Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum

Yanyan Hou, Satoshi Arai, Yoshiaki Takei, Atsushi Murata, Shinji Takeoka, Madoka Suzuki*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)


Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)–biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.

Original languageEnglish
Pages (from-to)293-299
Number of pages7
JournalScience and Technology of Advanced Materials
Issue number1
Publication statusPublished - 2016 Jan 1


  • Copper-free click reaction
  • N3-fura-2
  • calcium imaging
  • endoplasmic reticulum
  • nanosensor

ASJC Scopus subject areas

  • Materials Science(all)


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