DD301459A7 - Process for the immobilization of biomacromolecules - Google Patents

Abstract

The invention relates to a method for the immobilization of biomacromolecules. According to the invention, a sparingly soluble solid containing metal ions, excluding Al ions, in the matrix or in an intermediate layer is reacted with a bifunctional compound containing as functional groups one or optionally two geminal phosphonic acid groups and one functional group of high chemical reactivity.

Description

Field of application of the invention

The invention relates to a method for the immobilization of biomacromolecules on sparingly soluble solids. The field of application of the invention is the use of the products in bulk conversion (biotechnology), separation (affinity, immunochromatography) and analytics (solid-phase diagnostics).

Characteristic of the known state of the art

It is known that biomacromolecules are immobilized on poorly soluble solids by adsorption, ion exchange or kovalente bonding. The latter method is particularly well suited to the generation of stable bonds. Covalent bonding occurs after a chemical modification (activation) of the solid and / or the compound to be immobilized. As solids, natural substances (minerals, chitin, etc.) and synethetic inorganic substances (glass, silicates, etc.) or organic polymers are used. For continuous, technically performed processes, inorganic solids are selected as support materials because of their good mechanical properties. However, as a first step of immobilization they usually require functionalization by reactive coatings.

Methods are known for applying organic coatings to organic or inorganic solids which result in functionalization using bifunctional silanes. Here, a silane of the general formula (R ₁ O) ₃ SiR ₂ ImItR, an alkyl, R _{2 is} a vinyl, γ-aminopropyl, epoxy-alkyl-SH or other radical) in organic or aqueous-organic solution is used and condensing the primary product to form CO-Si, metal-O-Si and / or Si-O-Si bonds at elevated temperature. Both, the use of organic solvents and condensation at elevated temperature are relatively expensive as well as the production of silanes (U.D. DESCHLER, P. KLEINSCHMIT and P. PANSTER Angew. Chemie 9811986] 237-253).

Immobilization of biomakiomolecules to such silanized carriers is effected by direct coupling (eg reaction of amino groups of the biomacromolecule with iEpoxiilane) after modification of the coating layer (eg γ-aminopropylsilane by bifunctional compounds such as dialdehydes, carbodimides, quinones, etc.) or after modification of the biomacromolecule (eg, periodate oxidation of glycoproteins to aldehyde-containing proteins, introduction of polymerizable double bonds, etc.). Disadvantage of the known method is the multi-stage process of silanization and condensation. EP 218506 discloses aluminum compounds which are provided with functional phosphate groups and are carriers for biomolecules. Their production is complicated.

Object of the invention

The object of the invention is to develop a method for the immobilization of biomacromolecules, which allows the primary functionalization of a carrier material by simply treating with an aqueous solution of the functionalizing reagent with a large variety of functional groups.

Explanation of the essence of the invention

According to the invention, a sparingly soluble, polyvalent metal ion except Al-ion-containing solid is treated with a bifunctional compound containing one or two geminal phosphonic acid groups and a functional group of high chemical reactivity, which group then directly or after chemical modification with the biomacromolecule directly or reacts after the chemical modification. The solids used are metal oxides, salts, glasses or organic polymers. Suitable metal ions are those which form compounds or stable complexes which are sparingly soluble with phosphonic acids and which are contained a priori in the matrix or are applied as an intermediate layer. Biomacromolecules are preferably enzymes, antigens, antibodies or nucleic acids, optionally also polysaccharides. The inventive method is suitable for the immobilization of biomacromolecules after direct coating of

Metal oxides and salts, e.g. For example, silicates, phosphates and the like. or after treatment of non-metal-containing inorganic materials such as silica or organic polymers, e.g. Cellulose containing organic organotin compounds, salts, oxysalts, metal halides or oxyhalides for applying functional coatings to these substances. As bifunctional phosphoric acid derivatives are compounds of the general formula

5 'H ₂ PO ₃ -CH-R ₂

wherein R, = -H or -PO ₃ H ₂ and R _{2 is} an -OH, -NH ₂ or -SH substituted alkyl, alkylene, aryl, Alkylenaryl- or alkylaryl, an -O-acryloyl, -N -Acryloyl or epoxy group is.

In particular, the low price and easy accessibility of substituted phosphonic acids are of critical importance to the process of this invention, as broad variations of the alkyl and aryl radicals allow numerous specific applications.

The functional group R: is used for copolymerization (acryloyl or vinyl group), bifunctional compound activation, τ (dialdehydes, bisdiazo compounds, carbodiimides, carbonyldiimidazole, dicarboxylic acid chlorides, quinones

u. a.) In the case of ω-amino groups or for direct coupling (eg., Diazophosphonsäuren) used.

embodiments

example 1

10 g of hydroxyapatite are dissolved in 50 ml of a 6% solution of gem. Vinyl bisphosphonic acid suspended in water, filtered off after 1 h and washed. Subsequently, a solution of 0.5 g of acrolein and 0.5 g of acrylic acid in 50 ml of water is added, filtered off after 2 h and the wet carrier is coated by irradiation with a polymer layer ( ⁶⁰ Co-QUeIIe, 10 ⁶ rad). After washing with phosphate buffer (0.1 mol / l, pH 6.8), 100 mg of horseradish peroxidase antibody (rabbit) are added in 50 ml of buffer.

In 24 h at 4 ° C, the immobilization is completed. The product; is used for the purification of peroxidase.

Example 2

10 g of porous glass are treated with a solution of 2 g of titanium tetrachloride in 25 ml of carbon tetrachloride for 2 h, washed with carbon tetrachloride, acetone and water and then with 50 ml of a 6% solution of 3- (4-aminophenyl) propane-1,1-bisphosphonic acid functionalized. The product is suspended in ice-cold 10% hydrochloric acid 0-4 ⁰ C), treated with a solution of 200mg of sodium nitrite in 2 ml of cold hydrochloric acid and washed after 30min ice-cold dilute hydrochloric acid and ice water. Subsequently, the glass is placed in a solution of 200 mg glucose oxidase in acetate buffer (0.1 mol / l, pH 5.6) and shaken at 4 ^{C for} 24 h. Thereafter, the excess of enzyme is completely washed out and the product is used in an analytical reactor for glucose determination.

Example 3

10 g of macroporous glutaraldehyde crosslinked polyvinyl alcohol are heated to 80 ° C. in a solution of 2 g of tetraethoxyzirconate in 100 ml of toluene, washed, after cooling with toluene, acetone, ethanol and water, and then with 50 ml of a 5% solution of 2-aminoethane-1,1 -bisphosphonic stirred for 2h. After washing with water and phosphate buffer (0.1 mol / l, pH 7.0), the amino group is reacted with excess quinone (3 mmol / l in buffer). The product is washed thoroughly and stirred with 20 ml of a solution of penicillin acylase (100 units / ml) for 10 h at room temperature, washed (H ₂ O, buffer) and used to cleave penicillin G.

Document contemplated: EP-PS 218506

Claims (3)

A process for the immobilization of biomacromolecules characterized in that a sparingly soluble solid of the metal ions except aluminum ions in the matrix or in an intermediate layer which form sparingly soluble compounds or stable complexes with phosphonates is reacted with a bifunctional compound which contains as functional groups one or optionally two gemonal phosphonic acid groups and a functional group of high chemical reactivity, which then reacts directly or after chemical modification with the biomacromolecule directly or after its chemical modification. 2. The method according to claim 1, characterized in that enzymes, antigens, antibodies or nucleic acids are used as biomacromolecules, 3. The method according to claim 1, characterized in that as sparingly soluble solid metal oxides, salts, glasses or organic polymers are used.