Steric-dependent label-free and washing-free enzyme amplified protein detection with dual-functional synthetic probes.

Enzyme-catalyzed signal amplification with an antibody-enzyme conjugate is commonly employed in many bioanalytical methods to increase assay sensitivity. However, covalent labeling of the enzyme to the antibody, laborious operating procedures, and extensive washing steps are necessary for protein recognition and signal amplification. Herein, we describe a novel label-free and washing-free enzyme-amplified protein detection method by using dual-functional synthetic molecules to impose steric effects upon protein binding. In our approach, protein recognition and signal amplification are modulated by a simple dual-functional synthetic probe which consists of a protein ligand and an inhibitor. In the absence of the target protein, the inhibitor from the dual-functional probe would inhibit the enzyme activity. In contrast, binding of the target protein to the ligand perturbs this enzyme-inhibitor affinity due to the generation of steric effects caused by the close proximity between the target protein and the enzyme, thereby activating the enzyme to initiate signal amplification. With this strategy, the fluorescence signal can be amplified to as high as 70-fold. The generality and versatility of this strategy are demonstrated by the rapid, selective, and sensitive detection of four different proteins, avidin, O6-methylguanine DNA methyltransferase (MGMT), SNAP-tag, and lactoferrin, with four different probes.

Fluorescence switchable probes based on a molecular rotor for selective detection of proteins and small molecules.

In this communication, we report a general strategy to create fluorescence switchable probes, where a small molecule ligand is conjugated to a fluorescent molecular rotor, for the selective detection of proteins through a non-enzymatic process. In the presence of target proteins, bond rotation of the molecular rotor is restricted, thereby triggering the emission of strong fluorescence.

A rapid SNAP-tag fluorogenic probe based on an environment-sensitive fluorophore for no-wash live cell imaging.

One major limitation of labeling proteins with synthetic fluorophores is the high fluorescence background, which necessitates extensive washing steps to remove unreacted fluorophores. In this paper, we describe a novel fluorogenic probe based on an environment-sensitive fluorophore for labeling with SNAP-tag proteins. The probe exhibits dramatic fluorescence turn-on of 280-fold upon being labeled to SNAP-tag. The major advantages of our fluorogenic probe are the dramatic fluorescence turn-on, ease of synthesis, high selectivity, and rapid labeling with SNAP-tag. No-wash labeling of both intracellular and cell surface proteins was successfully achieved in living cells, and the localization of these proteins was specifically visualized.