We introduce a general design to construct fluorescence-switching probes. Upon the interaction of the ligand with the protein, the crowded surroundings restrict the bond rotation of the fluorescent molecular rotor to trigger a strong fluorescence signal, which is reduced upon the addition of a competitive ligand or after protein degradation.
In this communication, we report a simple albumin probe based on a fluorescent molecular rotor for the detection of trace albumin levels in urine. In the presence of albumin, the probe exhibits remarkable 400-fold fluorescence enhancement with high selectivity and sensitivity. The probe was successfully applied in the quantitative detection of urinary albumin.
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.
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