Imaging and Quantification of Secreted Peroxynitrite at the Cell Surface by a Streptavidin-Biotin-Controlled Binding Probe.

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.

Target-activated streptavidin-biotin controlled binding probe.

Target-activated chemical probes are important tools in basic biological research and medical diagnosis for monitoring enzyme activities and reactive small molecules. Based on the fluorescence turn-on mechanism, they can be divided into two classes: dye-based fluorescent probes and caged-luciferin. In this paper, we introduce a new type of chemical probe in which the fluorescence turn-on is based on controlled streptavidin-biotin binding. Compared to conventional probes, the streptavidin-biotin controlled binding probe has several advantages, such as minimal background at its "OFF" state, multiple signal amplification steps, and unlimited selection of the optimal dyes for detection. To expand the scope, a new synthetic method was developed, through which a wider range of analyte recognition groups can be easily introduced to construct the binding probe. This probe design was successfully applied to image and study secreted peroxynitrite (ONOO-) at the cell surface of macrophages where information on ONOO- is difficult to obtain. As the signals are generated upon the binding of streptavidin to the biotin probe, this highly versatile design can not only be used in fluorescence detection but can also be applied in various other detection modes, such as electrochemical and enzyme-amplified luminescence detection.