N-Aminofluorescein is a highly selective and sensitive fluorescent probe for Cu2+ and can be used to detect Cu2+ in biological systems (λex/em=495/516 nm).

N-Aminofluorescein consists of a fluorescein molecule and a hydrazide group that recognizes and binds to Cu2+, thereby promoting hydrolysis of the amide. [1]
N-Aminofluorescein (abbreviated as FG) is specific for Cu2+, and an absorption peak at 632 nm is observed in 70% HEPES buffer (pH 7.4) containing Cu2+. absorption peak and emission peak was observed at 515 nm. [2]

Cu2+ Assay
1. Prepare a 1.0 mM ethanol stock solution of helical fluorescein amidoxime.
2. Perform the fluorescence reaction in 0.01 M Tris-HCl buffer (pH 7.2) using 10 μM N-aminofluorescein.
3. Add an appropriate amount of sample solution to the solution so that the final Cu2+ concentration does not exceed 10 μM, and adjust the final volume to 10 mL with 0.01 M Tris-HCl buffer (pH 7.2).
4. After 2 hours of reaction, transfer 3 mL of the solution to a 1 cm quartz cuvette and measure the fluorescence intensity/spectrum at room temperature with an excitation wavelength of 495 nm and an emission wavelength of 516 nm, respectively, and an excitation and emission slit width of 5 nm.
5. At the same time, prepare a blank solution without Cu2+ and measure it under the same conditions for comparison.
6. Detection:
Use a fluorescence microscope, flow cytometer, or fluorescence spectrophotometer to detect the labeled samples.
Set the instrument parameters according to the excitation and emission wavelengths of N-aminofluorescein (e.g., excitation wavelength is about 496 nm, emission wavelength is about 519 nm).