Functionalized Nanopipette for Fluorescence Analysis of Protein Kinase Activity in Single Living Cells

Abstract

Evaluation of protein kinase activity in individual living cells is important for understanding intracellular phosphorylation-regulated signaling pathways. However, it remains challenging because most existing approaches require cell lysis and thereby disrupt the native intracellular environment. Herein, we developed a functionalized carbon nanopipette platform for fluorescence-based analysis of relative changes in intracellular protein kinase A (PKA) activity at the single-cell level. The outer quartz surface of the nanopipette was modified with a DNA-templated Dy³⁺ recognition interface, which selectively captured phosphorylated peptides through phosphate-lanthanide coordination. For intracellular analysis, a fluorescent non-phosphorylated peptide substrate was introduced into single living cells by electrochemical microinjection. The delivered substrate was subsequently phosphorylated by activated intracellular PKA. Following injection, the intracellular fluorescence stabilized within 10 min and remained nearly unchanged for at least 60 min, while cellular morphology was largely preserved. The resulting phosphopeptide products were selectively recognized by the Dy³⁺-functionalized nanopipette, producing a significant fluorescence enhancement at the nanopipette tip. This work establishes a nanopipette-based strategy for intracellular kinase activity analysis in single living cells and provides a modular platform that may be extended to other phosphorylation-related signaling events by changing the peptide substrate.

Keywords

Single-cell analysis / Protein kinase A (PKA) activity / Nanopipette / Fluorescence sensing / Electrochemical delivery

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