Pretreatment-integrated electrophoresis device and electrophoresis method

Abstract

The present invention provides an electrophoresis device and an electrophoresis method whereby the sample solution as a sample solution that has been subjected to electrophoresis can be subjected to electrophoresis again, and in which a drop in detection sensitivity with respect to a sample component contained in the sample solution and contamination between sample solutions are reduced. This electrophoresis device comprises: a pretreatment unit (102) that pretreats a sample to prepare a sample solution containing a sample component; a capillary (202); a chamber (701d); liquid feed channels (701a, 701b) that connect the pretreatment unit (102) and the chamber (701d); a liquid feed pump (112); and a power source that applies a voltage to the capillary (202). In this electrophoresis device and electrophoresis method, after the sample component is electrically injected into the capillary (202), the sample solution remaining in the chamber (701d) is reversely fed to the outside, and after the sample component has undergone electrophoresis in the capillary (202), the reversely fed sample solution is re-introduced into the chamber (701d) to perform the injection and the electrophoresis again.

Description

Pretreatment integrated electrophoresis device and electrophoresis method

[0001] The present invention relates to a pretreatment-integrated electrophoresis apparatus and an electrophoresis method for performing sample pretreatment and electrophoresis.

[0002] Applications of nucleic acid analysis have been put to practical use in fields such as forensic medicine, immigration control, and counterterrorism. In the field of forensic medicine, DNA identification using STR analysis has been put to practical use. STR analysis is a technique for analyzing short tandem repeats (STRs) present in the genome. The number of STR repeats varies from person to person. Therefore, analyzing the number of STR repeats makes it possible to identify individuals and determine paternity.

[0003] A typical STR analysis is performed according to the following procedure. First, nucleic acid is extracted from a sample collected from a tissue, site, etc. In most cases, genomic DNA is extracted as the nucleic acid. Next, the nucleic acid is amplified by PCR (Polymerase Chain Reaction) using the extracted nucleic acid as a template. Approximately 10 to 40 types of primer sets are used to amplify the nucleic acid. Multiple regions on the genome are amplified by multiplex PCR using many types of primer sets. During this PCR process, the nucleic acid is labeled with a fluorescent dye or the like.

[0004] The nucleic acid amplification product is mixed with a solution containing a variety of labeled fragments to form a sample solution for electrophoresis. The nucleic acid is then denatured into single strands using a denaturant such as formamide or by heating, and the sample solution for electrophoresis is then subjected to electrophoresis. The fragments contained in the sample solution are separated by electrophoresis according to their mobility, which is determined by size, and then optically detected. The STRs are then identified based on the size and sequence of the detected fragments.

[0005] Among the main steps in STR analysis, the pretreatment step corresponds to the process of preparing a sample solution for electrophoresis from a sample collected from a tissue, a site, etc. The electrophoresis step corresponds to the process of subjecting the sample solution for electrophoresis to electrophoresis and analyzing the results of the electrophoresis. Conventionally, an electrophoresis device with an integrated pretreatment step has been developed, which performs the pretreatment step and the electrophoresis step consecutively in a single device.

[0006] Patent Literature 1 describes a capillary electrophoresis device with integrated pretreatment that automatically performs all steps, including extracting nucleic acids from a biological sample, performing a PCR reaction on a target region, and performing a cycle sequencing reaction on the amplified nucleic acids, as well as analyzing the nucleic acids by capillary electrophoresis. This device is equipped with a liquid delivery pump that adjusts the delivery of the solution by pressurizing and depressurizing the flow channels and reservoirs within the cartridge. This liquid delivery pump delivers liquid in one direction only, and reverse delivery is prohibited.

[0007] Patent Document 2 describes an electrophoresis method for separating a sample on a microchip. This microchip has an inlet channel for introducing a sample and a separation channel for electrophoretically separating the sample. The inlet channel and the separation channel intersect at an intersection. The sample is introduced into a sample reservoir and then transported to the intersection through the inlet channel. The sample transported to the intersection is electrophoresed through the separation channel toward a buffer reservoir. In the first sub-step of the delay period introduction step, a return voltage is applied to the inlet channel to prevent excess sample from flowing into the separation channel.

[0008] Patent Document 3 describes a device that performs sample extraction, purification, PCR, etc., in an integrated manner with electrophoresis, analysis, etc. In this device, the outlet channel from the PCR chamber is connected to an archive chamber. The archive chamber is a chamber to which surplus sample is supplied, and is formed on a stub that can be separated from the cartridge. The device is designed to maintain its archive function by storing only the stub.

[0009] In general, pretreatment-integrated electrophoresis devices are designed to be usable with simple operations without requiring specialized knowledge. Even users unfamiliar with pretreatment and electrophoresis operations can easily use the devices. Conventionally, pretreatment-integrated electrophoresis devices have been used primarily to analyze swab samples collected from the oral cavity. However, in recent years, developments have been underway to expand the scope of analysis to include casework samples collected from crime victims, disaster victims, and the like.

[0010] JP 2014-021052 A JP 2008-233051 A International Publication No. 2010 / 091414

[0011] When a crime, disaster, or accident occurs, casework samples that cannot be used to identify individuals may be collected from the victim. DNA testing of the casework samples is required to determine the victim's identity. Generally, casework samples are collected in very small amounts, and recollection is often difficult. Therefore, it is necessary to pretreat limited casework samples to prepare a sample solution for electrophoresis, and then subject the sample solution to electrophoresis to ensure accurate interpretation of the electrophoresis results.

[0012] However, various defects can unexpectedly occur during electrophoresis, such as failure of sample component migration, poor separation of sample components, defects during detection after electrophoresis, and defects during analysis of the results. If the cause of the defect is something other than sample pretreatment, it is desirable to re-run the same sample solution and read the results. However, when only a small amount of the sample to be analyzed is collected or when re-collection of the sample is difficult, re-running can be difficult.

[0013] In Patent Document 1, the liquid is transferred unidirectionally within the cartridge. Unidirectional liquid transfer is effective from the viewpoint of preventing contamination. However, a unidirectional liquid transfer configuration makes it difficult to perform electrophoresis again if the electrophoresis results are unsatisfactory. If liquid were transferred in the reverse direction in the device of Patent Document 1, the entire flow path would be depressurized through each port. Backflow to areas other than the electrophoresis compartment would occur, and the sample solution would flow into the upstream compartments that are repeatedly used, potentially resulting in serious contamination.

[0014] In Patent Document 2, a return voltage is applied to the introduction channel to prevent excessive sample from flowing into the separation channel. However, when electrophoresis is performed while applying a return voltage, there is a risk that sample components contained in the sample solution will disperse to the ends of the separation channel or the introduction channel. If the sample components disperse, the amount of sample components introduced into the separation channel during re-electrophoresis will decrease. This reduces the detection sensitivity of sample components separated by electrophoresis, making it difficult to read the results of electrophoresis.

[0015] In Patent Document 3, excess samples are stored in an archive chamber. However, a portion of the sample solution containing the PCR amplification product must be stored in the archive chamber. Because the sample solution, which is a small amount, needs to be divided, less sample solution is used for the initial electrophoresis. This reduces the sensitivity of detecting sample components separated by electrophoresis, making it difficult to interpret the electrophoresis results. Furthermore, when re-running the sample solution stored in the archive chamber, a high degree of positional accuracy is required because the position of the sample solution determines the injection amount.

[0016] Therefore, an object of the present invention is to provide an electrophoresis apparatus and an electrophoresis method that enable the same sample solution that has been subjected to electrophoresis to be subjected to re-electrophoresis, thereby reducing a decrease in the detection sensitivity of sample components contained in the sample solution and reducing contamination between sample solutions.

[0017] In order to solve the above problems, the electrophoresis apparatus of the present invention comprises a pretreatment section that pretreats a sample to prepare a sample solution containing sample components, capillaries that perform electrophoresis of the sample components, a chamber into which the sample solution is introduced and into which the sample components are electrophoresed into the capillaries, a liquid delivery flow path that connects the pretreatment section and the chamber, a liquid delivery pump that delivers the sample solution through the liquid delivery flow path, and a power source that applies a voltage across the capillaries, and after the sample components contained in the sample solution introduced into the chamber are electrophoresed into the capillaries, the sample solution remaining in the chamber is sent back to the outside of the chamber, and the sample components electrophoresed into the capillaries are electrophoresed using the capillaries, and the sample solution that has been sent back to the outside of the chamber is reintroduced into the chamber to perform electrophoresis and electrophoresis.

[0018] Furthermore, the electrophoresis method according to the present invention includes the steps of: pretreating a sample to prepare a sample solution containing sample components; sending the sample solution to a chamber where electric field injection of the sample components into capillaries is performed; electric field injection of the sample components contained in the sample solution introduced into the chamber into the capillaries; sending the sample solution remaining in the chamber back to the outside of the chamber; electrophoresing the sample components electric field injected into the capillaries using the capillaries; and reintroducing the sample solution sent back to the outside of the chamber into the chamber to perform electric field injection and electrophoresis.

[0019] According to the present invention, it is possible to provide an electrophoresis apparatus and an electrophoresis method in which the same sample solution that has been subjected to electrophoresis can be subjected to re-electrophoresis, thereby reducing the decrease in detection sensitivity of sample components contained in the sample solution and the contamination of sample solutions.

[0020] 1 is a diagram illustrating a configuration of an electrophoresis device according to an embodiment of the present invention. FIG. 1 is a diagram illustrating a configuration of an electrophoresis device according to an embodiment of the present invention. FIG. 2 is a diagram illustrating a structure of a capillary cartridge connected to an electrophoresis device according to an embodiment of the present invention. FIG. 3 is a block diagram illustrating connections of flow paths between components in a pre-treatment integrated electrophoresis device. FIG. 4 is a block diagram illustrating connections of flow paths between components in a pre-treatment integrated electrophoresis device. FIG. 5 is a flowchart illustrating an example of a process performed by an electrophoresis device according to an embodiment of the present invention. FIG. 6 is a flowchart illustrating an example of a process performed by an electrophoresis device according to an embodiment of the present invention. FIG. 7 is a diagram illustrating an example of a configuration of main parts of an electrophoresis device according to an embodiment of the present invention. FIG. 8 is a diagram illustrating an example of a configuration of main parts of an electrophoresis device according to an embodiment of the present invention. FIG. 9 is a diagram illustrating the operation of an example of an in-line negative pressure mechanism. FIG. 10 is a diagram illustrating the operation of an example of an in-line negative pressure mechanism. FIG. 11 is a diagram illustrating an example of a configuration of main parts of an electrophoresis device according to an embodiment of the present invention. FIG. 12 is a diagram illustrating an example of a configuration of a sample cartridge with a built-in negative pressure mechanism. FIG. 13 is a block diagram illustrating connections of flow paths between components in a pre-treatment integrated electrophoresis device. FIG. 14 is a block diagram illustrating connections of flow paths between components in a pre-treatment integrated electrophoresis device. 1 is a diagram illustrating an example of a configuration of a main part of an electrophoretic device according to an embodiment of the present invention.

[0021] Hereinafter, an electrophoresis device and an electrophoresis method according to an embodiment of the present invention will be described. Note that common components in the following drawings are denoted by the same reference numerals, and duplicated explanations will be omitted.

[0022] 1A and 1B are diagrams showing the configuration of an electrophoresis device according to an embodiment of the present invention. Fig. 1A shows a front view of an electrophoresis device 100 according to this embodiment. Fig. 1B shows a side cross-sectional view of the electrophoresis device 100 according to this embodiment. Fig. 1B schematically shows the internal structure of the electrophoresis device 100.

[0023] As shown in FIGS. 1A and 1B , the electrophoresis apparatus 100 according to this embodiment includes a capillary cartridge 101, a sample cartridge 102, a power supply 103, an optical system 104, an injection pump 105, a control unit 107, an input / output unit 108, an interface unit 109, a hinge 110, a main body 111, a liquid delivery unit 112, a temperature control unit 113, a negative pressure mechanism 114, and the like.

[0024] The electrophoresis apparatus 100 according to this embodiment is an integrated pretreatment electrophoresis apparatus that performs sample pretreatment and electrophoresis. In this integrated pretreatment electrophoresis apparatus, a process of pretreating a sample to be analyzed to prepare a sample solution for electrophoresis and a process of subjecting the sample solution for electrophoresis to analysis are performed automatically and continuously by a single apparatus. During electrophoresis, sample components contained in the sample solution are separated and detected according to their mobility.

[0025] The electrophoresis device 100 can be used for nucleic acid sequencing such as DNA sequencing and RNA sequencing, and nucleic acid analysis such as STR analysis, SNP (Single Nucleotide Polymorphism) analysis, SSR (Simple Sequence Repeat) analysis, and CAPS (Cleaved Amplified Polymorphic Sequence) analysis.

[0026] The sample to be analyzed can be cells, tissues, or body fluids collected from living organisms, or cells, tissues, or body fluids collected from crime, disaster, or accident sites, etc. Sample components to be analyzed by electrophoresis include, for example, nucleic acid molecules such as DNA and RNA extracted from the sample.

[0027] Based on the results of detecting the sample components after electrophoresis, the electrophoresis apparatus 100 performs quantification of the sample components separated by mobility, size calling to identify the sizes of the sample components, base calling to identify the sequences of the sample components, identification of the attribution of the sample components, etc. Examples of attribution identification include allele calling to identify the type of allele encoded by a base sequence, and variant calling to identify polymorphisms in the base sequence.

[0028] The capillary cartridge 101 is a cartridge containing capillaries for performing electrophoresis. Electrophoresis of sample components to be analyzed is performed in the capillary cartridge 101. The capillary cartridge 101 is equipped with one or more capillaries, devices such as electrodes for performing electrophoresis, piping for transporting liquids such as sample solutions and reagents, reagents used for electrophoresis, etc. The capillary cartridge 101 can also be equipped with reagents used for pretreatment.

[0029] The capillary cartridge 101 is detachably attached to the electrophoresis apparatus 100. When the capillary cartridge 101 is placed on the interface unit 109, an electrical connection for transmitting an electric signal, a connection for a flow path through which a liquid can flow, and an optical connection for transmitting an optical signal are formed between the capillary cartridge 101 and the main body 111. The capillary cartridge 101 is a consumable item and is replaced after approximately 100 to 300 uses.

[0030] The sample cartridge 102 is a cartridge that performs pre-processing of the sample. The sample cartridge 102 constitutes a pre-processing section that pre-processes the sample to prepare a sample solution for electrophoresis containing sample components. The sample to be analyzed is loaded into the sample cartridge 102. The loaded sample is pre-processed to prepare a sample solution for electrophoresis. The sample cartridge 102 is equipped with processing tanks that perform various processes, storage tanks for storing liquids such as reagents, piping for transporting liquids such as the sample solution and reagents, valves for opening and closing the flow paths, reagents used for pre-processing, etc.

[0031] The sample cartridge 102 is detachably attached to the electrophoresis device 100. When the sample cartridge 102 is connected to a predetermined connector provided on the main body 111 of the electrophoresis device 100, an electrical connection for transmitting electrical signals and a connection of a flow path through which liquid can flow are formed between the sample cartridge 102 and the main body 111. The sample cartridge 102 is a consumable item and is usually replaced after each use.

[0032] 1A, a total of four sample cartridges 102 are connected to the main body 111. In this case, four samples can be processed in parallel.

[0033] The power supply 103 is a power supply that applies a voltage to the capillaries built into the capillary cartridge 101. The power supply 103 is electrically connected via an interface unit 109 or the like to electrodes installed near the ends of the capillaries built into the capillary cartridge 101. By applying a high voltage across the capillaries, electric field injection, in which sample components are injected into the capillaries, and electrophoresis of the sample components inside the capillaries are performed.

[0034] The optical system 104 is a unit that optically detects the results of electrophoresis. In order to detect sample components labeled with fluorescent dyes, the optical system 104 generates excitation light that excites the fluorescent dyes and detects fluorescence emitted from the excited fluorescent dyes. The optical system 104 includes a light source that generates excitation light, a processing circuit that performs signal processing on the information light extracted from the capillaries, and the like. The optical system 104 is optically connected to a detection unit built into the capillary cartridge 101 via an interface unit 109, optical fibers, and the like.

[0035] The injection pump 105 is a high-pressure pump that injects a polymer, which is a migration medium, into the capillaries built into the capillary cartridge 101. A solution in which the polymer, which is a migration medium, is dissolved is injected into the capillaries for each electrophoretic measurement. When a specific polymer is used as the migration medium, a molecular sieve effect is obtained during electrophoresis. Utilizing the separation sieve effect makes the mobility difference according to the size of the sample components significant. This makes it possible to separate the sample components by size and detect them with high sensitivity.

[0036] The control unit 107 controls each device built into the electrophoresis apparatus 100, controls the operation of the capillary cartridge 101, controls the operation of the sample cartridge 102, processes data related to electrophoresis, etc. The control unit 107 monitors the status of electrophoresis in the capillary cartridge 101, monitors the results of electrophoresis in the capillary cartridge 101, and analyzes the results of electrophoresis.

[0037] The input / output unit 108 inputs and outputs data to the electrophoresis device 100. The input / output unit 108 receives instructions from the user of the electrophoresis device 100, inputs data related to electrophoresis, outputs the status and results of electrophoresis, and outputs the results of analyzing the results of electrophoresis. The input / output unit 108 is configured with a touch panel or the like. The instructions and data input to the input / output unit 108 are transmitted to the control unit 107.

[0038] The interface unit 109 is a unit for connecting the capillary cartridge 101 to the main body 111 of the electrophoresis apparatus 100. The upper surface of the interface unit 109 is provided with connectors for forming electrical connections, ports for connecting flow paths, and connectors for forming optical connections. When the capillary cartridge 101 is connected to the interface unit 109, electrical connections for transmitting electrical signals, connections for flow paths through which liquid can flow, and optical connections for transmitting optical signals are formed between the capillary cartridge 101 and the main body 111.

[0039] 1 , an interface unit 109 is rotatably supported via a hinge 110 relative to a main body 111 of the electrophoresis device. The interface unit 109 is provided on the front side of the main body 111 so as to be openable and closable, with the lower side where the hinge 110 is axially supported as the axis of rotation. The interface unit 109 can be opened and closed between an open state in which it protrudes forward from the front side of the main body 111 and a closed state in which it stands upright and is in close contact with the front surface of the main body 111.

[0040] The interface unit 109 is in an open state when the electrophoresis apparatus 100 is in use. When the capillary cartridge 101 is placed at a predetermined position on the top surface of the interface unit 109, various connections are formed between the capillary cartridge 101 and the main body 111. On the other hand, the interface unit 109 can be in a closed state when the electrophoresis apparatus 100 is not in use. The top surface of the interface unit 109 is in close contact with the front surface of the main body 111, thereby protecting the connectors and ports formed on the top surface.

[0041] The main body 111 is a housing, a skeleton, or the like that constitutes the main body of the electrophoresis device 100. The main body 111 is formed of a structural material such as a metal bottom plate, a decorative plate, or the like. The main body 111 houses a power supply 103, an optical system 104, an injection pump 105, a control unit 107, an input / output unit 108, an interface unit 109, a liquid delivery unit 112, a temperature control unit 113, a negative pressure mechanism 114, and the like. Support legs for supporting these components may be provided on the bottom surface of the main body 111 and the bottom surface of the interface unit 109.

[0042] A flow path through which a liquid can flow is provided inside the main body 111. When the capillary cartridge 101 is connected to the main body 111, the flow path inside the main body 111 communicates with the flow path inside the capillary cartridge 101. Furthermore, when the sample cartridge 102 is connected to the main body 111, the flow path inside the main body 111 communicates with the flow path inside the sample cartridge 102.

[0043] When the capillary cartridge 101 and the sample cartridge 102 are connected to the main body 111, a flow path is formed that connects the flow path inside the sample cartridge 102 and the flow path inside the capillary cartridge 101 via the flow path inside the main body 111. This flow path functions as a liquid transfer flow path for transferring the sample solution for electrophoresis from the sample cartridge 102 to the capillary cartridge 101.

[0044] The liquid delivery unit 112 is a unit that delivers liquid such as a sample solution or a reagent. The liquid delivery unit 112 delivers liquid inside the sample cartridge 102, between the sample cartridge 102 and the main body 111, between the main body 111 and the capillary cartridge 101, and inside the capillary cartridge 101. The liquid delivery unit 112 incorporates a pump that transfers liquid.

[0045] The temperature control unit 113 is a unit that controls the temperature of pretreatment and the like. The temperature control unit 113 controls the temperature of reagents and the like delivered to the sample cartridge 102, and the temperature of a predetermined region of the sample cartridge 102. The temperature control unit 113 may control the temperature directly by contact with the temperature control target through heat conduction, or may control the temperature of the temperature control target indirectly by heat exchange using a heat exchange medium. The temperature control unit 113 may include, for example, a heating / cooling mechanism such as a Peltier element, a temperature sensor, etc.

[0046] The negative pressure mechanism 114 is a mechanism that creates a negative pressure in at least a portion of the flow path connecting the sample cartridge 102 and the cathode block 212 relative to the internal pressure of the chamber in which the end of the capillary is located. The negative pressure mechanism 114 creates a negative pressure in at least a portion of the flow path through which the sample solution for electrophoresis flows, the portion being downstream of the sample cartridge 102 and upstream of the chamber inside the cathode block 212 of the capillary cartridge 101. At this time, the flow path connecting the negative pressure mechanism 114 and the sample cartridge 102 is closed, so the negative pressure mechanism 114 does not change the pressure in the flow path connecting the sample cartridge 102 and the liquid delivery unit 112, and does not create a negative pressure inside the sample cartridge 102 or the liquid delivery unit 112.

[0047] The negative pressure mechanism 114 may be a pump that has a function of reducing the internal pressure of the flow path connected to the chamber inside the cathode block 212. The internal pressure of the liquid feed flow path may be reduced by any appropriate method, such as by suctioning the fluid in the liquid feed flow path, adjusting the cross-sectional area of ​​the flow path of the liquid feed flow path, or adjusting the flow path volume of the liquid feed flow path. The negative pressure mechanism 114 may be a decompression pump such as a vacuum pump, a bidirectional pump, a syringe pump, a piezoelectric pump, a membrane pump, or the like.

[0048] The negative pressure mechanism 114 applies a negative pressure to at least a portion of the liquid feed channel connecting the sample cartridge 102 and the cathode block 212 after electrophoresis of sample components contained in the sample solution into the capillaries and before electrophoresis of the sample components inside the capillaries. During electrophoresis, the sample solution containing the sample components is introduced near the ends of the capillaries inside the chamber of the capillary cartridge 101. When electrophoresis is performed, some of the sample components contained in the sample solution are injected into the capillaries. When the liquid feed channel is applied with a negative pressure after electrophoresis, the sample solution that has been subjected to electrophoresis and remains near the ends of the capillaries is sent back upstream of the chamber.

[0049] The sample solution sent back upstream of the chamber by the negative pressure mechanism 114 can be retained in the flow path upstream of the chamber by adjusting the internal pressure of the flow path, etc. Therefore, if the results of electrophoresis after electroinjection are poor, the sample solution used for electroinjection can be reintroduced from the flow path upstream of the chamber into the vicinity of the end of the capillary inside the chamber. In other words, the same type of sample solution used for electroinjection can be used for reinjection or re-electrophoresis. Since pretreated sample solution can be used for re-electrophoresis, pretreatment can be bypassed, thereby shortening the time required for analysis.

[0050] Fig. 2 is a diagram showing the structure of a capillary cartridge connected to an electrophoresis apparatus according to an embodiment of the present invention. Fig. 2 shows a perspective view of a capillary cartridge 101 connected to an electrophoresis apparatus 100. Fig. 2 shows a schematic view of the internal structure of the capillary cartridge 101, seen through the housing of the capillary cartridge 101.

[0051] As shown in FIG. 2, the capillary cartridge 101 includes a plurality of capillaries 202, a capillary head 204, a detection unit 205, a capillary connection port 208, a thermostatic bath 209, a polymer delivery unit 211, a cathode block 212, a migration reagent delivery pump 213, a migration reagent container 214, a cathode waste container 215, an anode waste container 219, flow path connection ports 220 and 221, a sample injection pipe 222, a pretreatment reagent container 223, a pretreatment reagent delivery pipe 224, and the like.

[0052] The capillary 202 is a thin tube for performing electrophoresis. The capillary 202 is formed of a glass tube with a coating applied to its surface. The glass tube has an inner diameter of several tens to several hundred micrometers and an outer diameter of several hundred micrometers. The glass tube is formed of fused silica, borosilicate glass, or the like. The glass tube is coated with polyimide or the like to ensure strength to withstand breakage, etc. However, the area of ​​the surface of the capillary 202 facing the detection unit 205 is provided with the coating removed.

[0053] The capillaries 202 are filled with a polymer, which is an electrophoretic medium. Generally, electrophoretic media are classified into fluid electrophoretic media such as gels and non-fluid electrophoretic media. The electrophoretic device 100 uses a fluid electrophoretic medium. When a fluid electrophoretic medium is used, the electrophoretic medium can be easily replaced by discharging and re-injecting it into the capillaries 202. This reduces contamination caused by components remaining inside the capillaries 202.

[0054] The capillary head 204 is a component that bundles together multiple capillaries 202. A capillary array is formed by the multiple capillaries 202. In Fig. 2, the capillary head 204 bundles together the anode-side ends of the capillaries 202. The capillary head 204 is connected to the polymer delivery part 211 by a structure that ensures pressure resistance and airtightness between the flow paths.

[0055] The capillaries 202 constituting the capillary array are provided to have the same length, with an error of millimeters or submillimeters or less. Multiple types of sample solutions with different compositions can also be prepared in the sample cartridge 102. By injecting multiple types of sample solutions into each capillary 202 constituting the capillary array, multiple types of sample solutions can be analyzed simultaneously.

[0056] It is preferable to replace the capillaries 202 with new ones when the number of electrophoresis runs reaches a predetermined number or when damage or deterioration is found. The capillaries 202 can be replaced in units of a capillary array bound to the capillary head 204. Alternatively, the capillaries 202 can be replaced in units of a capillary cartridge 101 containing a capillary array.

[0057] The detection unit 205 is a site for detecting sample components inside the capillary 202. The detection unit 205 is installed near the end of the capillary 202 in the longitudinal direction, opposite to the injection end 207 where the sample solution is injected. The detection unit 205 is arranged to face an uncoated area of ​​the surface of the capillary 202. The detection unit 205 is provided with a light irradiation port that irradiates the inside of the capillary 202 with excitation light and a light extraction port that extracts fluorescence from the inside of the capillary 202.

[0058] During electrophoresis in the capillary 202, excitation light generated by the optical system 104 is transmitted to the detection unit 205 via an optical fiber or the like. The detection unit 205 irradiates the excitation light onto the inside of the capillary 202. When the sample components labeled with fluorescent dyes move inside the capillary 202 by electrophoresis, they are irradiated with excitation light from the detection unit 205. The fluorescent dyes irradiated with excitation light emit fluorescence of a predetermined wavelength depending on the type of fluorescent dye. The fluorescence emitted from the fluorescent dyes that label the sample components is extracted by the detection unit 205 as information light.

[0059] The information light is transmitted to the optical system 104 via an optical fiber or the like. In the optical system 104, the information light is dispersed by a diffraction grating and detected by a photodiode or the like. By detecting the information light, the light intensity for each predetermined wavelength region is measured. As a result of the electrophoresis, the control unit 107 obtains a spectrum indicating the relationship between the migration time calculated from the start of the electrophoresis and the light intensity of the information light. Based on the spectrum obtained for each sample component, the control unit 107 performs base calling, size calling, and identification of the components.

[0060] The thermostatic bath 209 is a bath that maintains the capillaries 202 at a constant temperature. The thermostatic bath 209 accommodates a section of each capillary 202 excluding the ends. The thermostatic bath 209 is equipped with a heat insulating material that covers the periphery and a temperature control mechanism that controls the temperature of the internal space. The temperature inside the thermostatic bath 209 is controlled so that the temperature of the capillaries 202 is uniform across position and constant over time. The temperatures of the capillaries 202 that make up the capillary array are controlled to be equal to each other.

[0061] Methods for controlling the temperature of the thermostatic chamber 209 include a contact type in which heating or cooling is performed by contacting the capillary 202, which is the object of temperature control, and performing heating or cooling by thermal conduction, a space type in which heating or cooling is performed by heating or cooling the air present in the space in which the capillary 202 is installed, and a heat exchange type in which heating or cooling is performed by heat exchange between the structural material forming the capillary 202 or thermostatic chamber 209, which is the object of temperature control, and a heat exchange medium such as cooling water.

[0062] The polymer liquid delivery unit 211 is a component having a flow path formed therein through which a liquid can flow. The polymer liquid delivery unit 211 holds a buffer solution for electrophoresis near the anode-side end of the capillary 202 and injects a polymer, which is an electrophoresis medium, into the capillary 202. The anode-side end of the capillary 202 is connected to the polymer liquid delivery unit 211 so as to communicate with the internal flow path. The polymer liquid delivery unit 211 is connected via piping to an anode waste liquid container 219, an injection pump 105, and a container for storing a polymer solution in which a polymer is dissolved. For example, a syringe pump containing a syringe containing the polymer solution can be connected.

[0063] An anode electrode is installed in the flow path inside the polymer liquid delivery section 211. The anode electrode is electrically connected to the power supply 103. During electric field injection of sample components or electrophoresis, a high voltage is applied to the anode electrode by the power supply 103, and the anode electrode functions as an anode into which current flows from the capillary 202. On the anode side of the capillary 202, an electrophoresis medium is injected into the capillary 202, and the sample components that have migrated inside the capillary 202 by electrophoresis are discharged.

[0064] The cathode block 212 is a block-shaped member having a flow path formed therein through which a liquid can flow. The cathode block 212 holds a buffer solution for electrophoresis near the cathode end of the capillary 202 and injects sample components contained in a sample solution into the capillary 202. A chamber is formed inside the cathode block 212 into which the sample solution is introduced and into which electric field injection of the sample components into the capillary 202 is performed. The cathode end of the capillary 202 is inserted into the chamber. A migration reagent delivery pump 213, a migration reagent container 214, a cathode waste liquid container 215, and the sample cartridge 102 are connected to the cathode block 212 via piping.

[0065] Cathode electrodes corresponding to the number of capillaries 202 are installed in the flow path inside the cathode block 212. Each capillary 202 is individually inserted into a plurality of capillary connection ports 208 formed in the cathode block 212. The cathode electrodes are connected to ground. During electric field injection of sample components or electrophoresis, a high voltage is applied to the cathode electrodes by the power supply 103, and the cathode electrodes function as cathodes that cause current to flow through the capillaries 202. On the cathode side of the capillaries 202, electric field injection of sample components into the capillaries 202 is performed.

[0066] 2, a cathode electrode is provided on the injection end 207 side where the sample solution is injected, and an anode electrode is provided on the sample solution discharge side. However, the capillary cartridge 101 may be provided with a structure in which the polarity of the electrodes is reversed depending on the polarity of the sample components to be analyzed. The electrode on the injection end 207 side where the sample solution is injected may be electrically connected to the power supply 103. Furthermore, the electrode on the sample solution discharge side may be connected to ground.

[0067] Furthermore, the electrode on the side of the injection end 207 into which the sample solution is injected may be inserted into the flow channel in which the end of the capillary 202 is disposed, or may be attached to the end of the capillary 202. For example, a tubular hollow electrode made of a conductive metal or the like may be attached to the end of the capillary 202. The electrode attached to the end of the capillary 202 can be easily replaced together with the capillary 202.

[0068] The migration reagent delivery pump 213 is a pump that delivers the migration reagent. A migration reagent container 214 is connected to the migration reagent delivery pump 213 via piping. The migration reagent delivery pump 213 delivers the migration reagent stored in the migration reagent container 214 toward the cathode block 212. A migration buffer solution is prepared as the migration reagent. The migration buffer solution is delivered to the cathode block 212 at the start of electrophoresis and during electrophoresis to supply the charge required for electrophoresis. The migration buffer solution is also delivered to the cathode block 212 before the start of electrophoresis and after the end of electrophoresis to clean the flow path inside the cathode block 212.

[0069] The capillary cartridge 101 shown in Fig. 2 has a built-in pretreatment reagent container 223. The pretreatment reagent container 223 is a container for storing a reagent used in pretreatment and a channel cleaning liquid. The pretreatment reagent container 223 is connected to a channel connection port 220 via a pretreatment reagent supply pipe 224. The channel connection port 220 opens at the bottom of the capillary cartridge 101. The channel connection port 220 is an opening that forms a channel connection with the main body 111. The channel connection port 220 is connected to a corresponding port of the interface unit 109.

[0070] The pretreatment reagent container 223 communicates with the liquid delivery unit 112 via a pretreatment reagent delivery pipe 224, the interface unit 109, and a flow path through which liquid can flow. The liquid delivery unit 112 communicates with the sample cartridge 102 via a flow path through which liquid can flow. The reagent prepared in the pretreatment reagent container 223 is delivered to the sample cartridge 102 by the liquid delivery unit 112 via the pretreatment reagent delivery pipe 224, the interface unit 109, and the main body 111. In the sample cartridge 102, the supply of reagent performs pretreatment of the sample and preparation of a sample solution for electrophoresis.

[0071] The sample solution for electrophoresis prepared in the sample cartridge 102 is sent by the liquid sending unit 112 to the flow path inside the cathode block 212 through the main body 111, the interface unit 109, and the sample injection piping 222. A predetermined port of the interface unit 109 is connected to a flow path connection port 221. The flow path connection port 221 opens at the bottom of the capillary cartridge 101. The flow path connection port 221 is an opening that forms a flow path connection with the main body 111. The flow path connection port 221 is connected to the cathode block 212 via the sample injection piping 222.

[0072] During electric field injection to inject sample components into the capillary 202, a sample solution for electrophoresis is introduced into the flow path inside the cathode block 212. With the sample solution introduced, a high voltage is applied between both ends of the capillary 202 by the power supply 103. The application of the high voltage causes the sample components contained in the sample solution to be injected into the capillary 202.

[0073] During electrophoresis of the sample components inside the capillary 202, the flow path inside the cathode block 212 is replaced with a migration buffer solution by the migration reagent delivery pump 213. With the capillary 202 filled with the migration buffer solution, a voltage is applied across both ends of the capillary 202, causing electrophoresis of the sample components inside the capillary 202. After electrophoresis is completed, a new migration medium is injected into the capillary 202 by the polymer delivery unit 211.

[0074] The reagent prepared in the pretreatment reagent container 223 is also used to clean the sample injection piping 222 and the internal flow paths of the cathode block 212. After the pretreatment is completed and before the next pretreatment is started, the reagent prepared in the pretreatment reagent container 223 is delivered by the liquid delivery unit 112 to the sample injection piping 222 and the cathode block 212 through the pretreatment reagent delivery piping 224, the interface unit 109, and the main body 111. Replacing the liquid in the flow path and cleaning the inner walls by supplying the reagent reduces contamination between sample solutions.

[0075] The cathode waste liquid container 215 is a container for collecting waste liquid on the cathode side. After the electric field injection of the sample components or at the end of electrophoresis, waste liquid of the sample solution remaining inside the cathode block 212 is discharged from the cathode block 212 to the cathode waste liquid container 215.

[0076] The anode waste liquid container 219 is a container for collecting waste liquid on the anode side. At the end of electrophoresis, waste liquid remaining inside the polymer liquid delivery unit 211 is discharged from the polymer liquid delivery unit 211 to the anode waste liquid container 219. In addition, waste liquid generated when the inside of the polymer liquid delivery unit 211 is replaced with a new polymer solution and waste liquid generated when air bubbles inside the polymer liquid delivery unit 211 are removed are discharged from the polymer liquid delivery unit 211 to the anode waste liquid container 219.

[0077] 2, the capillary cartridge 101 may include a pretreatment reagent container for storing a pretreatment reagent, a cooling mechanism for cooling the polymer serving as the migration medium, etc. By cooling the polymer, it is possible to suppress a decrease in separation ability due to deterioration of the migration medium.

[0078] 3A and 3B are block diagrams showing the connections of channels between components in a pre-treatment integrated electrophoresis device. Fig. 3A shows the connections of channels between components in a conventional pre-treatment integrated electrophoresis device. Fig. 3B shows the connections of channels between components in a pre-treatment integrated electrophoresis device 100 according to an embodiment of the present invention.

[0079] 3A, in a conventional pretreatment-integrated electrophoresis apparatus, a liquid delivery unit 112, a sample cartridge 102, a cathode block 212, and a cathode waste liquid container 215 are arranged in this order from upstream to downstream. The flow path from the liquid delivery unit 112 to the cathode block 212 is a one-path type in which fluid flows in one direction. This structure in which the sample solutions flow in one direction prevents contamination due to mixing of the sample solutions.

[0080] In conventional electrophoresis devices with integrated pretreatment, reverse flow of the sample solution is prohibited, so that the sample solution that has been subjected to electrochemical injection cannot be re-injected or re-electrophoresed. If an attempt were made to reverse the sample solution, when the sample solution was aspirated from the upstream side, the sample solution would flow back upstream from the solution delivery unit 112, the sample cartridge 102, the cathode block 212, etc. As the sample solution flows into the solution delivery unit 112, the sample cartridge 102, etc., it may mix with the sample solution that has been pretreated, potentially causing serious contamination.

[0081] In particular, the sample solution in the sample cartridge 102 after PCR contains a high concentration of sample molecules. When such sample solution or air containing sample molecules flows back into the liquid delivery unit 112, the sample molecules adhere to the inner walls of the flow path and may not be completely removed by washing with liquid. If these adhered sample molecules flow into the sample cartridge 102 in the next run, even a small amount will be exponentially amplified in the PCR reaction, increasing the possibility of serious contamination.

[0082] 3B , the electrophoresis apparatus 100 according to this embodiment is provided with a negative pressure mechanism 114 in the liquid feed channel between the sample cartridge 102 and the cathode block 212. Operation of the negative pressure mechanism 114 creates a negative pressure in at least a portion of the liquid feed channel connecting the sample cartridge 102 and the cathode block 212, allowing the sample solution introduced into the chamber inside the cathode block 212 to be fed back upstream of the chamber. At this time, the sample solution or air containing sample molecules does not flow back into the sample cartridge 102 or the liquid feed unit 112, which are located upstream of the negative pressure mechanism 114.

[0083] The sample solution sent back by the negative pressure mechanism 114 can be held in the liquid sending channel connecting the sample cartridge 102 and the chamber inside the cathode block 212. If re-electrophoresis is required, the sample solution held in the liquid sending channel can be reintroduced near the end of the capillary inside the chamber for re-injection or re-electrophoresis. Since re-electrophoresis allows normal results to be obtained within a short time, the throughput and usability of the electrophoresis apparatus can be improved. Furthermore, since the sent back sample solution is held downstream of the sample cartridge 102, contamination due to mixing of sample solutions is reduced.

[0084] 4 is a flowchart showing an example of processing by the electrophoresis apparatus 100 according to the embodiment of the present invention, in which the negative pressure mechanism 114 is operated to reverse the sample solution subjected to electric field injection, and the reversed and stored sample solution is then re-injected and re-electrophoresed when poor electrophoresis results are found.

[0085] When a sample to be analyzed is analyzed by electrophoresis in the electrophoresis apparatus 100, the sample to be analyzed is first set in the sample cartridge 102. The sample cartridge 102 is connected to the main body 111 of the electrophoresis apparatus 100.

[0086] Next, a process of extracting nucleic acids from the sample is performed as a pretreatment of the sample (step S101). The nucleic acid extraction can be performed by a process of disrupting cells, a process of lysing cells, or the like. In addition to the process of extracting nucleic acids, the pretreatment may additionally include a process of purifying the extracted nucleic acids and a process of washing the extracted nucleic acids.

[0087] Next, a process of amplifying the nucleic acid extracted from the sample is performed (step S102). The nucleic acid is amplified by PCR using predetermined primers so that a predetermined region in the base sequence is amplified. At this time, the ends of the amplified nucleic acid molecules are fluorescently labeled. Depending on the purpose of the analysis, PCR may be performed as a singleplex using one primer set, or as a multiplex using multiple primer sets.

[0088] Next, the amplification product is mixed with reagents and the like (step S103). The nucleic acid fragments amplified by PCR are mixed with reagents, solvents, and the like to prepare a sample solution for electrophoresis. Reagents that can be used include size standards that serve as a reference for nucleic acid size and formamide, a denaturant.

[0089] Next, electrophoresis is performed using the sample solution for electrophoresis prepared by pretreatment as a load sample (step S104). The sample solution for electrophoresis is introduced near the ends of the capillaries 202 inside the capillary cartridge 101. Then, sample components contained in the sample solution are injected into the capillaries 202 under electric field injection to perform electrophoresis. After the sample components are injected under electric field injection, the negative pressure mechanism 114 is operated to send the sample solution near the ends of the capillaries 202 that have been subjected to electric field injection back upstream and store it.

[0090] Next, the results of the electrophoresis are analyzed (step S105). The sample components subjected to electrophoresis are separated into components with mobility according to their size, structure, etc. inside the capillary 202, and then optically detected in the detection unit 205. The results of the electrophoresis are obtained as a spectrum or the like showing the relationship between the migration time and the light intensity of the information light, based on the optically detected detection signal. The results of the electrophoresis are analyzed by base calling, size calling, identification of the attribution, etc. The analysis of the results of the electrophoresis may be performed after the electrophoresis has ended, or may be performed while the electrophoresis is being performed.

[0091] Next, the results of the electrophoresis are evaluated (step S106). The evaluation of the results of the electrophoresis involves determining whether or not there is a defect in the results of the electrophoresis. The evaluation of the results of the electrophoresis is performed from the perspective of whether or not there is a defect in the results of the electrophoresis due to factors other than the pretreatment of the sample. The evaluation of the results of the electrophoresis may be performed after the electrophoresis has ended, or may be performed while the electrophoresis is being performed.

[0092] If the determination reveals a defect due to factors other than sample pretreatment (step S106; NG), the process returns to step S104. In this case, the sample solution used for field injection or electrophoresis, which has been reversed and stored by the negative pressure mechanism 114, is re-injected and re-electrophoresed as a new load sample. If a defect is discovered during electrophoresis, the run in progress can be stopped and re-injection can be performed. On the other hand, if the determination reveals no defect due to factors other than sample pretreatment (step S106; OK), the electrophoresis is terminated.

[0093] In conventional electrophoresis devices with integrated pretreatment, if poor electrophoresis results were found, it was necessary to reprepare the sample solution for electrophoresis and perform the electrophoresis again. This poses the problem of increasing the time required for electrophoretic analysis because sample pretreatment must be repeated. Generally, it takes approximately 90 to 120 minutes to perform pretreatment and electrophoresis consecutively. Therefore, there is a problem in that an additional time of approximately 90 to 120 minutes, equivalent to one run, is required each time a reanalysis is performed.

[0094] In contrast, in the electrophoresis apparatus 100 according to this embodiment, after the sample components contained in the sample solution are electroinjected into the capillaries, the negative pressure mechanism 114 is activated to reverse the sample solution subjected to electroinjection and electrophoresis upstream and store it. This allows the same type of sample solution as the sample solution subjected to electroinjection and electrophoresis to be reinjected or re-electrophoresed, bypassing pretreatment. Since there is no need to re-perform pretreatment, the time required for electrophoretic analysis can be significantly reduced. Even if the electrophoresis results are found to be unsatisfactory, the analysis can be completed with only an additional time of approximately 30 to 45 minutes.

[0095] The causes of poor electrophoresis results can be broadly divided into those resulting from the pretreatment process leading up to the preparation of the sample solution for electrophoresis and those resulting from the electrophoresis process using the sample solution for electrophoresis. Whether the cause of the poor results is due to the pretreatment process or the electrophoresis process can be determined using the results of size standard detection as an indicator. The results of size standard detection can be confirmed in a spectrum showing the relationship between migration time and detection intensity, or in an electropherogram showing the positions of bands indicating the detection results on a two-dimensional image.

[0096] If the size standard is not detected as a result of electrophoresis, it is possible that the problem is due to a defect in the injection of the sample components into the capillary, a defect in the sample cartridge used to prepare the sample solution for electrophoresis, a defect due to an abnormality in the liquid delivery operation that delivers the sample solution for electrophoresis to the capillary, or a defect due to an abnormality in the liquid delivery operation that delivers the sample solution for electrophoresis.

[0097] If the results of electrophoresis show that part of the size standard detection result is missing, for example, if part of the peak on the spectrum is missing or part of the ladder-like band has disappeared, this may be due to a defect caused by an abnormal current during electrophoresis, a defect caused by an abnormal output from a laser light source that generates excitation light, a defect caused by an abnormality in the transmission and reception of information light detected for each sample component, or a defect caused by an abnormality in the transmission and reception of data showing the results of electrophoresis.

[0098] If the electrophoresis results show any abnormalities in the size standard detection results, for example, if the peak width on the spectrum is wide, the peak height on the spectrum is low, or there is an excessive difference in the peak height for each locus after fragment analysis, this may be due to a sudden electrophoresis failure or a failure caused by impurities contained in the sample solution used for electrophoresis.

[0099] If spikes or other artifacts appear on the baseline in the spectrum showing the relationship between migration time and detection intensity obtained as a result of electrophoresis, it is possible to infer defects such as air bubbles entering the capillary, foreign matter entering the capillary, or impurities contained in the sample solution used for migration.

[0100] When these defects are suspected, it is assumed that the defect is due to factors other than the pretreatment of the sample for electrophoresis. In such cases, it is highly likely that normal results can be obtained by reinjecting and re-running the sample solution subjected to field injection and electrophoresis. Therefore, by determining whether the electrophoresis results are defective due to factors other than the pretreatment of the sample and appropriately determining whether reinjection and re-run are necessary, normal electrophoretic analysis can be performed.

[0101] Specifically, whether or not the electrophoresis results are defective due to factors other than the sample pretreatment can be determined by a method of comparing the height or width of a size standard peak with a threshold in a spectrum showing the relationship between migration time and detection intensity, or a method of comparing the size, width, or height of a detected peak with a threshold to detect spikes or artifacts, etc. The threshold can be set in advance to indicate a height or width that is smaller or larger than that of a standard peak.

[0102] The determination of the electrophoresis results may be performed automatically by the control unit 107 of the electrophoresis device 100, or may be performed manually by the user of the electrophoresis device 100 via the input / output unit 108. The user of the electrophoresis device 100 can check the electrophoresis results output to the input / output unit 108 and determine whether or not the electrophoresis results are defective.

[0103] Furthermore, if the result of electrophoresis is found to be unsatisfactory (step S106; NG), it is also possible to determine whether or not to perform re-electrophoresis. The determination of whether or not to perform re-electrophoresis may be made automatically by the control unit 107 of the electrophoresis device 100, or may be made manually by the user of the electrophoresis device 100 via the input / output unit 108. When re-electrophoresis is started, the electrophoresis conditions may be changed.

[0104] 4, the sample solution subjected to electrochemical injection is transported back upstream from near the end of the capillary 202 by the operation of the negative pressure mechanism 114, and is retained in the upstream flow path. Because there is no need to remove the sample solution from the electrophoresis device 100, re-injection and re-electrophoresis can be performed with only an additional time of about 30 to 45 minutes, without re-passing the solution through the sample cartridge 102. This reduces the risk of contamination of the sample solution, and allows re-electrophoresis to be completed in a short time.

[0105] 5 is a flowchart showing an example of processing by the electrophoresis apparatus 100 according to the embodiment of the present invention, in which the negative pressure mechanism 114 is operated to reverse the sample solution that has been subjected to electric field injection, and the reversed sample solution that has been removed from the electrophoresis apparatus 100 is then re-injected and re-electrophoresed if the electrophoresis results are found to be unsatisfactory.

[0106] When a sample to be analyzed is analyzed by electrophoresis in the electrophoresis device 100, first, similar to the case shown in FIG. 4, a process of extracting nucleic acids (step S201), a process of amplifying the nucleic acids by PCR (step S202), a process of preparing a sample solution (step S203), electrophoresis (step S204), analysis of the electrophoresis results (step S205), and determination of the electrophoresis results (step S206) are performed.

[0107] If the result of the determination shows that no defect is due to factors other than the sample pretreatment (step S206; OK), the electrophoresis is terminated. On the other hand, if the result of the determination shows that a defect is due to factors other than the sample solution (step S206; NG), the process proceeds to step S207.

[0108] Next, the sample solution reversed by the negative pressure mechanism 114 is discharged to the outside of the electrophoresis apparatus 100 (step S207). The sample solution reversed by the negative pressure mechanism 114 can be discharged to the outside of the electrophoresis apparatus 100 via a pipe or the like from a flow path upstream of the chamber in which the end of the capillary 202 inside the capillary cartridge 101 is disposed.

[0109] Next, the sample solution discharged outside the electrophoresis apparatus 100 is re-introduced into the electrophoresis apparatus 100 (step S208). The sample solution discharged outside the electrophoresis apparatus 100 can be re-introduced into the sample cartridge 102 and transferred to the capillary cartridge 101 for re-injection and re-electrophoresis. The re-introduced sample solution does not need to be pre-treated, and therefore it is sufficient to simply pass it through the flow path inside the capillary cartridge 101.

[0110] Next, electrophoresis is performed using the recharged sample solution as a load sample (step S209). The recharged sample solution is introduced near the ends of the capillaries 202 inside the capillary cartridge 101. Then, sample components contained in the sample solution are injected into the capillaries 202 under electric field injection, and electrophoresis is performed. After the electric field injection of the sample components, the negative pressure mechanism 114 may be operated to remove the sample solution near the ends of the capillaries 202 that have been subjected to electric field injection to the outside of the electrophoresis apparatus 100.

[0111] Next, the results of the electrophoresis are analyzed (step S210). The sample components subjected to electrophoresis are separated into components with mobility according to their size, structure, etc. inside the capillary 202, and then optically detected in the detection unit 205. The results of the electrophoresis are obtained as a spectrum or the like showing the relationship between the migration time and the light intensity of the information light, based on the optically detected detection signal. The results of the electrophoresis are analyzed by base calling, size calling, identification of the attribution, etc. The analysis of the results of the electrophoresis may be performed after the electrophoresis has ended, or may be performed while the electrophoresis is being performed.

[0112] Next, the electrophoresis results are re-evaluated (step S211). In the re-evaluation of the electrophoresis results, it is determined again whether or not there is a defect in the electrophoresis results. The re-evaluation of the electrophoresis results is performed from the perspective of whether or not there is a defect in the electrophoresis results due to factors other than the pretreatment of the sample. The re-evaluation of the electrophoresis results may be performed after the electrophoresis has finished, or may be performed while the electrophoresis is being performed.

[0113] Thereafter, the electrophoresis is terminated. If the result of the determination indicates a defect in the electrophoresis results, it is assumed that the defect is due to factors other than the sample pretreatment or that there is a permanent defect in the electrophoresis device 100. In this case, it is preferable to perform analysis using another electrophoresis device, change the sample pretreatment method or conditions, or purify or recollect the sample.

[0114] The re-evaluation of the electrophoresis results may be performed automatically by the control unit 107 of the electrophoresis device 100, or may be performed manually by the user of the electrophoresis device 100 via the input / output unit 108. The user of the electrophoresis device 100 can check the electrophoresis results output to the input / output unit 108 and determine whether or not there is a defect in the electrophoresis results.

[0115] Furthermore, if the electrophoresis results are found to be unsatisfactory (step S211), it can be determined whether or not to perform re-electrophoresis. If re-electrophoresis is performed, the sample solution removed from the electrophoresis apparatus 100 can be re-injected and re-electrophoresed. The determination of whether or not to perform re-electrophoresis may be made automatically by the control unit 107 of the electrophoresis apparatus 100, or may be made manually by the user of the electrophoresis apparatus 100 via the input / output unit 108. When re-electrophoresis is started, the electrophoresis conditions may be changed.

[0116] 5, the sample solution subjected to electroinjection is reversed from near the end of the capillary 202 toward the upstream side by the operation of the negative pressure mechanism 114, and then removed to the outside of the electrophoresis device 100. The sample solution that has already been pretreated can be re-injected into the sample cartridge 102 for re-injection or re-electrophoresis. Since there is no need to retain the reversed sample solution in the upstream flow path, the flexibility of the flow path design is increased. Furthermore, there is no need for additional cleaning of the flow path or additional switching of the liquid flow direction. Therefore, re-electrophoresis can be performed in a short time without complicating the design of the electrophoresis device 100.

[0117] 6 is a diagram showing an example of the configuration of the main parts of an electrophoresis apparatus according to an embodiment of the present invention. Fig. 6 schematically shows an example of the cross-sectional structure and flow channel structure of the capillary cartridge 101 of the electrophoresis apparatus 100 according to this embodiment. As shown in Fig. 6, the negative pressure mechanism 114 can be connected to a branch flow channel 701c branched from the liquid supply flow channels 701a and 701b connecting the sample cartridge 102 and the cathode block 212.

[0118] 6, a liquid delivery unit 112 is connected to the pretreatment reagent container 223 via a flow path through which a liquid can flow. A sample cartridge 102 is connected to the other end of the liquid delivery unit 112 via a flow path through which a liquid can flow. A flow path switching unit 703a is connected to the other end of the sample cartridge 102 via a liquid delivery flow path 701a through which a liquid for delivering a sample solution for electrophoresis can flow.

[0119] One port of the flow path switching unit 703a is connected to the cathode block 212 via a liquid feed flow path 701b through which a liquid for feeding a sample solution for electrophoresis can flow. The other port of the flow path switching unit 703a is connected to a negative pressure mechanism 114 via a branch flow path 701c branched from the liquid feed flow paths 701a and 701b. When the negative pressure mechanism 114 is installed at the end of the branch flow path 701c, it is preferable to use a vacuum pump, a bidirectional pump, or the like.

[0120] The liquid supply flow path 701b is connected to the middle of a chamber 701d inside the cathode block 212. The injection end 207 of the capillary 202 is disposed in the chamber 701d. A migration reagent supply pump 213 is connected to one end of the chamber 701d via a flow path through which a liquid can flow. A migration reagent container 214 is connected to the migration reagent supply pump 213 via a flow path through which a liquid can flow. A cathode waste liquid container 215 is connected to the other end of the chamber 701d via a discharge flow path 701e through which a liquid can flow.

[0121] A gas intake flow path in which a check valve 705a is installed is formed at the other end of the chamber 701d. The gas intake flow path is a flow path through which gas can flow, and connects the interior of the chamber 701d with the outside of the cathode block 212. A gas exhaust flow path in which a check valve 705b is installed is formed in the cathode waste liquid container 215. The gas exhaust flow path is a flow path through which gas can flow, and connects the outside and inside of the cathode waste liquid container 215.

[0122] The flow path switching unit 703a switches the flow path between the liquid sending flow paths 701a and 701b that connect the sample cartridge 102 and the cathode block 212, and a branch flow path 701c that branches off from these flow paths and connects to the negative pressure mechanism 114. The flow path switching unit 703a is configured by, for example, a three-way valve or the like.

[0123] When sending sample solution from sample cartridge 102 to cathode block 212, flow path switching unit 703a opens flow paths 701a and 701b connecting sample cartridge 102 and cathode block 212, and closes branch flow path 701c connected to negative pressure mechanism 114. On the other hand, when negative pressure mechanism 114 is operating, flow path switching unit 703a opens branch flow path 701c connected to negative pressure mechanism 114 and flow path 701b connected to cathode block 212, and closes flow path 701a connected to sample cartridge 102. As a result, no pressure change occurs in flow path 701a, sample cartridge 102, or flow path sending unit 112, even when negative pressure mechanism 114 is operating.

[0124] In the following description, the direction of liquid flow from the liquid flow channel 701a to the liquid flow channel 701b formed by the flow channel switching unit 703a is referred to as the ab direction, and the direction of liquid flow from the liquid flow channel 701b to the branch channel 701c is referred to as the bc direction.

[0125] The check valve 705a is open when gas flows from the outside to the inside of the cathode block 212, and is closed when gas flows from the inside to the outside of the cathode block 212. The check valve 705a allows air to be taken in from the outside of the cathode block 212 through the gas intake flow path into the internal chamber 701d when the liquid supply flow path 701b etc. is negatively pressurized by the negative pressure mechanism 114. The air intake suppresses excessive pressure reduction caused by operation of the negative pressure mechanism 114, preventing backflow of liquid, generation of air bubbles, etc.

[0126] The check valve 705b is in an open state when gas flows from the inside to the outside of the cathode waste liquid container 215, and is in a closed state when gas flows from the outside to the inside of the cathode waste liquid container 215. The check valve 705b allows air to be discharged from the inside to the outside of the cathode waste liquid container 215 through the gas discharge flow path when liquid is sent from the liquid sending unit 112 to the cathode block 212.

[0127] In the capillary cartridge 101 having the structure shown in FIG. 6, the analysis by pretreatment-integrated electrophoresis is carried out in the following steps (1) to (8).

[0128] The control unit 107 controls the negative pressure mechanism 114, the liquid delivery unit 112, the flow path switching unit 703a, the electrophoresis reagent delivery pump 213, the sample cartridge 102, the power supply 103, the optical system 104, the injection pump 105, etc., to execute the process of electrophoresis analysis in the order of (1) to (8). The control unit 107 controls the electric field injection of the sample components into the capillary 202, the reverse transfer of the sample solution by the operation of the negative pressure mechanism 114, the electrophoresis of the sample components in the capillary 202, and the reintroduction of the reversed sample solution into the chamber 701d. The control unit 107 controls the reintroduction of the reversed sample solution into the chamber 701d based on the status or result of the electrophoresis in the capillary 202.

[0129] (1) Pretreatment First, the sample is pretreated using the sample cartridge 102. The pretreatment includes a process for extracting nucleic acids from the sample, a process for amplifying the nucleic acids extracted from the sample, and a process for mixing the amplified products with reagents, etc. A sample solution for electrophoresis is prepared by the sample pretreatment.

[0130] (2) Delivery of sample solution: Next, the sample solution for electrophoresis prepared by pretreatment is delivered. To deliver the sample solution, the flow path switching unit 703a is switched to the ab direction. The sample solution for electrophoresis is prepared in a processing tank inside the sample cartridge 102 where the sample solution for electrophoresis is prepared. By operating the liquid delivery unit 112, the sample solution for electrophoresis is delivered from the processing tank where the sample solution for electrophoresis is prepared through the liquid delivery paths 701a and 701b to the chamber 701d inside the cathode block 212.

[0131] When feeding the sample solution, the pump is operated while alternately feeding air and sample solution, creating a state in which segmented air is sandwiched between the front and rear of the plug-shaped liquid mass of sample solution. By feeding the liquid with segmented air sandwiched between the plug-shaped liquid mass of sample solution, mixing of the liquid mass of sample solution with other reagent solutions can be prevented.

[0132] The feeding of the sample solution is stopped when the sample solution to be analyzed for electrophoresis reaches the vicinity of the injection end 207 of the capillary 202 inside the chamber 701d. For example, the feeding is performed so that a liquid mass of the sample solution reaches the vicinity of the injection end 207 of the capillary 202. Arrival of the sample solution near the injection end 207 of the capillary 202 can be confirmed by installing an electrode near the injection end 207 and detecting an increase in the current value.

[0133] (3) Electric field injection of sample components Next, electric field injection of the sample components contained in the sample solution is performed into the capillary 202. In the electric field injection of the sample components, a high voltage is applied across the capillary 202 while the sample solution is introduced near the injection end 207 of the capillary 202 inside the chamber 701d. The application of the high voltage injects some of the sample components contained in the sample solution near the injection end 207 into the capillary 202.

[0134] (4) Reverse Transport of Sample Solution Subsequently, the sample solution subjected to electrochemical injection is transported in the reverse direction. In the reverse transport of the sample solution, the negative pressure mechanism 114 is operated to transport the sample solution remaining in the vicinity of the injection end 207 of the capillary 202 inside the chamber 701d in the reverse direction to the flow path upstream of the chamber 701d. When the liquid supply flow path 701b and the branch flow path 701c are made negative pressure relative to the chamber 701d, the sample solution inside the chamber 701d is transported in the reverse direction toward the liquid supply flow path 701b and the branch flow path 701c. At this time, the upstream of the liquid supply flow path 701a is not made negative pressure.

[0135] When the negative pressure mechanism 114 is activated, the flow path switching unit 703a is switched to a state in which the flow path 701a, which is one of the liquid supply paths 701a and 701b connecting the sample cartridge 102 and the cathode block 212 and is upstream of the flow path switching unit 703a, is closed. By closing the liquid supply path 701a, the negative pressure mechanism 114 can create a negative pressure in the liquid supply path 701b and the branch path 701c without changing the pressure in the sample cartridge 102, the liquid supply unit 112, the pretreatment reagent container 223, or the paths connecting these.

[0136] (5) Feeding of migration reagent Next, the migration reagent used for electrophoresis is fed. In feeding the migration reagent, the migration buffer solution prepared in the migration reagent container 214 is introduced into the chamber 701d and the like inside the cathode block 212 by operating the migration reagent feeding pump 213. The used migration reagent and excess migration reagent remaining in the chamber 701d are discharged from the chamber 701d and collected in the cathode waste liquid container 215.

[0137] (6) Electrophoresis Subsequently, the sample components injected under an electric field are electrophoresed inside the capillary 202. In electrophoresis, a high voltage is applied across both ends of the capillary 202 while the sample components are being injected under an electric field into the capillary 202. The application of the high voltage causes the sample components injected into the capillary 202 to electrophorese inside the capillary 202. The sample components that have migrated inside the capillary 202 are optically detected by a detection unit 205 installed on the anode side at intervals of a migration time according to their mobility. During electrophoresis, the delivery of the migration reagent may be stopped or may be continuously performed.

[0138] (7) Determination of the Electrophoresis Results Next, the electrophoresis results are determined. Based on the detection results in the detection unit 205, the electrophoresis results are obtained as a spectrum or the like showing the relationship between the migration time and the light intensity of the information light. In determining the electrophoresis results, it is determined whether or not the electrophoresis results are defective. The determination of the electrophoresis results may be performed after the electrophoresis has finished or while the electrophoresis is being performed. If it is determined that the electrophoresis results are defective, the electrophoresis may be interrupted.

[0139] (8-1) Re-electrophoresis after Judgment If the judgment of the electrophoresis result reveals a defect, re-electrophoresis is performed using the sample solution subjected to electric field injection. In the re-electrophoresis after judgment, the flow path switching unit 703a is switched to the ab direction, and the sample solution that has been reversely sent to and held in the liquid sending flow path 701b and branch flow path 701c by the operation of the liquid sending unit 112 is re-introduced into the chamber 701d inside the cathode block 212. Then, the operations following the electric field injection are repeated to perform re-electrophoresis.

[0140] (8-2) Cleaning after Determination If no defects are found in the electrophoresis results upon evaluation, the chamber 701d, the liquid sending flow path 701b, the branch flow path 701c, and the like inside the cathode block 212 are cleaned. In cleaning after determination, the flow path switching unit 703a is switched to the ab direction, and the liquid sending unit 112 is operated to send the flow path cleaning solution prepared in the pretreatment reagent container 223 to the chamber 701d, the liquid sending flow path 701b, the branch flow path 701c, and the like. By passing the flow path cleaning solution, the liquid inside the flow path is replaced and the inner walls are cleaned, and the remaining sample solution is discharged into the cathode waste liquid container 215. After cleaning, the series of runs is terminated.

[0141] 6, the sample solution subjected to electrophoresis can be reversed and retained in the liquid supply channel 701b or branch channel 701c upstream of chamber 701d, allowing the same sample solution as that subjected to electrophoresis to be re-used. The range in which the sample solution is reversed by the negative pressure mechanism 114 is limited to the channel between the sample cartridge 102 and the cathode block 212. This prevents sample solution or air containing sample molecules from flowing upstream of the sample cartridge, thereby preventing contamination in the next run.

[0142] Furthermore, with the structure shown in FIG. 6 , the entire amount of pretreated sample solution can be used for the first electrophoresis run without having to take an aliquot of the pretreated sample solution for storage. Therefore, second and subsequent re-electrophoresis runs can be performed without reducing the sensitivity of the first electrophoresis run. The sample components injected into the capillary 202 by the electric field are a portion of the sample components contained in the sample solution introduced near the end of the capillary 202. A sufficient amount of sample components remains in the sample solution that has been reversed upstream of chamber 701d. Therefore, even in second and subsequent re-electrophoresis runs, electrophoretic analysis can be repeated with minimal reduction in sensitivity.

[0143] Furthermore, what is electric-field-injected into the capillary 202 is a charged sample component contained in the sample solution introduced near the end of the capillary 202. Because the sample solution introduced near the end of the capillary 202 is not injected into the capillary 202, the amount of liquid sent back upstream of chamber 701d is hardly reduced compared to when the sample solution was introduced near the end of the capillary 202. Therefore, even in the second and subsequent re-electrophoresis runs, an equal amount of sample solution can be introduced near the end of the capillary 202 and the analysis by electrophoresis can be repeated.

[0144] In Figure 6, the liquid delivery paths 701b and 701c, the path switching unit 703a, and the negative pressure mechanism 114 are installed in the path between the sample cartridge 102 and the cathode block 212, but these components may be incorporated into the capillary cartridge 101 or into the main body 111.

[0145] The check valve 705b may be omitted as long as it can restore the negative pressure in the flow path. For example, if the cathode waste liquid container 215 is flexible, such as a plastic bag, the check valve 705b may not be installed. In such a case, when the migration reagent and the air inside are discharged into the cathode waste liquid container 215, the cathode waste liquid container 215 can expand, thereby allowing the air inside the flow path to be retained inside the cathode waste liquid container 215.

[0146] A check valve may be installed in the flow path between the cathode block 212 and the cathode waste liquid container 215 to prevent liquid from flowing from the cathode waste liquid container 215 to the cathode block 212. Installing such a check valve can prevent contamination due to backflow from the cathode waste liquid container 215 to the cathode block 212.

[0147] Fig. 7 shows the results of measuring the change in detection sensitivity with the number of repeated field injections. Fig. 7 shows the results of measuring the light intensity of the information light detected from the sample components after electrophoresis, after repeating the field injection and electrophoresis of the sample components a predetermined number of times. In Fig. 7, the horizontal axis represents the number of field injections, and the vertical axis represents the measurement results of the light intensity of the information light, which are the peak areas of the detection signals for each sample component.

[0148] The sample components were DNA fragments of STR loci derived from human genomic DNA. The DNA fragments were amplified using a PCR kit from the PowerPlex Fusion 6C System (Promega). Multiplex PCR was performed using 2800 M (10 ng / μL) of control DNA (Promega) as a template to amplify DNA fragments encoding each locus. PCR was performed for 29 cycles.

[0149] After PCR amplification, each DNA fragment was mixed with a size standard and formamide at a concentration ratio of 0.5:9.5:1.0 to prepare a sample solution for electrophoresis. WENIL S500 (Promega) was used as the size standard, and Hi-Di Formamide (Thermo Fisher Scientific) was used as the formamide.

[0150] 11 μL of the prepared sample solution for electrophoresis was introduced near the end of the capillary, and the DNA fragments contained in the sample solution were electrophoresed into the capillary. A capillary with a total length of 61 cm was used. The electrophoresis was performed by applying a voltage of 1.6 kV across the capillary for 15 seconds. After the electrophoresis, the DNA fragments injected into the capillary were electrophoresed within the capillary. Electrophoresis was performed by applying a voltage of 19.5 kV across the capillary.

[0151] After each DNA fragment was electrophoresed, the fluorescence emitted from the fluorescently labeled DNA fragment was detected, and the peak area of ​​the detection signal of the light intensity of the information light was quantified. Furthermore, the sample solution introduced near the end of the capillary was reversed upstream, and then the reversed sample solution was reintroduced near the end of the capillary, and re-injection and re-electrophoresis were repeated. This cycle was repeated a total of eight times, and the peak area of ​​the detection signal of the light intensity of the information light for each DNA fragment was quantified for each repetition.

[0152] Table 1 shows the details of the results of measuring the change in detection sensitivity with the number of repeated field injections. Table 1 also shows the peak area and CV (Coefficient of Variation) values ​​of the detection signals for measurement results other than the representative loci shown in Figure 7. The number of STR repeats is the number of repeats of the 2800M STR used as the template.

[0153]

[0154] As shown in Figure 7 and Table 1, no clear decrease in the peak area of ​​the detection signal was observed for each DNA fragment, even when electric field injection and electrophoresis were repeated. It can be seen that the effect of the decrease in sample components due to repeated electrophoresis is smaller than the variation in the amount injected into the capillary. Furthermore, the CV values ​​for each DNA fragment were 3-5%, indicating that the decrease in sample components due to repeated electrophoresis was less than 3%. Therefore, it can be said that the second and subsequent electrophoresis runs achieved a sensitivity of 97% or more compared to the first electrophoresis run.

[0155] 8 is a diagram showing an example of the configuration of the main parts of an electrophoresis device according to an embodiment of the present invention. Fig. 8 schematically shows an example of the cross-sectional structure and flow channel structure of the capillary cartridge 101 of the electrophoresis device according to this embodiment. As shown in Fig. 8, a flow channel switching unit 703b that can switch the flow channel connecting the cathode block 212 and the cathode waste liquid container 215 to an external flow channel may be provided.

[0156] 8, a flow path switching unit 703b is connected to the other end of chamber 701d via a discharge flow path 701e through which a liquid can flow. One port of flow path switching unit 703b is connected to cathode waste liquid container 215 via a discharge flow path 701f through which a liquid can flow. The other port of flow path switching unit 703b is connected to a gas discharge flow path branched from the discharge flow paths 701e and 701f. A check valve 705c is installed in the gas discharge flow path.

[0157] The flow path switching unit 703b switches the flow path between the exhaust flow paths 701e and 701f that connect the cathode block 212 and the cathode waste liquid container 215 and the gas intake flow path that branches off from the exhaust flow paths 701e and 701f and communicates with the outside. The flow path switching unit 703b is configured by, for example, a three-way valve or the like.

[0158] The flow path switching unit 703b opens the discharge flow paths 701e and 701f connecting the cathode block 212 and the cathode waste liquid container 215 and closes the gas intake flow path communicating with the outside when sending the sample solution from the sample cartridge 102 to the cathode block 212 or when cleaning the flow path inside the cathode block 212. On the other hand, when the negative pressure mechanism 114 is activated, it opens the gas intake flow path communicating with the outside and the discharge flow path 701e connected to the cathode block 212 and closes the discharge flow path 701f connected to the cathode waste liquid container 215.

[0159] The check valve 705c opens when gas flows from the outside to the inside of the gas intake channel, and closes when gas flows from the inside to the outside of the gas intake channel. The check valve 705c allows air to be taken in through the gas intake channel from the outside of the cathode block 212 to the internal chamber 701d when the liquid supply channel 701b, etc. is negatively pressurized by the negative pressure mechanism 114. The air intake suppresses excessive pressure reduction caused by the operation of the negative pressure mechanism 114, preventing backflow of liquid, generation of air bubbles, etc.

[0160] 8, the gas intake flow path for taking in air is connected to the exhaust flow paths 701e and 701f that connect the cathode block 212 and the cathode waste liquid container 215, so there is no need to form a gas intake flow path for taking in air in the cathode block 212. This prevents the sample solution for electrophoresis from entering the gas intake flow path, which could cause contamination between the sample solutions. Furthermore, backflow of waste liquid from the cathode waste liquid container 215 can be prevented when the negative pressure mechanism 114 is activated.

[0161] 9 is a diagram showing an example of the configuration of the main parts of an electrophoresis apparatus according to an embodiment of the present invention. Fig. 9 schematically shows an example of the cross-sectional structure and flow channel structure of a capillary cartridge 101 of the electrophoresis apparatus according to this embodiment. As shown in Fig. 9, a discharge flow channel 901 communicating with the outside of the electrophoresis apparatus 100 can also be connected to the negative pressure mechanism 114.

[0162] 9 , one port of the flow path switching unit 703a is connected to the cathode block 212 via a liquid feed flow path 701b through which a liquid for feeding a sample solution for electrophoresis can flow. One end of the negative pressure mechanism 114 is connected to the other port of the flow path switching unit 703a via a branch flow path 701c branching from the liquid feed flow paths 701a and 701b. One end of a discharge flow path 901 is connected to the other end of the negative pressure mechanism 114. The other end of the discharge flow path 901 is connected to the outside of the electrophoresis apparatus 100.

[0163] The discharge flow path 901 is a flow path that discharges fluid from the negative pressure mechanism 114 to the outside of the electrophoresis device 100. The discharge flow path 901 is used to discharge the sample solution and air that are sucked in when the negative pressure mechanism 114 is operating to the outside of the electrophoresis device 100. A user of the electrophoresis device 100 can collect the sample solution that is sucked in by the negative pressure mechanism 114 through the discharge flow path 901. The collected sample solution can be introduced into the electrophoresis device 100 or another device for reanalysis. Because the collected sample solution has been pretreated, pretreatment can be omitted when reanalyzing.

[0164] 9, the discharge flow path 901 is connected to the negative pressure mechanism 114, but the discharge flow path 901 may also be connected to a liquid supply flow path 701b that connects the flow path switching unit 703a and the cathode block 212, or to a discharge flow path 701e that connects the cathode block 212 and the cathode waste liquid container 215. When the discharge flow path 901 is connected to these locations, it is preferable to install an on-off valve in the discharge flow path 901.

[0165] A sample solution inlet may be provided upstream of the cathode block 212 for re-injecting the sample solution recovered through the discharge flow path 901. The sample solution inlet may be provided in the sample cartridge 102, the liquid feed flow path 701a connecting the sample cartridge 102 and the flow path switching unit 703a, or the liquid feed flow path 701b connecting the flow path switching unit 703a and the cathode block 212.

[0166] Fig. 10 is a diagram showing an example of the configuration of the main parts of an electrophoresis apparatus according to an embodiment of the present invention. Fig. 10 schematically shows an example of the cross-sectional structure and flow channel structure of the capillary cartridge 101 of the electrophoresis apparatus according to this embodiment. As shown in Fig. 10, the negative pressure mechanism 114 can also be provided inline so as to form at least a part of the flow channel connecting the sample cartridge 102 and the cathode block 212.

[0167] 10 , a liquid delivery unit 112 is connected to the pretreatment reagent container 223 via a flow path through which a liquid can flow. A sample cartridge 102 is connected to the other end of the liquid delivery unit 112 via a flow path through which a liquid can flow. A flow path switching unit 703a is connected to the other end of the sample cartridge 102 via a liquid delivery flow path 701a through which a liquid for delivering a sample solution for electrophoresis can flow.

[0168] One port of the flow path switching unit 703a is connected to the cathode block 212 via a liquid delivery flow path 701b through which a liquid for delivering a sample solution for electrophoresis can flow. The other port of the flow path switching unit 703a is connected to a branch flow path 701c branched from the liquid delivery flow paths 701a and 701b. An inline negative pressure mechanism 114 is installed on the branch flow path 701c.

[0169] The inline negative pressure mechanism 114 creates a negative pressure relative to the internal pressure of the chamber 701d in at least a portion of the flow path connected to the chamber 701d in which the end of the capillary 202 is located. The inline negative pressure mechanism 114 creates a negative pressure in at least a portion of the flow path through which the sample solution for electrophoresis flows, the portion being downstream of the sample cartridge 102 and upstream of the chamber 701d inside the cathode block 212 of the capillary cartridge 101.

[0170] The inline negative pressure mechanism 114 is installed on a flow path formed of a flexible material with a variable flow path cross-sectional area. In Fig. 10, at least a portion of a branch flow path 701c branching from liquid supply flow paths 701a and 701b connecting the sample cartridge 102 and the cathode block 212 is formed of a flexible material. The inline negative pressure mechanism 114 creates a negative pressure by adjusting the flow path cross-sectional area of ​​the flow path formed of a flexible material.

[0171] In Figure 10, the liquid supply flow path 701a, the liquid supply flow path 701b, the branch flow path 701c, and the inline negative pressure mechanism 114 are installed in the flow path between the sample cartridge 102 and the cathode block 212, but these components may be incorporated into the capillary cartridge 101 or into the main body 111.

[0172] A flow path switching unit that can switch to a discharge flow path 901 that communicates with the outside of the electrophoresis apparatus 100 can also be provided in the liquid supply flow path 701a between the sample cartridge 102 and the flow path switching unit 703a and the liquid supply flow path 701b between the flow path switching unit 703a and the cathode block 212. A user of the electrophoresis apparatus 100 can also collect the sample solution sucked by the inline negative pressure mechanism 114 through a discharge flow path connected to the other end of the inline negative pressure mechanism 114.

[0173] 11A and 11B are diagrams illustrating the operation of an example of an inline negative pressure mechanism. Figures 11A and 11B show a cross-sectional structure of the inline negative pressure mechanism 114, corresponding to the cross section taken along line II in Figure 10. Figure 11A shows a state in which the inline negative pressure mechanism 114 is not operating and is sending the sample solution for electrophoresis from the sample cartridge 102 toward the cathode block 212. Figure 11B shows a state in which the inline negative pressure mechanism 114 is operating and is sending the sample solution for electrophoresis backward toward the upstream side of the cathode block 212.

[0174] The inline negative pressure mechanism 114 includes a substrate 1101, a membrane 1102 made of elastomer, and a movable member 1103 that can press the membrane 1102. A linear groove 1104 is formed in the substrate 1101. The membrane 1102 is pressure-bonded so as to cover the groove 1104. The membrane 1102 covers the groove 1104, thereby forming liquid delivery channels 701a and 701b for delivering a sample solution for electrophoresis. The movable member 1103 is provided so as to be able to move forward and backward within the groove 1104 covered with the membrane 1102.

[0175] 11A , when the inline negative pressure mechanism 114 is not operating, the movable member 1103 is in an initial state in which it presses a predetermined section that forms the groove 1104 of the membrane 1102. In the initial state, the membrane 1102 is elastically deformed by the pressure, and the cross-sectional area of ​​the groove 1104 is reduced.

[0176] 11B , when the inline negative pressure mechanism 114 is activated, the movable member 1103 is driven by an actuator or the like in a direction away from the membrane 1102, thereby releasing the pressure on the membrane 1102 and setting the movable member 1103 in an activated state. In the activated state, the membrane 1102 is released from the pressure and elastically restores its original shape, thereby expanding the cross-sectional area of ​​the groove 1104. Therefore, the internal pressure of the liquid delivery channel 701b and the branch channel 701c is made negative relative to the internal pressure of the chamber 701d in which the end of the capillary 202 is disposed.

[0177] 11A and 11B, a portion of the branch flow path 701c is formed by the substrate 1101 and the membrane 1102, but a portion of the branch flow path 701c may be formed only by a flexible material whose cross-sectional area is variable. The inline negative pressure mechanism 114 is configured to switch between pressing and releasing the membrane 1102, but may also be configured to switch between pressing and releasing a pipe formed of a flexible material.

[0178] For example, some or all of the branch flow path 701c can be formed using flexible tubing. The inline negative pressure mechanism 114 can also be configured to switch between compressing and releasing the tubing. A pair of compressing members can be connected via an elastic member such as a compression coil spring, and the compressing members can be pressed by an actuator or the like to sandwich the tubing, thereby utilizing the contraction and restoration of the elastic member to adjust the cross-sectional area of ​​the flow path.

[0179] Fig. 12 is a diagram showing an example of the configuration of the main parts of an electrophoresis apparatus according to an embodiment of the present invention. Fig. 12 schematically shows an example of the cross-sectional structure and flow channel structure of a capillary cartridge 101 of an electrophoresis apparatus according to this embodiment. As shown in Fig. 12, the negative pressure mechanism 114 can also be built into the sample cartridge 102.

[0180] 12, the liquid delivery unit 112 is connected to the pretreatment reagent container 223 via a flow path through which a liquid can flow. The sample cartridge 102 is connected to the other end of the liquid delivery unit 112 via a flow path through which a liquid can flow. The cathode block 212 is connected to the other end of the sample cartridge 102 via a liquid delivery flow path 701a through which a liquid for delivering a sample solution for electrophoresis can flow.

[0181] The negative pressure mechanism 114 is connected to a liquid supply flow path that supplies the sample solution for electrophoresis inside the sample cartridge 102. The negative pressure mechanism 114 built into the sample cartridge 102 creates a negative pressure in at least a part of the section between a processing tank inside the sample cartridge 102 where the sample solution for electrophoresis is prepared and a chamber 701d inside the capillary cartridge 101 where the end of the capillary 202 is placed.

[0182] Fig. 13 is a diagram showing an example of the configuration of a sample cartridge incorporating a negative pressure mechanism. Fig. 13 shows a sample cartridge 102 equipped with a nucleic acid extraction tank 1301 for extracting nucleic acids from a sample, a PCR tank 1302 for performing PCR, and a mixing tank 1303 for mixing reagents. The mixing tank 1303 corresponds to a processing tank in which a sample solution for electrophoresis is prepared. As shown in Fig. 13, the negative pressure mechanism 114 can be incorporated inside the sample cartridge 102, downstream of the processing tank in which the sample solution for electrophoresis is prepared.

[0183] 13 , a PCR chamber 1302 is connected to a nucleic acid extraction chamber 1301 via a flow path through which a liquid can flow. A mixing chamber 1303 is connected to the PCR chamber 1302 via a flow path through which a liquid can flow. A flow path switching unit 703c is connected to the mixing chamber 1303 via a liquid sending flow path 701s for sending a sample solution for electrophoresis toward the capillary cartridge 101.

[0184] The capillary cartridge 101 is connected to one port of the flow path switching unit 703c via a liquid delivery flow path 701a through which a liquid for delivering a sample solution for electrophoresis can flow. The other port of the flow path switching unit 703c is connected to a negative pressure mechanism 114 via a branch flow path 701t branching from the liquid delivery flow paths 701s and 701a. When the negative pressure mechanism 114 is installed at the end of the branch flow path 701t, it is preferable to use a syringe pump or the like as the negative pressure mechanism 114.

[0185] When the sample solution is sent from the mixing chamber 1303 to the cathode block 212, the flow path switching unit 703c opens the flow paths 701s and 701a connecting the mixing chamber 1303 and the cathode block 212, and closes the branch flow path 701t connected to the negative pressure mechanism 114. On the other hand, when the negative pressure mechanism 114 is operating, the flow path switching unit 703c opens the branch flow path 701t connected to the negative pressure mechanism 114 and the flow path 701a connected to the cathode block 212, and closes the flow path 701s connected to the mixing chamber 1303.

[0186] The sample introduced into the sample cartridge 102 is introduced into the nucleic acid extraction chamber 1301, PCR chamber 1302, and mixing chamber 1303 in this order for pretreatment. The sample solution for electrophoresis prepared in the mixing chamber 1303 is sent to the capillary cartridge 101 through the liquid sending flow paths 701s, 701a, etc., and introduced near the end of the capillary 202 inside the chamber 701d, where it is subjected to electric field injection into the capillary 202.

[0187] The negative pressure mechanism 114 built into the sample cartridge 102 applies negative pressure to at least a portion of the liquid feed channel 701a after electrophoresis of sample components contained in the sample solution into the capillaries 202 and before electrophoresis of the sample components within the capillaries 202. The sample solution subjected to electrophoresis and remaining near the end of the capillaries 202 is reversed upstream of the chamber 701d. At this time, the pressure upstream of the channel switching unit 703c remains unchanged, and no reverse flow occurs within the liquid feed channel 701s, the nucleic acid extraction chamber 1301, the PCR chamber 1302, the mixing chamber 1303, and the channels connecting these, the liquid feed unit 112, the pretreatment reagent container 223, and the channels connecting these. The reversed sample solution can be retained in the liquid feed channel 701a.

[0188] The negative pressure mechanism 114 built into the sample cartridge 102 allows the negative pressure mechanism 114 and the piping connected to the negative pressure mechanism 114 to be replaced when the sample cartridge 102 is replaced. This reduces contamination caused by components remaining on the negative pressure mechanism 114 and the piping.

[0189] 13, the sample cartridge 102 includes a nucleic acid extraction chamber 1301, a PCR chamber 1302, and a mixing chamber 1303, but the sample cartridge 102 may include one or more of these. The sample cartridge 102 may also include a processing chamber for performing other processing, such as a purification chamber for performing purification.

[0190] 13, the negative pressure mechanism 114 is connected to a branch flow path 701t branching off downstream of the mixing chamber 1303, but it may also be connected to a branch flow path branching off upstream of the mixing chamber 1303 and downstream of the PCR chamber 1302. When the mixing chamber 1303 is not provided, it is preferable to connect the negative pressure mechanism 114 to a branch flow path branching off downstream of the PCR chamber 1302.

[0191] 14A and 14B are block diagrams showing the connection of channels between components in a pretreatment-integrated electrophoresis device. Fig. 14A shows the connection of channels between components in an electrophoresis device 100 according to an embodiment of the present invention, in which a negative pressure is directly applied to the solution delivery channel between the sample cartridge 102 and the cathode block 212. Fig. 14B shows the connection of channels between components in an electrophoresis device 100 according to an embodiment of the present invention, in which a negative pressure is directly applied to the inside of the cathode block 212.

[0192] 14A and 14B, the negative pressure mechanism 114 can also be installed in a circulation flow path that bypasses a portion of the liquid feed flow path between the sample cartridge 102 and the cathode block 212. The circulation flow path connects the intermediate portion of the liquid feed flow path connecting the sample cartridge 102 and the cathode block 212 to the cathode block 212, bypassing the downstream section of the liquid feed flow path. The direction of circulation in the circulation flow path can be either direction.

[0193] 14A, the negative pressure mechanism 114 installed in the circulation flow path can be configured to directly apply negative pressure to the liquid feed flow path between the sample cartridge 102 and the cathode block 212, thereby sending the sample solution introduced into the chamber inside the cathode block 212 back into the circulation flow path. When re-electrophoresis is required, the sample solution sent back into the circulation flow path can bypass the downstream section of the liquid feed flow path and be directly introduced into the cathode block 212 through the circulation flow path.

[0194] 14B, the negative pressure mechanism 114 installed in the circulation flow path can also be configured to directly create a negative pressure in the chamber inside the cathode block 212, and to send the sample solution introduced into the chamber inside the cathode block 212 back to the circulation flow path, bypassing the downstream section of the liquid feed flow path. When re-electrophoresis is required, the sample solution sent back to the circulation flow path can be introduced into the liquid feed flow path between the sample cartridge 102 and the cathode block 212 through the circulation flow path. In this case, operation of the negative pressure mechanism 114 does not cause any pressure change in the sample cartridge 102 or the liquid feed unit 112.

[0195] The circulation flow path in which the negative pressure mechanism 114 is installed may be connected between the intermediate part of the liquid supply flow path connecting the sample cartridge 102 and the cathode block 212 and the cathode block 212, or may be connected between the intermediate part of the liquid supply flow path connecting the sample cartridge 102 and the cathode block 212 and the discharge flow path connecting the cathode block 212 and the cathode waste liquid container 215.

[0196] 15 is a diagram showing an example of the configuration of the main parts of an electrophoresis apparatus according to an embodiment of the present invention. Fig. 15 schematically shows an example of the cross-sectional structure and flow channel structure of the capillary cartridge 101 of the electrophoresis apparatus according to this embodiment. As shown in Fig. 15, the negative pressure mechanism 114 can be installed in the circulation channel by connecting it to bypass channels 701g and 701h outside the cathode block 212.

[0197] 15 , a liquid delivery unit 112 is connected to the pretreatment reagent container 223 via a flow path through which a liquid can flow. A sample cartridge 102 is connected to the other end of the liquid delivery unit 112 via a flow path through which a liquid can flow. A flow path switching unit 703c is connected to the other end of the sample cartridge 102 via a liquid delivery flow path 701a through which a liquid for delivering a sample solution for electrophoresis can flow.

[0198] One port of the flow path switching unit 703c is connected to the cathode block 212 via a liquid sending flow path 701b through which a liquid for sending a sample solution for electrophoresis can flow, and one end of the negative pressure mechanism 114 is connected to the other port of the flow path switching unit 703c via a bypass flow path 701h branching from the liquid sending flow paths 701a and 701b.

[0199] The liquid supply flow path 701b is connected to the middle of a chamber 701d inside the cathode block 212. The injection end 207 of the capillary 202 is disposed in the chamber 701d. A migration reagent supply pump 213 is connected to one end of the chamber 701d via a flow path through which a liquid can flow. A migration reagent container 214 is connected to the migration reagent supply pump 213 via a flow path through which a liquid can flow. A flow path switching unit 703d is connected to the other end of the chamber 701d via a discharge flow path 701e through which a liquid can flow.

[0200] One port of the flow path switching unit 703d is connected to the cathode waste liquid container 215 via a discharge flow path 701f through which a liquid can flow, and the other end of the negative pressure mechanism 114 is connected to the other port of the flow path switching unit 703d via a bypass flow path 701g branching off from the discharge flow paths 701e and 701f.

[0201] The bypass flow paths 701g and 701h are located outside the cathode block 212 and are connected between the intermediate portions of the liquid supply flow paths 701a and 701b that connect the sample cartridge 102 and the cathode block 212 and the intermediate portions of the discharge flow paths 701e and 701f that connect the cathode block 212 and the cathode waste liquid container 215. The bypass flow paths 701g and 701h and the chamber 701d inside the cathode block 212 form a circulation flow path that allows liquid to circulate.

[0202] The negative pressure mechanism 114 is installed in the middle of the bypass flow paths 701g and 701h, and applies negative pressure to at least a portion of the section between the chamber 701d in which the end of the capillary 202 inside the capillary cartridge 101 is disposed and the end of the bypass flow path 701g connected to the negative pressure mechanism 114. When the negative pressure mechanism 114 is activated, the bypass flow path 701h connected to the negative pressure mechanism 114 and the like are applied with positive pressure. When the negative pressure mechanism 114 is installed in the bypass flow paths 701g and 701h, it is preferable to use a piezo pump or the like as the negative pressure mechanism 114.

[0203] The flow path switching unit 703c switches the flow path between the liquid sending flow paths 701a and 701b that connect the sample cartridge 102 and the cathode block 212, and a bypass flow path 701h that branches off from these flow paths and connects to the negative pressure mechanism 114. The flow path switching unit 703c is configured, for example, by a three-way valve or the like.

[0204] When the sample solution is sent from the sample cartridge 102 to the cathode block 212, the flow path switching unit 703c opens the liquid sending flow paths 701a and 701b connecting the sample cartridge 102 and the cathode block 212, and closes the bypass flow path 701h connected to the negative pressure mechanism 114. On the other hand, when the negative pressure mechanism 114 is operating, the flow path switching unit 703c opens the bypass flow path 701h connected to the negative pressure mechanism 114 and the liquid sending flow path 701b connected to the cathode block 212, and closes the liquid sending flow path 701a connected to the sample cartridge 102.

[0205] The flow path switching unit 703d switches the flow path between discharge flow paths 701e and 701f that connect the cathode block 212 and the cathode waste liquid container 215, and a bypass flow path 701g that branches off from these flow paths and connects to the negative pressure mechanism 114. The flow path switching unit 703d is configured, for example, by a three-way valve or the like.

[0206] When sending the sample solution from the sample cartridge 102 to the cathode block 212 or when cleaning the internal flow paths of the cathode block 212, the flow path switching unit 703d opens the discharge flow paths 701e and 701f connecting the cathode block 212 and the cathode waste liquid container 215 and closes the bypass flow path 701g connected to the negative pressure mechanism 114. On the other hand, when the negative pressure mechanism 114 is operating, the flow path switching unit 703d opens the bypass flow path 701g connected to the negative pressure mechanism 114 and the discharge flow path 701e connected to the cathode block 212 and closes the discharge flow path 701f connected to the cathode waste liquid container 215.

[0207] In the following description, the direction of liquid supply from the liquid supply flow path 701a to the liquid supply flow path 701b formed by the flow path switching unit 703c is referred to as the ab direction, the direction of liquid supply from the bypass flow path 701h to the liquid supply flow path 701b is referred to as the hb direction, the direction of liquid supply from the discharge flow path 701d formed by the flow path switching unit 703d to the cathode waste liquid container 215 is referred to as the dw direction, and the direction of liquid supply from the chamber 701d to the bypass flow path 701g is referred to as the dg direction.

[0208] The check valve 705b is in an open state when gas flows from the inside to the outside of the cathode waste liquid container 215, and is in a closed state when gas flows from the outside to the inside of the cathode waste liquid container 215. The check valve 705b allows air to be discharged from the inside to the outside of the cathode waste liquid container 215 through the gas discharge flow path when liquid is sent from the liquid sending unit 112 to the cathode block 212.

[0209] In the capillary cartridge 101 having the structure shown in FIG. 15, the analysis by pretreatment-integrated electrophoresis is carried out in the following steps (1) to (8).

[0210] The control unit 107 controls the negative pressure mechanism 114, the solution delivery unit 112, the flow path switching units 703c and 703d, the electrophoresis reagent delivery pump 213, the sample cartridge 102, the power supply 103, the optical system 104, the injection pump 105, etc., to execute the process of electrophoresis analysis in the order of (1) to (8). The control unit 107 controls the electric field injection of the sample components into the capillary 202, the reverse transfer of the sample solution by the operation of the negative pressure mechanism 114, the electrophoresis of the sample components in the capillary 202, and the reintroduction of the reversed sample solution into the chamber 701d. The control unit 107 controls the reintroduction of the reversed sample solution into the chamber 701d based on the status or result of the electrophoresis in the capillary 202.

[0211] (1) Pretreatment First, the sample is pretreated using the sample cartridge 102. The pretreatment includes a process for extracting nucleic acids from the sample, a process for amplifying the nucleic acids extracted from the sample, and a process for mixing the amplified products with reagents, etc. A sample solution for electrophoresis is prepared by the sample pretreatment.

[0212] (2) Feeding of sample solution Next, the sample solution for electrophoresis prepared by pretreatment is fed. For feeding of the sample solution, the flow path switching unit 703c is switched to the ab direction. The flow path switching unit 703d is switched to the dw direction. The sample solution for electrophoresis is prepared in a processing tank inside the sample cartridge 102 where the sample solution for electrophoresis is prepared. By operating the liquid feeding unit 112, the sample solution for electrophoresis is fed from the processing tank where the sample solution for electrophoresis is prepared through the liquid feeding flow paths 701a and 701b to the chamber 701d inside the cathode block 212.

[0213] When feeding the sample solution, the pump is operated while alternately feeding air and sample solution, creating a state in which segmented air is sandwiched between the front and rear of the plug-shaped liquid mass of sample solution. By feeding the liquid with segmented air sandwiched between the plug-shaped liquid mass of sample solution, mixing of the liquid mass of sample solution with other reagent solutions can be prevented.

[0214] The feeding of the sample solution is stopped when the sample solution to be analyzed for electrophoresis reaches the vicinity of the injection end 207 of the capillary 202 inside the chamber 701d. For example, the feeding is performed so that a liquid mass of the sample solution reaches the vicinity of the injection end 207 of the capillary 202. Arrival of the sample solution near the injection end 207 of the capillary 202 can be confirmed by installing an electrode near the injection end 207 and detecting an increase in the current value.

[0215] (3) Electric field injection of sample components Next, electric field injection of the sample components contained in the sample solution is performed into the capillary 202. In the electric field injection of the sample components, a high voltage is applied across the capillary 202 while the sample solution is introduced near the injection end 207 of the capillary 202 inside the chamber 701d. The application of the high voltage injects some of the sample components contained in the sample solution near the injection end 207 into the capillary 202.

[0216] (4) Reverse Flow of Sample Solution Subsequently, the sample solution subjected to electroinjection is reversed. For the reverse flow of the sample solution, the flow path switching unit 703c is switched to the hb direction. The flow path switching unit 703d is switched to the dg direction. By operation of the negative pressure mechanism 114, the bypass flow path 701g becomes negative pressure relative to the chamber 701d, and the bypass flow path 701h becomes positive pressure relative to the chamber 701d, and the sample solution remaining near the end of the capillary in the chamber 701d, etc., is reversed to the bypass flow path 701g. The sample solution present in the bypass flow path 701g is reversed to the bypass flow path 701h. At this time, the upstream of the liquid supply flow path 701a is not negatively pressurized.

[0217] When the negative pressure mechanism 114 is activated, the flow path switching unit 703c is switched to a state in which the flow path 701a, which is one of the liquid supply paths 701a and 701b connecting the sample cartridge 102 and the cathode block 212 and is upstream of the flow path switching unit 703a, is closed. By closing the liquid supply path 701a, the negative pressure mechanism 114 can create a negative pressure in the bypass flow path 701g without changing the pressure in the sample cartridge 102, the liquid supply unit 112, the pretreatment reagent container 223, or the flow paths connecting these.

[0218] (5) Feeding of migration reagent Next, the migration reagent used for electrophoresis is fed. In feeding the migration reagent, the migration buffer solution prepared in the migration reagent container 214 is introduced into the chamber 701d and the like inside the cathode block 212 by operating the migration reagent feeding pump 213. The used migration reagent and excess migration reagent remaining in the chamber 701d are discharged from the chamber 701d and collected in the cathode waste liquid container 215.

[0219] (6) Electrophoresis Subsequently, the sample components injected under an electric field are electrophoresed inside the capillary 202. In electrophoresis, a high voltage is applied across both ends of the capillary 202 while the sample components are being injected under an electric field into the capillary 202. The application of the high voltage causes the sample components injected into the capillary 202 to electrophorese inside the capillary 202. The sample components that have migrated inside the capillary 202 are optically detected by a detection unit 205 installed on the anode side at intervals of a migration time according to their mobility. During electrophoresis, the delivery of the migration reagent may be stopped or may be continuously performed.

[0220] (7) Determination of the Electrophoresis Results Next, the electrophoresis results are determined. Based on the detection results in the detection unit 205, the electrophoresis results are obtained as a spectrum or the like showing the relationship between the migration time and the light intensity of the information light. In determining the electrophoresis results, it is determined whether or not the electrophoresis results are defective. The determination of the electrophoresis results may be performed after the electrophoresis has finished or while the electrophoresis is being performed. If it is determined that the electrophoresis results are defective, the electrophoresis may be interrupted.

[0221] (8-1) Re-electrophoresis after Determination: If the electrophoresis results are found to be unsatisfactory, re-electrophoresis is performed using the sample solution subjected to electric field injection. In the re-electrophoresis after determination, with the flow path switching unit 703c switched to the hb direction and the flow path switching unit 703d switched to the dg direction, the negative pressure mechanism 114 is activated to reintroduce the sample solution, which has been reversely transported and held in the bypass flow paths 701g and 701h, into the chamber 701d inside the cathode block 212. Then, the operations following the electric field injection are repeated to perform re-electrophoresis.

[0222] (8-2) Cleaning after Determination: If no defects are found in the electrophoresis results upon evaluation, the chamber 701d, the liquid feed flow path 701b, the bypass flow paths 701g and 701h, and the like inside the cathode block 212 are cleaned. In cleaning after determination, with the flow path switching unit 703c switched to the hb direction and the flow path switching unit 703d switched to the dg direction, the negative pressure mechanism 114 is operated to transfer the sample solution remaining in the bypass flow paths 701g and 701h to the liquid feed flow path 701b and the chamber 701d. Then, with the flow path switching unit 703c switched to the ab direction and the flow path switching unit 703d switched to the dw direction, the liquid feed unit 112 is operated to send the flow path cleaning solution prepared in the pretreatment reagent container 223 to the chamber 701d, the liquid feed flow path 701b, and the like inside the cathode block 212. By passing the channel cleaning liquid, the liquid in the channel is replaced and the inner walls are cleaned, and the remaining sample solution is discharged into the cathode waste liquid container 215 .

[0223] Next, the flow path switching unit 703c is switched to the hb direction and the flow path switching unit 703d is switched to the dg direction, and the flow path cleaning solution remaining in the liquid supply flow path 701b and chamber 701d is introduced into the bypass flow paths 701g and 701h by operating the negative pressure mechanism 114. Thereafter, the flow path switching unit 703c is switched to the ab direction and the flow path switching unit 703d is switched to the dw direction, and the liquid supply unit 112 is operated to send the flow path cleaning solution prepared in the pretreatment reagent container 223 to the chamber 701d and the liquid supply flow path 701b inside the cathode block 212. The liquid in the flow path is replaced and the inner walls are cleaned by passing a buffer solution through the flow path, and the remaining sample solution is discharged into the cathode waste liquid container 215. After cleaning, the series of runs is completed.

[0224] 15, the sample solution subjected to electrophoresis can be transported back to the bypass flow paths 701g and 701h outside the chamber 701d and held there, allowing the same sample solution as that subjected to electrophoresis to be re-used. The range in which the sample solution is transported back by the negative pressure mechanism 114 is limited to the circulation flow path formed by the bypass flow paths 701g and 701h. This prevents sample solution or air containing sample molecules from flowing upstream of the sample cartridge, thereby suppressing contamination in the next run.

[0225] In Figure 15, the liquid delivery paths 701a and 701b, the bypass paths 701g and 701h, the path switching unit 703c, and the negative pressure mechanism 114 are installed in the path between the sample cartridge 102 and the cathode block 212, but these components may be incorporated into the capillary cartridge 101 or into the main body 111.

[0226] Fig. 16 is a diagram showing an example of the configuration of the main parts of an electrophoresis apparatus according to an embodiment of the present invention. Fig. 16 schematically shows an example of the cross-sectional structure and flow channel structure of a capillary cartridge 101 of an electrophoresis apparatus according to this embodiment. As shown in Fig. 16, a discharge flow channel 902 communicating with the outside of the electrophoresis apparatus 100 can also be connected to the circulation flow channel in which the negative pressure mechanism 114 is installed.

[0227] 16, a liquid delivery unit 112 is connected to the pretreatment reagent container 223 via a flow path through which a liquid can flow. A sample cartridge 102 is connected to the other end of the liquid delivery unit 112 via a flow path through which a liquid can flow. A flow path switching unit 703c is connected to the other end of the sample cartridge 102 via a liquid delivery flow path 701a through which a liquid for delivering a sample solution for electrophoresis can flow.

[0228] One port of the flow path switching unit 703c is connected to a flow path switching unit 703e via a liquid sending flow path 701b through which a liquid for sending a sample solution for electrophoresis can flow, and one end of a negative pressure mechanism 114 is connected to the other port of the flow path switching unit 703c via a bypass flow path 701h branching from the liquid sending flow paths 701a and 701b.

[0229] One port of the flow path switching unit 703e is connected to the cathode block 212 via a liquid delivery flow path 701i through which a liquid for delivering a sample solution for electrophoresis can flow. One end of a discharge flow path 902 is connected to the other port of the flow path switching unit 703e. The other end of the discharge flow path 902 is connected to the outside of the electrophoresis apparatus 100.

[0230] The flow path switching unit 703c switches the flow path between the liquid sending flow paths 701a and 701b that connect the sample cartridge 102 and the cathode block 212, and a bypass flow path 701h that branches off from these flow paths and connects to the negative pressure mechanism 114. The flow path switching unit 703c is configured, for example, by a three-way valve or the like.

[0231] When the sample solution is sent from the sample cartridge 102 to the cathode block 212, the flow path switching unit 703c opens the liquid sending flow paths 701a and 701b connecting the sample cartridge 102 and the cathode block 212, and closes the bypass flow path 701h connected to the negative pressure mechanism 114. On the other hand, when the negative pressure mechanism 114 is operating, the flow path switching unit 703c opens the bypass flow path 701h connected to the negative pressure mechanism 114 and the liquid sending flow path 701b connected to the cathode block 212, and closes the liquid sending flow path 701a connected to the sample cartridge 102.

[0232] The flow path switching unit 703e switches the flow path between the liquid supply flow paths 701b and 701i that connect the sample cartridge 102 and the cathode block 212, and the discharge flow path 902 that branches off from the liquid supply flow paths 701b and 701i and communicates with the outside of the electrophoresis device 100. The flow path switching unit 703e is configured, for example, by a three-way valve or the like.

[0233] When the sample solution is sent from the sample cartridge 102 to the cathode block 212 or when the negative pressure mechanism 114 is operating to send the sample solution in the reverse direction, the flow path switching unit 703e opens the liquid sending paths 701b and 701i connecting the sample cartridge 102 and the cathode block 212 and closes the discharge path 902 communicating with the outside of the electrophoresis device 100. On the other hand, when the liquid sending pump 112 is operating to recover the sample solution, the flow path switching unit 703e opens the liquid sending path 701b connected to the flow path switching unit 703c and the discharge path 902 communicating with the outside of the electrophoresis device 100 and closes the liquid sending path 701i connected to the cathode block 212.

[0234] The discharge flow path 902 is a flow path that discharges fluid from the circulation flow path toward the outside of the electrophoresis device 100. The discharge flow path 902 is used to discharge the sample solution that is reversed when the negative pressure mechanism 114 is activated to the outside of the electrophoresis device 100. A user of the electrophoresis device 100 can recover the sample solution that is reversed by the negative pressure mechanism 114 through the discharge flow path 902. The recovered sample solution can be introduced into the electrophoresis device 100 or another device for reanalysis. Because the recovered sample solution has been pretreated, pretreatment can be omitted when reanalyzing.

[0235] 16, the discharge flow path 902 is connected to the intermediate portion of the liquid supply paths 701b and 701i connected to the cathode block 212, but the discharge flow path 902 may also be connected to the discharge flow path 701e connecting the cathode block 212 and the cathode waste liquid container 215, the bypass flow path 701g connected to one end of the negative pressure mechanism 114, or the bypass flow path 701h connected to the other end of the negative pressure mechanism 114. When the discharge flow path 902 is connected to these locations, it is preferable to install an on-off valve in the discharge flow path 902.

[0236] A sample solution inlet may be provided upstream of the cathode block 212 for re-injecting the sample solution recovered through the discharge flow path 902. The sample solution inlet may be provided in the sample cartridge 102, the liquid feed flow path 701a connecting the sample cartridge 102 and the flow path switching unit 703c, the liquid feed flow path 701b connecting the flow path switching unit 703c and the flow path switching unit 703e, or the liquid feed flow path 701i connecting the flow path switching unit 703e and the cathode block 212.

[0237] Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the present invention. For example, the present invention is not necessarily limited to those having all of the configurations of the above-described embodiments. It is possible to replace part of the configuration of an embodiment with another configuration, add part of the configuration of an embodiment to another form, or omit part of the configuration of an embodiment.

[0238] 100 Electrophoresis apparatus 101 Capillary cartridge 102 Sample cartridge (pretreatment section) 103 Power supply 104 Optical system 105 Injection pump 107 Control section 108 Input / output section 109 Interface unit 110 Hinge 111 Main body section 112 Liquid delivery unit (liquid delivery pump) 113 Temperature control unit 114 Negative pressure mechanism 202 Capillary 204 Capillary head 205 Detection section 207 Injection end 208 Capillary connection port 209 Thermostatic bath 211 Polymer liquid delivery section 212 Cathode block 213 Migration reagent delivery pump 214 Migration reagent container 215 Cathode waste container 219 Anode waste container 220 Flow path connection port 221 Flow path connection port 222 Sample injection piping 223 Pretreatment reagent container 224 Pretreatment reagent liquid supply pipe 701 Flow path 703 Flow path switching unit 705 Check valve 901 Discharge flow path 1101 Substrate 1102 Membrane 1103 Movable member 1301 Nucleic acid extraction chamber 1302 PCR chamber 1303 Mixing chamber

Claims

1. An electrophoresis device comprising: a pretreatment section that pretreats a sample to prepare a sample solution containing sample components; capillaries that perform electrophoresis of the sample components; a chamber into which the sample solution is introduced and into which the sample components are electroinjected into the capillaries; a liquid transfer flow path that connects the pretreatment section to the chamber; a liquid transfer pump that transfers the sample solution through the liquid transfer flow path; and a power source that applies a voltage across the capillaries, wherein after the sample components contained in the sample solution introduced into the chamber are electroinjected into the capillaries, the sample solution remaining in the chamber is sent back to the outside of the chamber; and after the sample components electroinjected into the capillaries are electrophoresed using the capillaries, the sample solution that has been sent back to the outside of the chamber is reintroduced into the chamber to perform electroinjection and electrophoresis.

2. An electrophoresis device according to claim 1, comprising a negative pressure mechanism that creates a negative pressure relative to the chamber in at least a portion of the liquid delivery flow path, or in at least a portion of a circulation flow path that bypasses a portion of the liquid delivery flow path and connects the chamber to an intermediate portion of the liquid delivery flow path, and the reverse transfer of the sample solution is performed by the negative pressure mechanism.

3. An electrophoresis device according to claim 2, wherein the negative pressure mechanism creates a negative pressure in the section without changing the pressure in the flow path connecting the pre-treatment section and the liquid feed pump.

4. An electrophoresis device according to claim 2, wherein the negative pressure mechanism is connected to a branch flow path branching off from the middle of the liquid delivery flow path, or is connected onto the circulation flow path.

5. An electrophoresis device according to claim 4, comprising a flow path switching unit in the intermediate section of the liquid supply flow path that can freely open and close the liquid supply flow path, and the flow path switching unit closes the flow path of the liquid supply flow path that is upstream of the flow path switching unit while the section is under negative pressure.

6. An electrophoresis apparatus according to claim 2, wherein the sample solution is reintroduced into the chamber by the liquid feed pump or the negative pressure mechanism.

7. An electrophoresis apparatus according to claim 2, wherein the negative pressure mechanism is a pump having a function of reducing the internal pressure of a flow channel connected to the chamber.

8. An electrophoresis device according to claim 1, wherein at least a portion of the liquid transport flow path is formed from a flexible material whose flow path cross-sectional area is variable.

9. An electrophoresis apparatus according to claim 1, wherein the pretreatment section comprises a nucleic acid extraction chamber for extracting nucleic acids from the sample, and a PCR chamber for carrying out PCR to amplify the nucleic acids.

10. An electrophoresis apparatus according to claim 9, wherein the negative pressure mechanism is connected downstream of the PCR chamber, or is connected to a branch flow path branching off from downstream of the PCR chamber.

11. An electrophoresis device according to claim 1, wherein a gas intake flow path is connected to the chamber or downstream of the chamber, and the gas intake flow path takes in air from the outside while the sample solution is being reversed.

12. An electrophoresis apparatus according to claim 1, comprising a control unit that controls the electric field injection of the sample components into the capillaries, the reverse transport of the sample solution, the electrophoresis of the sample components in the capillaries, and the reintroduction of the sample solution into the chamber, wherein the control unit controls the reintroduction of the sample into the chamber based on the status or result of electrophoresis in the capillaries.

13. An electrophoresis method comprising the steps of: pretreating a sample to prepare a sample solution containing sample components; sending the sample solution to a chamber where electric field injection of the sample components into capillaries is performed; electric field injection of the sample components contained in the sample solution introduced into the chamber into the capillaries; sending the sample solution remaining in the chamber back to the outside of the chamber; electrophoresing the sample components electric field injected into the capillaries using the capillaries; and reintroducing the sample solution sent back to the outside of the chamber into the chamber to perform electric field injection and electrophoresis.

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