Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells

• Published in: Analytical biochemistry Vol. 324; no. 1; pp. 60 - 67
• Main Authors: Sukhanova, Alyona, Devy, Jérôme, Venteo, Lydie, Kaplan, Hervé, Artemyev, Mikhail, Oleinikov, Vladimir, Klinov, Dmitry, Pluot, Michel, Cohen, Jacques H.M, Nabiev, Igor
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2004; Elsevier Masson
• Subjects: Antibodies - chemistry; Antibodies - immunology; ATP Binding Cassette Transporter, Subfamily B, Member 1 - analysis; ATP Binding Cassette Transporter, Subfamily B, Member 1 - chemistry; Cancer tissue; Cell Membrane - chemistry; Confocal microscopy; Engineering Sciences; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique - methods; Fluorescent Dyes - chemistry; Human health and pathology; Humans; Immunohistochemistry; Immunolabeling; Keratins - analysis; Keratins - chemistry; Life Sciences; MCF7 cancer cells; Membrane Proteins - analysis; Membrane Proteins - chemistry; Membrane Proteins - immunology; Micro and nanotechnologies; Microelectronics; Microscopy, Confocal - methods; Microscopy, Electron - methods; Nanocrystals; Nanotechnology; Optics; p-Glycoprotein; Photonic; Polyamines; Quantum dots; Quinolinium Compounds; Selenium Compounds - chemistry; Selenium Compounds - immunology; Sulfides - chemistry; Sulfides - immunology; Tumor Cells, Cultured; Zinc Compounds - chemistry; Zinc Compounds - immunology; Immunohistochemistry; 3D; Nanocrystals; Quantum dots; p-Glycoprotein; Cancer tissue; Confocal microscopy; MCF7 cancer cells; Immunolabeling
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2003.09.031

A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shell nanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluorescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys were stabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs conjugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We further demonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocal analysis of p-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant to photobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively. The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D images of the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained by the method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, using immunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studies of membrane proteins and cells.