New class of fluorogenic probes for antigens based on DARPin-silicon pyronine conjugates

Fluorogenic probes for antigens are useful for various purposes, including medical diagnostics and imaging, but achieving a rapid, large fluorescence increase is difficult. Here, we report a new class of fluorogenic probes for antigens based on a conjugate of an antibody-mimetic DARPin bearing a site-specifically incorporated cysteine and silicon-pyronine (SiP), which reacts reversibly with thiols. By using a library-screening approach, we found that the absorbance and fluorescence of SiP conjugated to a DARPin are quenched by an addition reaction of the cysteine installed in the DARPin to SiP, and the equilibrium of this reaction is shifted to dissociation by antigen binding, leading to rapid, large increases of absorption and fluorescence. As a proof of concept of this chemical design principle, we constructed fluorogenic probes targeting GFP and EpCAM, which showed 25- and 12-fold fluorescence increases upon binding, respectively. The latter probe enabled wash-free cancer cell imaging with a low background.