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Journal for Biophysical Chemistry

A comparative investigation of methods for protein immobilization on self-assembled monolayers using glutaraldehyde, carbodiimide, and anhydride reagents


Three different approaches to the immobilization of proteins at surfaces have been compared. All rely on the creation of surface groups that bind primary amines on lysine residues. Carboxylic acid terminated self-assembled monolayers (SAMs) have been activated using a water soluble carbodiimide to yield an active ester functionalized surface and with trifluoroacetic anhydride to yield a surface anhydride, and amine terminated SAMs have been activated using glutaraldehyde. Although the degree of surface derivatization by n-alkylamines was greater using the carbodiimide and anhydride methods under anhydrous conditions, the glutaraldehyde activation of amine terminated SAMs yielded significantly greater attachment of streptavidin than is achieved using either of the other methods. This is attributed to the susceptibility to hydrolysis of the active species formed by activation of the carboxylic acid terminated monolayers. Patterned protein structures may be formed by using both glutaraldehyde activation of amine terminated thiols and carbodiimide activation of carboxylic acid terminated thiols, in conjunction with selective photo-oxidation of oligo(ethylene glycol) terminated SAMs.


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Ducker, R.E., Montague, M.T. & Leggett, G.J. A comparative investigation of methods for protein immobilization on self-assembled monolayers using glutaraldehyde, carbodiimide, and anhydride reagents. Biointerphases 3, 59–65 (2008).

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