JP4892843B2 - Liquid handling device - Google Patents

Description

The present invention separates a specific component from a liquid having a plurality of solutes, fractionated relates to liquid handling equipment to concentrate or purified.

  In the fields of biochemistry, pharmacy, cell biology, and molecular biology, for example, a specific protein is separated and purified from a biological tissue extract, or a protein in a cell culture produced by a cell is purified. An operation for separating a target substance from a mixture, such as separating a gene from a cell extract, has been performed. Means used for such operation is disclosed in Non-Patent Document 1. Of these, gel chromatography is the most versatile. Gel filtration is based on differences in molecular weight, ion exchange chromatography is based on differences in charge state, and separation is based on differences in affinity for ligands. There is affinity chromatography. In recent years, a technology for producing proteins useful for cells and bacteria has been developed due to advances in molecular biology. As shown in Patent Document 1, a target protein is separated from a culture supernatant using a hollow fiber membrane. Techniques for purification are known. In these separation technologies, a column equipped with a membrane or a column filled with gel and a liquid feed pump are connected by a silicon tube or the like, and a mobile phase such as a buffer solution is conveyed by the liquid feed pump, A stock solution containing the target substance is introduced into the membrane and passed through a membrane or column to separate the target substance.

  As a configuration for accurately separating using these technologies, a series of systems including an extraction liquid supply mechanism, a plurality of separation columns, a column for concentrating the separated filtrate, and a mechanism for collecting the concentrated liquid are considered. It is done.

However, since the circuit is complicated, a large space occupied by the liquid operation device is required, and it takes time to assemble. Also, since the system such as a tube becomes long, a lot of mobile phases are required. The separation and recovery efficiency deteriorates as the length increases.
Japanese Patent No. 3439503 Edited by the Japanese Biochemical Society, “Seminar in Experimental Chemistry for the New Chemistry (Vol. 1) Protein (1) Separation, Purification, and Properties”, Tokyo Chemical Doujin, 1990

  Recently, the above-mentioned technology has been actively used for research to search for pathological substances, and is used to process a large amount of specimens coming from the clinical field. However, liquid manipulation is possible even with a small amount of stock solution and mobile phase. Therefore, it is a problem to be solved to shorten the processing time, increase the separation / recovery efficiency, and create a compact liquid handling device that can be used in the laboratory.

That is, the present invention
“(1) A part of a circuit for performing a liquid operation is built in, and a removable cartridge having a plurality of liquid feeding tubes corresponding to a part of the circuit on the outside, a plurality of rotatable rotors, and a connection to the rotor It has a drive mechanism and a roller rotatably installed on the outer periphery of the rotor, and exists in the inside of the plurality of tubes by rotating at least one roller compressing the tube by the rotational drive of the rotor. A liquid pumping system comprising a tube pump for transferring liquid, the tube pump capable of independently adjusting the rotational speed of at least two or more rotors, wherein a part of the outside of the cartridge is connected to the tube pump rotor. confronting, has become a pressing block for pressing the liquid feed tube, the guide hole drilled in the cartridge, the rotor axis When mounting the cartridge through the guide shaft that is installed in the sliding base in a parallel positional relationship, the slide table is moved to the pump rotor direction, the pump tube is mounted in a position where the crush amount suitable are crimped to the pump roller A liquid operating device.
(2) The liquid operation device according to (1), wherein the liquid operation is one or more operations obtained from separation, fractionation, concentration, and purification of a liquid containing a biological component.
(3) The liquid manipulation device according to (2), wherein the biological component is protein . It consists of.

By configuring the liquid body operating device of the present invention, the liquid handling enables liquid handling in the mobile phase with a small amount of stock solution to shorten the processing time, increase the efficiency of the separation and recovery, the compact liquid handling device It can be configured.

The liquid operation device of the present invention will be described first. The liquid operation device of the present invention includes a part of a circuit for performing liquid operation, and a removable cartridge having a plurality of liquid supply tubes corresponding to a part of the circuit on the outside, and a tube pump described later. In this liquid operation system, a part of the outside of the cartridge is a squeezing block for confronting the rotor of the tube pump and compressing the liquid feeding tube. The tube pump to be used has a plurality of rotatable rotors, a drive mechanism for the rotors, and a roller rotatably installed on the outer periphery of the rotor, and at least one roller is driven by the rotation of the rotor. Is a tube pump that transfers liquid existing inside a plurality of tubes by rotating while compressing the tubes, and is capable of independently adjusting the rotational speed of at least two or more rotors. .

  The liquid operation circuit is preferably at least one kind of operation of separation, fractionation, concentration and purification of biological components. This device can be used to prepare samples for proteome analysis. In proteome analysis applications, a protein having a molecular weight higher than albumin, which is present in a large amount in serum, inhibits MS analysis. Therefore, it must have means for removing components having a molecular weight higher than albumin by membrane separation or adsorption. Is preferred.

  The term “fraction” as used in the present invention refers to separation of solutes in a solution, and when a plurality of solutes are contained, it refers to separation of all or part of the solutes. In particular, in the case of pretreatment of proteome analysis by MS of body fluid, it means that a protein for recovery is discriminated from a protein for disposal. The solution here refers to a liquid containing biological components such as proteins, nucleic acids, sugars, lipids, vitamins, inorganic salts, body fluids such as blood, serum, plasma, urine, lymph, brain spinal fluid, and fluids containing body fluids. There is no particular limitation as long as it is present, but it is preferable not to contain cells.

  The circuit formed by connecting the tubes of the present invention is a flow path through which a mobile phase liquid is conveyed. In order to smoothly perform a series of steps such as separation, fractionation, concentration, or purification after collection of body fluid or biological component-containing liquid, the mobile phase introduction tube and the A unit for supplying a body fluid or a biological component-containing liquid arranged so as to merge, a unit for performing liquid operations such as separation, fractionation, concentration, and purification downstream of the unit, and a unit for recovering the biological component are arranged in a circuit. It is preferable to comprise. Specifically, the units may be connected using plastic tubes such as silicon, Teflon (registered trademark), polyethylene, polypropylene, and polyvinyl chloride. If the circuit is a closed system, not only will it become more compact, but the experimenter will not be in contact with the liquid and safety will be ensured.

  The squeezing surface of the cartridge containing such a circuit and the rotor attached to the tube pump squeeze a part of the tube in the circuit so that liquid feeding is performed, for example, a device for fractionating proteins Can do. A body fluid or a biological component-containing liquid (hereinafter also referred to as “stock solution”) is input from the outside in the supply unit. The method of charging the stock solution is not particularly limited, but the stock solution collected in the syringe can be manually or by using a syringe pump or the like through a three-way stopcock or rubber button installed in the circuit, For example, a method of connecting a pipe connected to a container to the inside of a circuit and feeding it with a pump can be considered. The speed at which the stock solution is introduced is determined from the resistance in the unit such as separation and the circulating flow rate of the circuit defined by the pressure resistance of the membrane and circuit. If the charging speed is too high, the circuit pressure will rise, causing leakage from the circuit and damage to the membrane. If it is too slow, it takes a long time to process the stock solution.

  For separation, fractionation, concentration or purification, a hollow fiber separation membrane (hollow fiber membrane) is particularly preferably used because it has a large surface area per amount of processing liquid and a small operational pressure loss. Filters equipped with hollow fiber membranes (hollow fiber membrane modules) have been widely used as artificial kidneys (dialysis columns) in the past for proteins, but all are retained so as not to leak proteins such as albumin, and creatinine It is used for the purpose of purifying blood flowing in the hollow fiber lumen side by leaking low molecular components such as urea and urea. On the other hand, in this device, the fraction leaking from the hollow fiber lumen side is used for the analysis, and the molecular weight is mainly 5 kDa while retaining high molecular weight components such as albumin on the hollow fiber lumen side. The following protein components are leaked. Albumin refers to albumin derived from humans, cows, other mammals and birds. A large molecular weight mainly means a protein having a higher molecular weight than albumin (molecular weight 60,000 to 70,000). Whether it has a higher molecular weight than albumin can be determined by a method called SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis).

  In this apparatus, a circuit including a hollow fiber membrane module is preferably used as a means for liquid operation, and a stock solution supplied from the outside is first sent to a unit including the hollow fiber membrane module. As described above, a hollow fiber membrane is more preferably used because of its high throughput and low pressure loss. Regarding the molecular fractionation performance of the membrane, the molecular fractionation ability (cutoff value) can be appropriately selected in consideration of the molecular weight of the solute to be collected and the molecular weight of the solute to be removed. It is preferable that a liquid feed pump is installed in this circuit, and the pump can circulate the treatment liquid through the hollow fibers in the column. When a multi-stage configuration is used in order to increase the separation efficiency, a plurality of circuits are arranged horizontally, the filtrate outlet of the hollow fiber column of the front stage and the rear stage circuit are connected by a feeding pipe, and the treatment liquid is a multi-stage column. Are sequentially sent to the printer and separated.

  Regarding the molecular fractionation performance of the membrane in the case of concentration, a membrane or an ultrafiltration membrane having a molecular fractionation ability (cut-off value: 0.01 to 0.5 kDa or less) that does not allow peptides to pass through in physiological saline is used. As described above, a hollow fiber membrane is more preferably used because it has a high throughput and a small pressure loss. It is preferable that a liquid feed pump is installed in this circuit, and the pump can circulate the treatment liquid through the hollow fibers in the column.

  In this apparatus, it is desirable that the concentrated liquid is recovered by a recovery unit having a recovery container.

As described above, the required amount of liquid delivery and the timing of liquid delivery are different for the mobile phase supply, the circulation in the unit such as separation, and the concentrated liquid recovery. Therefore, in the tube pump of the present invention, it is preferable that the liquid feeding speed can be controlled independently.
Further, the tube pump has an effect that there is no leakage and that a minute flow rate can be easily controlled. If it is a system using a plurality of pump tubes, each pump tube is arranged on a common compression plate, and a circuit can be arranged on the back of the compression plate, so that a cartridge can be formed, and a compact system is constructed. Is possible.

  Each pump rotor must be concentric. If the envelope surface of the roller attached to each rotor is made to have the same radius, the pressing surface of the pressing plate can be made to have the same radius curved surface. However, this does not matter when the tube diameter is different.

  If two rotors are concentric, the two rotors can be positioned densely by arranging the drive shafts symmetrically outside, but the drive shafts come out on both sides and the width of the device is large. Will be.

  Further, in the case where a three-axis concentric rotor is required in this apparatus, at least two drive shafts interfere with each other when the three axes are arranged concentrically.

  In a preferred embodiment of the apparatus of the present invention, the first and second rotors are configured such that the driving shaft of the second rotor is used as a sheath tube and the driving shaft of the first rotor is passed, and the driving shaft of the third rotor is used as a sheath tube. All drive shafts are combined on one side through the drive shafts. A similar structure can be adopted with four or more axes. Here, there is a method of arranging the drive shaft of the third rotor symmetrically with the drive shaft of the first rotor, but it is preferable to combine the drive shafts in one direction in consideration of the width of the apparatus.

  The compression plate and circuit in the form of a cartridge are mounted on the guide shaft provided on the slide base with the guide hole provided in the compression plate, and then pressed against the pump roller, and the pump tube sandwiched between them is suitable for crushing. It is mounted at the position where the amount is reached.

  Hereinafter, one embodiment of the method for separating protein components in an aqueous solution and the protein fractionation apparatus of the present invention will be described with reference to the drawings, but the present invention is not limited thereto.

(Example)
1, 2, 3, and 4 are views of the liquid operating device of the present invention. FIG. 1 is a partial circuit of a liquid operating device in which a separation unit is constituted by a three-stage circuit. The circuit assembly was as follows.

  A three-way joint 2a and a joint 2c were connected to the rubber button 2b. Using a flexible tube, the joint 2c and the lower nozzle 6a of the hollow fiber membrane column 5a were connected to the curved surface of the multichannel pressing plate 8. Furthermore, the back 13 with a tube was connected to the three-way joint 2a. The hollow fiber membrane columns 5a, 5b, 5c constituting the separation unit, and the hollow fiber membrane column 5d constituting the water removal unit and the multi-channel type pressing plate 8 are integrated, and the hollow fiber membrane columns 5a, 5b, 5c, A flexible tube is connected to the upper nozzles 4a, 4b, 4c and 4d provided at the upper end of 5d, and the respective tubes are installed so as to run over the curved surface of the multi-channel type compression plate 8, and the lower nozzles 6a, 6b and 6c. , 6d, respectively. Next, the body nozzle 7a of the hollow fiber membrane column 5a and the lower nozzle 6b of the hollow fiber membrane column 5b are connected, and the body lower nozzle 7b of the hollow fiber membrane column 5b and the lower nozzle 6c of the hollow fiber membrane column 5c are connected to the hollow fiber. The lower body nozzle 7c of the membrane column 5c and the lower nozzle 6d of the hollow fiber membrane column 5d were connected by piping. The trunk lower nozzle 7d of the hollow fiber membrane column 5d and the three-way joint 2a were connected by piping. Furthermore, the lower nozzle 6d of the hollow fiber membrane column 5d and the recovery container cap 12 were connected, and the upper nozzle 4d of the hollow fiber membrane column 5d and the recovery container cap 12 were connected. The collection container cap 12 of the collection container 11 was sealed. All the hollow fiber membrane columns, nozzles, tubes, joints, backs with tubes, collection containers, and collection container caps described above formed a circuit that was not open to the atmosphere, that is, a closed circuit. In this closed circuit, the collection container 11 was filled with sterilized air, the tube-back 13 was empty, and the other was filled with an aqueous buffer which was a mobile phase.

  As shown in FIG. 2, the circuit is stored in a storage box 14 to form a cartridge C.

FIG. 3 is a sectional view of the tube pump constituting the rotor 9 shown in FIG. The rotor 9a, the rotor 9b, and the rotor 9c are arranged concentrically, and eight (not shown) rollers are attached to the outer periphery of each roller. The rotor 9a is driven from the side by a drive pulley 16a. The rotor 9b is driven by the drive pulley 16b through a through hole provided in the shaft center of the rotor 9a and the drive pulley 16a. Furthermore, the rotor 9c is driven by the drive pulley 16c through a through hole provided in the shaft center of the rotor 9b and the drive pulley 16b.
As shown in FIG. 4, the liquid operating device 15 is equipped with a multi-channel rotor 9, and the cartridge C is provided with guide holes of the multi-channel pressing plate 8 on two guide shafts provided vertically on the slide base 18. 8a and 8b were penetrated and fixed. The fixed cartridge C is moved in parallel to the rotor 9 side, and the rollers 10a, 10b, 10c, the pressing plate 8 and the seven tubes (not shown) installed on the curved surface of the pressing plate 8 are arranged on the rotor 9. ) To form a liquid feed pump. At the same time, due to the parallel movement of the cartridge C, the needle of the syringe 1 is stuck in the rubber button 2b.

  Operation of the device assembled as described above was started. Returning to FIG. The liquid flow is indicated by arrows. The specimen in the syringe 1 enclosing a stock solution such as serum was introduced at a speed controlled by a syringe pump. The administered stock solution was transported to the hollow fiber membrane column 5a of the separation unit while being transported by the rotor 9a driven by a motor while being mixed with the mobile phase. The filtrate produced while circulating through the hollow fiber membrane column 5a by the rotor 9b driven by the motor exited from the body nozzle 7a and was conveyed by the rotor 9b to the subsequent hollow fiber membrane column 5b. The filtrate of the hollow fiber membrane column 5b was further transported to the subsequent hollow fiber membrane column 5c. Thus, the solute of the stock solution was fractionated by the hollow fiber membrane columns 5a, 5b, and 5c forming the separation unit. The filtrate of the hollow fiber membrane column 5c was conveyed to the hollow fiber membrane column 5d of the water removal unit. The filtrate produced while circulating through the hollow fiber membrane column 5d exited from the body nozzle 7d and was returned to the supply unit via the joint 2a. The liquid circulation and liquid feeding in the separation unit and the water removal unit were performed by the rotor 9b. After the designated time has elapsed, the rotor 9a is stopped and the rotor 9c driven by the motor is started. As a result, the air in the recovery container 11 pushed out the concentrated liquid in the circuit in the water removal unit, and the concentrated liquid was recovered in the recovery container 11 through the lower nozzle 6d.

  The volume of the undiluted solution injected with the syringe 1 into the closed circuit was absorbed by the bag with tube 13.

A rotor which is a part of a liquid operation device and is a component of a liquid feeding circuit, a pressure plate and a pump comprising a supply unit, a separation unit, a concentration unit, and a recovery unit Explanatory drawing which shows a mode that the apparatus of FIG. 1 was accommodated in the storage box and it was set as the cartridge. Sectional drawing of the tube pump which is an example of the tube pump of this invention. The external view which shows an example of the fractionation apparatus of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Syringe 2a 3 way joint 2b Rubber button 2c Joint 3 Tube 4a Upper nozzle 4b Upper nozzle 4c Upper nozzle 4d Upper nozzle 5a Hollow fiber membrane column 5b Hollow fiber membrane column 5c Hollow fiber membrane column 5d Hollow fiber membrane column 6a Lower nozzle 6b Below Nozzle 6c Lower nozzle 6d Lower nozzle 7a Body nozzle 7b Body nozzle 7c Body nozzle 7d Body nozzle 8 Multi-channel squeezing plate 8a Guide hole 8b Guide hole 9 Multi-channel rotor 9a Rotor 9b Rotor 9c Rotor 11 Collection container 12 Collection container cap 13 Tube with tube 14 Storage box 15 Liquid operation device 16a Drive pulley 16b Drive pulley 16c Drive pulley 17 Bearing 18 Slide base

Claims (3)

A part of a circuit for performing liquid operation is incorporated, and a removable cartridge having a plurality of liquid feeding tubes corresponding to a part of the circuit on the outside, a plurality of rotatable rotors, and a drive mechanism for the rotors, A roller rotatably disposed on the outer periphery of the rotor, and at least one roller is rotated while pressing the tube by the rotational drive of the rotor, thereby transferring the liquid existing in the plurality of tubes. A liquid operation system having a tube pump and a tube pump capable of independently adjusting the rotational speed of at least two or more rotors, wherein a part of the outside of the cartridge faces the rotor of the tube pump and feeds it. has become a pressing block for pressing the liquid tube, the guide hole drilled in the cartridge, parallel to the rotor axis When mounting the cartridge through the guide shaft that is installed in the sliding base in location relationship, Rukoto slide table is moved to the pump rotor direction, the pump tube is mounted in a position where the crush amount suitable are crimped to the pump roller A liquid operation device characterized by the above. The liquid operation apparatus according to claim 1, wherein the liquid operation is one or more operations selected from separation, fractionation, concentration and purification of a liquid containing a biological component. The liquid manipulation device according to claim 2, wherein the biological component is a protein .

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