JPH04358527A - Dialysis method and device for trace solutions - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for dialysis of trace amounts of solutions. Dialysis technology using semipermeable membranes is used to purify polymeric substances.

[0002] A semipermeable membrane is a membrane with micropores that allows low molecular weight components to pass through but not high molecular weight components of the components that make up a solution, and includes membranes such as bladder membranes, collodion membranes, and cellophane membranes. This includes membranes, etc. If a mixed solution of high-molecular weight components and low-molecular weight components, such as a protein electrolyte solution, is wrapped in a semi-permeable membrane and placed in pure water, the osmotic pressure will increase to equalize the concentration difference. Due to this action, low molecular weight components and electrolytes in the solution permeate into the pure water through the semipermeable membrane, while pure water permeates into the semipermeable membrane, making it possible to purify proteins.

[0003] A well-known application of dialysis is blood dialysis using an artificial kidney, in which blood is passed through a semipermeable membrane on one side and dialysate is passed on the other side to eliminate unnecessary low levels in the blood. Molecular waste products and water are removed to the dialysate side to purify the blood and adjust its water content.

0004

[Prior Art] With the progress of biotechnology (bioengineering), processing to purify macromolecules such as deoxyribonucleic acids and proteins, and purification after culturing nerve tissue, etc., is commonly performed. The unit amount of samples handled in the field is extremely small, such as microliters (μl), and the efficiency of using conventional chemical handling processes is extremely low.

[0005] Conventionally, a method has been adopted in which a sample solution is placed in a commercially available tube-shaped dialysis membrane, tightly tied at both ends to seal the membrane, and the membrane is immersed in the target solvent for dialysis. It was getting worse.

However, in this method, the area of contact between the dialysis membrane and the sample solution increases, resulting in a large amount of adhesion, which not only reduces the amount recovered, but also causes problems such as adsorption of polymer components to the dialysis membrane. rate is very low.

[0007] Methods for buffer exchange include gel filtration and ultrafiltration, but these are not suitable for processing small amounts of samples.

[0008]

[Problems to be Solved by the Invention] In recent years, advances in equipment have made it possible to perform trace analysis, and as a result, a trace amount of sample is often sufficient, and opportunities to handle trace quantities of samples are increasing.

However, the conventional dialysis method is not suitable for practical use because the yield is too low for a small amount of sample. Therefore, it was necessary to put into practical use a dialysis method for a trace amount of sample solution.

0010

[Means for solving the problem] The above problem is solved by fixing only the dialysis membrane of a cylindrical container equipped with a dialysis membrane at one end so that it is in contact with the buffer solution, and then dropping a sample solution onto the dialysis membrane in this container. This problem can be solved by configuring a method for dialysis of a trace amount of solution, characterized in that the dialysis is performed while stirring the buffer solution.

[0011]

[Operation] As a dialysis method for microsolutions, the present invention involves dropping a sample solution onto the dialysis membrane while the lower surface of the dialysis membrane is in contact with a buffer solution (solvent solution), which takes on a hemispherical shape due to surface tension. Dialysis is performed while maintaining the patient's condition.

[0012] When such a method is adopted, adsorption of polymeric components to the dialysis membrane can be reduced, and dialysis can be carried out efficiently. FIG. 1 shows the dialysis process of the present invention.

That is, the dialysis membrane 2 is brought into contact with the lower end of the cylindrical container 1 made of glass or plastic, tightly closed to prevent wrinkles from forming on the lower surface, and fixed using a rubber band 3 or the like to form the dialysis container 4. Configure. (A in the same figure) Next, a fixing fitting 5 for fixing the cylindrical container 1 to the buffer solution is attached to the dialysis container 4. (B of the same figure) Next, as shown in (C) of the same figure, after fixing the dialysis container 4 to a container 7 equipped with a stirrer 6 (for example, a magnetic stirrer) at the bottom using a fixing fitting 5,
A buffer solution 8 is supplied, and the lower surface of the dialysis membrane 2 is exposed to the buffer solution 8.
so that it is in contact with

[0014] Next, the sample solution 9 is gently dropped onto the dialysis membrane 2 using a pipette or the like and held so that it swells due to surface tension. Then, dialysis is performed while stirring the buffer solution 8 using the stirrer 6.

In this way, the sample solution 9 and the dialysis membrane 2
Since the contact area can be kept to a minimum, it is possible to perform dialysis of trace amounts of solutions with high yields.

[0016]

[Example] A 5 cm square dialysis membrane (Union
A dialysis container was made by cutting open a dialysis tube manufactured by Carbide Corp.

On the other hand, a buffer solution for dialysis (Tris hydrochloric acid with a concentration of 10 mM, pH 8.0) was placed in the container, and the container was fixed so that the dialysis membrane of the dialysis container was in contact with the buffer solution and kept horizontal. Next, on top of this dialysis membrane, sodium thiocyanide (NaSCN, manufactured by Wako Pure Chemical Industries, Ltd.) with a concentration of 3M and 3.66
CAT (chloramphenicol acetyltransferase, Escherichia coli H
B101 (derived from pYEJ001)) containing 50 μl
The sample solution was gently dropped onto the plate and held in a hemispherical shape.

Then, dialysis was carried out all day and night while stirring the buffer solution with a magnetic stirrer to diffuse and transfer NaSCN into the buffer solution. As a reference, dialysis was also performed under the same conditions using the conventional method of dialysis, in which both ends of a dialysis tube (manufactured by Sanko Junyaku) were tied together.

After the dialysis was completed, the sample solution was collected into a minitube using a micropipette, and the total amount was measured, and the enzyme activity of CAT was also measured. Here, the method for measuring CAT activity is as follows: 0.15mM chloralphenicol, 0.3mM acetyl-CoA, 0.
．． 100mM Tris H containing 5mM DTNB
The reaction rate when an appropriate amount of CAT was added to Cl (pH 8.0) and enzymatically reacted was determined by spectrophotometric measurement.

Table 1 shows the results. In the method of the present invention, the volume of the sample increases, but in the conventional method, the volume of the collected sample decreases because the sample solution adheres to the dialysis membrane. ing.

[0021] Furthermore, both the concentration and amount of CAT of the collected sample are far superior to those of the conventional method.

[0022]

[Table 1]

[0023]

Effects of the Invention By implementing the dialysis method according to the present invention, buffer exchange of a trace amount of sample solution can be carried out efficiently with high yield.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of a dialysis process according to the present invention.

[Explanation of symbols]

1 Cylindrical container 2 Dialysis membrane 4 Dialysis container 8 Buffer liquid 9 Sample solution

Claims (2)

[Claims] Claim 1: After fixing only the dialysis membrane of a cylindrical container equipped with a dialysis membrane at one end so that it is in contact with a buffer solution, a sample solution is dropped onto the dialysis membrane in the container, and the buffer solution is stirred. A method for dialysis of a trace amount of solution, characterized by performing dialysis while 2. A dialysis apparatus comprising a dialysis container 4 having a dialysis membrane 2 at one end and a container 7 having a stirrer 6, the dialysis container 4 being detachably held within the container 7. .