Spectroscopic Characterization of Coumarin-Stained Beads: Quantification of the Number of Fluorophores Per Particle with Solid-State¹⁹F-NMR and Measurement of Absolute Fluorescence Quantum Yields

Abstract

The rational design of nano- and micrometer-sized particles with tailor-made optical properties for biological, diagnostic, and photonic applications requires tools to characterize the signal-relevant properties of these typically scattering bead suspensions. This includes methods for the preferably nondestructive quantification of the number of fluorophores per particle and the measurement of absolute fluorescence quantum yields and absorption coefficients of suspensions of fluorescent beads for material performance optimization and comparison. Here, as a first proof-of-concept, we present the first time determination of the number of dye molecules per bead using nondestructive quantitative ((19)F) NMR spectroscopy and 1000 nm-sized carboxylated polystyrene particles loaded with varying concentrations of the laser dye coumarin 153 containing a CF(3) group. Additionally, the signal-relevant optical properties of these dye-loaded particles were determined in aqueous suspension in comparison to the free dye in solvents of different polarity with a custom-built integrating sphere setup that enables spectrally resolved measurements of emission, transmission, and reflectance as well absolute fluorescence quantum yields. These measurements present an important step toward absolute brightness values and quantitative fluorescence analysis with particle systems that can be exploited, for example, for optical imaging techniques and different fluorescence assays as well as for the metrological traceability of fluorescence methods.

Selected Standard Protocols for the Synthesis, Phase Transfer, and Characterization of Inorganic Colloidal Nanoparticles

“…In other words, the quantum yield describes the probability that a photo-excited state gets relaxed by fluorescence rather than by another, non-radiative mechanism. As photons may be emitted in all directions, in principle, a 4-detection geometry is required to detect absolute quantum yields 123,124 . Instead, often relative quantum yields are measured in standard 90° detection To demonstrate how to determine the quantum yield of NPs ( NP ), hydrophobic Au NPs (d c  4 nm) were synthetized (cf.…”

Quantification of amine functional groups on silica nanoparticles: a multi-method approach

“…A wide range of methods is available for the identication and quantication of surface functional groups on nanomaterials. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] They vary in their sensitivity, applicability to specic classes of nanomaterials, and time and resource requirements. Methods such as NMR, thermogravimetric analysis (TGA), and elemental analysis measure total functional group content.…”

“…[21][22][23] In the present study we compare the results from two colorimetric assays for quantication of accessible surface amines to results obtained by labeling amines with tri-uoromethylated probes that provide high sensitivity for XPS and quantitative solid state 19 F NMR. 2,5,19,24,25 The two colorimetric assays are based on the reaction of ninhydrin with primary amines to generate a dye that is released from the surface and quantied in the supernatant and the reaction of an aldehyde with surface amines to generate an imine product that can be cleaved for quantication aer isolation of the NPs. These two assays may differ in their ability to detect surfaceaccessible amines, particularly at high surface coverage where steric effects are expected to become important.…”

Nanoparticles for Bioimaging

“…With the commercial availability of the integrating spheres, the absolute method has increasingly been used to measure quantum yield of a variety of nanoparticles including silica-covered nanocrystals [69], unconverted nanoparticles [70], core-shell nanoparticles [56], polystyrene beads [71], and fluorescent nanopigments [72].…”