Circulating type free flow isoelectric focusing electrophoresis device and method

Abstract

The invention discloses a circulating type free flow isoelectric focusing electrophoresis device and method.The circulating type free flow isoelectric focusing electrophoresis device is mainly composed of a gas-liquid buffer chamber, liquid storage pipes, a separation chamber, a collecting pipe, a cooling device and a driving pump, liquid inlets of the separation chambers are connected with liquid storage pipes in the gas-liquid buffer chambers in a one-to-one correspondence manner through switches, liquid outlets of the separation chambers are connected with a collecting pipe through a three-way valve, another outlet of the three-way valve corresponds to the liquid storage pipes in the gas-liquid buffer chambers in a one-to-one correspondence manner, and a switch is connected between the three-way valve and the liquid storage pipes. According to the device and the method, required reagents are low in price, a constant flow pump is adopted to replace a multi-channel driving pump, the effect of infinite plate length can be achieved under continuous circulation, heat dissipation is facilitated, and a solution without a sample can be recycled in a pipeline with the corresponding pH value.

Description

Technical Field

[0001] The present invention relates to the field of preparative free-flow electrophoresis separation technology, specifically, a circulating free-flow isoelectric focusing electrophoresis device and method, which are mainly used for free-flow isoelectric focusing electrophoresis separation of macromolecular charged substances such as proteins. Background Art

[0002] Free flow electrophoresis (FFE) is a continuous separation technology without solid support medium, which has both preparation and analysis functions, and has the advantages of high recovery rate, mild separation conditions, and continuous separation and purification. At present, FFE has formed a variety of electrophoresis modes, such as free flow zone electrophoresis (FFZE), free flow isoelectric focusing electrophoresis (FFIEF), free flow isotachophoresis electrophoresis (FFITP) and free flow field gradient electrophoresis (FFFSE). FFIEF is the one with the highest separation efficiency and the most extensive application.

[0003] FFIEF is a separation technique that utilizes the different isoelectric points (pI) of samples. The carrier amphoteric electrolyte forms a stable pH gradient in the electric field. When the charged protein or peptide moves to the position corresponding to its isoelectric point (pI), the net charge becomes zero and the movement stops, thus achieving the focusing separation of the amphoteric substances. FFIEF technology can be divided into continuous free flow isoelectric focusing technology and cyclic free flow isoelectric focusing technology. At present, there are many studies on continuous free flow isoelectric focusing electrophoresis technology. During operation, the carrier amphoteric electrolyte solution and protein sample are continuously pumped into the separation chamber, and the focusing separation is carried out under the action of the electric field, and then the components are collected. This method often has the problem of incomplete focusing, which usually requires a longer separation chamber to solve. However, an overly long separation chamber will bring problems of heat dissipation and device stability. Bier et al. developed the recycling free flow isoelectric focusing (RFFIEF) technology in 1979. When performing RFFIEF, a multi-channel drive pump is used to drive the carrier ampholyte solution and protein sample to flow into the separation chamber. The carrier ampholyte and protein achieve partial isoelectric focusing under the action of the transverse electric field, flow out from the separation chamber outlet, and flow into the separation chamber again for refocusing after external circulation. This process is repeated until sufficient focusing separation is achieved, effectively solving the problem of incomplete focusing in continuous free-flow isoelectric focusing electrophoresis.

[0004] However, the above-mentioned circulating free-flow isoelectric focusing technology has the following problems: first, the device requires a multi-channel drive pump, which is relatively expensive; second, the number of channels of the multi-channel drive pump limits the number of inlets and outlets of the device; third, it is more troublesome to discharge bubbles from the separation chamber, and the rotation direction of the drive pump needs to be repeatedly adjusted. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention proposes a circulating free-flow isoelectric focusing electrophoresis device and method, which uses a single-channel drive pump instead of a multi-channel drive pump, and the gas-liquid buffer chamber adopts a separate liquid storage tube design.

[0006] The technical solutions adopted in this application are as follows:

[0007] A circulating free-flow isoelectric focusing electrophoresis device mainly consists of a gas-liquid buffer chamber 1, a liquid storage tube 2, a gas-liquid buffer chamber-separation chamber switch 3, an electrode chamber 4, a separation chamber 5, a separation chamber-collection tube switch 6, a collection tube 7, a collection tube-gas-liquid buffer chamber switch 8, an electrode liquid circulation pump 9, a medium drive pump 10 and a cooling device 28; wherein the liquid storage tube is connected to a gas-liquid buffer chamber liquid outlet 16, the gas-liquid buffer chamber liquid outlet is connected to a separation chamber liquid inlet 13 through a switch 3, the separation chamber liquid outlet 14 is connected to a collection tube through a switch 6, the collection tube is connected to the liquid storage tube through a switch 8, the medium drive pump drives a medium liquid 19 to flow into the gas-liquid buffer chamber from the gas-liquid buffer chamber liquid inlet, an air hole is provided above the gas-liquid buffer chamber to connect a manual air pump 11 for discharging bubbles, and a liquid outlet 12 is provided at the bottom for discharging the medium liquid.

[0008] The separation chamber liquid outlet 14 is connected to the tee 15, and the other two ports of the tee are respectively connected to the collecting pipe and the liquid storage pipe. A switch and a switch are respectively arranged between the separation chamber liquid outlet and the tee, and between the liquid storage pipe and the tee.

[0009] The gas-liquid buffer chamber 1 is a closed structure, the upper plate 17 of the gas-liquid buffer chamber can be opened, and a silicone gasket 18 is used to seal the middle. The liquid storage tube 2 is independently distributed inside the gas-liquid buffer chamber, and the lower end of the liquid storage tube is connected to the liquid outlet of the gas-liquid buffer chamber. The upper plate of the gas-liquid buffer chamber is provided with a gas-liquid buffer chamber liquid inlet 23 and air pump 10, 11 connection ports.

[0010] The internal volume of the gas-liquid buffer chamber is more than twice the total volume of the liquid storage tube, ensuring that the medium liquid level will not exceed the tube opening of the liquid storage tube.

[0011] The liquid storage tube 2 is independently bonded to the bottom plate of the gas-liquid buffer chamber, the gas-liquid buffer chamber shell and the bottom plate are sealed with glue, the gas-liquid buffer chamber upper plate can be opened, and when in use, the gas-liquid buffer chamber 1 and the gas-liquid buffer chamber upper plate 17 can be clamped with the silicone gasket 18 to seal.

[0012] It also includes a cooling device connected to a refrigeration circulation bath, located below the separation chamber, using water-ethylene glycol as the cooling medium, and a layer of thermal conductive silica gel is placed in the middle to remove air between the cooling device and the lower plate of the separation chamber.

[0013] The running mobile phase is an amino acid solution, wherein the amino acid concentration is 0.001 mol / L-0.1 mol / L.

[0014] The positive electrode solution 20 is an acidic solution with a pH value ranging from 1 to 7 and a concentration of 0.01 mol / L to 2 mol / L; the negative electrode solution 21 is an alkaline solution with a pH value ranging from 7 to 14 and a concentration of 0.01 mol / L to 2 mol / L.

[0015] The specific operation steps are as follows: Step 1: add running buffer solution to the liquid storage tube, seal the gas-liquid buffer chamber, and keep the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection tube switch (6) open, and the collection tube-gas-liquid buffer chamber switch (8) closed; Step 2: after the bubbles in the separation chamber are exhausted, start the electrode liquid drive pump, fill the electrode chamber with electrode liquid to immerse the electrode and keep circulating, apply appropriate voltage to the two electrodes, start the medium drive pump, run stably for a period of time, and form a stable pH gradient at the rear of the separation chamber; Step 3: when the gas-liquid buffer chamber liquid storage tube is When the liquid is nearly empty, turn off the medium-driven pump, and turn off the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection tube switch (6), and turn on the collection tube-gas-liquid buffer chamber switch (8). The liquid in the collection tube flows back to the liquid storage tube under the action of gravity, completing one cycle. Step 4: turn off the collection tube-gas-liquid buffer chamber switch (8), turn on the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection tube switch (6), turn on the medium-driven pump, and start the second cycle. Repeat the above process until the sample is separated and the solution in the collection tube that does not contain the sample is recovered for next use.

[0016] Compared with the existing circulating free-flow isoelectric focusing electrophoresis technology, the beneficial effects of the present invention are:

[0017] The device uses a single-channel driving pump instead of a multi-channel driving pump, has a simple structure and is relatively cheap.

[0018] There is no need to use carrier ampholytes as running buffer solutions to form a pH gradient; small molecule amino acid solutions can achieve the same effect.

[0019] After the cycle is completed, the collected liquid without samples can be reused and placed in the corresponding liquid storage tube for next use, so as to quickly achieve pH gradient stability and reduce the generation of waste liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0021] Figure 1 It is a schematic diagram of the principle of the circulating free-flow isoelectric focusing electrophoresis device provided by the present invention; Figure 2 is a schematic diagram of the separation chamber structure; Figure 3 It is a schematic diagram of the separation chamber and cooling device.

[0022] Among them, 1 is a gas-liquid buffer chamber, 2 is a liquid storage tube, 3 is a gas-liquid buffer chamber-separation chamber switch, 4 is an electrode chamber, 5 is a separation chamber, 6 is a separation chamber-collection tube switch, 7 is a collection tube, 8 is a collection tube-gas-liquid buffer chamber switch, 9 is an electrode liquid circulation pump, 10 is a medium drive pump, 11 is a manual air pump, 12 is a medium liquid discharge port, 13 is a separation chamber liquid inlet, 14 is a separation chamber liquid outlet, 15 is a three-way, 16 is a gas-liquid buffer chamber liquid outlet, 17 is a gas-liquid buffer chamber upper plate, 18 is a silicone gasket, 19 is a medium liquid storage tank, 20 is a positive electrode liquid, 21 is a negative electrode liquid, 22 is a high-voltage power supply, 23 is a gas-liquid buffer chamber circulation liquid inlet, 24 is an ion exchange membrane, 25 is a graphite electrode rod, 26 is a silicone gasket, 27 is a high thermal conductivity silicone, and 28 is a cooling plate. DETAILED DESCRIPTION

[0023] The detailed technical contents and preferred implementations of the present invention are as follows, but they cannot limit the contents of the present invention. The contents of the accompanying drawings are described as follows.

[0024] See also Figure 1 As shown, a schematic diagram of a circulating free-flow electrophoresis device used in the present application. This device comprises: a high-voltage power supply (22), a gas-liquid buffer chamber (1), a liquid storage tube (2), a gas-liquid buffer chamber-separation chamber switch (3), an electrode chamber (4), a separation chamber (5), a separation chamber-collection tube switch (6), a collection tube (7), a collection tube-gas-liquid buffer chamber switch (8), an electrode liquid circulation pump (9), and a medium drive pump (10). The gas-liquid buffer chamber liquid outlet and the separation chamber liquid inlet are connected in a one-to-one correspondence through the switch (3), and a tee (15) is provided between the separation chamber liquid outlet and the collection tube, which are connected in a one-to-one correspondence through the switch (6), and the other end of the tee is connected to the gas-liquid buffer chamber circulation liquid inlet (23) through the switch (8). The liquid level of the liquid storage tube should be lower than the separation chamber liquid inlet, and the lowest point of the collection tube should be higher than the separation chamber liquid outlet. The medium drive pump (10) is used to drive the medium liquid in the medium liquid storage bottle (19) into the gas-liquid buffer device (1) to drive the mobile phase circulation. A manual air pump (11) is used to repeatedly suck out bubbles in the separation chamber (5) so that the separation chamber is filled with the running mobile phase; an electrode liquid circulation pump (9) is used to drive the circulation of the electrode liquid in the positive and negative electrode liquid storage tanks (20) (21); and a high-voltage power supply (22) is used to provide voltage for the device.

[0025] See also Figure 2 The schematic diagram of the separation chamber structure in the free-flow electrophoresis device used in the present invention is shown. The device comprises: a separation chamber liquid inlet (13), a separation chamber liquid outlet (14); positive and negative electrode chambers (41) (42) are separated from the free-flow electrophoresis separation chamber by cation and anion exchange membranes (24), respectively; a graphite electrode rod (25); and a silicone sealing pad (26) for clamping and sealing the separation chamber.

[0026] See also Figure 3The schematic diagram of the separation chamber and cooling device used in the present invention is shown in FIG. A high thermal conductivity silica gel (27) is placed under the separation chamber, and then connected to a cooling plate (28) to remove the air in the middle. The cooling plate is externally connected to a refrigeration circulation bath.

[0027] The operation steps are as follows: Step 1: Turn on the cooling device, set the cooling temperature to 4°C, turn on the electrode liquid driving pump to keep the electrode liquid circulating; Step 2: Add the sample solution to one of the liquid storage tubes, add the running buffer solution to the other liquid storage tubes, seal the gas-liquid buffer chamber, and keep the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection tube switch (6) open, and the collection tube-gas-liquid buffer chamber switch (8) closed; Step 3: Repeatedly press the manual air pump until the bubbles in the separation chamber are exhausted, turn on the medium driving pump, apply a voltage of 1000V to the two electrodes, run stably for a period of time, and form a stable pH gradient at the rear of the separation chamber. degree; Step 4: when the liquid in the liquid storage pipe of the gas-liquid buffer chamber is nearly emptied, turn off the medium-driven pump, and turn off the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection pipe switch (6), and turn on the collection pipe-gas-liquid buffer chamber switch (8), so that the liquid in the collection pipe flows back to the liquid storage pipe under the action of gravity, completing one cycle; Step 5: turn off the collection pipe-gas-liquid buffer chamber switch (8), turn on the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection pipe switch (6), turn on the medium-driven pump, and start the second cycle. Repeat the above process until the sample is separated, and recover the solution in the collection pipe that does not contain the sample for next use.

[0028] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the concept of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A circulating free-flow isoelectric focusing electrophoresis device, characterized in that: The invention mainly comprises a gas-liquid buffer chamber (1), a liquid storage pipe (2), a gas-liquid buffer chamber-separation chamber switch (3), an electrode chamber (4), a separation chamber (5), a separation chamber-collection pipe switch (6), a collection pipe (7), a collection pipe-gas-liquid buffer chamber switch (8), an electrode liquid circulation pump (9), a medium-driven pump (10) and a cooling device; wherein the liquid storage pipe is connected to the gas-liquid buffer chamber liquid outlet (16), the gas-liquid buffer chamber liquid outlet is connected to the separation chamber liquid inlet (13) through the switch (3), the separation chamber liquid outlet (14) is connected to the collection pipe through the switch (6), the collection pipe is connected to the liquid storage pipe through the switch (8), the medium-driven pump drives the medium liquid to flow from the gas-liquid buffer chamber liquid inlet into the gas-liquid buffer chamber, an air hole is provided above the gas-liquid buffer chamber to be connected to a manual air pump (11) for discharging bubbles, and a liquid outlet (12) is provided at the bottom for discharging the medium liquid.

2. The circulating free-flow isoelectric focusing electrophoresis device according to claim 1, characterized in that: The liquid outlet of the separation chamber is connected to a tee (15), and the other two ports of the tee are respectively connected to a collecting pipe and a liquid storage pipe. A switch (6) and a switch (8) are respectively arranged between the liquid outlet of the separation chamber and the tee, and between the liquid storage pipe and the tee.

3. The circulating free-flow isoelectric focusing electrophoresis device according to claim 2, characterized in that: The gas-liquid buffer chamber is a closed structure. The upper plate (17) of the gas-liquid buffer chamber can be opened and sealed in the middle with a silicone gasket (18). The liquid storage pipe is independently distributed inside the gas-liquid buffer chamber. The lower end of the liquid storage pipe is connected to the liquid outlet of the gas-liquid buffer chamber. The upper plate of the gas-liquid buffer chamber is provided with a gas-liquid buffer chamber liquid inlet and an air pump connection port.

4. The circulating free-flow isoelectric focusing electrophoresis device according to claim 2 or 3, characterized in that: The internal volume of the gas-liquid buffer chamber is 2 to 10 times the total volume of the liquid storage tube, and the medium liquid level in the gas-liquid buffer chamber does not exceed the tube opening of the liquid storage tube.

5. The circulating free-flow isoelectric focusing electrophoresis device according to claim 4, characterized in that: The liquid storage tubes are independent of each other and are independently bonded to the bottom plate of the gas-liquid buffer chamber. The gas-liquid buffer chamber shell and the bottom plate are sealed with glue. The upper plate of the gas-liquid buffer chamber can be opened and clamped for sealing during use.

6. The circulating free-flow isoelectric focusing electrophoresis device according to claim 5, characterized in that: It also includes a cooling device connected to a circulating cooler, located below the separation chamber, using water-ethylene glycol as the cooling medium, and a layer of thermally conductive silica gel is placed in the middle to remove air between the cooling device and the lower plate of the separation chamber.

7. The circulating free-flow isoelectric focusing electrophoresis device according to claim 6, characterized in that: The positive electrode liquid is an acidic solution with a pH value ranging from 1 to 7 and a concentration of 0.01 mol / L to 2 mol / L; the negative electrode liquid is an alkaline solution with a pH value ranging from 7 to 14 and a concentration of 0.01 mol / L to 2 mol / L.

8. The circulating free-flow isoelectric focusing electrophoresis device according to claim 7, characterized in that: The running mobile phase is an amino acid solution, wherein the amino acid concentration is 0.001 mol / L-0.1 mol / L.

9. The circulating free-flow isoelectric focusing electrophoresis device according to claim 1 or 8, characterized in that: The specific operation steps are as follows: Step 1: Add running buffer solution into the liquid storage tube, seal the gas-liquid buffer chamber, and keep the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection tube switch (6) open, and the collection tube-gas-liquid buffer chamber switch (8) closed; Step 2: After the bubbles in the separation chamber are exhausted, the electrode liquid driving pump is turned on to fill the electrode chamber with electrode liquid to immerse the electrodes and keep circulating, and an appropriate voltage is applied to the two electrodes, and the medium driving pump is turned on to run stably for a period of time to form a stable pH gradient at the rear of the separation chamber; Step 3: When the liquid in the liquid storage pipe of the gas-liquid buffer chamber is close to being emptied, the medium driving pump is turned off, and the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection tube switch (6) are turned off, and the collection tube-gas-liquid buffer chamber switch (8) is turned on, and the liquid in the collection tube flows back to the liquid storage pipe under the action of gravity, completing one cycle; Step 4: Close the collection tube-gas-liquid buffer chamber switch (8), open the gas-liquid buffer chamber-separation chamber switch (3) and the separation chamber-collection tube switch (6), start the medium drive pump, start the second cycle, repeat the above process until the sample is separated, and recover the solution in the collection tube that does not contain the sample for next use.

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