal applied voltage due to a short circuit between the reservoirs due to the microchip surface getting wet with the cleaning liquid, and the like. In particular, when a cleaning liquid having a low surface tension containing an organic solvent or a surfactant is used, there is a higher risk of the cleaning liquid leaking from the reservoir. However, such a problem does not occur when the cleaning liquid is introduced into the flow path in the cleaning step by capillary action as in the present invention.

  A microchip electrophoresis apparatus of the present invention that realizes this microchip electrophoresis method includes a flow path formed of a fine tube including at least a separation flow path inside a plate-shaped member, and a plate-shaped member at an end of the flow path. A microchip having a reservoir opened on the surface thereof, a chip holding section for holding the reservoir so that the reservoir faces upward and the flow path is horizontal, a cleaning liquid holding section, and a separation buffer solution in the microchip flow path A buffer liquid filling mechanism for filling the liquid, a dispensing probe for inhaling the liquid and injecting the liquid into a predetermined reservoir of the microchip, a suction position of the liquid to be dispensed, and a dispensing position on the microchip. A probe moving mechanism that moves between the injection position, a suction nozzle for sucking the liquid from the reservoir, and a high pressure for electrophoresis for applying a voltage for electrophoresis between the reservoirs A source unit, the buffer solution filling mechanism, the dispensing probe, the probe moving mechanism includes at least a control unit, the at least control the operation of the high-voltage power supply unit for the suction nozzle and electrophoresis. And the said control part was hold | maintained at the washing | cleaning liquid holding | maintenance part and the electrophoresis control part which perform the buffer liquid removal process in the microchip electrophoresis method of this invention, a buffer liquid filling process, a sample dispensing process, and an electrophoretic separation process A cleaning control unit is provided that executes a cleaning step in the microchip electrophoresis method using a cleaning liquid.

  By providing such a cleaning step, it is possible to suppress the deterioration of the performance of the microchip due to the adsorbate during repeated use of the microchip.

  The microchip electrophoresis method of the present invention including the washing process is automatically executed by incorporating a program for performing the process of each process into the control unit in the microchip electrophoresis apparatus of the present invention.

  As a result of further examination of performance degradation due to repeated use of the microchip, it was found that the performance degradation due to adsorbed material in the flow path is affected by the sample components to be analyzed and analysis conditions.

  Therefore, in a preferred embodiment, at least one of the number of cleaning repetitions, the cleaning liquid holding time in the flow path, and the cleaning liquid supply amount can be changed as a cleaning condition in the cleaning process.

  Therefore, the cleaning control unit in the control unit of the microchip electrophoresis apparatus includes a cleaning condition setting unit in which the number of cleaning repetitions, the cleaning liquid holding time in the flow path, and the cleaning liquid supply amount are set as the cleaning conditions in the cleaning process. In the cleaning condition setting unit, a plurality of types are set for at least one of the cleaning conditions. Then, the cleaning control unit executes the cleaning process based on the cleaning conditions set in the cleaning condition setting unit and the cleaning conditions set for a plurality of types based on the cleaning conditions selected from the cleaning conditions. It is configured as follows.

  By selecting the cleaning conditions, it is possible to realize an appropriate cleaning operation according to the type of sample solution analyzed. In addition, by performing appropriate cleaning conditions according to the type of sample solution and the degree of degradation of microchip performance, if the degree of performance degradation is minor, cleaning is performed so that it can be completed in a short time. The efficiency of the cleaning operation can be dramatically improved.

  In a preferred embodiment, when supplying the cleaning liquid to one reservoir in the cleaning process, the other reservoirs are emptied or set to an amount that does not leak from the reservoir even if liquid is contained. The amount that does not leak from the reservoir can be determined in advance by experiments.

  In a preferred embodiment, the cleaning process is automatically performed using a cleaning liquid containing an organic solvent or surfactant. A cleaning liquid containing an organic solvent or a surfactant has a lower surface tension than water, and there is a high possibility that the cleaning liquid leaks from the reservoir. When such a cleaning liquid having a low surface tension is used, there is a phenomenon in which the cleaning liquid leaks from the reservoir to the microchip surface even when the liquid level of the injected cleaning liquid is lower than the microchip surface. If the cleaning liquid leaks from the reservoir to the microchip surface, there is a possibility that the number of theoretical plates in the subsequent electrophoresis will decrease, or that the applied voltage may be abnormal due to a short circuit between the reservoirs due to the microchip surface getting wet with the cleaning liquid.

  The electrophoresis medium is not particularly limited as long as it can perform electrophoresis and can discharge the one filled in the flow path and replace it with a new one. If the electrophoresis buffer solution injected into the reservoir is used when applying, it becomes possible to dispense the electrophoresis medium and the electrophoresis buffer solution with the same mechanism, simplify the device, and reduce the device cost. Can do.

  If the chip holding unit is arranged to fix a plurality of microchips and the control unit is configured to execute the microchip electrophoresis method of the present invention for each microchip, Occupancy rate improves.

  In the present invention, when replacing the electrophoresis medium after the analysis of one sample is completed, after removing the electrophoresis medium used for the previous analysis, before filling with a new electrophoresis medium, the flow path is washed with a cleaning solution, In addition, since the cleaning liquid is introduced into the flow path by capillary action at the time of cleaning, it is possible to maintain a high analytical performance in the microchip electrophoresis method in which the microchip is repeatedly used. It is possible to increase the number of times that can be used repeatedly. In addition, the analysis cost can be reduced and the amount of waste can be reduced by increasing the number of times the microchip can be used repeatedly, and the work efficiency can be improved by reducing the frequency of chip replacement.

  The microchip electrophoresis apparatus of the present invention is configured to carry out this microchip electrophoresis method.

  Further, when the cleaning liquid holding part is provided on the microchip, it is difficult to obtain a sufficient capacity, so that it is not possible to hold a sufficient cleaning liquid amount for cleaning in the flow path. Since the microchip electrophoresis apparatus includes the cleaning liquid holding unit, a sufficient amount of cleaning liquid can be held.

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 block diagram which shows the control part of the 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 a separation buffer liquid in the same microchip electrophoresis apparatus, and discharging | emitting a washing | cleaning liquid. It is a perspective view which shows the washing | cleaning process in one Example in order of a process. It is a perspective view which shows the start part of the analysis process in one Example in order of a process. It is a perspective view which shows the subsequent part of the analysis process in the Example in order of a process. It is a perspective view which shows the further subsequent part of the analysis process in the Example in order of a process. It is a flowchart which shows the process sequence of the analysis process in the Example. It is a figure which shows the electrophoretic separation result by the microchip in which the analytical performance deteriorated. It is a figure which shows the electrophoretic separation result by the microchip after giving the washing | cleaning by the Example.

  FIG. 1 is a block diagram schematically showing a portion related to a control unit in one embodiment of a microchip electrophoresis apparatus.

  The dispensing unit 2 includes a probe moving mechanism that moves between the suction position of the liquid to be dispensed and the dispensing position on the microchip. The dispensing probe of the dispensing unit 2 sucks the separation buffer solution or sample by the syringe pump 4 and injects it into the reservoir of the electrophoresis channel of the microchip, and is provided in common for the plurality of electrophoresis channels. ing. The separation buffer solution is also used as a separation medium, and includes, for example, at least one of a pH buffer and a water-soluble polymer (such as a cellulose polymer).

  As a buffer liquid filling mechanism for filling the microchip flow path with the separation buffer liquid, a separation buffer liquid filling / discharging unit 16 and a suction pump unit 23 are provided. The separation buffer liquid filling / discharging unit 16 injects a certain amount of the separation buffer liquid into one reservoir of the electrophoresis flow path, and fills the electrophoresis flow path with the injected separation buffer liquid from the reservoir by air pressure. The suction pump unit 23 discharges unnecessary separation buffer liquid overflowing into other reservoirs. The separation buffer liquid filling / discharging unit 16 and the suction pump unit 23 are also provided in common for a plurality of electrophoresis channels to be processed.

  The high voltage power supply unit for electrophoresis 26 applies the voltage for electrophoresis between the reservoirs so that the voltage for electrophoresis is applied independently to each of the electrophoresis channels.

  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. Further, the microchip is arranged in a fixed state, and the control unit 38 repeatedly uses the microchip before filling the electrophoresis channel after the analysis of the previous sample is completed with the separation buffer solution. The washing operation of the electrophoresis channel is also controlled.

  As shown in FIG. 2, the control unit 38 includes a migration control unit 70 and a cleaning control unit 72.

  The electrophoresis control unit 70 includes a buffer liquid filling step for filling the empty channel with the electrophoresis medium, a sample dispensing step for dispensing the sample to the sample supply reservoir, and a separation channel by applying an electrophoresis voltage between the reservoirs. Electrophoretic separation step for performing electrophoretic separation of the sample, and supply of pressurized gas from one reservoir and aspiration of the migration medium and migration buffer solution from the other reservoir, thereby removing the migration medium and migration buffer solution in the flow path and reservoir. The buffer solution removal process to be removed is automatically repeated.

  The cleaning control unit 72 uses a cleaning liquid held in a cleaning liquid holding unit, which will be described later, (1) a step of supplying a certain amount of cleaning liquid to one reservoir with the flow channel and the reservoir empty, and (2) a constant amount thereafter A step of maintaining the time and introducing the cleaning liquid into the flow path by capillary action; and (3) supplying the pressurized gas from one reservoir and sucking the cleaning liquid from the other reservoir, thereby cleaning the flow path and the reservoir. The cleaning process including the process of removing the is automatically performed.

  The cleaning control unit 72 includes a cleaning condition setting unit 74. In the cleaning condition setting unit 74, the number of cleaning repetitions, the cleaning liquid holding time in the flow path, and the cleaning liquid supply amount are set as the cleaning conditions of the cleaning process. As a preferred example, the cleaning condition setting unit 74 has a plurality of types set for at least one of the cleaning conditions. Then, the cleaning control unit 72 executes the cleaning process based on the cleaning conditions set in the cleaning condition setting unit 74 and, based on the cleaning conditions selected from among the cleaning conditions set for a plurality of types. Is configured to do.

  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.

  FIG. 3 schematically shows a main part of a microchip electrophoresis apparatus according to one embodiment. Four microchips 5-1 to 5-4 are held by the holding unit 7 and are used repeatedly. 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 as a cleaning liquid holding part, 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 wells 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 by the syringe pump 4 to the reservoir | reserver of any electrophoresis channel of the microchips 5-1-5-4. 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.

  When washing the electrophoresis channels of the microchips 5-1 to 5-4, after switching the three-way solenoid valve 6 to the direction in which the syringe pump 4 and the container 10 for the washing liquid are connected and sucking the washing liquid into the syringe pump 4 Then, the dispensing probe 8 is moved to the reservoir of the microchips 5-1 to 5-4, and a predetermined amount of cleaning liquid is dispensed into the reservoir. The washing liquid dispensed into the reservoir enters the electrophoresis channel by capillary action.

  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 is also used when discharging the cleaning liquid after holding the cleaning liquid in the electrophoresis channel for a predetermined time. When filling the electrophoresis channel with the separation buffer solution, the separation buffer solution filling / discharging unit 16 moves onto the microchips 5-1 to 5-4, and the electrophoresis flow of the microchips 5-1 to 5-4 is performed. The air supply port 18 is pressed on the reservoir (reservoir into which the separation buffer solution has been dispensed) while being airtight, the suction nozzle 22 is inserted into the other reservoir, and air is blown through the air supply port 18 to separate the separation buffer solution. Is pushed into the electrophoresis channel, and the separation buffer liquid overflowing from the other reservoir is sucked from the nozzle 22 by the suction pump and discharged to the outside. The same applies when the washing liquid in the electrophoresis channel is discharged, and the air supply port 18 is pressed onto the reservoir at one end of the electrophoresis channel of the microchips 5-1 to 5-4 while keeping airtightness, and is applied to the other reservoir. The suction nozzle 22 is inserted, air is blown from the air supply port 18 to push out the cleaning liquid in the electrophoresis channel, and the cleaning liquid that has come out to the other reservoir is sucked from the nozzle 22 by 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.

  4 and 5 show an example of a microchip in this embodiment. The microchip in the present invention refers to such a medium for electrophoresis in which an electrophoresis channel is formed in a substrate, and is not necessarily limited to a small size.

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

  The microchip 5 is basically the one shown in FIG. 4, but in order to facilitate the handling, as shown in FIG. 5, electrode terminals for applying a voltage are previously formed on the chip. Is used. FIG. 5 shows 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. 6 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 is 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 or washing solution overflowing from the flow path is sucked and discharged.

  In this microchip electrophoresis apparatus, the microchip is repeatedly used in a state of being fixed to the holding unit 7 without moving.

  The microchip processing is automatically executed by a program held by the control unit 38. The program includes a washing process and an analysis process by electrophoretic separation. The analysis process is repeated for each microchip. The cleaning step is performed for each microchip before a series of analysis steps, once for each analysis step, or after the end of a series of analysis steps.

The cleaning process is shown in FIGS.
(A) Washing is performed on a microchip with an empty flow path and reservoir. The dispensing probe 8 is moved to the reservoir 53-4, and the cleaning liquid is dispensed. Various cleaning liquids can be used. For example, an aqueous solution containing about 70 vol% of ethanol. The dispensing amount of the cleaning liquid is set as a cleaning condition, and preferably, a cleaning liquid of a dispensing amount selected from a plurality of set values set in the cleaning condition setting unit 74 is dispensed.

  When automatic cleaning is to be realized in a microchip electrophoresis apparatus, when water is used as the cleaning liquid, especially when there are two or more branch flow paths, such as a cross-injector type microchip, the branch portions should be evenly distributed. Although it is difficult to clean, branching channels can be cleaned by using a cleaning solution containing an organic solvent or a cleaning solution containing a surfactant, such as a cleaning solution made of an aqueous solution containing ethanol as described above. Become. In a microchip electrophoresis apparatus equipped with an automatic cleaning mechanism, water is generally used as a cleaning liquid. In a microchip electrophoresis apparatus equipped with an automatic cleaning mechanism, a cleaning liquid or surfactant containing the above organic solvent is used. Although what uses the contained washing | cleaning liquid is not implement | achieved, this invention also includes the microchip electrophoresis apparatus carrying the automatic washing | cleaning mechanism using such washing | cleaning liquids other than water.

  (B) The reservoir 53-4 is left in a state where the cleaning liquid is dispensed and is held for a predetermined time. In this state, the cleaning liquid dispensed into the reservoir 53-4 enters the flow path by capillary action. The holding time of this step is also set as a cleaning condition, and preferably, the holding time is left for a holding time selected from a plurality of set values set in the cleaning condition setting unit 74.

  (C) After a predetermined holding time elapses, the air supply port 18 is pressed on 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 cleaning liquid pushed out from the flow paths to the respective reservoirs 53-1 to 53-3 is sucked and removed. The

  Steps (a) to (c) constitute a one-cycle cleaning step. The cleaning cycles (a) to (c) are repeated a predetermined number of times. This number of repetitions is also set as the cleaning condition, and is preferably repeated for the number of repetitions selected from a plurality of set values set in the cleaning condition setting unit 74.

  The analysis process by electrophoretic separation will be described with reference to the process charts of FIGS. 8 to 10 and the flowchart of FIG. Reference numerals (A to U) in the flowchart of FIG. 11 mean the reference numerals of the steps in FIGS. The processing performed here uses a microchip for the first analysis step or a microchip in which the separation medium is discharged from the microchip used in the previous analysis, and a separation buffer as a separation medium in the flow path. The solution is filled, and each reservoir is filled with the separation buffer solution, and an electrophoresis test is performed to check whether the current flows normally. Then, the sample is dispensed and the analysis is performed by electrophoresis. After completion, the electrophoresis medium is discharged, and then the dispensing probe and the suction nozzle are washed. This example process is repeated a plurality of times.

  (A) shows one microchip 5. The microchip 5 is shown in FIGS. 4 and 5, 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. 5 are denoted by reference numerals 53-1 to 53-4 here.

  The microchip 5 is a microchip in a state in which the separation medium is discharged from the microchip for the first analysis step or the microchip used in the previous analysis.

  The dispensing probe 8 is moved to the reservoir 53-4, and the separation buffer solution is dispensed from the dispensing probe 8.

  (B) The air supply port 18 is pressed onto the reservoir 53-4 while maintaining airtightness, and suction nozzles 22-1 to 22-3 are inserted into the other reservoirs 53-1 to 53-3, respectively. Air is supplied to the flow path from the air supply port 18 via the reservoir 53-4, and the separation buffer liquid overflowing from the flow path to the respective reservoirs 53-1 to 53-3 is sucked by the suction nozzles 22-1 to 22-3. Aspirated and removed.

  (C) 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.

  (D) to (G) The separation buffer solution is sequentially dispensed to the reservoirs 53-1 to 53-4 by the dispensing probe 8.

  (H) 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 (I) to inject the sample for analysis. If it is not determined that the buffer has been normally filled, the process returns to step (A) in order to refill the flow path with the separation buffer solution.

  The number of times (n) at which refilling of the separation buffer liquid into the flow path is allowed is set in advance, and the separation buffer is normally filled into the flow path even if refilling of the separation buffer liquid is performed that many 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 then the process (A) is started. The number n of times that the refilling of the separation buffer solution is allowed is not particularly limited, but for example, 2 or 3 is appropriate.

  (I) 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.

  (J) The cleaning liquid is supplied from the dispensing nozzle 8 to the sample supply reservoir 53-1.

  (K) The suction nozzle 22-1 is inserted into the sample supply reservoir 53-1, and the cleaning liquid is sucked and removed.

  Steps (J) and (K) are washing steps for removing the separation buffer solution remaining in the sample supply reservoir 53-1. This cleaning operation may be repeated a plurality of times as necessary.

  (L) A sample is injected from the dispensing probe 8 into the sample supply reservoir 53-1. Although not essential, the internal standard substance may be dispensed from the dispensing probe 8 into the sample supply reservoir 53-1 following the sample injection.

  (M) An electrode is inserted into each of the reservoirs 53-1 to 53-4, a voltage for introducing a sample is applied, and the sample is guided to the intersection of the flow paths 54 and 55.

  (N) The applied voltage is switched to the voltage for electrophoretic separation, and the sample is electrophoresed and separated in the direction of the reservoir 53-4 in the separation channel 55, and detected by a detection device (not shown).

  (O) After completion of the analysis, the air supply port 18 is pressed onto the reservoir 53-4 while keeping airtightness, and suction nozzles 22-1 to 22-3 are inserted into the other reservoirs 53-1 to 53-3, respectively. The Air from the air supply port 18 is supplied from the reservoir 53-4 to the flow path, and the separation buffer liquid pushed out from the flow path to the respective reservoirs 53-1 to 53-3 is sucked and removed.

  (P) Thereafter, the suction nozzles 22-1 to 22-4 are inserted into the phosphorus spool 102 and the cleaning liquid is sucked to clean the inside and outside of the nozzle, and the probe 8 is inserted into the rinse port 100 to clean the inside and outside. The

  This completes the electrophoresis analysis cycle of one sample on one microchip. When electrophoretic analysis of a new sample is repeated for the microchip, the operation is repeated after returning to step (A).

  In the first embodiment, the microchip cleaning step shown in FIG. 7 is executed before the series of electrophoresis analysis cycles of the above steps (A) to (P).

  In the third embodiment, the microchip cleaning step shown in FIG. 7 is executed after a series of electrophoresis analysis cycles of the above steps (A) to (P).

  In the third embodiment, the microchip cleaning step shown in FIG. 7 is executed before the step (A) in each of the series of electrophoretic analysis cycles of the steps (A) to (P). Is.

  The analysis performance of the microchip electrophoresis by providing the washing step was evaluated using a microchip electrophoresis apparatus (Shimadzu MCE-202 MultiNA). This microchip electrophoresis apparatus realizes what is shown in the examples.

  As the evaluation conditions for analysis performance, the separation buffer solution is DNA-1000 kit (product of Shimadzu Corporation), the sample solution is φ × 174-HaeIII marker (product of Promega), TE buffer solution (Tris) and ethylenediamine. A 100-fold diluted solution of tetraacetic acid (EDTA) (Nacalai Tesque) was used. In addition, as a cleaning liquid, a chip cleaning kit RA (a product of Shimadzu Corporation) was dispensed into a polypropylene container and placed at a predetermined position of the reagent holder. This cleaning solution contains about 70 vol% of ethanol and has a lower surface tension than pure water.

  As an effect verification technique, a change in the number of theoretical plates of a polymer marker (hereinafter referred to as UM), which is an internal standard substance, before and after the microchip was cleaned by a predetermined procedure was used as a criterion.

The theoretical plate number N was calculated by the half width method defined by the following equation.
N = 5.54 (tr / W _0.5h ) ²
tr is the peak retention time obtained by electrophoretic analysis, and W _0.5h ) is the half width of the peak.

The washing conditions were set as follows. The correspondence with FIG. 7 is as follows.
(A) Dispensing cleaning solution: The dispensing volume is 2.0 μL.
Here, the cleaning liquid is dispensed only to the reservoir 53-4, and the other reservoirs 53-1 to 53-3 are empty. However, the reservoirs 53-1 to 53-3 may be dispensed with an amount of cleaning liquid that does not leak from the respective reservoirs.
(B) Holding time for allowing the cleaning liquid to stand in the flow path: 30 seconds.
During this time, the cleaning liquid enters the flow path by capillary action.
(C) Removal of cleaning solution.

  Steps (a) to (c) are defined as one cycle, and cleaning is performed once in 20 cycles. In the microchip electrophoresis apparatus of the embodiment, a plurality of types of cleaning are set in the cleaning condition setting unit 74, and any one of the set cleaning times can be selected.

  FIG. 12 shows an analysis example of a microchip whose analytical performance has deteriorated due to adsorption of sample solution components and the like. The peak width of the UM peak (two peaks indicated by ▼ in the figure) is large, the average value of the number of theoretical plates is about 47,000, which is significantly lower than the initial performance. Moreover, no improvement in the number of theoretical plates was observed even when the microchip was repeatedly washed with pure water.

  FIG. 13 shows the result of analyzing the microchip under the same conditions as in FIG. 12 after washing the microchip with the above washing solution 5 times (100 cycles). The width of the UM peak was sharpened, and the average value of the theoretical plate number was improved to about 113,000.

  Tables 1 and 2 show changes in the number of UM theoretical plates when cleaning was performed on eight microchips with deteriorated analytical performance.

  Table 1 shows a result of an average value of the number of UM theoretical plates of the two UM peaks shown in FIG. 13 when the number of washing repetitions is 1 (20 cycles). While there is a chip in which the number of UM theoretical plates is remarkably improved, there is a case where a large change is not recognized in the number of theoretical plates.

  Table 2 shows the UM of the two UM peaks shown in FIG. 13 when the number of times of washing back-up is 5 times (100 cycles) using the function of setting the washing conditions for the microchip whose analytical performance has deteriorated. It is the result of the average value of the number of theoretical plates. Although the cleaning time increases as the number of cleaning repetitions increases, unlike the case shown in Table 1, an improvement in the number of UM theoretical plates is recognized for all microchips.

2 Dispensing unit 4 Syringe pump 5, 5-1 to 5-4 Microchip 8 Dispensing probe 10 Washing liquid container 16 Separation buffer solution filling / discharging unit 22-1 to 22-4 Suction nozzle 26 (26-1 to 26-1 26-4) High-voltage power supply unit for electrophoresis 38 Control unit 70 Electrophoresis control unit 72 Cleaning control unit 74 Cleaning condition setting unit

Claims (8)

A microchip having a flow path composed of a fine tube including at least a separation flow path inside the plate-shaped member, and having a reservoir opened at the surface of the plate-shaped member at an end of the flow path, In the microchip electrophoresis method, in which the flow path is arranged in a horizontal direction and the analysis process by electrophoresis separation in each microchip is repeated,
A cleaning step including the following steps (1) to (3) is performed before the start of a plurality of analysis steps on the microchip, for each analysis step, or after the end of the plurality of analysis steps. A microchip electrophoresis method.
(1) supplying a fixed amount of cleaning liquid to one reservoir in a state where the flow path and the reservoir are empty;
(2) Thereafter, a process of holding the cleaning liquid for a certain time and introducing the cleaning liquid into the flow path by capillary action; and (3) supplying pressurized gas from one reservoir and sucking the cleaning liquid from the other reservoir. Removing the cleaning liquid of the flow path and the reservoir.   2. The microchip electrophoresis method according to claim 1, wherein at least one of a repetition number of cleaning, a cleaning liquid holding time in the flow path, and a cleaning liquid supply amount can be changed as a cleaning condition in the cleaning step.   The microchip electrophoresis method according to claim 1, wherein the cleaning step is automatically executed using a cleaning liquid containing an organic solvent or a surfactant as a cleaning liquid.   4. When supplying the cleaning liquid to one reservoir in the cleaning step, the other reservoir is emptied or the amount is not leaked from the reservoir even if liquid is contained. 5. The microchip electrophoresis method as described.   The microchip electrophoresis method according to claim 1, wherein an electrophoresis buffer solution is used as the electrophoresis medium. A microchip having a flow path composed of a fine tube including at least a separation flow path inside the plate-shaped member, and having a reservoir opened at the surface of the plate-shaped member at an end of the flow path, A chip holding unit for holding the flow path in a horizontal direction;
A cleaning liquid holding part;
A buffer solution filling mechanism for filling a separation buffer solution into the flow path of the microchip;
A dispensing probe for inhaling and injecting a liquid into a predetermined reservoir of the microchip;
A probe moving mechanism for moving the dispensing probe between a suction position of a liquid to be dispensed and a dispensing position on the microchip;
A suction nozzle for sucking liquid from the reservoir;
A high voltage power supply unit for electrophoresis for applying a voltage for electrophoresis between the reservoirs;
A control unit that controls at least the operation of the buffer solution filling mechanism, the dispensing probe, the probe moving mechanism, the suction nozzle, and the high-voltage power supply unit for electrophoresis,
The control unit according to claim 1, using an electrophoresis control unit that automatically repeats a buffer solution filling step, a sample dispensing step, an electrophoretic separation step, and a buffer solution removal step, and a cleaning solution held in the cleaning solution holding unit. A microchip electrophoresis apparatus that includes a cleaning control unit that automatically executes the cleaning process, and is configured to execute a microchip electrophoresis method. The cleaning control unit includes a cleaning condition setting unit in which the number of cleaning repetitions, the cleaning liquid holding time in the flow path, and the cleaning liquid supply amount are set as the cleaning conditions of the cleaning process. There are multiple types for at least one of
The cleaning control unit executes the cleaning step based on the cleaning conditions set in the cleaning condition setting unit and the cleaning conditions selected from among the cleaning conditions set with a plurality of types. The microchip electrophoresis apparatus according to claim 6, configured as described above. The chip holding part is for fixing and arranging a plurality of microchips,
The microchip electrophoresis apparatus according to claim 6 or 7, wherein the control unit is configured to execute the microchip electrophoresis method for each microchip.

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