Cytoplasmic Protein-Powered In Situ Fluorescence Amplification for Intracellular Assay of Low-Abundance Analyte.

Fluorescence amplification is critical for in situ and real-time detection of intracellular low abundance biological species. However, current intracellular amplification techniques mainly rely on synthetic nucleic acid-based nanodevices, manipulating them in living cells remains challenging. To solve this problem, herein, a new signal amplification concept named cytoplasmic protein-powered in situ fluorescence amplification (CPFA) is proposed. CPFA takes cytoplasmic protein as cell-self-power for signal amplification enabling it to operate in living cells. To establish a prototype of CPFA, an amplifiable sensor for hydroxyl radicals (•OH) was designed by entrapping the screened cytoplasmic protein-enhanced fluorophore (PBF1) inside mesoporous silica (MSN) nanocontainer with ssDNA/PTAD-based signal switch. When encountered with •OH in living cells, the ssDNA was cleaved to separate PTAD from MSN, liberating multiple copies of the loaded PBF1 to light up the fluorescence. Furthermore, these released PBF1 molecules can instantly bind with cytoplasmic proteins to amplify their fluorescence signals. Take advantage of this two-stage amplification mode, the sensor in response to •OH exhibited remarkable fluorescence enhancement (near 400-fold) in cell lysates, and the •OH was linearly determined from 0 to 800 nM with a detection limit of 6.4 pM. Moreover, this sensor can track basal level and fluctuation of •OH in living cells on account of its high sensitivity. To our knowledge, this is the first effort to use cytoplasmic protein for amplifying detection signals, which will provide a new dimension to current methodologies for low-abundance biomarkers discovery and regulation for chemical biology and medical diagnostics.