Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-06-07
2003-05-13
Barts, Samuel (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S030000, C536S043000, C536S044000, C536S055300, C536S057000, C536S090000, C536S099000, C536S120000, C536S124000, C424S488000
Reexamination Certificate
active
06562961
ABSTRACT:
The invention relates to novel polysaccharide derivatives, such as cellulose ethers, i.e. in particular aminoalkyltrialkylsilylcelluloses, their preparation and their use in a process for coating surfaces with defined molecular layers.
The immobilization of biomolecules on solid carriers plays a decisive role in a large number of modern analysis and separation techniques, such as affinity chromatography, bioreactor technique and, in particular, bio- and chemosensory analysis.
For example, a detection in immunological tests and hybridization tests is carried out by specific reaction of receptor molecules adsorbed on a solid substrate surface with the species to be determined. In particular in the field of the highly sensitive detection of individual DNA, RNA, antigen and/or protein molecules, it is very important that the molecules in question are bound specifically and firmly to surfaces, and that unspecific adsorption of molecules at these substrate surfaces is prevented.
Various processes for binding biomolecules on solid substrate surfaces are known.
A simple option is adsorptive immobilization in which the binding on a substrate surface is purely by adsorption via non-covalent interaction. This method has various disadvantages. Immobilization is limited to substrates whose surface properties permit adsorptive binding and ensure sufficient stability. Gaps in the biomolecule layer may be formed by incomplete coating or desorption processes. Finally, control of both orientation and the amount of receptor molecules is unsatisfactory, so that it is difficult to achieve a reproducible preparation.
To avoid these disadvantages, there has been a quest for covalent immobilization processes which allow biomolecules to be bound covalently to solid substrates via functional groups. These covalent processes are based on bifunctional bridge reagents reacting both with the substrate surface and with the biomolecule.
These immobilization processes, which frequently proceed in several steps, are very time-and material-consuming. A further disadvantage of these methods is the formation of inhomogeneous polymeric surface structures, for example of silane films generated in practice, in the presence of moisture; see, for example, Joachim Renken et al., Anal. Chem. 1996, 68, pp. 176 to 182 in “Multifrequency Evaluation of Different Immunosorbents on Acoustic Plate Mode Sensors”.
A further technique for coupling biomolecules on solid substrate surfaces is the self-assembly (SA) technique (see, for example, Kevin L. Prime et al., J. Amer. Chem. Soc. 1993, 115, pp. 10714 to 10721 in “Adsorption of Proteins onto Surfaces Containing End-Attached Oligo(ethylenoxide): A Model System Using Self-Assembled Monolayers”). Here, stable films of organic substances are formed by spontaneous self-assembly of the molecules during adsorption on solid substrates. The best-known SA systems are organic disulphides and thiols on gold surfaces. This method has the disadvantage that it is limited to only a few types of substrate, such as metals or specific oxides.
Related to the SA technique is the Langmuir-Blodgett (LB) technique. If suitable substances are spread on a surface of water, they spread out to form a monomolecular film. With the aid of this technique developed by Langmuir and Blodgett, it is possible to transfer these monomolecular films to solid substrates (see Katharine B. Blodgett et al., Physical Review, Vol. 51, June 1937, pp. 964 to 982 in “Built-Up Films of Barium Stearate and Their Optical Properties”). This process involves a very limited input of time and material.
Particularly ordered and stable films are obtained by using so-called non-arnphiphilic Stab-Haar polymers (for example M. Schaub et al., Thin Solid Films, 210/211, 1992, pp. 397 to 400 in “Investigation of molecular superstructures of hairy rodlike polymers by X-ray reflection”).
During transfer to the solid substrate, the polymer rods orientate themselves in parallel to the dipping direction. A particularly high stability of the films can additionally be achieved by crosslinking alkene substituents present in the polymer in a [2+2]-cycloaddition with UV irradiation (Gerhard Wegner, Thin Solid Films, 216, 1992, pp. 105 to 116 in “Ultrathin Films of polymers: architecture, characterization and properties”).
Cellulose derivatives having olefinic side chains have been used with particular success. After the film has been transferred to the substrate, these can be converted in a modified Lemieux oxidation into carbonyl groups to which biomolecules are coupled covalently as “Schiff bases” (WO-A 95/08770 or Frank Löscher et al., Proc. SPIE Vol. 2928, 1996, pp. 209 to 219).
By varying the length of time of exposure, it is additionally possible to vary the density of coverage. It is possible to couple biomolecules covalently to other cellulose derivatives having free amino or hydroxyl groups by using a bifunctional bridge reagent such as cyanuric chloride (see reference above).
This process has the disadvantage that it is limited to hydrophobic or hydrophobicized substrate types. Thus, hydrophilic glass or quartz substrates, for example, have to be hydrophobicized in complicated wet-chemical steps with the aid of, for example, silane derivatives prior to coating with hydrophobic LB substances.
Accordingly, it is the object of the invention to provide a process and a chemical compound suitable for this process allowing the application of at least one molecule layer onto a wide variety of different surfaces, independently of the hydrophilicity of surfaces.
This object is achieved on the one hand by polysaccharide derivatives, in particular cellulose derivatives having a degree of polymerization of >5, preferably mixed cellulose ethers comprising a) at least one hydrophobic and b) at least one nitrogen-containing substituent.
In preferred embodiments, the mixed cellulose ethers have, as substituent a), a trialkylsilyl and, as substituent b), an aminoalkyl group, where the alkyl radical in particular in the substituent a) has 1 or 2 C atoms and in the substituent b) has 2 to 8 C atoms. Additionally, the polysaccharide derivative may also comprise c) at least one further substituent which carries a group which is crosslinkable photochemically, by a free-radical reaction or thermally.
According to the present invention, the preferred mixed cellulose ethers are to be understood as compounds in which the H of individual OH groups of the cellulose skeleton is replaced by organic or organosilyl groups, i.e. the atom directly adjacent to the O is a C or Si. Furthermore, this expression can also include derivatives which additionally carry further substituents (in particular at the O of the OH group), an example of such an additional substituent being the substituent c). In the actual molecules (as information base, see Lothar Brandt in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A5, 2nd edition, keyword “cellulose ethers”, p. 461 ff.), it is not necessary for each individual molecule unit (anhydroglucose unit) in the cellulose ether molecule to be substituted at one or more OH groups, but the designation of the compound refers to the entirety of the molecules or molecule units, i.e. designates an average value; in general, a maximum of 3 OH groups per molecule unit can be substituted. For the preparation and/or the behaviour of cellulose derivatives comprising the substituents a) or c) (but not b)), reference is made to the literature reference above by Frank Löscher et al. and to Dieter Klemm et al., Z. Chem., 24 (1984), Vol. 2, p. 62 in “4-Dimethylamino-pyridin-katalysierte Synthese von Celluloseestern über organolösliche Synthese von Celluloseestern über organolösliche Trimethylcellulose”.
The object of the invention is furthermore achieved starting with the known process for immobilizing biomolecules on a coated sheet-like carrier material in which the biomolecules are attached at or in the coating. In this case, the process according to the invention is characterized in that the coated sheet-like carrier material c
Loescher Frank
Seeger Stefan
Barts Samuel
Khare Devesh
Molecular Machines & Industries GmbH
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