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A Class of Biocompatible Dye–Protein Complex Optical Nanoprobes

Xindong Wang,Xinyu Wang,Bo Qu,Nuernisha Alifu,Ji Qi,Ruiyuan Liu,Qinrui Fu,Ruifang Shen,Qi Xia,Lijun Wu,Bing Sun,Jibin Song,Youping Lin,Xin Huang,Anjun Qin,Jun Qian,Ben Zhong Tang,Guanying Chen

Molecular organic dyes are classic fluorescent nanoprobes finding tremendous uses in biological and life sciences. Yet, they suffer from low brightness, poor photostability, and lack of functional groups for bioconjugation. Here, we describe a class of biocompatible dye–protein optical nanoprobes, which show long-time photostability, superbrightness, and enriched functional groups. These nanoprobes utilize apoferritin (an intracellular protein for iron stores and release) to encase appropriate molecular organic dyes to produce on-demand fluorescence in aqueous solution. A pH-driven dissociation–reconstitution process of apoferritin subunits allows substantial incorporation of hydrophilic (aggregation caused quenching, ACQ) or hydrophobic (aggregation induced enhancement, AIE) dye molecules into the protein nanocavity (8 nm), producing monodispersed dye–apoferritin nanoparticles (apo-dye-NPs, ∼12 nm). As compared with single dye monomer, single apo-dye-NPs possess hundreds of times larger molar extinction coefficient and 2 orders of magnitude higher absolute luminescence quantum yield (up to 45-fold), multiplying fluorescence brightness up to 2778-fold. We show that varying the type of incorporated dyes entails a precise control over nanoprobe emission profile tunable in a broad spectral range of 370–1300 nm. Mechanical investigations indicate that the diversified microstructures of nanocavity inner surface are able to conform ACQ dyes at reasonable space interval while providing protein-guided-stacking for AIE dyes, thus enhancing fluorescence quantum yield through confining intermolecular quenching and intramolecular rotation. Moreover, apo-dye-NPs are able to emit stable fluorescence (over 13 min) without quenching in confocal imaging of HepG2 cancer cell under ultrahigh laser irradiance (1.3 × 106 W/cm2). These superb properties make them suitable, as demonstrated in this work, for long-term super-resolved structured illumination microscopic cell imaging (spatial resolution, 117 nm) over 48 h, near-infrared (NIR) fluorescence angiography imaging of whole-body blood vessels (spatial resolution, 380 μm), and NIR photoacoustic imaging of liver in vivo.