Efficiency scale for scattering luminescent particles linked to fundamental and measurable spectroscopic properties

Abstract

Comparing the performance of molecular and nanoscale luminophores and luminescent micro- and nanoparticles and estimating achievable signal amplitudes and limits of detection requires a standardizable intensity scale. This initiated the development of the relative MESF (number of molecules of equivalent soluble fluorochromes) and ERF (equivalent reference fluorophores) scales for flow cytometry and fluorescence microscopy. Both intensity scales rely on fluorescence intensity values assigned to fluorescent calibration beads by an intensity comparison to spectrally closely matching fluorophore solutions of known concentration using a spectrofluorometer. Alternatively, the luminophore or bead brightness (B) can be determined that equals the product of the absorption cross section (σa) at the excitation wavelength (σa(λex)) and the photoluminescence quantum yield (Φpl). Thereby, an absolute scale based on fundamental and measurable spectroscopic properties can be realized which is independent of particle size, material, and luminophore staining or labeling density and considers the sensitivity of the optical properties of luminophores to their environment. Aiming for establishing such a brightness scale for light-scattering dispersions of luminescent particles with sizes exceeding a few ten nanometers, we demonstrate how the brightness of quasi-monodisperse 25 nm, 100 nm, and 1 µm sized polystyrene particles (PSP), loaded with two different dyes in varying concentrations, can be obtained with a single custom-designed integrating sphere setup that enables the absolute determination of Φpl and transmittance and diffuse reflectance measurements. The resulting Φpl, σa(λex), imaginary parts of the refractive index, and calculated B values of these samples are given in dependence of the number of incorporated dye molecule per particle. Finally, a unitless luminescence efficiency (LE) is defined allowing for the direct comparison of luminescence efficiencies of particles with different sizes.

Exploring Simple Particle-Based Signal Amplification Strategies in a Heterogeneous Sandwich Immunoassay with Optical Detection

“…Only for noninteracting dye molecules, as typically found for low loading concentrations, the particle's absorption cross section σ abs (λ exc ) can be directly calculated from ε(λ exc ) of the loading fluorophore determined in a matrix modeling the polarity and refractive index of the particle matrix, and the (average) number of dye molecules per particle. 21 These dyeand matrix-specific considerations as well as the prevention of dye-specific and concentration-dependent aggregation-induced fluorescence quenching have to be considered for the signal intensity and sensitivity optimization of FIAs using particle reporters. 6,22 An elegant, yet rarely utilized, approach to enhance the intensity of fluorescence signals, circumventing dye aggregation-related signal losses, is the release of the signal-generating fluorescent dyes from the particles.…”

“…In this case, reporter brightness can be controlled by the number of absorbing and fluorescent molecules per NP and thus, also by NP size. , For this signal enhancement strategy, B of the particle reporter depends on the particle size, particle matrix, and dye loading concentration, particularly for fluorophores that can interact with each other and form dye aggregates. Only for noninteracting dye molecules, as typically found for low loading concentrations, the particle’s absorption cross section σ abs (λ exc ) can be directly calculated from ε­(λ exc ) of the loading fluorophore determined in a matrix modeling the polarity and refractive index of the particle matrix, and the (average) number of dye molecules per particle . These dye- and matrix-specific considerations as well as the prevention of dye-specific and concentration-dependent aggregation-induced fluorescence quenching have to be considered for the signal intensity and sensitivity optimization of FIAs using particle reporters. , …”