Using the recent polarized resonance synchronous spectroscopic (PRS2) technique, we reported the quantification of photon extinction, absorption, scattering cross-section spectra, and scattering depolarization spectra for AuNPs of different sizes and shapes. The effects of the solvent composition, ligand functionalization, and nanoparticle aggregation on the AuNP photon absorption and scattering have also been experimentally quantified. The light scattering depolarization is close to 0 for gold nanospheres (AuNSs) crossing the entire UV-vis region but is strongly wavelength-dependent for gold nanorods (AuNRs). Increasing the dielectric constant of the medium surrounding AuNPs either by solvents or ligand adsorption increases photon absorption and scattering but has no significant impact on the AuNP scattering depolarization. Nanoparticle aggregation increases AuNP photon scattering. However, even the extensively aggregated AuNPs remain predominantly photon absorbers with photon scattering-to-extinction ratios all less than 0.03 for the investigated AuNP aggregates at the AuNP peak extinction wavelength. The AuNP scattering depolarization initially increases with the AuNP aggregation but decreases when aggregation further progresses. The insights from this study are important for a wide range of AuNP applications that involve photon/matter interactions, while the provided methodology is directly applicable for experimental quantification of optical properties for nanomaterials that are commonly simultaneously photon absorbers and scatterers.

Presented herein is the ratiometric bandwidth-varied polarized resonance synchronous spectroscopy (BVPRS2) method for quantification of material optical activity spectra. These include the sample light absorption and scattering cross-section spectrum, the scattering depolarization spectrum, and the fluorescence emission cross-section and depolarization spectrum in the wavelength region where the sample both absorbs and emits. This ratiometric BVPRS2 spectroscopic method is a self-contained technique capable of quantitatively decoupling material fluorescence and light scattering signal contribution to its ratiometric BVPRS2 spectra through the linear curve-fitting of the ratiometric BVPRS2 signal as a function of the wavelength bandwidth used in the PRS2 measurements. Example applications of this new spectroscopic method are demonstrated with materials that can be approximated as pure scatterers, simultaneous photon absorbers/emitters, simultaneous photon absorbers/scatterers, and finally simultaneous photon absorbers/scatterers/emitters. Because the only instruments needed for this ratiometric BVPRS2 technique are the conventional UV-vis spectrophotometer and spectrofluorometer, this work should open doors for routine decomposition of material UV-vis extinction spectrum into its absorption and scattering component spectra. The methodology and insights provided in this work should be of broad significance to all chemical research that involves photon/matter interactions.

The sample inner filter effect (IFE) induces spectral distortion and affects the linearity between intensity and analyte concentration in fluorescence, Raman, surface enhanced Raman, and Rayleigh light scattering measurements. Existing spectrofluorometric-based measurements treat light scattering and absorption identically in their sample IFEs. Reported herein is the finding that photon scattering and absorption differ drastically in inducing the sample IFE in Stokes-shifted fluorescence (SSF) spectrum, resonance synchronous spectrum (RS2), and the polarized resonance synchronous spectrum (PRS2) measurements. Absorption with an absorption extinction as small as 0.05 imposes significant IFE on SSF, RS2, and PRS2 measurements. However, no significant IFE occurs even when the scattering extinction is as high as 0.9. For samples that both absorb and scatter light, one should decompose their UV-vis extinction spectra into absorption and scattering extinction component spectra before correcting the sample IFE. An iteration PRS2 method was introduced for the experimental decoupling of the photon absorption and scattering contribution. The methodology presented in this work can be easily implemented by researchers with access to one conventional UV-vis spectrophotometer and one spectrofluorometer equipped with a pair of excitation and detection polarizers. This work should be of broad significance in chemical research given the popularity of fluorescence spectroscopy in material characterization applications.

Optical properties of fluorescent materials including their UV–vis absorption, scattering, and on-resonance fluorescence activities are strongly wavelength-dependent. Reported herein is a divide-and-conquer strategy for experimental quantification of fundamental optical constants of fluorescent nanomaterials including their UV–vis absorption, scattering, and on-resonance-fluorescence (ORF) cross-section spectra and ORF fluorescence and light scattering depolarization spectra. The fluorophore UV–vis extinction spectrum is first divided into a blue and a red wavelength region. The UV–vis extinction cross-section spectrum in the blue wavelength region is decomposed into its absorption and scattering extinction spectra straightforwardly using the established polarized resonance synchronous spectroscopic technique. In its red wavelength region, however, the fluorophores can be simultaneous photon absorbers, scatterers, and anti-Stokes-shifted, on-resonance, and Stokes-shifted fluorescence emitters under the resonance excitation and detection conditions. A polarized anti-Stokes’-shifted, on-resonance, and Stokes’-shifted spectroscopic method is developed for quantifying fluorophore absorption, scattering, one-resonance fluorescence (ORF) cross-section spectra, and scattering and ORF fluorescence depolarization spectra in this wavelength region. Example applications of the presented techniques were demonstrated with fluorescent polystyrene nanoparticles, fluorescent quantum dots, and molecular fluorophores Rhodamine 6G and Eosin Y.

An effective intensity-based reference is a cornerstone for quantitative nuclear magnetic resonance (NMR) studies, as the molecular concentration is encoded in its signal. In theory, NMR is well suited for the measurement of competitive protein adsorption onto nanoparticle (NP) surfaces, but current referencing systems are not optimized for multidimensional experiments. Presented herein is a simple and novel referencing system using 15 N tryptophan (Trp) as an external reference for 1 H− 15 N 2D NMR experiments. The referencing system is validated by the determination of the binding capacity of a single protein onto gold NPs. Then, the Trp reference is applied to protein mixtures, and signals from each protein are accurately quantified. All results are consistent with previous studies, but with substantially higher precision, indicating that the Trp reference can accurately calibrate the residue peak intensities and reduce systematic errors. Finally, the proposed Trp reference is used to kinetically monitor in situ and in real time the competitive adsorption of different proteins. As a challenging test case, we successfully apply our approach to a mixture of protein variants differing by only a single residue. Our results show that the binding of one protein will affect the binding of the other, leading to an altered NP corona composition. This work therefore highlights the importance of studying protein−NP interactions in protein mixtures in situ, and the referencing system developed here enables the quantification of binding kinetics and thermodynamics of multiple proteins using various 1 H− 15 N 2D NMR techniques.