Our paper has been published in ACS Nano!

Our new article has been published in ACS Nano (doi: 10.1021/acsnano.5c13869). 

In this work, the group of prof. Bednarkiewicz together with the group of prof. Gorris from Masaryk University in Brno, Czech Republic, optimized the concentration of Tm³⁺ ions in for efficient FRET and compared two classical approaches for evaluating FRET efficiency: donor emission quenching and donor luminescence lifetimes shortening in the presence of an acceptor. By directly attaching the dyes to the nanoparticle surface, they achieved high energy transfer efficiencies of approximately 90% and 40%, respectively, for ATTO 488. However, these approaches yield different numerical values due to the complex nature of upconversion processes, energy migration within the Yb³⁺/Tm³⁺ network and the repopulation of excited Tm³⁺ states, therefore, they cannot be interpreted independently.  The most sensitive measure of FRET proved to be ratiometric detection, based on the ratio of acceptor to donor emission intensities in well-defined spectral windows (500 – 614 nm and 435 – 485 nm). Although this method does not provide mechanistic insight into the FRET process, it enabled the lowest limits of detection. The wide spectral separation between the blue and red Tm³⁺ emission bands was further exploited to develop a simple multiplexing system capable of distinguishing four ATTO dyes using only a pair of optical filters. This approach demonstrated the potential of Tm³⁺ - based UCNPs for constructing multicolor, highly sensitive FRET biosensing platforms.

ABSTRACT

Upconverting nanoparticles (UCNPs) have emerged as promising alternative donors for resonance energy transfer (FRET)-based biosensing. However, employing UCNPs in FRET assays remains challenging because they display relatively small absorption cross sections and are relatively large as compared to the Förster distance. Thousands of individual donor ions in each UCNP are located within various distances from surface-bound acceptors, complicating the data analysis. While previous studies have explored how the composition and architecture of UCNPs influence FRET, many reports remain qualitative, and multicolor UC-FRET systems involving a single donor and multiple acceptors are less commonly studied than single-donor-single-acceptor systems. To address these challenges, we synthesized UCNPs with an absorbing core (Yb³⁺-doped)/active shell (Yb³⁺, Tm³⁺-doped) nanoparticles systematically varying Tm³⁺ concentrations to optimize the FRET efficiency to surface-bound organic acceptors. A shell composition containing 4% Tm³⁺ yielded the highest FRET efficiency. Moreover, four distinct ATTO dyes showing spectral overlap with the blue emission of Tm³⁺ were used as acceptor dyes on the surface of UCNPs to evaluate FRET efficiencies in spectral and time domains. The differentiation of the four ATTO dyes on one type of upconverting donor nanoparticles using a simple ratiometric approach lays the foundation for the design of multiplexed bioassays. Our results offer a strategy for improving UC-FRET sensitivity through smart core–shell UCNPs designs, donor concentration tuning, and provide important insights into the rational design of more efficient, multicolor, and wash-free UC biosensing platforms.