title: Label-free electrochemical monitoring of protein addressing through electroactivated "click" chemistry on gold electrodes.
abstract: In this work, using electrochemical impedance spectroscopy (EIS), we have, for the first time, label-free monitored protein immobilization on a gold surface through a strategy of electroaddressing, compatible with the production of microarrays for multi-detection. This functionalization is achieved via the alkyne/azide cycloaddition, better known as the "click" reaction. The electroaddressing was applied to a polythiol hexynyl derivative previously grafted onto the gold surface. This compound consists of two dithiol phosphate groups and a hexynyl function and was synthesized through a supported synthesis approach, from a dithiol reagent, phosphoramidite (DTPA), and a hexynyl phosphoramidite. Next, an azide-PEG3-biotin derivative was grafted onto the modified gold surface by electro-chronocoulometry. The "click" reaction was controlled by electrochemical impedance spectroscopy, showing the change in impedance only when the electroaddressing was performed at -300 mV. No effect on the EIS signal was observed when a positive potential was applied, confirming the specificity of the electroactivation. Biotin-modified electrodes were used to fix streptavidin and the immobilization was monitored using EIS. Fluorescent streptavidin-functionalized silica nanoparticles were also specifically grafted onto the biotinylated gold surface in order to confirm the "click" reaction using fluorescence microscopy. The obtained streptavidin platform was used to detect the surface coverage by biotinylated human serum albumin (HSA). The lowest detectable concentration is 10 pg/mL, and surface saturation is obtained with concentrations higher than 100 ng/mL.
authors.affiliation: Institute of Analytical Sciences, University of Lyon, UMR CNRS 5280, 5 rue de la Doua, Villeurbanne 69100, France; Laboratory of Environmental Engineering, Badji Mokhtar University of Annaba, BP 12, Annaba 23000, Algeria.
authors.forename: Nadir
authors.initials: N
authors.lastname: Meini
authors.affiliation: Institute of Analytical Sciences, University of Lyon, UMR CNRS 5280, 5 rue de la Doua, Villeurbanne 69100, France.
authors.forename: Micaël
authors.initials: M
authors.lastname: Ripert
authors.affiliation: Institute of Analytical Sciences, University of Lyon, UMR CNRS 5280, 5 rue de la Doua, Villeurbanne 69100, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Chaix
authors.affiliation: Institute of Analytical Sciences, University of Lyon, UMR CNRS 5280, 5 rue de la Doua, Villeurbanne 69100, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Farre
authors.affiliation: Institute of Analytical Sciences, University of Lyon, UMR CNRS 5280, 5 rue de la Doua, Villeurbanne 69100, France.
authors.forename: Gabriel
authors.initials: G
authors.lastname: De Crozals
authors.affiliation: Laboratory of Environmental Engineering, Badji Mokhtar University of Annaba, BP 12, Annaba 23000, Algeria.
authors.forename: Rochdi
authors.initials: R
authors.lastname: Kherrat
authors.affiliation: Institute of Analytical Sciences, University of Lyon, UMR CNRS 5280, 5 rue de la Doua, Villeurbanne 69100, France. Electronic address: nicole.jaffrezic@univ-lyon1.fr.
authors.forename: Nicole
authors.initials: N
authors.lastname: Jaffrezic-Renault
mesh_terms: D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002138:Calibration; D057930:Click Chemistry / Q000379:methods*; D058266:Dielectric Spectroscopy; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D006046:Gold / Q000737:chemistry*; D006801:Humans; D008856:Microscopy, Fluorescence; D004369:Pentetic Acid / Q000737:chemistry; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D013194:Staining and Labeling; D019809:Streptavidin / Q000378:metabolism; D013499:Surface Properties
chemical_list: D012709:Serum Albumin; D001710:Biotin; D006046:Gold; D004369:Pentetic Acid; D019809:Streptavidin
keywords: Electroaddressing; Electrochemical impedance spectroscopy; Gold chip; Protein immobilization; Streptavidin; “Click” reaction