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The mesoporous nature of silica nanoparticles provides a novel platform for the development of ultrabright fluorescent particles, which have organic molecular fluorescent dyes physically encapsulated inside the silica pores. The close proximity of the dye molecules, which is possible without fluorescence quenching, gives an advantage of building sensors using FRET coupling between the encapsulated dye molecules. Here we present the use of this approach to demonstrate the assembly of ultrabright fluorescent ratiometric sensors capable of simultaneous acidity (pH) and temperature measurements. FRET pairs of the temperature-responsive, pH-sensitive and reference dyes are physically encapsulated inside the silica matrix of ~50 nm particles. We demonstrate that the particles can be used to measure both the temperature in the biologically relevant range (20 to 50 °C) and pH within 4 to 7 range with the error (mean absolute deviation) of 0.54 °C and 0.09, respectively. Stability of the sensor is demonstrated. The sensitivity of the sensor ranges within 0.2–3% °C−1 for the measurements of temperature and 2–6% pH−1 for acidity. 

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.