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

Abstract

International audienceEnhancement 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

Impact of Side Chain Polarity on Non-Stoichiometric Nano-Hydroxyapatite Surface Functionalization with Amino Acids

“…The impact of the different reaction conditions on amino acid affinity for the ns-nHA surface was also quantified using a ninhydrin protocol 20 which characterizes the amine groups free at the ns-nHA surface, and a fluoraldehyde method 21 which involves the digestion of ns-nHA and subsequent accounting of all amine groups present with ns-nHA (attached or free at the ns-nHA surface). The fraction of moles of amino acid attached to moles of sites originally available for grafting on ns-nHA is shown in Fig.…”

“…The type of amino acid may also impact the size of these respective cone projections 9 . In addition, the model used to calculate the theoretically expected number of grafting sites is based on a fully crystalline material 20 . As our ns-nHA is not fully crystalline, it is important to recognize that the ninhydrin and fluoraldehyde assays in effect present a scaled comparison of the success of different amino acid attachment to the surface of ns-nHA.…”

“…The success of amino acid adsorption was further quantified relative to the theoretical availability of grafting sites using both a pre-digestion ninhydrin colourimetric method and a post-digestion fluoraldehyde method. Firstly, the ninhydrin method involves the reaction of free amine groups with a ninhydrin-ethanol solution, and the formation of a Ruhemann’s purple dye anion under basic conditions which can then be photometrically quantified 20 . Utilizing a modified version of an existing ninhydrin staining protocol by Poli et al .…”

Use of a Ceramic Membrane to Improve the Performance of Two-Separate-Phase Biocatalytic Membrane Reactor

“…The mixtures were heated at 100˝C for 5 min and then cooled down in a cool water bath [31]. Blank experiments were performed also by using Al-fibers in order to check that no interference occurs with the membrane matrix and to confirm that the purple color is only due to the presence of amino groups.…”

Solvent-free functionalisation of graphene oxide with amide and amine groups at room temperature

“…In order to minimise the large deviations usually observed in the measurements, we have carried out a modified Kaiser test procedure based on a calibration curve as previously proposed by Poli et al for the quantification of primary amino groups present in ceramic materials. 25 The calibration curve was initially prepared by reaction of hexylamine with ninhydrin, leading to the formation of coloured solutions of RB with known concentrations. The resulting calibration curve (Fig.…”