Cell-penetrating nanobiosensors for pointillistic intracellular Ca2+-transient detection

Nano Lett. 2014 Jun 11;14(6):2994-3001. doi: 10.1021/nl500733g. Epub 2014 May 7.


Small-molecule chemical calcium (Ca(2+)) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca(2+) entry. Nanobiosensors incorporating functionalized quantum dots (QDs) have emerged as promising alternatives but their intracellular use remains a major challenge. We designed cell-penetrating FRET-based Ca(2+) nanobiosensors for the detection of local Ca(2+) concentration transients, using commercially available CANdot565QD as a donor and CaRuby, a custom red-emitting Ca(2+) indicator, as an acceptor. With Ca(2+)-binding affinities covering the range of 3-20 μM, our CaRubies allow building sensors with a scalable affinity for detecting intracellular Ca(2+) transients at various concentrations. To facilitate their cytoplasmic delivery, QDs were further functionalized with a small cell-penetrating peptide (CPP) derived from hadrucalcin (HadUF1-11: H11), a ryanodine receptor-directed scorpion toxin identified within the venom of Hadrurus gertschi. Efficient internalization of QDs doubly functionalized with PEG5-CaRuby and H11 (in a molar ratio of 1:10:10, respectively) is demonstrated. In BHK cells expressing a N-methyl-d-aspartate receptor (NMDAR) construct, these nanobiosensors report rapid intracellular near-membrane Ca(2+) transients following agonist application when imaged by TIRF microscopy. Our work presents the elaboration of cell-penetrating FRET-based nanobiosensors and validates their function for detection of intracellular Ca(2+) transients.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques / methods*
  • Calcium / metabolism*
  • Calcium Signaling / physiology*
  • Cell-Penetrating Peptides / chemistry*
  • Cricetinae
  • Fluorescence Resonance Energy Transfer*
  • HEK293 Cells
  • Humans
  • Quantum Dots / chemistry*
  • Scorpion Venoms / chemistry


  • Cell-Penetrating Peptides
  • Scorpion Venoms
  • hadrucalcin protein, Hadrurus gertschi
  • Calcium