Rapid balancing dialysis device

Abstract

The invention belongs to the technical field of balancing dialysis experimental equipment, and discloses a rapid balancing dialysis device which comprises a bottom plate, a dialysis pool is arranged in the bottom plate, an insertion piece can be installed in the dialysis pool, a dialysis membrane is arranged on the insertion piece, an independent dialysis membrane chamber is defined by the insertion piece and the dialysis membrane in the dialysis pool, and the insertion piece is arranged in the dialysis pool. 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 invention relates to the technical field of equilibrium dialysis experimental equipment, and more specifically to a rapid equilibrium dialysis device. Background Art

[0002] In order to analyze the free concentration of drugs rather than the total concentration in biological matrices (serum, plasma, blood or tissues), specific pretreatment techniques must be used to separate free drugs from plasma protein-bound drugs. Currently, the existing pretreatment technologies include equilibrium dialysis (ED), ultrafiltration (UF), ultracentrifugation (UC), etc.

[0003] Among them, Figure 2 As shown in Figure 2, equilibrium dialysis (ED) is a concentration-driven process that separates free protein-bound drugs from small volumes of plasma by diffusion through selective and permeable membrane pores with different molecular weight cutoffs (MWCO), such as 8K or 12K MWCO. ED is considered the authentic gold standard for studying protein interactions because no external forces (such as pressure) are applied.

[0004] To control environmental conditions, ED is performed at 37°C to simulate in vivo conditions and preferably in a carbon dioxide (usually 5%) controlled incubator to control pH.

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

[0006] Advantages: ① No need to apply pressure; ② High throughput potential; ③ Easy to operate; ④ Controllable temperature and pH; ⑤ Low sample volume.

[0007] Disadvantages: ① Requires a large amount of plasma and a long equilibrium time; ② Not suitable for unstable compounds; ③ Cannot be used for highly hydrophobic compounds.

[0008] Based on the above problems, a rapid equilibrium dialysis device is proposed to solve the problem that traditional ED technology requires a large amount of plasma and a long equilibrium time. Summary of the invention

[0009] The purpose of the present invention is to solve the shortcomings in the prior art and to propose a rapid equilibrium dialysis device.

[0010] In order to solve the above-mentioned background technical problems, the present invention adopts the following technical solutions:

[0011] A rapid equilibrium dialysis device comprises a bottom plate, a dialysis pool and a plug-in are arranged in the bottom plate, a dialysis membrane is arranged on the plug-in, an independent dialysis membrane chamber is formed in the dialysis pool by enclosing the plug-in and the dialysis membrane, and the peripheral part of the dialysis pool and the dialysis membrane chamber is a buffer chamber;

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

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

[0014] As a further solution of the present invention: the material of the dialysis pool is medical grade plastic.

[0015] As a further solution of the present invention: the wall thickness of the dialysis pool is 1-3 mm.

[0016] As a further solution of the present invention: the dialysis membrane is a semipermeable membrane, and the molecular weight cutoff of the semipermeable membrane 4 is different according to the customer's application. Dialysis membranes with different molecular weight cutoffs can be selected, such as 3.5KD, 6-8KD, 12-14KD, etc.

[0017] As a further solution of the present invention: the flattened width of the semipermeable membrane is 9 to 11 mm, and the permeability coefficient of the dialysis membrane to water is not less than 1×10 -6 cm / s.

[0018] As a further solution of the present invention: the cross-sectional shape of the dialysis membrane chamber is elliptical or polygonal.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] The rapid equilibrium dialysis device of this scheme 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 dialysis is rapid, the equilibrium time is greatly reduced, potential problems such as volume shift and protein leakage can be minimized, and non-specific binding in the device is also reduced. It can be specifically divided into the following points:

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

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

[0023] ③ High-throughput-fast equilibrium dialysis device design makes it suitable for scale-up and automation;

[0024] ④Flexibility—Any number of tests (1-48 tests / board) can be performed without wasting the entire board;

[0025] ⑤ Certainty - The disposable base plate, packaging and partition design of the pre-set cannula eliminate the possibility of cross contamination or leakage;

[0026] ⑥ Reproducibility and accuracy - The validated plasma protein binding assay generates results that are consistent with those reported in the literature. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a structural schematic diagram of the present invention;

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

[0029] Figure 3 This is a data diagram of an example of the present invention.

[0030] Description of the numbers in the figure:

[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 invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention; it is obvious that the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments, and all other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] See also Figure 1 A rapid equilibrium dialysis device comprises a bottom plate 2, a dialysis pool 1 is arranged in the bottom plate 2, a plug-in 3 can be installed in the dialysis pool 1 for plugging in the plug-in 3, a dialysis membrane 4 is arranged on the plug-in 3, an independent dialysis membrane chamber 5 is formed in the dialysis pool 1 by enclosing the plug-in 3 and the dialysis membrane 4, the cross-sectional shape of the dialysis membrane chamber 5 is elliptical or polygonal, and the outer part of the dialysis pool 1 and in the outer periphery of the dialysis membrane chamber 5 is a buffer chamber 6.

[0035] Before equilibrium dialysis, the bottom plate 2 was placed in a 20% ethanol solution for 10 minutes and then rinsed with deionized water.

[0036] Insert 3 does not need to be soaked prior to equilibration dialysis.

[0037] The invention combines equilibrium dialysis (ED) technology, which is also driven by concentration. It separates free protein-bound drugs in a small volume of plasma through selectivity with different molecular weight cutoff points (MWCO) and diffusion through permeable membrane pores. The difference between it and traditional ED is that the rapid equilibrium dialysis of this scheme shortens the dialysis time through a high surface-to-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 device.

[0038] Working principle:

[0039] After the drug binds to plasma proteins at a certain ratio, both bound and free forms will exist in the plasma. The free form has drug activity, while the bound form temporarily loses its pharmacological activity and is stored in the blood, acting as a drug depot, which is of great significance to the drug's effect and how long it lasts.

[0040] The dialysis membrane chamber 5 is separated from the buffer chamber 6 by a dialysis membrane 4, and the plasma is placed in the dialysis membrane chamber 5. Macromolecules such as proteins cannot pass through the dialysis membrane 4, but free drugs can pass freely. When equilibrium is reached, the concentrations of free drugs on both sides of the dialysis membrane 4 are equal. If the total amount of drugs in the system is known, the concentration of drugs in the protein-free membrane chamber can be measured to deduce the plasma protein binding rate of the drugs.

[0041] The material of the dialysis pool 1 is medical grade plastic, which has good chemical stability and physical properties. The top of the plug 3 can visually observe the solution state in the dialysis membrane chamber 5 and the buffer chamber 6. In addition, the wall thickness of the dialysis pool 1 is 1-3 mm to ensure sufficient mechanical strength to ensure that it will not break due to internal pressure changes or external minor collisions during normal use and operation.

[0042] Furthermore, the dialysis membrane 4 is a semipermeable membrane. The molecular weight cutoff of the semipermeable membrane 4 can be selected according to different customer applications, such as 3.5KD, 6-8KD, 12-14KD, etc. The flattened width of the semipermeable membrane 4 is 9-11mm to ensure that it has sufficient mechanical strength and can achieve efficient material exchange. The permeability coefficient of the dialysis membrane 4 to water is not less than 1×10 -6 cm / s, ensuring that water can reasonably migrate on both sides of the dialysis membrane 4 during the dialysis process and maintaining the osmotic pressure balance in the dialysis membrane chamber 5 and the buffer chamber 6.

[0043] Example 2

[0044] Based on the above embodiment, the method of rapid equilibrium dialysis includes the following steps.

[0045] Step 1: Preparation of equipment and reagents:

[0046] 1. Base plate preparation: Soak the base plate in 20% ethanol solution for 10 minutes, then rinse with deionized water;

[0047] 2. Plug-in preparation: Assemble the plug-in and the base plate;

[0048] 3. Plasma sample requirements: The pH value of plasma samples should be 7.4±0.1;

[0049] 4. Preparation of positive substrate.

[0050] Step 2: Experimental design:

[0051] 1. Test group setting: add the test sample to the plasma, set the final concentration of the test substance to 5 μM, and keep the organic reagent content no more than 5%;

[0052] 2. Positive control group setting: warfarin sodium was added to the plasma and its final concentration was set to 5 μM to verify the normal operation of the incubation system;

[0053] 3. Negative control group setting: Add the test substance or positive control into PBS to verify that the test substance or positive control can pass through the semipermeable membrane and is evenly distributed on both sides of the membrane.

[0054] Step 3: Incubation experiment:

[0055] 1. After the rapid equilibrium dialysis device is assembled, add an appropriate amount of PBS to the buffer chamber and an appropriate amount of incubated plasma sample to the dialysis membrane chamber to keep the final liquid levels on both sides consistent;

[0056] 2. Cover the top of the dialysis pool with a sealing film to prevent evaporation to maintain pH value, and place the entire device at 37°C and 5% CO 2 Incubate in an incubator with shaking for 4-6 hours until both sides of the membrane reach equilibrium;

[0057] 3. After the sample incubation is completed, acetonitrile is used to terminate the incubation. After termination, the supernatant of the sample to be tested is taken for LC-MS / MS.

[0058] Step 4: Data analysis:

[0059] 1. Use analytical methods such as LC-MS / MS to determine the concentration or peak area of ​​the test sample;

[0060] 2. Treat and analyze the PBS in the buffer chamber by using acetonitrile for termination and pre-processing, and take the supernatant for analysis on the machine for free test sample concentration (Cf);

[0061] 3. Process and analyze the plasma in the dialysis membrane chamber, and take the supernatant for analysis on the machine for the determination of the total concentration (Cp) of the test sample;

[0062] 4. The plasma protein binding of the test substance in the plasma is determined by calculating the binding rate. The plasma protein binding rate is calculated according to the formula: binding rate (%) = (1-Cf / Cp) × 100.

[0063] Embodiment 3:

[0064] Taking the SD rat plasma protein binding test as an example, Figure 3 As shown;

[0065] Rat-6h was the test group, and the results showed that its plasma protein binding rate was higher than 99%, meeting the test plasma protein binding rate requirement of ≥98%.

[0066] Rat-6h-PBS was the negative control group. The results showed that the positive controls on both sides could pass through the semipermeable membrane, and the data on both sides were nearly consistent, further verifying the accuracy of the test.

[0067] The above is only a preferred specific implementation of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved conception within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A rapid equilibrium dialysis device, comprising a base plate (2), a dialysis pool (1) being arranged in the base plate (2), a plug-in unit (3) being installed in the dialysis pool, and a dialysis membrane (4) being arranged on the plug-in unit (3); Features: An independent dialysis membrane chamber (5) is formed in the dialysis pool (1) by enclosing the plug-in unit (3) and the dialysis membrane (4); the peripheral portion of the dialysis pool (1) and the dialysis membrane chamber (5) is a buffer chamber (6); The bottom plate (2) is placed in a 20% ethanol solution and soaked for 10 minutes before equilibrium dialysis, and then rinsed with deionized water; The insert (3) does not need to be soaked before equilibrium dialysis; The flattened width of the semipermeable membrane (4) is 9 to 11 mm, and the water permeability coefficient of the dialysis membrane (4) is not less than 1×10 - 6 cm / s.

2. The rapid equilibrium dialysis device according to claim 1, characterized in that: The material of the dialysis pool (1) is medical-grade plastic.

3. The rapid equilibrium dialysis device according to claim 1, characterized in that: The wall thickness of the dialysis pool (1) is 1-3 mm.

4. The rapid equilibrium dialysis device according to claim 1, characterized in that: The dialysis membrane (4) is a semipermeable membrane.

Images