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We here describe a fluorescent signal amplification method termed "Click-based amplification" that can be well integrated with various click-labelling modes, including chemical labelling, genetic incorporation and covalent inhibitor probe mediated target labelling. Picolyl azide (pAz) was used as a functional group of a streptavidin-based amplifier to enhance the efficiency of click chemistry. Click-based amplification provided 3.0-12.7 fold amplification on fixed HeLa cells with different click-labelling modes. Click-based amplification has proven to be superior to tyramide signal amplification (TSA) in view of its low nonspecific amplification and high signal-to-noise ratio. Moreover, in terms of the challenging signal amplification of tissue specimens, Click-based amplification successfully achieved remarkable fluorescence enhancement on intestinal tissue slices of afatinib-N 3 treated mice, which provided direct evidence of the presence of afatinib-N 3 in the intestinal tissues and helped in revealing the off-target toxicity of afatinib. Collectively, these results illustrate that Click-based amplification could serve as a promising method for bioimaging studies.

Click-based amplification: designed to facilitate various target labelling modes with ultralow background amplification