EDC/sulfo-NHS

In addition to the potential side reactions of EDC as mentioned previously (Section 1.1, this chapter), the additional efficiency obtained by the use of a sulfo-NHS intermediate in the process may cause other problems. In some cases, the conjugation actually may be too efficient to result in a soluble or active complex. Particularly when coupling some peptides to carrier proteins, the use of EDC/sulfo-NHS often causes severe precipitation of the conjugate. Scaling back the amount of EDC/sulfo-NHS added to the reaction may be done to solve this problem. However, eliminating the addition of sulfo-NHS altogether may have to be done in some instances to preserve the solubility of the final product. 

A number of BODIPY derivatives that contain reactive groups able to couple with amine-containing molecules are commonly available. The derivatives either contain a carboxy-late group, which can be reacted with an amine in the presence of a carbodiimide to create an amide bond, or an NHS ester derivative of the carboxylate, which can react directly with amines to form amide linkages. The three discussed in this section are representative of this amine-reactive BODIPY family. The two NHS ester derivatives react under alkaline conditions with primary amines in molecular targets to form stable, highly fluorescent derivatives. The carboxylate derivative can be coupled to an amine using the EDC/sulfo-NHS reaction discussed in Chapter 3, Section 1.2. 

BODIPY 530/550 C3 is insoluble in aqueous solution, but it may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction. Coupling to amine-containing molecules may be done using the EDC/sulfo-NHS reaction as discussed in Chapter 3, Section 1.2 (Figure 9.29). However, modification of proteins with this fluorophore probably won t yield satisfactory results, since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry. For labeling molecules which contain only one amine group, such as DNA probes modified at the 5 end to contain an amine (Chapter 27, Section 2.1), BODIPY 530/550 C3 will give intensely fluorescent derivatives. 

Figure 9.61 QDs containing carboxylate groups can be coupled to amine-containing proteins or other molecules using the EDC/sulfo-NHS reaction to form amide bond linkages. The intermediate sulfo-NHS ester is negatively charged and will help maintain particle stability due to like charge repulsion between particles.

QD nanoparticles containing carboxylate groups also may be reacted in a two-step EDC/ sulfo-NHS reaction to couple proteins and other molecules containing both amines and car-boxylates. This type of reaction is designed to remove excess EDC activating agent before addition of protein, so protein polymerization cannot occur. 

To each ml of QD solution, add 50 pi of the EDC/sulfo-NHS stock solution. Maintain the pH at 7.0 by the addition of base, if necessary. Small volume reactions may be controlled using a pH stat. 

The following protocol can be used to biotinylate carboxylate-containing molecules in aqueous solution using the EDC/sulfo-NHS reaction. 

Figure 18.22 Biotin-PEG -amine can be used to add a biotin label to carboxylate-containing molecules using the EDC/(sulfo)NHS reaction, which forms a stable amide linkage.

Add EDC (Pierce) to the above solution to obtain at least a 10-fold molar excess of EDC to the protein. Alternatively, a 0.05—0.1 M EDC concentration in the reaction usually works well. Also, add sulfo-NHS (Pierce) to the reaction to bring its final concentration to 5 mM. To make it easier to add the correct quantity of EDC or sulfo-NHS, higher concentration stock solutions may be prepared if they are dissolved and used rapidly. Mix to dissolve. If this ratio of EDC/sulfo-NHS to peptide or protein results in precipitation, scale back the amount of addition until a soluble conjugate is obtained. 

Another method of NHS ester mediated hapten—carrier conjugation is to create reactive sulfo-NHS esters directly on the carboxylates of the carrier protein using the EDC/sulfo-NHS reaction described in Chapter 3, Section 1.2. A carbodiimide reaction in the presence of sulfo-NHS activates the carboxylate groups on the carrier protein to form amine-reactive sulfo-NHS esters. The activation reaction is done at pH... 

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...

Fig. 5). Furthermore, RGD peptides were covalently attached to EDC/sulfo-NHS activated carboxylic acid functionalities of heparin. A wide variety of growth factors (such as FGF-2) can be incorporated through noncovalent interactions with heparin (Freudenberg et al. 2009). Biofunctionalisation was found to correlate well with the heparin content of the structurally different materials (type A to C, Figs. 5 and 7), as shown for the attachment of adhesive ligands. This allows for an independent variation of mechanical properties at constant biomolecular characteristics. 

A companion approach is to form copolymers using pyrrole and pyrrole butyric acid. The copolymer presents available carboxylic acid groups at the surface that may serve to covalently immobilize the IgG. Figure 6 illustrates the covalent tethering of amine-functionalized biotin following EDC/sulfo-NHS activation of the pendant carboxylic acid groups of poly(pyrrole-co-pyrrolylbutyric acid). Subsequent incubation in streptavidin or neutravidin followed by incubation in biotinylated IgG immobilizes the primary antibody to the device surface. This format has been used to build sandwich immunoassays for antigens that are detected by the indirect action of the oxidoreductase enzyme-linked antibodies on the conductivity of the polymer film. 

PDA coatings on filter membranes were prepared and used in assays as previously described 9,12). Antibodies were conjugated to PDA coating surfaces with carboxylic acids incorporated using EDC/sulfo-NHS according to the literature (75). Coatings were stored at 4 °C. Attached liposomes were prepared and used in assays as previously described 14). 

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