Ultrabright fluorescent silica particles with a large number of complex spectra excited with a single wavelength for multiplex applications

Abstract

We report on a novel approach to synthesize ultrabright fluorescent silica particles capable of producing a large number of complex spectra. The spectra can be excited using a single wavelength which is paramount in quantitative fluorescence imaging, flow cytometry and sensing applications. The approach employs the physical encapsulation of organic fluorescent molecules inside a nanoporous silica matrix with no dye leakage. As was recently demonstrated, such an encapsulation allowed for the encapsulation of very high concentrations of organic dyes without quenching their fluorescent efficiency. As a result, dye molecules are distanced within ∼5 nm from each other; it theoretically allows for efficient exchange of excitation energy via Förster resonance energy transfer (FRET). Here we present the first experimental demonstration of the encapsulation of fluorescent dyes in the FRET sequence. Attaining a FRET sequence of up to five different dyes is presented. The number of distinguishable spectra can be further increased by using different relative concentrations of encapsulated dyes. Combining these approaches allows for creating a large number of ultrabright fluorescent particles with substantially different fluorescence spectra. We also demonstrate the utilization of these particles for potential multiplexing applications. Though fluorescence spectra of the obtained multiplex probes are typically overlapping, they can be distinguished by using standard linear decomposition algorithms.

Ultrabright Fluorescent Silica Nanoparticles for Multiplexed Detection

“…However, one can see from the previous paragraph that the contribution of FRET is substantially different for these two types of particles. It is worth noting that a similar nontrivial behavior of FRET was observed when mixing these dyes in micron size nanoporous particles [26]. Investigation of FRET between encapsulated dye molecules is beyond the scope of this work, which is the demonstration of the ability to obtain complex spectra by mixing dyes inside particles, and the potential use of these particles for demultiplexing applications.…”

“…Assuming all particles of the same diameter (Z average is defined by the DLS technique), one can find the average number of the dye molecules of each type encapsulated inside of each particle. To calculate the fluorescence brightness of the particles, the MESF units (molecules of equivalent soluble fluorochrome) are frequently used [14,26,[31][32][33][34][35]. These units are popular in applications in which the quantitative nature of measurements is important, such as flow cytometry.…”

“…Synthesis of Star-dots with five encapsulated dyes (Silica multi-color (Si-MC)): A previously reported procedure was modified for making Star dots encapsulated with five dyes based on mesoporous silica particles [24,26]. The molar ratio was 1 TEOS:8.2 TEA:0.23 CTAB:142 H 2 O:0.1 ETES.…”

Data on ultrabright fluorescent cellulose acetate nanoparticles for imaging tumors through systemic and topical applications

“…Simulations utilizing linear combinations of component spectra (CA and PF-127) were performed to minimize residuals and match nanoparticle spectra (see Ref. [5] for detail). It was determined that CA nanoparticles assembled at a 1:1 CA to guest polymer molar ratio, produces a particle concentration of roughly 1–2 mg/mL with a guest polymer composition of 25% by weight ( Fig.…”

Ultrabright fluorescent nanothermometers

“…Besides ultrahigh brightness (considerably exceeding those of quantum dots), it was found that the distance between the dye molecules became as small as 3-5nm. As a result, one can have an effective Forster resonance energy transfer (FRET) between encapsulated dye molecules 69,70 . The nanothermometers presented in this work are built by utilizing this FRET, which takes place between the reference (donor) and temperature-sensitive (acceptor) dyes.…”