Reduction-Activatable Fluorogenic Nanobody for Targeted and Low-Background Bioimaging

Environment-sensitive fluorogenic antibodies enable target-specific bioimaging with reduced unspecific background signal and improved spatiotemporal resolution. However, current strategies for the construction of fluorogenic antibodies are hard to handle due to challenges that lie in the prior design of fluorogenic probes and subsequent antibody labeling. Here, we report a simple strategy to generate a fluorogenic nanobody, which we term D-body, by in situ incorporation of a reduction-responsive Nile blue foldamer which is self-quenched via a dimerization-caused quenching mechanism. The D-body can be efficiently internalized by cells with high epidermal growth factor receptor expression levels and is highly fluorogenic upon lysosomal activation, allowing wash-free cell imaging with exquisite specificity and fast in vivo imaging with a high tumor-to-background ratio. The modular D-body is readily available and easy to handle, offering a platform that is highly tunable for bioimaging applications.