title: Selective conjugation of proteins by mining active proteomes through click-functionalized magnetic nanoparticles.
abstract: Superparamagnetic iron oxide nanoparticles (SPIONs) coated with azide groups were functionalized at the surface with biotin (biotin@SPIONs) and cysteine protease inhibitor E-64 (E-64@SPIONs) with the purpose of developing nanoparticle-based assays for identifying cysteine proteases in proteomes. Magnetite particles (ca. 6 nm) were synthesized by microwave-assisted thermal decomposition of iron acetylacetonate and subsequently functionalized following a click chemistry protocol to obtain biotin and E-64 labeled particulate systems. Successful surface modification and covalent attachment of functional groups and molecules were confirmed by FT-IR spectroscopy and thermal gravimetric analysis. The ability of the surface-grafted biotin terminal groups to specifically interact with streptavidin (either horseradish peroxidase [(HRP)-luminol-H2O2] or rhodamine) was confirmed by chemiluminescent assay. A quantitative assessment showed a capture limit of 0.55-1.65 μg protein/100 μg particles. Furthermore, E-64@SPIONs were successfully used to specifically label papain-like cysteine proteases from crude plant extracts. Owing to the simplicity and versatility of the technique, together with the superparamagnetic behavior of FeOx-nanoparticles, the results demonstrate that click chemistry on surface anchored azide group is a viable approach toward bioconjugations that can be extended to other nanoparticles surfaces with different functional groups to target specific therapeutic and diagnostic applications.
authors.affiliation: Institute of Inorganic Chemistry, University of Cologne , Greinstraße 6, D-50939 Cologne, Germany .
authors.forename: Shaista
authors.initials: S
authors.lastname: Ilyas
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ilyas
authors.forename: Renier A L
authors.initials: RA
authors.lastname: van der Hoorn
authors.forename: Sanjay
authors.initials: S
authors.lastname: Mathur
mesh_terms: D001386:Azides / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D057930:Click Chemistry / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D057225:Data Mining / Q000379:methods*; D005290:Ferric Compounds / Q000737:chemistry; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry; D049449:Luminescence; D008280:Magnetics; D008872:Microwaves; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods; D010447:Peptide Hydrolases / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D020543:Proteome; D012235:Rhodamines / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D019809:Streptavidin / Q000737:chemistry; D013818:Thermogravimetry
chemical_list: D001386:Azides; D005290:Ferric Compounds; D010940:Plant Proteins; D020543:Proteome; D012235:Rhodamines; C000499:ferric oxide; D001710:Biotin; D019809:Streptavidin; D010447:Peptide Hydrolases; D007930:Leucine; D003545:Cysteine; C024974:E 64