Representation of the azurin (K27C) structure and schematic view of the covalent coupling of azurin to a SAM-modified Au surface. The distance between the dye label at the N-terminus and the Au surface is denoted by d.
10 × 10 μm2 fluorescence images of immobilized reduced K27C azurin (20 mM Hepes buffer, pH 7). A) on a 100 nm Au film coated with C10d SAM. B) on silanized glass (see the Experimental Methods). The scale bars show the intensity counts in 1.8 msec bin time. The fluorescence intensities of individual spots on the 100 nm thick coated Au film are brighter than on glass (notice different scale bars).
Fluorescence time traces obtained from immobilized reduced Cu-Az (K27C azurin) on 100 nm Au film coated with C10d SAM (black) and from reduced Cu-Az on a silanized (see the Experimental Methods) glass surface (gray).
Intensity count rate histograms for reduced K27C azurin molecules immobilized on silanized glass (A) and on Au films (B: 20 nm, C: 50 nm, and D: 100 nm thickness) coated with a C10d SAM.
Fluorescence decay curves of reduced azurin immobilized on silanized glass (purple), immobilized directly on 50 nm Au film (red) and on 50 nm Au films coated with C4d (green), C6d (blue), C8d (magenta), or C10d (cyan). IRF (black) is also included. Each decay curve represents an average over more than 500 individual molecules.
Fluorescence lifetimes of reduced Cu-Az immobilized on Au films of different film thicknesses (from 10 to 100 nm) that are coated with C4d (olive), C6d (green), C8d (blue-green), and C10d (bright blue) SAMs and of reduced Cu-Az adsorbed directly on 50 nm Au (red). Also presented is the lifetime of reduced Cu-Az immobilized on silanized glass (red). The lifetimes were obtained by monitoring the total emission of a 10 × 10 μm2 surface area. For each bar the lifetimes measured for a number of fluorescence images (varying from 2 to 16) were averaged. The error bars correspond to standard deviations.
Time traces for two individual labeled K27C azurin molecules immobilized on a 20 nm Au film coated with C10d SAM, one trace for azurin in the reduced state (black, τred = 2.6 nsec), and one for azurin in the oxidized state (gray, τoxid = 1.6 nsec). Data refer to two different samples. Reduction/oxidation was controlled by addition of 10 mM ascorbate (reduced sample) or 1 mM [Fe(CN)6]3− (oxidized sample).
Fluorescence lifetime histograms of labeled K27C azurin in the oxidized (grey histograms at the left) and the reduced state (black histograms at the right) immobilized on 20 nm Au coated with a C10d SAM (bottom) and immobilized on glass (top).
Dependence of the energy transfer rate γabs on the inverse cubic distance, 1/d.3 The straight line is a linear fit to the data points. Data taken from Table III.
Root-mean square (rms) values of height variations for various Au films, as determined by AFM.
Fluorescence lifetimes, τ, of azurin immobilized on glass and on 50 nm Au films coated with SAMs of varying thicknesses.
Distance dependence of the fluorescence lifetime τ and the metal energy transfer rate γabs.
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