Emitters with different dimensionality: 2D cadmium chalcogenide nanoplatelets and 0D quantum dots in non-specific cell labeling and two-photon imaging.

Since CdSe nanoplatelets were reported to have a ten-fold higher two-photon (2P) absorption coefficient as compared to quantum dots, we examined their applicability for cell labeling and 2P imaging. CdSSe/ZnCdS core-shell nanoplatelets and CdSe/ZnS quantum dots, both emitting at 585 nm were encapsulated with an amphiphilic zwitterionic polymer having slightly positive zeta potential. As measured with flow cytometry, glioma C6 cells demonstrated equally efficient uptake of nanoplatelets and quantum dots, despite the different sizes of these two types of nanoparticles. 2P fluorescence microscopy revealed ca. two orders of magnitude higher fluorescence response from nanoplatelets thus offering a chance to use them as highly efficient 2P fluorescent labels in biomedicine.

Highly luminescent Zn-Cu-In-S/ZnS core/gradient shell quantum dots prepared from indium sulfide by cation exchange for cell labeling and polymer composites.

Gradient core-shell Zn-Cu-In-S/ZnS quantum dots (QDs) of small size and with highly efficient photoluminescence were synthesized via a multi-step high-temperature method involving cation exchange. The procedure starts with the preparation of indium sulfide nanoparticles followed by the addition of Cu and Zn precursors. At this stage, Zn replaces Cu atoms and as a result the concentration of Cu ions in the final QDs is only about 5% of the total In content in a QD. Zn incorporation and the formation of a gradient ZnS shell dramatically increases the photoluminescence quantum yield. Furthermore, the formation of the ZnS shell improves the chemical stability of Cu-In-S QDs, as demonstrated by the preparation of polystyrene-QD composites and labeling of glioma cells. This work provides an effective strategy for obtaining efficient and stable fluorophores free of heavy metals.

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes.

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR: The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.

Efficiency of energy transfer from organic dye molecules to CdSe-ZnS nanocrystals: nanorods versus nanodots.

We report on comparative experimental study of FRET efficiency in two different systems: organic dye molecules (donors) and CdSe-ZnS core-shell nanodots or nanorods (acceptors). Fluorescein isothiocyanate was bound chemically to the surface of nanocrystals using cysteine as a linker and the conjugates were embedded into the polymeric films. Contrary to intuitive presumptions based on the order of magnitude larger molar absorption coefficient for nanorods, the experiment demonstrated almost equal efficiency in the energy transfer from FITC to nanorods and nanodots. This effect is attributed to a distance-limited region of nanorod to which an efficient FRET from dye molecule can be achieved. These results may pave the way to hybrid materials with FRET efficiency controlled by the geometry of nanocrystals.

Functionalized nanocrystal-tagged fluorescent polymer beads: synthesis, physicochemical characterization, and immunolabeling application.

A methodology for incorporating solubilized CdSe/ZnS core/shell nanocrystals (NCs) into functionalized carboxylated polystyrene latexes 0.3-1 microm in diameter via a swelling procedure was developed and used for the production of homogeneous, highly fluorescent polymeric beads (HFPBs), which were found to be comparable in brightness to standard polymeric microspheres doped with organic fluorophores and more photostable than the latter by more than 50 times (Fluoresbrite yellow-orange microspheres were used as an example). The three-dimensional (3D) confocal analysis of individual 1-microm HFPB demonstrated that the beads were doped with the NCs almost homogeneously. HFPBs 0.3 microm in diameter were conjugated with anti-mouse polyvalent immunoglobulins and used for immunofluorescent detection of p-glycoprotein, a mediator of the multidrug resistance phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. The photostability of NCs-tagged HFPBs offers obvious advantages for the reconstruction of 3D confocal fluorescence images of antigen distribution, and their exceptionally high brightness combined with photostability permits the detection of a single antigen molecule using a standard epifluorescence microscope.

Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells.

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.