Here we address an important roadblock that prevents the use of bright fluorescent nanoparticles as individual ratiometric sensors: the possible variation of fluorescence spectra between individual nanoparticles. Ratiometric measurements using florescent dyes have shown their utility in measuring the spatial distribution of temperature, acidity, and concentration of various ions. However, the dyes have a serious limitation in their use as sensors; namely, their fluorescent spectra can change due to interactions with the surrounding dye. Encapsulation of the d, e in a porous material can solve this issue. Recently, we demonstrated the use of ultrabright nanoporous silica nanoparticles (UNSNP) to measure temperature and acidity. The particles have at least two kinds of encapsulated dyes. Ultrahigh brightness of the particles allows measuring of the signal of interest at the single particle level. However, it raises the problem of spectral variation between particles, which is impossible to control at the nanoscale. Here, we study spectral variations between the UNSNP which have two different encapsulated dyes: rhodamine R6G and RB. The dyes can be used to measure temperature. We synthesized these particles using three different ratios of the dyes. We measured the spectra of individual nanoparticles and compared them with simulations. We observed a rather small variation of fluorescence spectra between individual UNSNP, and the spectra were in very good agreement with the results of our simulations. Thus, one can conclude that individual UNSNP can be used as effective ratiometric sensors.
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Ultrabright Fluorescent Silica Nanoparticles for Multiplexed Detection
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.
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- NSF-PAR ID:
- 10280892
- Date Published:
- Journal Name:
- Nanomaterials
- Volume:
- 10
- Issue:
- 5
- ISSN:
- 2079-4991
- Page Range / eLocation ID:
- 905
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/nano10050905
