Abstract
Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging.
Highlights
Microdomains of intracellular ion concentrations are central to the compartmentalization and specificity of cellular signalling pathways
We constructed fluorescence resonance energy transfer (FRET)-based nanobiosensors from a central quantum dots (QDs) donor (D) and 1 to 10 ion-sensitive acceptor (A) dye molecules (Figure 1A)
The fluorescent acceptor combines an extended rhodamine fluorophore, an ion specificchelating group and a linker arm allowing its conjugation to the QD surface (Figure 1B–D)
Summary
Microdomains of intracellular ion concentrations are central to the compartmentalization and specificity of cellular signalling pathways. In either case the precise spatio-temporal characterization of such microdomain signaling events with fluorimetric ion indicators has been difficult because the measurement of the local ion concentrations is hampered by the rapid diffusion of the ion and the indicator, the rapidity of the event, the small spatial extent of the microdomain and the relatively low number of ions and photons involved. The broad absorption spectrum, the well-defined symmetric emission spectrum and the large brightness and high resistance to photobleaching compared to small-molecule organic fluorophores make QDs good energy donors for fluorescence resonance energy transfer (FRET) and facilitate the detection of single QDs inside live cells. The FRET efficiency of this assembly is given by the overlap of the emission spectrum of the QD with the absorbance spectrum of the fluorescent ion indicator, as well as their number, orientation and proximity with the QD surface
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
