Dye Encapsulation in Fluorescent Core–Shell Silica Nanoparticles as Probed by Fluorescence Correlation Spectroscopy

Abstract

Synthetic advances in the formation of ultrasmall (<10 nm) fluorescent poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles (SNPs), enabling improved particle size and surface chemical property control have led to successful clinical translation of SNPs as diagnostic probes in oncology. Despite the success of such probes, details of the dye incorporation and resulting silica architecture are still poorly understood. Here, we employ afterpulse-corrected fluorescence correlation spectroscopy (FCS) to monitor fast fluorescence fluctuations (lag times <10−5 s) of the negatively charged cyanine dye Cy5 as a probe to study such details for dye encapsulation in 5 nm silica cores of PEGylated core-shell SNPs (C dots). Upon deposition of additional silica shells over the silica core we find that the amplitude of photo-induced cis-trans isomerization decreases, suggesting that the Cy5 dyes are located near or on the surface of the original SNP cores. In combination with time correlated fluorescence decay measurements we deduce radiative and non-radiative rates of the Cy5 dye in these particles. Results demonstrate that FCS is a well-suited tool to investigate aspects of the photophysics of fluorescent nanoparticles, and that conformational changes of cyanine dyes like Cy5 are excellent indicators for the local dye environment within ultrasmall SNPs.

Ultrasmall, Bright, and Photostable Fluorescent Core–Shell Aluminosilicate Nanoparticles for Live‐Cell Optical Super‐Resolution Microscopy

“…This effect has been quantitatively analyzed in earlier static and dynamic spectroscopy studies and is due to an increase in radiative rate and decrease in non‐radiative rate. [ 30–32 ] As shown previously, this brightness enhancement relative to free dye carries over into photon emission enhancements during dye blinking, which is crucial for a potential probe for STORM for which localization precision is proportional to the square root of brightness. [ 4–6 ] Confocal fluorescence correlation spectroscopy (FCS) measurements on the diffusing PEG‐Cy5‐aC’ dots yielded one‐component autocorrelation functions (ACF) suggesting an average particle diameter of 4.6 nm and corroborating the narrow size dispersity reflected in the TEM results (Figure 1d).…”

“…We determined an average of 2.5 Cy5 dyes per particle from the combination of FCS derived sample concentration and absorption spectroscopy results. [ 30,32 ] Furthermore, photobleaching studies based on previously published methods (see Supporting Information) showed that Cy5 encapsulated in aC’ dots is 2.8 times more photostable than in free form (Figure 1e). [ 33 ] Since we already showed in two separate previous studies that Al is fourfold coordinated in aC’ dots, replacing Si in the silica network, we did not repeat the same solid‐state 27 Al nuclear magnetic resonance (NMR) spectroscopy experiments on aC’ dot samples here.…”

Molecular Engineering of Ultrasmall Silica Nanoparticle–Drug Conjugates as Lung Cancer Therapeutics

“…The resulting reaction solution of C' dots was purified by centrifuging with a 30K MWCO PES membrane spin filter, and finally by GPC purification through Superdex 200 resin on a Bio-Rad fast protein liquid chromatography. The purified particles were characterized using fluorescence correlation spectroscopy (FCS) on a home-built setup, equipped with a 633-nm solid-state laser as the excitation source for Cy5(þ) dye, and UV/Vis spectroscopy on a Cary 5000 spectrometer as described previously (43,49). TEM images were taken using a FEI Tecnai T12 Spirit microscope operated at an acceleration voltage of 120 kV.…”

“…As-synthesized DFO-Gef-C' dots were then subjected to morphology, size, and surface payload density characterizations using a combination of transmission electron microscopy (TEM), fluorescence correlation spectroscopy (FCS), and UV/Vis spectroscopy. The hydrodynamic particle size, particle brightness, and concentration were obtained by fitting the FCS autocorrelation curves (43,49). The number of Cy5 dyes (or functional groups of DFO, DBCO) per DFO-Gef-C' dot was calculated using a similar method we reported previously (43,49).…”

“…The hydrodynamic particle size, particle brightness, and concentration were obtained by fitting the FCS autocorrelation curves (43,49). The number of Cy5 dyes (or functional groups of DFO, DBCO) per DFO-Gef-C' dot was calculated using a similar method we reported previously (43,49). Surface payload density (or DPR) of DFO-Gef-C' dot was estimated by measuring and comparing the absorption peak intensity of gefitinib at 346 nm and Cy5 dye at 651 nm.…”