RU183227U1 - A device for long-term storage of a biosample with DNA - Google Patents

Abstract

A device for long-term storage of a biological sample with DNA, containing a shockproof sealed container with a removable lid, a capsule for placement of a stored DNA sample enclosed in a hard protective shell, adapted for installation inside the container, a container for a disinfectant solution having a removable lid, on the inner surface of which is fixed with the cap can be detached in the shape of the capsule inlet, with a rod fixed on its inner surface and holding at its end Spruce biological sample. The capsule inlet in the initial state is sealed with a removable protective membrane and preservative material is placed in the capsule cavity in a viscous-flowing state, in a volume sufficient to completely immerse the holder with the DNA sample, capable of forming a solid protective shell of the DNA sample under normal environmental conditions. 5 ill., 7 wp f-ly.

Description

The utility model relates to the field of biotechnology and more specifically relates to a device for long-term storage of biological samples containing DNA molecules, mainly of human origin, which are the carriers of the characteristic genes of each individual.

The treatment of most diseases, organ transplantation, anti-aging therapy - all this is already impossible to imagine today without genetic analysis. In the foreseeable future, the use of gene therapy will go into everyday practice. However, despite the fact that the DNA molecule is relatively stable, adverse environmental effects can lead to its destruction. Among the factors negatively affecting DNA and leading to its damage, we can name the effects of oxygen, ultraviolet and X-ray radiation, chemical hydrolysis, etc. At the same time, having available after 10, 20 or more years of genetic material that has not undergone the destructive effects of the environment, we will have an excellent source for use in the technology of genetic “restoration” of the human body and prolong life.

In addition, stored DNA samples are indispensable for mass emergencies, disasters and natural disasters to search for those in distress and identify victims.

Currently, the most common devices for long-term storage of DNA are cryopreservation devices that store biological samples in liquid nitrogen while maintaining a constant boiling point of nitrogen (-195.8 ° C). These devices allow you to freeze biosamples in the most gentle mode and save them without any damage during essentially unlimited time. However, the implementation of the cryopreservation method requires laboratory conditions, the use of sophisticated refrigeration equipment, the involvement of professionally trained employees, as well as the high costs of maintaining the smooth operation of this equipment and the stability of thermal storage.

Also known is a device for collecting, storing and transporting biological samples, disclosed in the patent of Russian Federation 2616242 and containing a carrier card with a sorption porous material placed on it, onto which a liquid bio-sample containing DNA molecules is applied. Before applying the biosample, the sorption material is impregnated with a preservative composition based on sucrose and ethylenediaminetetraacetic acid, after which the biosample is applied to the carrier card, and it is dried. The device does not require large manufacturing costs, provides storage for a sufficiently long time of liquid bio-samples without losing their original properties, however, it is characterized by low reliability (a carrier card is made of paper) and requires the use of laboratory equipment for the preparation and processing of the bio-sample. Also, this device does not provide a protective barrier and the biomaterial is subject to the action of destroyers and oxidizing agents over time.

In addition, it is known that the device for the immobilization of DNA samples described in the patent of the Russian Federation 2346984, as well as the above, containing a paper carrier, onto which is applied pre-isolated and purified DNA dissolved in bidistilled water. After drying in a thermostat, the DNA carrier is placed in a protective shell, which is used as a laminate film. This device provides reliable long-term storage of DNA molecules, however, for the preparation of DNA preparations and placement of a carrier with DNA in a protective shell requires the use of laboratory equipment and expensive materials.

And finally, it is known (see RF patent 2228358) a device for long-term storage of DNA molecules, which is the closest analogue, which contains a detachable capsule for placing a biosample containing DNA molecules enclosed in a solid protective shell of the corresponding polymer and adapted for installation inside a sealed shockproof container with removable lid. In this device, the capsule has an oval shape and is made of a malleable, oxidation-resistant and non-DNA polluting metal, in particular gold or platinum. In addition, this patent states that before putting DNA into a capsule, it is necessary to undergo a multi-stage purification process, including cell disaggregation, protein removal, DNA isolation, amplification by polymerization, as a result of which the DNA material is obtained in the form of a suspension in an alcohol solution. A portion of the suspension is then introduced into a solution of acrylic or polyacrylic acid in methyl alcohol, the alcohol is slowly evaporated to obtain a viscous gel, and the whole is dried at 50 ° C until a solid block is formed.

This device provides long-term preservation of genetic information for tens of thousands of years, provided that the integrity of the capsule is preserved, but it is expensive and time-consuming and requires special laboratory equipment and personnel to prepare the DNA material for packaging in the capsule.

The basis of this utility model is the task of developing a device for long-term storage of biological samples containing DNA, which would be simple and convenient to use, would not require laboratory equipment to prepare the sample for preservation, and could be used in any, including field conditions and at the same time would not require large expenditures for its manufacture and ensuring long-term storage of genetic material without loss of its quality.

The problem is solved in that the device for long-term storage of a biological sample with DNA, containing a shockproof sealed container with a removable lid and a detachable capsule for placement of a stored DNA sample enclosed in a hard protective shell, adapted for installation inside the container, according to the proposed utility model, further comprises a container for a disinfectant solution having a removable cover, on the inner surface of which a cover is fixed with the possibility of detachment e of the inlet of the capsule, with a rod fixed on its inner surface, bearing a biological sample holder at its end.

In a preferred embodiment, the capsule has a glass-like shape, preserving material is placed in its cavity in a viscous-flowing state, in a volume sufficient to completely immerse the holder with the DNA sample, capable of forming a protective shell of the DNA sample under given conditions, while the inlet of the capsule is sealed by means of a removable protective membrane.

It is possible that the preserving material is an ether-paraffin mixture containing at least two volume fractions of paraffin with a melting point exceeding 42 ° C and no more than one volume fraction of ether with a boiling point not exceeding 25 ° C.

Instead of ether, the preservative material may contain any other volatile substance, for example, liquefied gas, which has the ability to dissolve paraffin and with a boiling point not exceeding 25 ° C.

It is advisable to carry the holder of the DNA sample in the form of a detachable mesh drum, the cell size of which is sufficient for free passage of a viscous flowing ether - paraffin mixture.

It is also desirable to make at least one opening for ester vapor in the capsule lid.

To increase the protection of the capsule from external influences, the container wall can be multilayer, containing, in addition to the external impact-resistant layer, an intermediate heat-insulating layer and an inner layer of elastic material capable of adsorbing ether vapor.

It is advisable to equip the capsule with a unique identification tag.

To use the device as an identifier for an individual, a ring can be fixed on the outer surface of the lid of the container, allowing the owner to constantly carry the container with the DNA sample in the form of a keychain or medallion.

The technical result achieved by using the proposed utility model consists in the fact that with a fairly simple design of the components, the device does not require the involvement of highly qualified personnel for its maintenance, as well as sophisticated laboratory equipment for the selection and preservation of the bio-sample, and besides, it can be successfully used in the field. The device provides long-term (for decades) DNA preservation without loss of initial properties and at the same time does not require large expenditures for its manufacture and subsequent storage (storage occurs in a natural environment)

In the future, the utility model is illustrated by a description of specific options for its implementation and the accompanying drawings, in which:

FIG. 1 shows a capsule for placement of a biological sample in its initial state, sealed with a membrane, isometry;

FIG. 2 - capacity with a disinfectant solution, isometry;

FIG. 3 - a cover with a biosample holder, isometry;

FIG. 4 - capsule with a biosample immersed in a preservative mixture, isometry;

FIG. 5 - container, in general form, partial section, isometry.

A device for long-term storage of a biosample with DNA, according to the present utility model, includes a capsule 1 (Fig. 1) of a glass-like, in this case, cylindrical shape, having an inlet in the initial position hermetically closed by a removable membrane 2 made, for example, from a film or metal foil. The capsule 1 itself can be made of any impact-resistant material that is inert with respect to the bio-sample - metal, glass, ceramic, polymer, etc. In addition, the device includes a container 3 (Fig. 2) with a disinfectant solution 4, hermetically closed by a removable cover 5, connected to the container 3 with a threaded connection. On the inner surface of the lid 5 (Fig. 3) there is a central hollow annular protrusion 6, which is detachably mounted, for example, by means of a tight fit, the lid 7 in the shape of the inlet of the capsule 1, provided with an internal thread corresponding to an external thread made on the neck capsules 1. On the inner surface of the lid 7, a rod 8 is fixed at its center, bearing at its end a biosample holder 9 made in the form of a detachable mesh drum.

In the initial state of the device in the capsule 1 there is a preservative material in a viscous-flowing state, capable of under the given conditions (in this case, at normal ambient temperature and pressure) pass into the solid state. The volume of preserving viscous fluid material was selected taking into account the complete immersion of the holder 9 with the DNA sample and enveloping the entire surface of the biosample with the subsequent formation of a solid protective shell.

In a preferred embodiment, the preservative material is an ether-paraffin mixture containing not less than two volume fractions of paraffin with a melting point of 42-65 ° C. and not more than one volume fraction of ether with a boiling point of not higher than 25 ° C. In addition, the mixture may contain 1.5-3% natural wax as a plasticizer.

The mesh size of the mesh drum of the holder 9 is selected taking into account the free passage of the viscous flowing preservative mixture and enveloping the biosample inside the drum. In the lid 7 (Fig. 4) of the capsule 1, at least one, in the described example, four capillary openings 10 is made for the release of ether vapor, expanding along the vapor.

The composition of the device, according to the utility model, also includes a shockproof sealed container 11 (Fig. 5) with a removable lid 12, designed to place a capsule 1 with a bio-sample in it.

The container 11 has a cylindrical shape and is preferably made of anti-allergenic steel. The wall of the container 11 is multilayered and contains, in addition to the outer impact-resistant steel layer 13, an intermediate heat-insulating layer 14, for example, of felt, and an inner layer 15 of elastic, capable of adsorbing vapors of ether, material, for example, foamed polyurethane, sponge rubber or other material, not absorbing water and having cushioning properties. The bottom of the container 11, as well as its side wall, has a similar multilayer structure.

On the outer surface of the lid 12, a ring 16 is fixed, allowing the container to be used as a medallion or keychain for the owner to carry the DNA continuously.

The capsule 1 is provided with a unique identification, preferably an electronic tag, for example, an electronic chip embedded in the wall of the capsule 1. Also, unique information can be applied in any other suitable way, for example, glued in the form of a label on the wall of the capsule.

The device is used as follows.

Remove the lid 5 (Fig. 2) from the container 3 with a disinfecting solution, for example, 96% alcohol or other disinfecting and dehydrating solution, and a solid sample of biological material containing DNA, for example, is placed inside the detachable mesh drum-holder 9 attached to the lid a fragment of the nail plate or hair follicle. Then the lid 5 is returned into place, while the holder 9 with the biosample is completely immersed in the disinfectant solution 4, after which the container 3 is vigorously shaken for "5 seconds. As a result of these actions, the sample is cleaned and dehydrated. Next, from the capsule 1 (Fig. 1) containing a viscous flowing ether-paraffin mixture, remove the removable sealing membrane 2, unscrew the lid 5 (Fig. 2) of the container 3 and, holding the lid 5, move the decontaminated and dehydrated bio-sample located in the mesh drum , into the capsule 1 (Fig. 4), completely immersing the holder 9 with the biosample in a preservative mixture. In this case, the viscous-fluid mixture freely passes through the cells of the mesh drum and envelops the bio-sample from all sides. The capsule 1 is tightly closed, engaging the thread of the cap 7 with the thread of the inlet of the capsule 1, and the cap 5 is disconnected from the cap 7. The capsule 1 is clamped in the hand and paraffin cures for several minutes (2-3 minutes) due to the heat of the hand and evaporation of the ether through the openings 10 in the lid 7. The formed solid paraffin shell on the bio-sample creates a protective barrier that excludes the access of oxidizing agents and moisture to it.

Capsule 1 closed by lid 7 is placed in container 11 (Fig. 5), which is then hermetically closed with lid 12 (screwed, glued, welded, etc.). The elastic inner adsorbing layer 15 absorbs the remaining ether vapor and softens the load on the capsule during sudden movements of the container, the intermediate thermal insulation layer 14 allows the temperature to be comfortable for the bio-sample in the container, preventing the paraffin shell from melting and the outer impact-resistant layer 13 protects the capsule 1 from mechanical damage.

To test the effectiveness of the DNA storage methodology used in the proposed device, an experiment was conducted on 10 experimental samples of biomaterial.

For this, first, under conditions of low temperature (minus 4-7 C), a preserving ether-paraffin mixture was prepared. To a solution of methyl vinyl ether (UN1087) weighing 32 parts, with constant stirring, grains of paraffin (refractory paraffin 65 ° C) weighing 62 parts and wax weighing 6 parts were added. As a result, a viscous flowing substance resembling in appearance a slightly flowing gel was obtained. Next, in a test tube with a capacity of 10 ml, the prepared mixture was placed in an amount of less than half the volume of the tube, which was then sealed by sealing the inlet openings of the tubes with removable foil membranes. The strength parameters of the tubes and sealing membranes were selected taking into account the withstanding of 2-fold excess of internal pressure (1052 mm r.p. X 2 = 2104 mm r.p.). Sealed tubes were stored at room temperature. DNA bio-images in the form of a fragment of a nail plate were placed in mesh detachable holders. Then the holders with the biosample were placed and kept in a container with 96% ethanol for 5 seconds, as a result of which the biosamples were dehydrated and disinfected. After that, each biosample treated with a disinfectant solution was kept in the open air for 5-7 seconds in order to evaporate alcohol residues. A membrane was removed from tubes containing a viscous fluid mixture of methyl vinyl ether and paraffin, and a holder containing a dehydrated and purified bio-sample was immersed in each tube. In case of leakage in the test tube, the reaction of ether evaporation occurred. In the experiments, the ether evaporation period did not exceed 3 minutes (the preserving mixture was selected taking into account the required evaporation time of no more than 3 minutes at an ambient temperature of 22 ° C).

As a result of the evaporation reaction in test tubes, a solid precipitate was obtained, in the volume of one third of the volume of the tube, completely covering and sealing the bio-sample in itself.

Prototypes of biomaterial were mothballed in a similar way in 1987 on the basis of the Department of Genetics of the Moscow Veterinary Academy named after K. Scriabin. Tubes with preserved biomaterial were placed in a separate metal box and stored from the time of preparation in a laboratory cabinet at an average temperature for such rooms (+18 - + 26 ° C). In 2012, the stored biosamples were dewaxed by dissolution in an appropriate solvent and subsequent lysis to isolate the DNA. Multiple studies conducted over the entire period using the samples stored in 1987 showed that, during DNA lysis, the amount of material obtained and its integrity did not essentially differ from similar parameters for freshly harvested biosamples.

The experiment confirmed the effectiveness of this method of preservation of biosamples and the possibility of their long-term (20 years or more) storage without loss of quality in environmental conditions.

The device and the method of its application described in this utility model allow outside the laboratory, without the use of any special equipment and without attracting significant costs, to ensure reliable long-term storage of biomaterial without loss of its quality.

The embodiment of the device, adapted for constant wear by the owner and equipped with a unique electronic tag containing information about the owner, will be indispensable in the case of mass emergency situations, in catastrophes and natural disasters to search for victims and identify victims. In this case, an ordinary smartphone with the appropriate program for reading information or detecting the capsule is sufficient, including from a remote database associated with the chip embedded in the capsule.

Also, it should be noted that it is possible when changing the materials mentioned in this utility model to similar in their properties, as well as changing the composition of the preserving mixture to a similar one, adapted to create a protective polymer shell, outside the laboratory and specialized institutions, for a short period of time ( no more than 5 minutes) to achieve the cumulative above effect.

Claims (8)

1. Device for long-term storage of a biological sample with DNA, containing a shockproof sealed container with a removable lid and a detachable capsule for placement of a stored DNA sample enclosed in a hard protective shell, adapted for installation inside the container, characterized in that it further comprises a container for a disinfectant solution, having a removable lid, on the inner surface of which is fixed with the possibility of detachment of the lid in the form of the inlet of the capsule, with fixed on it the inner surface of the rod, bearing at its end the holder of the biological sample. 2. The device according to p. 1, characterized in that the capsule has a glass-like shape, preserving material is placed in its cavity in a viscous-flowing state, in a volume sufficient to completely immerse the holder with the DNA sample, capable of forming a protective shell of the DNA sample under given conditions, while the inlet of the capsule is sealed by a removable protective membrane. 3. The device according to p. 2, characterized in that the preservative material is an ether-paraffin mixture containing at least two volume fractions of paraffin with a melting point of 42-65 ° C and not more than one volume fraction of ether with a boiling point not higher than 25 ° FROM. 4. The device according to p. 3, characterized in that the holder of the DNA sample is made in the form of a detachable mesh drum, the cell size of which is sufficient for free passage of a viscous flowing ether-paraffin mixture. 5. The device according to p. 3, characterized in that at least one hole for the exit of ether vapor is made in the capsule lid. 6. The device according to p. 5, characterized in that the wall of the container is multilayer and contains, in addition to the outer impact-resistant layer, an intermediate heat-insulating layer and an inner layer of elastic material capable of adsorbing ether vapor. 7. The device according to any one of the preceding paragraphs, characterized in that the capsule is equipped with a unique identification mark. 8. The device according to p. 7, characterized in that on the outer surface of the lid of the container is fixed a ring for the possibility of constant individual wearing of the container in the form of a keychain or medallion.

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