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.
Lateral flow assay (LFA) has been a valuable diagnostic tool in many important fields where rapid, simple, and on-site detection is required, for applications such as pregnancy tests and infectious disease prevention. Currently, two types of LFAs are available: lateral flow immunoassay (LFIA) and nucleic acid lateral flow assay (NALFA). Both are generally used for the testing of proteins and nucleic acids. However, enzyme activities and small molecules without the corresponding binding partner cannot be detected by the existing LFAs. In this paper, we introduce a LFA approach termed affinity-switchable lateral flow assay (ASLFA) to overcome the limitations. The detection principle is based on the switchable binding between the affinity-switchable biotin (ASB) probe and avidin protein. In the presence of the target molecule, the activated ASB probe would be captured by the avidin, thereby leaving a distinct test line on the membrane. The ASLFA concept was demonstrated by testing the F ion, NADH cofactor, and nitroreductase activity. Thus, this general ASLFA can be used for the rapid detection of molecules that cannot be accessed by the classical LFAs.
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.
Although many protein labeling probes have been developed to elucidate the trafficking and turnover processes of cell surface proteins, real-time tracking of intracellular proteins remains a challenging task. Herein, we describe a new design to construct a cell-permeable, photostable, and far-red fluorescent turn-on probe to enable no-wash, organelle-specific, and long-term visualization of intracellular SNAP-tagged proteins in living cells. When the probe was used in dual-color pulse chase labeling experiments to differentiate between preLamin and mature Lamin, our results reveal that the shape of mature Lamin can be altered by the newly synthesized preLamin and that this alteration is progressive, cumulative, and due to a concentration-dependent dominant-negative effect of preLamin. We believe that this probe can also be applied to other intracellular proteins whose cellular localization and synthesis changes dynamically in response to external stimuli.
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