Selective detection of inorganic phosphates in live cells based on a responsive fluorescence probe

Abstract

In this work, we have designed and synthesized a new 4-phenylthiazole-Cu²⁺ ensemble, L-Cu²⁺, as a fluorescence probe for the quantitative detection of inorganic phosphates (Pi) in aqueous and live cells. A fluorescent ligand, 2-((4-phenylthiazole-2-imino)methyl)-5-diethylaminophenol (L), was synthesized and its chemical structure was well characterized. L exhibited a high affinity (K_a = 3.27 × 10⁵ M⁻¹) and specificity for Cu²⁺ ions through a 1 : 1 stoichiometry to form a fluorescence sluggish L-Cu²⁺ ensemble. This L-Cu²⁺ ensemble was then employed as a turn-on fluorescence probe for the detection of Pi. Spectroscopic studies revealed that L-Cu²⁺ possesses excellent tolerance to other common interference anions, and in particular to AMP, ADP, ATP, PPi. The detection limit of L-Cu²⁺ to Pi was determined to be 0.2 μM. The desirable features of L-Cu²⁺, such as high specificity and sensitivity and low cytotoxicty, enable its application in the detection of Pi in biological samples. Fluorescence imaging of Pi in live MDA-MB-231 cells was conducted to demonstrate its capability in bioimaging applications. Quantitative analysis of Pi in single cells was then demonstrated using flow cytometry.