Hybrid silica-nanocrystal-organic dye superstructures as post-encoding fluorescent probes.

The demand for multiplex bioassays without complex instrumentation and processing has driven the development of encoding fluorescent nanoprobes. Organic dyes and luminescent semiconductor nanocrystals (or quantum dots, QDs) are representative fluorescent encoding elements, which have been incorporated into various nanoor microsized spherical supports, such as silica and polymer latex spheres, for highcapacity spectral encoding. Organic-dye-doped nanoparticles give an intense fluorescence signal together with highly improved photostability, which makes them especially suitable for ultrasensitive bioassays. However, the number of available organic dyes with the same excitation wavelength and distinguishable emission spectra is limited, which restricts the number of spectrally distinct codes that can be generated. QDs are bright, highly photostable, and have continuous excitation spectra along with narrow, symmetric, sizetunable fluorescence emission. These unique optical properties make them ideal luminophores for wavelength and intensity encoding. Various approaches have been employed in the preparation of encoding fluorescent probes. To the best of our knowledge, three major approaches are typically adopted to incorporate fluorescent encoding elements, such as organic dyes and QDs. In the first, the encoding elements are encapsulated inside nanoor microsized spheres through electrostatic and hydrophobic interactions, covalent linkage, hydrogen bonding, and physical encapsulation. 4] The second route is to directly assemble the encoding elements on the external surface of spherical supports. In the third approach, the encoding elements are located in concentric shells surrounding a solid core or alternating with nonfluorescent spacer shells through layer-by-layer techniques. Although these approaches have been successfully used to prepare encoding fluorescent nanoor microsized spheres, the process of preparation is rather laborious and time-consuming when the number of codes required for multiplex analysis is large. As the codes manufactured by these methods are based on the incorporation of different fluorescent encoding elements with distinguishable ratios or concentration, the amounts of different encoding elements must be controlled precisely in the preparation of each code. The complex and diverse encoding elements also influence the process of the preparation of spherical supports. Thus, the quest to prepare encoding fluorescent spheres with a more labor-saving process and higher efficiency is of extreme significance. Herein, we report the synthesis and characterization of a well-defined hybrid superstructure that comprises a fluorescein isothiocyanate (FITC)-doped silica core coated with a dense monolayer of nanocrystalline CdTe QDs, followed by a silica shell. We define this superstructure as a hybrid silica– nanocrystal–organic dye superstructure (HSNOS). The HSNOS has shown unique, well-resolved, dual fluorescence emission under a single excitation wavelength. To endow HSNOSs with multicolor fluorescence combinations, which extend their use in multicolor imaging applications, they were encoded subsequently by incubation with different concentrations of Cu ions as an efficient quencher for CdTe QDs. Such a post-encoding strategy, which is different from existing pre-encoding methods, makes the preparation process of multicolor fluorescent spheres rather more labor saving and highly efficient. HSNOSs were synthesized by a multistep procedure (shown in Figure 1), which involved the synthesis of FITCdoped silica cores, modification of the silica surface with amino groups, self-assembly of CdTe QDs on the particle