Rapid balancing dialysis device

Abstract

The utility model belongs to the technical field of balancing dialysis experiment equipment, and discloses a rapid balancing dialysis device which comprises a bottom plate, a dialysis pool is arranged in the bottom plate, an inserting piece can be installed in the dialysis pool, a dialysis membrane is arranged on the inserting piece, an independent dialysis membrane chamber is defined by the inserting piece and the dialysis membrane in the dialysis pool, and the dialysis membrane chamber is arranged in the bottom plate. A buffer solution chamber is arranged in the dialysis pool and at the periphery of the dialysis membrane chamber; the bottom plate is placed in a 20% ethanol solution to be soaked for 10 minutes before balance dialysis and then is washed with deionized water; the insert does not need to be pre-soaked prior to equilibrium dialysis. The rapid balance dialysis device is suitable for high-throughput analysis, and due to the high surface volume ratio of the membrane chamber, the contact area is increased, so that the dialysis is rapid, the balance time is greatly reduced, the potential problems of volume displacement, protein leakage and the like can be reduced to the maximum extent, and non-specific binding in the device is also reduced.

Description

TECHNICAL FIELD

[0001] The utility model relates to balanced dialysis experimental equipment technical field more specifically, relate to a quick balanced dialysis device. BACKGROUND

[0002] In order to analyze the free concentration of drug instead of total concentration in biological matrix (serum, plasma, blood or tissue), specific pre-treatment techniques must be used to separate free drugs from plasma protein-bound drugs. At present, the existing pre-treatment techniques include equilibrium dialysis (ED), ultrafiltration (UF), ultracentrifugation (UC) and the like.

[0003] Among them, as shown in formula (I), equilibrium dialysis (ED) is a concentration-driven process, and free protein-bound drugs in small volume plasma are separated out by diffusion through selective and permeable membrane pores (such as 8K or 12K MWCO) with different molecular weight cutoff points (MWCO). Since no external force (such as pressure) is applied, ED is considered to be the true gold standard for studying protein interactions. Figure 2

[0004] In order to control the environmental conditions, ED is carried out at 37 DEG C to simulate in vivo conditions, and it is best to carry out ED in a carbon dioxide (usually 5%) controlled incubator to control the pH value.

[0005] Advantages and disadvantages of equilibrium dialysis (ED):

[0006] Advantages: ① no need to apply pressure; ② has high potential of throughput; ③ easy to operate; ④ temperature and pH value are controllable; ⑤ low sample volume.

[0007] Disadvantages: ① requires a large amount of plasma and a long equilibration time; ② not suitable for unstable compounds; ③ cannot be used for highly hydrophobic compounds.

[0008] Based on the above problems, a quick equilibrium dialysis device is proposed to solve the problem of a large amount of plasma and a long equilibration time required by the traditional ED technology. UTILITY MODEL CONTENT

[0009] The utility model aims at solving the shortcomings in the prior art and proposes a quick equilibrium dialysis device.

[0010] To solve the above background technical problems, the utility model adopts the following technical scheme:

[0011] The quick equilibrium dialysis device comprises a bottom plate, a dialysis cell and an insert are arranged in the bottom plate, a dialysis membrane is arranged on the insert, an independent dialysis membrane chamber is formed by the insert and the dialysis membrane in the dialysis cell, and the dialysis cell is a buffer solution chamber at the peripheral part of the dialysis membrane chamber. ​

[0012] The bottom plate is placed in a 20% ethanol solution for 10 minutes before equilibrium dialysis and then rinsed with deionized water;

[0013] The insert does not need to be soaked before equilibrium dialysis.

[0014] As a further aspect of the present application: the material of the dialysis cell is medical grade plastic.

[0015] As a further aspect of the present application: the wall thickness of the dialysis cell is 1-3 mm.

[0016] As a further aspect of the present application: the dialysis membrane is a semi-permeable membrane, and the molecular weight cut-off of the semi-permeable membrane can be selected according to different customer uses, such as 3.5KD, 6-8KD, 12-14KD, etc.

[0017] As a further aspect of the present application: the flattened width of the semi-permeable membrane is 9-11mm, and the permeability coefficient of the dialysis membrane for water is not less than 1x10 -6 cm / s.

[0018] As a further aspect of the present application: the cross-sectional shape of the dialysis membrane chamber is oval or polygonal.

[0019] Compared with the prior art, the present application has the following advantages:

[0020] The rapid equilibrium dialysis device of the present application is suitable for high-throughput analysis, and due to the high surface-to-volume ratio of the membrane chamber, the contact area is increased, thereby rapid dialysis, which greatly reduces the equilibrium time, can minimize potential problems such as volume shift and protein leakage, and also reduces non-specific binding in the equipment. Specifically, it can be divided into the following points:

[0021] ①Easy to use - disposable tubes do not need to be pre-wetted;

[0022] ②Fast solution - the high surface area-to-volume ratio of the membrane allows it to reach equilibrium within 4-6 hours;

[0023] ③High throughput - the design of the rapid equilibrium dialysis device makes it suitable for scaling and automation;

[0024] ④Flexibility - any number of tests (1-48 tests per plate) can be performed without wasting the entire plate;

[0025] ⑤Determination - the disposable bottom plate and packaging and partition design of the pre-inserted cannula eliminate the possibility of cross-contamination or leakage;

[0026] ⑥Reproducibility and accuracy - the results generated by the validated plasma protein binding test are consistent with the results reported in the literature. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a structural schematic view of the present application;

[0028] Figure 2 is a schematic view of the prior art;

[0029] Figure 3 is a data view of the present application example.

[0030] Reference numerals in the drawings:

[0031] 1, dialysis pool; 2, bottom plate; 3, plug-in; 4, dialysis membrane; 5, dialysis membrane chamber; 6, buffer chamber. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. EMBODIMENT

[0033] Please refer to Figure 1 , the rapid equilibrium dialysis device comprises a bottom plate 2, the bottom plate 2 is provided with a dialysis pool 1, the dialysis pool 1 can be loaded with a plug-in 3, which is used for inserting the plug-in 3, the plug-in 3 is provided with a dialysis membrane 4, an independent dialysis membrane chamber 5 is formed in the dialysis pool 1 through the plug-in 3 and the dialysis membrane 4, the cross-sectional shape of the dialysis membrane chamber 5 is oval or polygonal, and the peripheral part of the dialysis membrane chamber 5 in the dialysis pool 1 is a buffer chamber 6.

[0034] The bottom plate 2 is soaked in 20% ethanol solution for 10 minutes before equilibrium dialysis and then washed with deionized water.

[0035] The plug-in 3 does not need to be soaked before equilibrium dialysis.

[0036] The present application combines the equilibrium dialysis (ED) technology, and the rapid equilibrium dialysis is also driven by concentration, and free protein binding drugs in small volume plasma are separated out through diffusion of selective and permeable membrane holes with different molecular weight cut-off points (MWCO). The difference between the rapid equilibrium dialysis and the traditional ED is that the rapid equilibrium dialysis of the present application shortens the dialysis time through a high surface volume ratio, greatly reduces the equilibrium time, can minimize potential problems such as volume shift and protein leakage, and also reduces non-specific binding (NSB) in the equipment.

[0037] Working principle:

[0038] After the drug binds to plasma protein at a certain ratio, both bound and free types exist in the plasma. The free type has drug activity, the bound type temporarily loses pharmacological activity and is stored in the blood, acting as a drug depot, which is important for drug action and the length of time it is maintained.

[0039] The dialysis membrane chamber 5 is separated from the buffer chamber 6 by the dialysis membrane 4. The plasma is placed in the dialysis membrane chamber 5. Proteins and other macromolecules cannot pass through the dialysis membrane 4, but free drugs can pass freely. When both sides reach equilibrium, the concentrations of free drugs on both sides of the dialysis membrane 4 are equal. If the total amount of drug in the system is known, the concentration of drug in the protein-free membrane chamber can be measured to calculate the plasma protein binding rate of the drug.

[0040] The material of the dialysis cell 1 is medical-grade plastic, which has good chemical stability and physical properties. The top of the insert 3 can directly observe the solution state in the dialysis membrane chamber 5 and the buffer chamber 6. The wall thickness of the dialysis cell 1 is 1-3 mm to ensure sufficient mechanical strength to prevent rupture due to internal pressure changes or external minor collisions during normal use and operation.

[0041] Further, the dialysis membrane 4 is a semi-permeable membrane. The molecular weight cut-off of the semi-permeable membrane can be selected according to different customer applications, such as 3.5KD, 6-8KD, 12-14KD, etc. The flat width of the semi-permeable membrane is 9-11mm to ensure sufficient mechanical strength while achieving efficient material exchange. The permeability coefficient of the dialysis membrane 4 to water is not less than 1x10 -6 cm / s to ensure that water can migrate reasonably between the two sides of the dialysis membrane 4 during dialysis, maintaining the osmotic pressure balance in the dialysis membrane chamber 5 and the buffer chamber 6. Embodiment

[0042] Based on the above embodiment, the rapid equilibrium dialysis method comprises the following steps.

[0043] Step 1, device and reagent preparation:

[0044] 1. Base plate preparation: soak the base plate in a 20% ethanol solution for 10 minutes, then rinse with deionized water.

[0045] 2. Insert preparation: assemble the insert and base plate.

[0046] 3. Plasma sample requirements: the pH value of the plasma sample should be 7.4±0.1.

[0047] 4. Positive substrate preparation.

[0048] Step 2, test design:

[0049] 1. Test group setting: Add the test sample to the plasma, and set the final concentration of the test substance to 5 μM, and keep the organic reagent content not more than 5%;

[0050] 2. Positive control group setting: Add warfarin sodium to the plasma, and set its final concentration to 5 μM, for verifying the normal work of the incubation system;

[0051] 3. Negative control group setting: Add the test substance or positive control to PBS, for verifying that the test substance or positive control can pass through the semi-permeable membrane and be uniformly distributed on both sides of the membrane.

[0052] Step three, incubation experiment:

[0053] 1. After assembling the rapid equilibrium dialysis device, add appropriate amount of PBS to the buffer chamber, and add appropriate amount of incubation plasma sample to the dialysis membrane chamber, so that the final liquid level on both sides is consistent;

[0054] 2. Cover a layer of sealing film on the top of the dialysis cell to prevent evaporation and maintain the pH value, and place the entire device in a 37℃, 5% CO2 incubator for shaking incubation for 4-6 hours, until the equilibrium of both sides of the membrane is reached;

[0055] 3. After the sample incubation is completed, terminate with acetonitrile, and after termination, shake and centrifuge, take the supernatant of the test sample for LC-MS / MS machine.

[0056] Step four, data analysis:

[0057] 1. Use LC-MS / MS analysis method to determine the concentration or peak area of the test sample;

[0058] 2. Treat and analyze the PBS in the buffer chamber, the treatment method is to terminate with acetonitrile and process before machine, take the supernatant for machine analysis for free test sample concentration (Cf) analysis;

[0059] 3. Treat and analyze the plasma in the dialysis membrane chamber, take the supernatant for machine analysis for determination of the total concentration of the test sample (Cp);

[0060] 4. Calculate the binding rate to determine the plasma protein binding of the test substance in the plasma, the plasma protein binding rate is calculated according to the formula: binding rate (%) = (1 - Cf / Cp) x 100. Example

[0061] Take the SD rat plasma protein binding test as an example, as shown in Figure 3 ;

[0062] Rat-6h is the test group, and the results show that its plasma protein binding rate is higher than 99%, which meets the requirement of plasma protein binding rate ≥98%.

[0063] Rat-6h-PBS is a negative control group, and the results show that the two positive controls can pass through the semi-permeable membrane, and the data on both sides are close to each other, further verifying the accuracy of the test.

[0064] The above merely describes a preferred specific embodiment of the present application; however, the scope of protection of the present application is not limited thereto. Any person skilled in the art, within the technical scope disclosed by the present application, according to the technical scheme and improvement concept of the present application, can make equivalent replacement or change, which should be covered within the scope of protection of the present application.

Claims

1. A rapid equilibrium dialysis device, comprising a base plate (2) in which a dialysis cell (1) is arranged, and a plug-in (3) is installed in the dialysis cell, and a dialysis membrane (4) is arranged on the plug-in (3); characterized in that An independent dialysis membrane chamber (5) is formed by the plug-in (3) and the dialysis membrane (4) in the dialysis cell (1), and a buffer solution chamber (6) is arranged in the dialysis cell (1) and at the peripheral part of the dialysis membrane chamber (5); The base plate (2) is soaked in a 20% ethanol solution for 10 minutes and then washed with deionized water before equilibrium dialysis; The plug-in (3) does not need to be soaked before equilibrium dialysis; The flattened width of the dialysis membrane (4) is 9-11 mm, and the permeability coefficient of the dialysis membrane (4) to water is not less than 1x10 - 6 cm / s.

2. The rapid equilibrium dialysis device of claim 1, wherein: The material of the dialysis cell (1) is medical grade plastic.

3. The rapid equilibrium dialysis device of claim 1, wherein: The wall thickness of the dialysis cell (1) is 1-3 mm.

4. The rapid equilibrium dialysis device of claim 1, wherein: The dialysis membrane (4) is a semi-permeable membrane.

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