Ensemble multicolour FRET model enables barcoding at extreme FRET levels

Abstract

Quantitative models of Förster resonance energy transfer (FRET)-pioneered by Förster-define our understanding of FRET and underpin its widespread use. However, multicolour FRET (mFRET), which arises between multiple, stochastically distributed fluorophores, lacks a mechanistic model and remains intractable. mFRET notably arises in fluorescently barcoded microparticles, resulting in a complex, non-orthogonal fluorescence response that impedes their encoding and decoding. Here, we introduce an ensemble mFRET (emFRET) model, and apply it to guide barcoding into regimes with extreme FRET. We further introduce a facile, proportional multicolour labelling method using oligonucleotides as homogeneous linkers. A total of 580 barcodes were rapidly designed and validated using four dyes-with FRET efficiencies reaching 76%-and used for multiplexed immunoassays with cytometric readout and fully automated decoding. The emFRET model helps to expand the barcoding capacity of barcoded microparticles using common organic dyes and will benefit other applications subject to stochastic mFRET.

A single quantum dot-based nanosensor with multilayer of multiple acceptors for ultrasensitive detection of human alkyladenine DNA glycosylase

“…The total FRET efficiency can be derived from three QD-Cy5 subsystems ( i.e. , the QD/Cy5 pairs with Cy5 in position-1, Cy5 in position-2, and Cy5 in position-3, respectively) based on eqn (4)–(6),46 where is the FRET efficiency of the QD/Cy5 pair with Cy5 in position-1, is the FRET efficiency of the QD/Cy5 pair with Cy5 in position-2, and is the FRET efficiency of the QD/Cy5 pair with Cy5 in position-3. The calculated results are as follows: , and the total FRET efficiency is theoretically estimated to be 85.1%, consistent with the value obtained experimentally by fluorescence spectroscopy measurement (84.9%) and the single QD-based nanosensor (85.3%), further confirming the formation of the QD-dsDNA-Cy5 nanosensor with multilayer of multiple Cy5 molecules.…”

Surface Plasmon Enhanced Light Scattering Biosensing: Size Dependence on the Gold Nanoparticle Tag

“…The carboxylate end group of the PEG molecules was activated with standard carbodiimide chemistry to conjugate goat anti-mouse IgG. Goat anti-mouse and mouse anti-goat IgG were used in this study as a model immunoassay [31,32] to investigate the size-dependence of SP-LS. As shown in the UV-Vis spectra (Figure 2), an 8 nm blue shift of the absorbance peak was measured after PEGylation in the case of 17 nm AuNPs; there was no significant shift in the absorbance peak after the conjugation of antibodies.…”

A clinical role for Förster resonance energy transfer in molecular diagnostics of disease

“…Another promising application of FRET in microarrays is to create different barcodes for probing distinct binding events. Due to the innumerable available luminophores (e.g., organic dyes, NPs, lanthanide complexes) and the different properties of luminescence, namely color, lifetime, intensity, and polarization, FRET between luminophores with different emission spectra, lifetimes, and brightness placed at defined distances to each other has the potential to provide extremely high numbers of barcodes for higher-order multiplexed microarray application in diagnostics [24,25]. Theoretically, the combination of only five colors, five lifetimes, and five intensities can result in 5 5 × 5 = 5 25 = 298 × 10 15 (~300 quadrillion) distinguishable codes, if these codes can be spatially separated.…”