DD232066A1 - PROCESS FOR THE CHEMICAL BINDING OF BIOLOGICAL MATERIALS TO SOLID PHASES - Google Patents

Abstract

The invention relates to a method for the immobilization of biological cells, tissues and viruses on siliziumdioxidhaltige surfaces. In this case, the silicon dioxide-containing surface is chemically modified with silane coupling agents and, if appropriate, additionally with other suitable reagents. The biological materials can be chemically bound to the silicic acid-containing surface modified in this way via functional groups contained on them. The advantage of the method is that in this type of immobilization neither morphological nor functional impairments of the cells, tissues and viruses occur.

Description

The chemical bonding between the biological materials and the modified siliceous solid phase can be carried out at pH values between 2.5 and 11.5, depending on the nature of the modification and / or the biological material. Other additives may be necessary to prevent or prevent changes in biological materials. The concentration of the biological materials in the dispersion is suitably chosen so that complete binding to the modified silica-containing solid phase can take place. The biological materials may be animal and / or plant cells and / or tissues and / or microorganisms

be jj.n_d / oryiren. - -

The size of the moldings has no influence on the process, but should suitably between 0.01 cm ³ and 100 m ³ or move between 0.001 cm ² and 1 m ² . The shape of the moldings can assume all conceivable geometric configurations while being open or closed. The modification of the shaped body surfaces takes place in a manner known per se with silane coupling agents and / or hetero- or homobifunctional reagents.

The binding of cells and tissues and viruses to the solid phase occurs via functional groups on the surfaces of the biological materials. In this case, buffers, electrolytes and / or other additives (example: phosphate buffered saline = PBS) are used, neither the functional groups of the biological materials nor those of the modified

block solid phases. ~

The cells, tissues and viruses bound in this way can be used for diagnostic, scientific and biotechnological purposes without further after-treatment while retaining their biological function and their structure.

Surprisingly, there is such a strong fixation that subsequent steps such as several washing operations do not lead to detachment of the cells.

It is also conceivable use of the method according to the invention for the artificial replacement of organs.

Embodiment 1

Commercially available glass microscope slides (20 x 80mm) are in a 1% solution of Aminopropyläthoxysilan in ethanol / water (1: 1) immersed and incubated at 5O ⁰ C for 4 hours. After washing three times in PBS, pH 7.2, the slides are immersed in a 2% glutaraldehyde solution in PBS, incubated for a further 2 hours at 37 ⁰ C and washed again three times in PBS. After drying, 0.1 ml of a suspension of human lymphocytes (1.5 × 10 ⁶ cells / ml, isolated from whole blood by gradient centrifugation) is applied to each slide. Subsequently, the cells are allowed to sediment for 15 minutes at 37 ⁰ C in a humid chamber. Upon impact of the cells on the modified glass surfaces, covalent bonding between amino groups of the cell surfaces and glutaric dialdehyde bonded to the glass occurs. After rinsing the slides is dyed without further treatment in the usual way. Once controls are unmodified, intensively cleaned slides as well as analogously modified method slides, the aldehyde groups were saturated with glycine. These carriers are charged with the same lymphocyte suspension in the above-mentioned manner. The following table shows the number of bound cells in the three specified procedures:

Number of bound number of bound number of bound

Cells on fiction cells on fiction cells on normal,

according to modified according to modified intensively purified

Slides slide slides, aldehyde slides

groups satisfied

χ s χ s xs

73.05 8.24 21.40 8.70 29.90 5.67

(light microscopic evaluation of 5 visual fields on 5 preparations per batch)

Embodiment 2

Glass slide modified according to Example 1 are coated with a suspension of Brucella mellitensis in PBS, pH 7.2, (10 ⁶ cells per ml) and incubated for 15 minutes at 37 ^° C. in a moist chamber. Subsequently, the staining according to GRAM is carried out in the usual methodology. In contrast to conventionally produced control preparations (heat fixation), bacteria show better parameters both in terms of morphological structure and color brilliance.

Embodiment 3

Commercially available Petri dishes (010 cm) are charged with 25 ml of a 1% solution of glycidoxysilane in ethanol / water (1: 1) and incubated for 18 hours at 4 ° C, then washed and then heat sterilized. Thereafter, 25 ml of a suspension of Escherichia coli K12 in PBS, pH 5.5, bacterial count 10 ⁸ / ml, entered and incubated again for 4 hours at room temperature. After washing under sterile conditions with PBS, pH 7.2, the bacteria bound to the bottom of the Petri dish are overlaid with nutrient agar and then incubated at 37 ° C for 18 hours. The result is a dense colonial growth. "

Claims (7)

Invention claim: 1. A process for the chemical bonding of biological materials, preferably of cells, tissues and viruses, to solid phases, characterized in that are used as solid phase moldings having siliceous, chemically modified surfaces and that these moldings with an aqueous dispersion in contact which contains the biological materials, electrolytes and / or buffers and / or other additives, wherein the aqueous dispersion contains up to 50% of organic solvents and the pH of the aqueous dispersion is between 2.5 and 11.5. 2. The method according to item 1, characterized in that are used as the molded body glassy, glassy and ceramic and / or coated with the aforementioned materials body. 3. The method according to item 1 and 2, characterized in that the used three-dimensionally useful shaped body hemispherical, spherical, cylindrical, cubic, conical, spiral and / or combined form of the aforementioned forms that they are both compact and hollow body, that the hollow bodies are open or closed and that the volume of the shaped bodies is 0.01 cm ³ to 100 m ³ . 4. The method according to item 1 and 2, characterized in that the 2dimensional usable moldings can be planned, the surface can be divided into regularly divided districts, wherein the surface of the planar moldings is 0.001 cm ² to 1 m ² . 5. The method according to item 1 to 4, characterized in that the modification of the moldings with silane coupling agents and / or hetero- and homobifunctional reagents takes place. 6. The method according to item 1 to 5, characterized in that the cells, tissues and viruses are all (but not exclusively) human, animal and plant cells, tissues and viruses and bacteria, parasites and fungi. 7. The method according to item 1 to 6, characterized in that at the same time several and / or different cells, tissues and viruses and / or combinations of the aforementioned biological materials are bound. Field of application of the invention The invention relates to a method for the immobilization of biological cells, tissues and viruses on siliziumdioxidhaitige surfaces, preferably made of glass. This technique is used in medical research, diagnostics and therapy, biotechnology and life science research. Characteristic of the known technical solutions The binding of cells and tissues to surfaces is u. a. for working with cell cultures, in particular in biotechnological and diagnostic methods, for the cultured cells are the prerequisite, eg. As virus propagation, production of monoclonal antibodies, artificial organ replacement and in morphological studies (light or fluorescence microscopy, electron microscopy) are used. The fixation of microorganisms and viruses is of great importance for both diagnostic and biotechnological processes. The bond o. G. Materials on suitable surfaces are once solved by nonspecific adsorption and subsequent fixation, which inevitably leads to changes and death of biological materials. A second way is to coat the corresponding surface with a matrix (gelatin, agarose, dextran, proteins and the like), which is adsorbed or bound in a second step. The two patent applications DD-WP G 01 N / 2499925 and DD-WP G 01 N / 2538484 describes the binding of proteins to glass surfaces and their use in immunoassay. With the methodology disclosed there, it is not possible to covalently bind biological cells and tissues as well as viruses via the functional groups located on their surface to chemically modified siliceous materials. U.S. Patent 4,357,142 et al. described the covalent binding of bacteria and fungi to glass surfaces. However, the authors use an adsorptively attached to the glass surface matrix and bind it to bacteria and fungi, so that there is no complete covalent bond between glass and biological material. The GDR patent WP C 12 N / 262260 describes the covalent binding of microorganisms to paper. However, no siliceous carrier is used here. Methods for the chemical covalent binding of biological cells and tissues as well as viruses on siliceous surfaces by means of silane coupling agents are not known. Object of the invention The object of the invention is to bind biological materials in a simple manner to solid phases so that these materials can be repeatedly used in diagnostic, therapeutic, scientific and biotechnological processes. Explanation of the essence of the invention The object of the invention is to develop a method for the chemical bonding of biological materials to silicon dioxide-containing solid phases, in which structural and functional impairments of the bound materials are avoided and, where appropriate, the viability of these materials is maintained. The advantages of such a procedure would be that A more exact statement can be made in morphological examinations (for example statements about cell membrane receptors with the aid of labeled antibodies), since fixation of the objects is a prerequisite for such examinations and a change of the materials takes place with conventional methods (eg organic solvents). In biotechnological methods, the fixation of the living cells (eg animal cells or bacteria) is possible, so that a less expensive separation of the products (eg genetically engineered proteins, monoclonal antibodies) can be carried out by their producers. - In silica-containing solid phases (eg, glass, ceramics) a nearly ideal carrier with respect to mechanical and chemical stability would be used, which guarantees both a wide range of successor methods as well as the frequent reusability. The object of the invention is achieved in that the biological materials are brought into an aqueous solution containing electrolytes and optionally buffer and / or other additives, and this dispersion is brought into contact with moldings having silicon dioxide-containing, chemically modified surfaces.