EDC-NHS cross-linking

Pieper, J. S., Hafmans, T., Veerkamp, J. H., and van Kuppevelt, T. H. (2000). Development of tailor-made collagen-glycosaminoglycan matrices EDC/NHS cross-linking, and ultrastructural aspects. Biomaterials 21, 581-593. 

Surface immobilization of the capture molecules follows standard procedures that are commonly practiced in many biosensor applications and some are discussed in the previous section. Layers of carboxymethyl dextran. Protein A or Protein G, streptavidin-coated surface, or EDC [N-ethyl-N-(diethylaminopropyl) carbidimide]/NHS (N-hydroxysuccmimide)-based amine coupling through amide bond are used for protein (antibody, receptor, etc.) cross-linking. 

CMC should be able to participate in the two-step reaction using a sulfo-NHS ester intermediate similar to EDC however, there are no reports in the literature to this effect. Protocols for the use of this reagent in biological cross-linking applications should be essentially the same as those given previously for EDC, except substituting a molar equivalent quantity of CMC. See Chapter 3, Sections 1.1 and 1.2 for additional information concerning carbodiimide reactions. 

Fig. 4 Reaction of EDC/sulfo-NHS activated heparin with amine-end-functionahsed star-PEG to form biohybrid gels. Gel materials are additionally modified with adhesion ligands (integrin binding RGD peptides) and loaded with soluble signalling molecules (growth factors, e.g. FGF-2). The covalent cross-links (dashed lines) could he replaced by use of enzymatically cleavable crosslinks (e.g. matrix metalloprotease sensitive peptide sequences) to allow for remodelling of the matrix by invading cells...

Certain proteins are imstable and may deteriorate once they are on the sensor surface, even if the immobihzation procedure has worked efficiently. HIV protease, for example, dissociates into its component monomers over time and the gradual reduction in molecular weight on the sensor surface is seen as a basehne drift on the interaction profile. This makes the kinetic analysis of interactions, particularly those involving small molecules, problematic. An extra post-immobilization activation/cross-linking step with EDC/NHS may be considered in order to achieve a stable basehne. The use of cross-hnking must be empirically determined for each interaction and the process must not interfere with the activity of the protein. 

The efficiency of cross-linking wheat gluten with EDC/NHS was evaluated by measuring ... 

Figure 3. Proposed mechanism of cross-linking proteins with EDC/NHS at different pH.

In covalent immobilization of biomolecules onto the fibers, chemical modifications are made in electrospun polyester in order to produce reactive functional groups in its chain. Primary amine and carboxylate are chemical groups frequently used as intermediates of reaction. Through this strategy, the amino or carboxyl groups present on biomolecules are cross-linked to free carboxyl or amino groups on activated electrospun polyesters. The l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and M-hydroxysuccinimide (NHS) are the most used intermediary reagents in activation reactions of polyesters. 

Immobilization of a protein via the carboxyl group can be easily accomplished by simultaneous reaction of EDC with the protein. The formed product is then incubated with the matrices in the presence of NHS. Using this reaction, the intermolecular cross-linking is substantially reduced. 

Altogether, it can be concluded from these studies that such biofabricated scaffolds should not only solve the problem of severe donor shortfall in case of comeal allograft, but also can be considered as a better and more practical alternative to donor transplantation. Collagen can also be used to develop novel blends suitable for ocular application. For example, collagen has been blended with gelatin and hyaluronic acid (HA) and cross-linked with EDC and NHS to produce films that may have potential use as a corneal repair material [122]. These films were shown to have appropriate hydrophilicity, optical performance, mechanical properties, and good biocompatibility. In addition, the diffusion properties were found to be similar to the human cornea. 

GOD is immobilized on nanospheres Carboxymethyl CDs are bond through EDC/NHS as coupling agents and EP as cross-linker Acyl chloride linking reaction [59]... 

Fig.1 Schematic representation of a physical and a chemical gelatin - ChS network. A physical gelatin network contains triple-helix junctions dotted gray color lines). By chemical cross-linking of this physical network with EDC and NHS, chemical junctions are introduced (O) (Reproduced from Kuijpers et al. 2000b)...

Collagen was mixed with HCI in the aim to dissolve the fibrils and was then blended either with the pluronic cross-linker. The pH of the reaction mixture was adjusted to 5.5 to obtain the optimal pH for EDC activation. The mixture was then activated with EDC and NHS and blended until the solution viscosity began to increase. Subsequently, the CL reaction mixture was compressed between two glass plates. The resulting material was a cross-linked CL hydrogel with sufficient mechanical strength to be manipulated... 

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