title: Oriented Bioconjugation of Unmodified Antibodies to Quantum Dots Capped with Copolymeric Ligands as Versatile Cellular Imaging Tools.
abstract: Distinctive optical properties of inorganic quantum dot (QD) nanoparticles promise highly valuable probes for fluorescence-based detection methods, particularly for in vivo diagnostics, cell phenotyping via multiple markers or single molecule tracking. However, despite high hopes, this promise has not been fully realized yet, mainly due to difficulties at producing stable, nontoxic QD bioconjugates of negligible nonspecific binding. Here, a universal platform for antibody binding to QDs is presented that builds upon the controlled functionalization of CdSe/CdS/ZnS nanoparticles capped with a multidentate dithiol/zwitterion copolymer ligand. In a change-of-paradigm approach, thiol groups are concomitantly used as anchoring and bioconjugation units to covalently bind up to 10 protein A molecules per QD while preserving their long-term colloidal stability. Protein A conjugated to QDs then enables the oriented, stoichiometrically controlled immobilization of whole, unmodified antibodies by simple incubation. This QD-protein A immobilization platform displays remarkable antibody functionality retention after binding, usually a compromised property in antibody conjugation to surfaces. Typical QD-protein A-antibody assemblies contain about three fully functional antibodies. Validation experiments show that these nanobioconjugates overcome current limitations since they retain their colloidal stability and antibody functionality over 6 months, exhibit low nonspecific interactions with live cells and have very low toxicity: after 48 h incubation with 1 μM QD bioconjugates, HeLa cells retain more than 80% of their cellular metabolism. Finally, these QD nanobioconjugates possess a high specificity for extra- and intracellular targets in live and fixed cells. The dithiol/zwitterion QD-protein A nanoconjugates have thus a latent potential to become an off-the-shelf tool destined to unresolved biological questions.
authors.affiliation: Laboratoire de Physique et d'Étude des Matériaux, ESPCI ParisTech, PSL Research University, CNRS, Sorbonne Universités, UPMC Univ. Paris 6; LPEM , 10 rue Vauquelin, F-75231 Paris Cedex 5, France.
authors.forename: Mariana
authors.initials: M
authors.lastname: Tasso
authors.affiliation: Laboratoire Aimé Cotton, CNRS, ENS Cachan, Univ. Paris-Saclay , Bâtiment 505, Campus d'Orsay, 91405 Orsay, France.
authors.forename: Manish K
authors.initials: MK
authors.lastname: Singh
authors.affiliation: Laboratoire de Physique et d'Étude des Matériaux, ESPCI ParisTech, PSL Research University, CNRS, Sorbonne Universités, UPMC Univ. Paris 6; LPEM , 10 rue Vauquelin, F-75231 Paris Cedex 5, France.
authors.forename: Emerson
authors.initials: E
authors.lastname: Giovanelli
authors.affiliation: Laboratoire de Physique et d'Étude des Matériaux, ESPCI ParisTech, PSL Research University, CNRS, Sorbonne Universités, UPMC Univ. Paris 6; LPEM , 10 rue Vauquelin, F-75231 Paris Cedex 5, France.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Fragola
authors.affiliation: Laboratoire de Physique et d'Étude des Matériaux, ESPCI ParisTech, PSL Research University, CNRS, Sorbonne Universités, UPMC Univ. Paris 6; LPEM , 10 rue Vauquelin, F-75231 Paris Cedex 5, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Loriette
authors.affiliation: Laboratoire de Biotechnologies et Pharmacologie Appliquée, CNRS, ENS Cachan, Univ. Paris-Saclay , 61, Avenue du Président Wilson, 94235 Cachan, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Regairaz
authors.affiliation: Laboratoire de Biotechnologies et Pharmacologie Appliquée, CNRS, ENS Cachan, Univ. Paris-Saclay , 61, Avenue du Président Wilson, 94235 Cachan, France.
authors.forename: François
authors.initials: F
authors.lastname: Dautry
authors.affiliation: Laboratoire Aimé Cotton, CNRS, ENS Cachan, Univ. Paris-Saclay , Bâtiment 505, Campus d'Orsay, 91405 Orsay, France.
authors.forename: François
authors.initials: F
authors.lastname: Treussart
authors.affiliation: Brain Plasticity Unit, PSL Research University, ESPCI ParisTech, CNRS UMR 8249 , 10 rue Vauquelin, 75005 Paris, France.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Lenkei
authors.affiliation: Laboratoire de Physique et d'Étude des Matériaux, ESPCI ParisTech, PSL Research University, CNRS, Sorbonne Universités, UPMC Univ. Paris 6; LPEM , 10 rue Vauquelin, F-75231 Paris Cedex 5, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Lequeux
authors.affiliation: Laboratoire de Physique et d'Étude des Matériaux, ESPCI ParisTech, PSL Research University, CNRS, Sorbonne Universités, UPMC Univ. Paris 6; LPEM , 10 rue Vauquelin, F-75231 Paris Cedex 5, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Pons
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism; D000067493:Dynamic Light Scattering; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D008024:Ligands; D061986:MCF-7 Cells; D057054:Molecular Imaging / Q000379:methods*; D058726:Nanoconjugates / Q000737:chemistry*; D010316:Particle Size; D045663:Quantum Dots / Q000737:chemistry*; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism
chemical_list: D000906:Antibodies; D015820:Cadherins; D055767:Immobilized Proteins; D008024:Ligands; D058726:Nanoconjugates; D043884:Receptor, Cannabinoid, CB1; D013205:Staphylococcal Protein A
keywords: antibodies; oriented bioconjugation; quantum dots; receptor tracking; toxicity