RU2176530C1 - Device for applying electrophoresis - Google Patents

Abstract

FIELD: medical engineering. SUBSTANCE: device has holder having two U-shaped parts joined with locks. The holder is mounted on cell partition and has at least two parallel ribs all along the whole length. One or several membranes are stretched on the lower part. The membranes are clamped between the ribs of the upper and lower parts by means of locks. The upper part of the holder has a row of windows and one or several rows of guiding slits to enable applicator device to pass and to be fixed. The electrodes are L-shaped and manufactured from titanium. The positive electrode is coated with platinum. EFFECT: enhanced effectiveness in setting early stage diagnosis; improved quality of membrane fastening. 7 dwg

Description

 The invention relates to medical equipment and can be used in all medical institutions.

Known devices for electrophoresis in gel and on membrane carriers (hereinafter referred to as membranes) according to patents, copyright certificates, certificates for industrial designs and GOSTs issued in the name of Sobolev V.I., patent N 1780513 dated 11/20/90, "Device for gel electrophoresis, "A.S. N 967470 dated 04/26/07, A.S. N 997672 from 10.23.79, A. S. N 1517938 from 10.06.87, certificates for industrial samples N 11918, N 11919, N 11920 from 04.30.80, GOST 27153-86 "Medical devices and apparatus for processing samples by electrophoresis. "
All of them contain a number of chambers, each of which includes a cuvette with a partition dividing it into two buffer cavities, on which a holder with a membrane or gel is placed, and a lid with electrodes. Membranes and gel are used for applying samples to them, as well as for the electrical connection of the anode-cathode space in turn buffer.

 However, using these devices it is impossible to carry out a number of diagnostic biochemical methods indicated below.

 For the implementation of new methods, new structural solutions with the use of membranes and holders of other structures are needed, which would make it possible to simply, quickly and reliably fasten membranes, apply samples, connect the camera to a power source and get clear fractions.

 The prototype of the claimed device is a device (patent N 1780513 dated November 20, 1990) containing a number of chambers, each of which includes a cell with a partition dividing it into two buffer cavities, on which a gel holder with a working surface is placed, on both sides of which made parallel vertical slots, as well as a cover with electrodes. On the holder, on two sides of the working surface, strips are made, the upper surface of which is parallel to the working surface of the holder. At least one cellulose acetate membrane is superimposed on the slats, the ends of which are inserted into the vertical slots of the holder provided with clamps from the side of its non-working surface. The device is equipped with a template with two parallel shoulders that are in contact with the membrane and the upper surfaces of the strips. The template has a number of windows, two sides of each of which are made with recesses for applying samples.

 This device is convenient for attaching single strips of membranes with a width of 28 mm if it is necessary to study a small number of samples, but it is inconvenient when attaching membranes of standard sizes 90x90 mm and 180x90 mm. Therefore, the disadvantage of this device is that the time spent on attaching six strips of membranes with a width of 28 mm is 15 minutes The time spent on fastening the membrane 90x90 mm and 180x90 mm in the inventive device is 0.5 minutes

The aim of the invention is the ability to conduct the following methods:
- electrophoretic fractionation of serum proteins and lipoproteins;
- electrophoretic separation of isoenzymes of lactate dehydrogenase and alkaline phosphatase;
- determination of homogeneity of serum γ-globulin preparations;
- determination of the uniformity of the precipitated β-lipoprotein fraction.

 All these techniques have been tested on the inventive device at the Department of Biochemistry of the St. Petersburg Sanitary and Hygienic Academy.

 The proposed device and methods are necessary for the diagnosis of lipid metabolism that occurs with coronary heart disease and atherosclerosis, cancer, acute and chronic inflammatory processes in the early stages.

 The proposed device is necessary in determining the purity in the manufacture and use of γ-globulin preparations, for example, at blood transfusion stations.

 This goal is achieved by the fact that in the proposed device, the holder consists of two U-shaped parts fastened with locks. The lower part is mounted on the cell wall and has at least two parallel collars along the entire length of the holder, with one or more membranes stretched to the lower part, which are fastened by the upper part of the holder and locks that hold the collars in the pressed state and the membranes are in tension on throughout the process of electrophoresis. Moreover, the upper part of the holder has a number of windows and contains one or more rows of guide slots for a fixed passage of the applicator. The electrodes are made of titanium and have a L-shape, with the positive electrode covered with platinum.

 The proposed device for electrophoresis (hereinafter “device”) contains one or more electrophoretic cameras and a power supply (not shown).

 In FIG. 1 shows a chamber for electrophoresis; in FIG. 2 and 3 - the lower part of the holder; in FIG. 4 and 5 - the upper part of the holder; in FIG. 6 - electrode; in FIG. 7 - sectional lock.

 The chamber (Fig. 1) contains a cuvette 1 with a partition 2 dividing it into two cavities for buffer 3, on which a holder 4 with a cellulose acetate membrane 6 is placed. The holder consists of two U-shaped parts fastened with locks 13. The lower part is mounted on the partition 2 of the cuvette 1 and has at least two parallel collars 5 along the entire length of the holder. One or several membranes 6 are tensioned on the lower part, which are fastened by the collars 8 of the upper part of the holder 7. Thus, the collars of the upper and lower parts are clamped together the membrane, and the locks 13 keep them in this state until the end of the electrophoresis process. The upper part of the holder has a number of windows 9 and contains one or more rows of guide slots 10 for a fixed passage of the applicator. The electrodes 12 (Fig. 1 and 6) are L-shaped. The positive electrode is platinum coated.

 The U-shaped form of the lower and upper parts of the holder gives the U-shape to the membrane 6, as a result of which the membrane provides electrical contact between the two buffer-electrode spaces 3 of the cell 1.

 A membrane 6 pre-soaked in the buffer is pulled onto the lower part of the holder 4.

 The upper part of the holder 7 is mounted on top, and when pressed, the locks snap into place. In this case, the collars 5 of the lower part and the collars 8 of the upper part are superimposed on each other and the locks 13 hold the membrane 6 in tension until the electrophoresis process is completed.

 Windows 9 of the upper part of the holder are necessary for visual observation of the moment of contact of the applicator with the membrane during application of the sample in order to prevent damage to the membrane and ensure that the sample is applied in the same place. Guide slots 10 are used for fixed application of samples to the membrane, since the sample can be applied in the same place from one to three times.

 The aim of the research was to develop commercially suitable electrodes for electrophoresis, reduce their cost, increase mechanical strength while maintaining resistance to aggressive environments.

 When choosing the materials of electrodes for electrophoresis, we proceeded from the fact that the electrode should have a low polarization resistance, be commercially suitable, mechanically strong, and at the same time it should be resistant to aggressive acid-base environment under the influence of ionization and electrolysis.

 It is known that platinum, palladium and titanium possess the minimum polarization resistance. Electrodes with low polarization resistance can reduce the heating of the electrodes and buffer, so that the destructive effects of electrolysis and ionization can be reduced and more favorable conditions for electrophoresis can be created. Electrodes with low polarization resistance can reduce power consumption and increase the efficiency of the device as a whole.

 The increase in serial suitability, including the efficiency of titanium electrodes, compared to platinum and palladium electrodes is explained by the fact that titanium is 400 times cheaper than platinum and 100 times cheaper than palladium.

 Titanium electrodes have significantly greater mechanical strength, in addition, the diameter of the electrodes and their length can be increased to the required structural dimensions. Calculations show that the diameter of the electrodes should be in the range from 4 to 10 mm, which was confirmed in working chambers. In addition, titanium allows you to develop electrodes of any structural form: round, plate, curly, etc. from a bar.

 The device uses electrodes from a bar of a L-shaped form. It is advisable to use larger diameter electrodes for electrophoresis, since this reduces the density of the electric current when the electrodes contact the buffer, due to the fact that with a slight increase in the diameter of the electrode, the working area of the electrode increases significantly. With a decrease in the density of electric current, the heating of the electrodes and the buffer decreases. This is especially important at high currents. So, when used as a gel carrier, the electric current can reach 200 mA. The use of membranes as a carrier allows one to reduce the electric current by an order of magnitude. So, when using the standard VLADIPOR cellulose acetate membrane with a size of 180x90 mm, the current is 15-20 mA. With a membrane width of 90 mm, the current is 7.2 mA, with a strip of width 28 mm, the current is 1.3 - 1.5 mA.

 Until now, platinum wire with a diameter of 0.1-0.3 mm is used as electrodes in electrophoretic chambers. It connects to the live pin using a screw. The junction due to electrolysis quickly oxidizes, collapses and fails.

 The proposed L-shaped shape of the electrode eliminates any mechanical connection of the electrode with live parts, which significantly (tens of times) increases the durability of the electrodes and the chamber as a whole and provides electrophoresis with stable parameters.

 Platinum plating of electrodes eliminates their corrosion and erosion. Experiments show that when using pure titanium as electrodes, the positive electrode is destroyed more quickly, and the greater the electric current passing through the electrodes, the greater the destruction. Therefore, only the positive electrode can be platinized, which saves twice the platinum chloride acid when applying the platinum coating.

 The device operates as follows.

 1. The device uses a VLADIPOR cellulose acetate membrane MFA-MA N 4 or N 5, which has standard sizes 90x90 mm and 180x90 mm TU 6-05-1903-87. The holder allows you to fix the membrane of any width from 180 mm or less almost instantly.

2. A buffer of the following composition is used:
- sodium barbital - 12.75 g
- barbital - 2.3 g is filled with distilled water to 1000 ml and put in a water bath.

 3. Prepare a 7% solution of acetic acid, for which 7 ml of glacial acetic acid is diluted with distilled water to 100 ml.

 4. For staining proteins, prepare a 0.25% solution of amidochem, for which 250 mg of amidochem dye is dissolved in 100 ml of 7% acetic acid.

 5. For staining lipoproteins use Sudan black, Sudan 3 or Sudan 4 (red). A small amount of dye (at the tip of the scalpel) is placed in 5 ml of isopropyl alcohol. After complete dissolution of the dye, 10 ml of 5% NaOH is added.

 6. To clarify the cellulose acetate membranes, a solution is used consisting of methanol - 87 ml, glacial acetic acid 12 ml and glycerol - 1 ml. This solution is suitable only for fractionation of proteins.

Operating procedure
1. In each part 3 of the cuvette 1, a buffer of 45 to 50 ml is poured.

 2. Moisten the membrane by placing it in the buffer for 1-3 minutes.

 3. The membrane is removed from the buffer and placed between two sheets of filter paper.

 4. The membrane is placed in the holder, which is installed on the partition 2 of the cuvette 1.

 5. Through slots 10 with an applicator, samples are applied to the membrane.

 6. The cuvette with holder, membrane and buffer is closed by a lid and immediately connected to a power source.

7. A constant stabilized voltage is applied to the camera, the value of which is determined depending on the methodology used:
- when fractionating serum proteins and lipoproteins ... 100-150V, at a time of 20-30 minutes.

 - when fractionating lactate dehydrogenase and alkaline phosphatase ... 270V at a time of 50 minutes

 - in determining the homogeneity of serum γ-globulin preparations. .. 100-200V at a time of 30-40 minutes.

 9. After electrophoresis, the membrane is placed in a fixing solution for 10 minutes, then in a staining solution. The buffer from both parts of the cuvette is poured into one container for reuse.

10. To count the obtained fractions, the membrane is placed in glycerol or processed as follows. The membrane is washed in water for 10-15 minutes. Residual moisture is removed with filter paper. Place in ethyl alcohol for 2-3 minutes for the final displacement of water. Then the membrane is placed on the glass, squeezing air from under it, and placed in a clarifying liquid for 3-4 minutes. Then the membrane is placed in a thermostat at 100 ^o C for 4-5 minutes

Device benefits
1. The main advantage of the device is the implementation of the above diagnostic methods.

 2. Improved the quality of fastening membranes, reduced the time spent on their fastening. Membrane of any width is fixed instantly.

 3. Increased durability of the device due to the proposed design of the camera and electrodes, the device is simple and reliable in operation.

Process automation
1. The device is connected to the power source by simply sliding the camera into the connector. The voltage is supplied by one turn of the timer knob, while the time of electrophoresis is set. The camera turns off automatically.

 2. Among the available methods for processing electrophoregrams, the densitometry method is most preferable. However, in recent years, a computer processing method has been widely introduced.

 Currently, on the basis of the St. Petersburg Research Institute of Cardiology of the Ministry of Health of the Russian Federation, we have developed a computer system for electrophoretic research of blood serum.

 The system includes an electrophoretic device, an optical unit, a Pentium 200-mmh computer, a software tool for automated processing of electrophoregrams.

 The system allows you to automatically determine the percentage ratio of fractions and their mass concentration in g / l, detect the presence of abnormal proteins, "paraproteins" that are not characteristic of the blood of a healthy person, detect the absence of fractions, detect changes in the structure of any protein, approximate electrophoregrams depending on amendment program, etc.

3. The device can be included in the package of devices that are serially produced and successfully used for more than ten years:
- electrophoretic device PEFAG-1 TU 64-1-3174-80, reg. Certificate N 78 / 626-82,
- Immunoelectrophoretic devices PIEF-01, PIEF-01-1, PIEF-01-2, PIEF-01-3 TU 64-1-3945-85, reg. Certificate N 96 / 386-178.

 These devices are entered in the State Register of medical devices of the Ministry of Health and the medical industry of the Russian Federation, M., 1996, p. 124.

Claims (1)

 An electrophoresis device comprising a series of chambers, each of which includes a cuvette with a partition dividing it into two buffer cavities, on which a holder with a cellulose acetate membrane is placed, and a cover with electrodes, characterized in that the holder consists of two parts -shaped, which is installed on the partition of the cell and has at least two parallel shoulders along the entire length of the holder, with one or more membranes stretched to the lower part, which are clamped between the shoulders neither of the upper part of the holder has a series of windows and contains one or more rows of guide slots for a fixed passage of the applicator, and the electrodes are made of titanium and have a L-shaped shape, with the positive electrode covered with platinum.

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