Cell-Penetrating Nanobiosensors for Pointillistic Intracellular Ca super(2+)-Transient Detection
Small-molecule chemical calcium (Ca super(2+)) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca super(2+) entry. Nanobiosensors incorporating functionalized quantum dots (QDs) have emerged as promising alternatives but...
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Published in | Nano letters Vol. 14; no. 6; pp. 2994 - 3001-2994-3001 |
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Main Authors | , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
01.06.2014
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Subjects | |
Online Access | Get full text |
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Summary: | Small-molecule chemical calcium (Ca super(2+)) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca super(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 super(2+) nanobiosensors for the detection of local Ca super(2+) concentration transients, using commercially available CANdot565QD as a donor and CaRuby, a custom red-emitting Ca super(2+) indicator, as an acceptor. With Ca super(2+)-binding affinities covering the range of 3-20 mu M, our CaRubies allow building sensors with a scalable affinity for detecting intracellular Ca super(2+) transients at various concentrations. To facilitate their cytoplasmic delivery, QDs were further functionalized with a small cell-penetrating peptide (CPP) derived from hadrucalcin (Had sub(UF1-11): 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 super(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 super(2+) transients. Keywords: Quantum dot biosensors; nanoparticle surface chemistry; FRET-based calcium probes; red-emitting calcium indicator; intracellular calcium fluorimetry; cell penetrating peptide |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 content type line 23 ObjectType-Feature-2 |
ISSN: | 1530-6984 1530-6992 |
DOI: | 10.1021/nl500733g |