Fluofit version 4

Fluorescence lifetimes of all the samples were measured using FluoTime 200 (PicoQuant, GmbH, Berlin, Germany) time resolved spectrofluorometer. This instrument contains a multichannel plate detector (Hamamatsu, Japan) and a 470 nm laser diode was used as the excitation source. The front face geometry as shown in scheme 2 was used for these measurements as well. The fluorescence intensity decays were measured under magic angle conditions and data was analyzed with FluoFit version 4.5.3 software (PicoQuant GmbH, Berlin, Germany) using both the exponential reconvolution procedure using non-linear regression (multiexponential deconvolution model) and by the lifetime distribution model (Lorentzian model) [32 (link)]. In the case of multiexponential analysis, the fluorescence decay was analyzed using: $$I\left(t\right)={\int }_{-\infty }^{t}IRF\left(t^{\prime }\right)\sum _i{\alpha }_i{e}^{\frac{-t-t^{\prime }}{{\tau }_i}}$$
Where IRF (t^’) is the instrument response function at time t^’, α is the amplitude of the decay of the i^th component at time t and τ_i is the lifetime of the i^th component. In case of Lorentzian lifetime distribution, all the data were analyzed using the following equation. $$I\left(t\right)={\int }_{-\infty }^{\infty }\rho \left(\tau \right)e^{-\frac{t}{\tau }}{d}_{\tau }$$ Where, $$\rho \left(\tau \right)={\sum }_{i=1}^n\frac{A_i}{\pi }\frac{\frac{{\Delta }_{FWHMi}}{2}}{{(\tau -{\tau }_i)}^2+{({\Delta }_{FWHMi}∕2)}^2}$$ Where A_i is the amplitude of the i^th component, τ_i is the central lifetime value of the i^th distribution. The use of the continuous distribution ρ(τ) minimizes the number of floating parameters in the fitting algorithms.

Absorption spectra of native BSA and BSA Au nanocluster were measured using Carry 50 Bio UV-Vis spectrophotometer. Fluorescence emission spectra were obtained using the Carry Eclipse spectrofluorometer (Varian Inc.). All measurements were done in 1cm × 1cm cuvette. Emission from the tryptophan of native BSA and BSA Au nanocluster were measured using 280 nm excitation. To measure the peak emission of the BSA Au nanoclusters a 360 nm excitation was used. Both measurements were done using appropriate filers on the emission side.
Time resolved intensity decay were measured using the FluoTime 200 (PicoQuant, GmbH, Berlin, Germany) time resolved spectrofluorometer. This instrument contains a multichannel plate detector (Hamamatsu, Japan) and a 290 nm LED was used as an excitation source to measure intensity decay for tryptophan in native BSA and BSA Au nanocluster. To measure the peak emission (650 nm) of BSA Au nanocluster, a 375 nm laser diode was used. The fluorescence intensity decays were measured in magic angle conditions and data was analyzed with FluoFit version 4.5.3 software (PicoQuant GmbH, Berlin, Germany) using the exponential reconvolution procedure using non-liner regression (multiexponential deconvolution model).