In this paper, we present a novel charge-free fluorescence-switchable near-infrared (IR) dye based on merocyanine for target specific imaging. In contrast to the typical bathochromic shift approach by extending πconjugation, the bathochromic shift of our merocyanine dye to the near-IR region is due to an unusual S-cis diene conformer. This is the first example where a fluorescent dye adopts the stable S-cis conformation. In addition to the novel bathochromic shift mechanism, the dye exhibits fluorescence-switchable properties in response to polarity and viscosity. By incorporating a protein-specific ligand to the dye, the probes (for SNAP-tag and hCAII proteins) exhibited dramatic fluorescence increase (up to 300-fold) upon binding with its target protein. The large fluorescence enhancement, near-IR absorption/ emission, and charge-free scaffold enabled no-wash and site-specific imaging of target proteins in living cells and in vivo with minimum background fluorescence. We believe that our unconventional approach for a near-IR dye with the S-cis diene conformation can lead to new strategies for the design of near-IR dyes.

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.

Quantitative detection of trace amounts of a biomarker in protein rich human blood plasma using fluorescent probes is a great challenge as the real signal is usually obscured by nonspecific fluorescence. This problem occurs because most of the fluorescent dyes bind very tightly with blood proteins to produce a large fluorescence increase, resulting in overestimation of the biomarker concentrations and false positive diagnosis. In this paper, we report that biotinylated fluorescent probes encapsulated in avidin protein can generate very specific fluorescence in blood serum by blocking out nonspecific dye-protein interactions. We applied our novel probe design to detect two different types of biomolecules, hydrogen sulfide and nitroreductase. Our Avidin conjugated probes achieved quantitative analyte detection in blood serum; whereas concentrations were overestimated up to 320-fold when bare fluorescent probes were employed. As compared to conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple approach successfully overcomes many key issues such as dye leakage, long preparation steps, inconsistent dye-host ratios, difficulty in constructing in situ in a complex medium, and limited application to detect only small metabolites.

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.