The streptavidin–biotin controlled binding probe has several advantages for the detection of enzymes and reactive small molecules, such as minimal background, multiple signal amplification steps, and wide selection of the optimal dyes for detection.

We study the effects of metal-enhanced fluorescence (MEF) on rhodamine B fluorophore by nanoparticles of varied shapes. The avidin−biotin system was used as spacer to connect the fluorophore to the surface of nanoparticles. Fluorescence lifetime image microscopy (FLIM) was used to detect emission lifetime for dye molecules on single nanoparticles. Spherical gold particles diameter of 60 and 170 nm, respectively, cube length of 70 nm, and rhombic dodecahedron (RD) diameter of 63 nm were used. In the measured emission curves of rhodamine B, we obtained a short component with lifetime 16−26 ps attributed to the fluorophore under influence of the local electric field of gold nanoparticle and energy dissipation to nanoparticle. The second lifetime is 200, 270, 280, and 330 ps for 170 nm sphere, 70 nm cube, 63 nm RD, and 60 nm sphere, respectively. This component is referred to as the bright mode of nanoparticle which is coupled to the excited dye molecule and transfers energy back to the fluorophore. On the basis of the large amplitude obtained for the short lifetime component, the effect of MEF was great. The avidin−biotin assembly serves as a biospacer in the MEF applications. Moreover, the MEF effect on the Ag shell gold nanoparticle is studied. Various thicknesses of Ag shell around 40 nm diameter gold core nanoparticles were synthesized. In these Au@Ag−R nanoparticles, time constants obtained in rhodamine B emission curves are τ 1 /τ 2 = 15/210, 23/240, 26/251, 21/246, 25/255 ps for Ag shell thickness of 4, 6, 7.5, 13, and 16 nm, respectively. Because of this multiple exponential decay behavior, we derived a kinetic model for the MEF process and calculated the rate constants of energy transfer between the dye molecules and the Au@Ag nanoparticle. As rhodamine B is excited, it can transfer energy to Au nanoparticle and also dissipates energy to Ag shell via nanosurface energy transfer (NSET), leading to severe fluorescence quenching. This results in low enhancement factors of fluorescence in this core−shell system. According to the experimental lifetime data, the NSET rate constant for the energy dissipation to Ag surface is estimated to be (4−6.6) × 10 10 s −1 .

The ability to detect and image secreted peroxynitrite (ONOO−) along the extracellular surface of a single cell is biologically significant, as ONOO− generally exerts its function for host defense and signal transductions at the plasma membrane. However, as a result of the short lifetime and fast diffusion rate of small ONOO−, precise determination of the ONOO− level at the cell surface remains a challenging task. In this paper, the use of a membrane‐anchored streptavidin–biotin‐controlled binding probe (CBP), ONOO‐CBP, to determine quantitatively the ONOO− level at the cell surface and to investigate the effect of different stimulants on the production of ONOO− along the plasma membrane of macrophages is reported. Our results revealed that the combination of NO synthase (iNOS) and NADPH oxidase (NOX) activators was highly effective in inducing ONOO− secretion, achieving more than a 25‐fold increase in ONOO− relative to untreated cells. After 1 h of phorbol‐12‐myristate‐13‐acetate (PMA) stimulation, the amount of ONOO− secreted by RAW264.7 macrophages was similar to the condition treated with 25 μm 3‐morpholinosydnonimine hydrochloride (SIN‐1), which was estimated to release about 20 μm of ONOO− into Dulbecco's modified Eagle's medium (DMEM) in 1 h. This novel approach should open up new opportunities to image various reactive oxygen and nitrogen species secreted at the plasma membrane that cannot be simply achieved by conventional analytical methods.