Intracellular Zinc Quantification by Fluorescence Imaging with a FRET System

Abstract

Fluorescence imaging of cellular metals is widely reported. However, the quantification of intracellular metals with fluorescence imaging is so far not feasible and highly challenging. In this work, a ratiometric probe with two fluorescently labeled complementary DNA strains is designed for intracellular zinc quantification via fluorescence imaging, based on fluorescence resonance energy transfer (FRET) from carbon dots (CDs) to fluorescein (FAM). The donor CDs are modified with a Zn²⁺ aptamer, whereas the receptor FAM is conjugated with the complementary DNA sequence to ensure selectivity. MCF-7 cells are cultured sequentially with Zn²⁺ (20, 40, 55, 70, 85, and 100 μmol L^-1) and CDs-FAM (100 μg mL^-1), which is used for fluorescence imaging (at λ_ex = 405 nm and λ_em = 440-490 nm for CDs, λ_em = 500-550 nm for FAM) to provide a relative fluorescence ratio (( F - F₀)/ F₀, F = I_CDs/ I_FAM), followed by quantifying intracellular zinc with ICPMS. A linear correlation is achieved between the relative fluorescence ratio in fluorescence images and the intracellular zinc content derived by ICPMS, which facilitates intracellular zinc quantification via fluorescence imaging. It is especially useful for real-time tracing of intracellular zinc during the cell culturing process or in vivo. The cellular uptake of Zn²⁺ by MCF-7 cells is further evaluated with this approach by culturing with 100 μmol L^-1 of Zn²⁺ for different times, and a maximum uptake of 60.5 fg per cell is observed at an incubation time of 60 min. This value is further demonstrated well by ICPMS detection.