Efficient quantification of primary amine functions grafted onto apatite ceramics by using two UV-Vis spectrophotometric methods

Abstract

Enhancement of osteogenic properties of bone substitutes is best achieved by the covalent attachment of appropriate biomolecules, e.g. proteins and peptides, on amino-functionalized implant materials. Measurement of the free amino surface density of these materials is mandatory before derivatization. Two photometric methods were developed to assay the free amino groups of organosilane molecules covalently grafted onto hydroxyapatite and silicon-substituted hydroxyapatite ceramic surfaces. Reaction of amino-derivatized apatites with ninhydrin led to the formation of Ruhemann's purple quantified by the absorbance at 586 nm. Alternatively, ceramics were reacted with Fmoc-derivatized amino acids in the presence of piperidine, leading to the dibenzofulvene–piperidine adduct measured at 301 nm. Both methods allow the detection of free amino groups down to 1.0 × 10⁻⁸ mol g⁻¹ of apatite-ceramics and, since they are conducted under non-acid conditions, preserve the acid-sensitive structure of hydroxyapatite.