Abstract
Fluorescent tagging is a popular method in biomedical research. Using multiple taggants of different but resolvable fluorescent spectra simultaneously (multiplexing), it is possible to obtain more comprehensive and faster information about various biochemical reactions and diseases, for example, in the method of flow cytometry. Here we report on a first demonstration of the synthesis of ultrabright fluorescent silica nanoporous nanoparticles (Star-dots), which have a large number of complex fluorescence spectra suitable for multiplexed applications. The spectra are obtained via simple physical mixing of different commercially available fluorescent dyes in a synthesizing bath. The resulting particles contain dye molecules encapsulated inside of cylindrical nanochannels of the silica matrix. The distance between the dye molecules is sufficiently small to attain Forster resonance energy transfer (FRET) coupling within a portion of the encapsulated dye molecules. As a result, one can have particles of multiple spectra that can be excited with just one wavelength. We show this for the mixing of five, three, and two dyes. Furthermore, the dyes can be mixed inside of particles in different proportions. This brings another dimension in the complexity of the obtained spectra and makes the number of different resolvable spectra practically unlimited. We demonstrate that the spectra obtained by different mixing of just two dyes inside of each particle can be easily distinguished by using a linear decomposition method. As a practical example, the errors of demultiplexing are measured when sets of a hundred particles are used for tagging.
Highlights
Optical microscopy and detection methods, which employ a wide variety of non-invasive optical techniques, are fundamental to understanding the structural, organizational, and dynamic properties of biological systems
Individual dye molecules are encapsulated inside of nanoscopic cylindrical channels formed by the nematic phase of the liquid crystal, which in turn is created by the templating surfactant molecules
Because flow cytometry is expected to be one of the immediate applications of the developed particles, we demonstrate multiplexing using a phantom system, which is similar to the fluorescence detection in flow cytometry
Summary
Optical microscopy and detection methods, which employ a wide variety of non-invasive optical techniques, are fundamental to understanding the structural, organizational, and dynamic properties of biological systems. Among those optical detection modalities, fluorescence microscopy and flow cytometry are important because they facilitate highly sensitive and specific imaging and detection. The most developed (and commercially available) fluorescent nanoparticles are quantum dots (QDs) [12,13,14,15]. Polymer dots [11,17,18], an organic version of QDs, are promising bright particles that are still under development and have a limited number of available semiconductor polymers. Solid dye-doped silica particles were used for a while [5] which are not that bright and require chemical modification of dye, which limits the number of available dyes and adds to the cost
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