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. 2005 Apr 15;89(1):572–580. doi: 10.1529/biophysj.104.049627

FIGURE 2.

FIGURE 2

Sensitized photoreduction of Ag+ in the presence of ThT resulted in the generation of hybrid Ag-ThT nanoclusters. (a) Emission spectra in aqueous solution of ThT alone (10 μM, solid line) and immediately after addition of AgNO3 (0.1 μM, dashed line) collected upon excitation at 330 nm.(b) Emission spectra of aqueous ThT (10 μM)/AgNO3 (0.1 μM) solution recorded during repetitive cycles of illumination; the bottommost spectrum corresponds to time zero and the topmost spectrum to 180 s of illumination. Each cycle included illumination of the ThT/AgNO3 solution in a quartz cuvette (10 × 10 × 40 mm) for 10 s at 312 nm with approximate irradiance 0.1 W/cm2 from a mercury lamp (Fotodyne, Hartland, WI), model 3-300). (c) Kinetics of photoreduction in aqueous solutions of ThT (10 μM)/AgNO3 (0.1 μM, dashed line) and ThT (10 μM)/AgNO3 (10 μM, solid line) monitored as a change of ThT fluorescence at 450 nm upon excitation at 330 nm. Photoreduction was achieved by illumination of the cuvette directly in the chamber of a FluoroMax-3 spectrometer, under excitation wavelength at 330 nm with a slit width of 1 nm and approximate irradiance of 1 W/cm2. ThT is known to have abnormal photophysical properties, in particular the ThT excitation maximum (∼330 nm) does not correspond to the ThT absorption peak (∼400 nm; (31)); therefore, in these experiments we used the excitation wavelength that corresponds to the excitation maximum of 330 nm (see panels d and e). (d) The absorption spectra of 10 μM ThT alone (solid line) and in the presence of 0.5 μM AgNO3 (dashed line) monitored after UV illumination at 312 nm for 3 min. To correct the last spectrum for scattering, the absorption spectrum for 0.5 μM AgNO3 alone was subtracted. Sensitized photoreduction was accompanied by the appearance of a peak at 520 nm (inset), a characteristic of silver nanoclusters (5). (e) The excitation spectra of 10 μM ThT alone (solid line) and in the presence of fibrillar rPrP, 1 μM (dashed line). λem = 482 nm. The spectra were corrected for scattering.