Dual-emission fluorescence detection of histidine using carbon dots and calcein/Ni²⁺ complexes

Histidine (His) is a natural amino acid that plays very important roles in biota. However, the low concentrations of His in biological fluids and the similar structures and properties of other amino acids mean it is difficult to selectively determine His concentrations in biological fluids with a high degree of sensitivity. A novel ratiometric fluorescence probe for detecting His in aqueous solutions is described here. The method involves carbon dots (CDs) and calcein/Ni²⁺ complexes. At an excitation wavelength of 480 nm, the CD/calcein system emits green fluorescence (maximum emission from calcein at 512 nm) and red fluorescence (maximum emission from CDs at 617 nm). The presence of Ni²⁺ decreases the calcein fluorescence intensity because of static quenching caused by the formation of calcein/Ni²⁺ complexes but the CD fluorescence intensity remains almost unchanged. Fluorescence of calcein/Ni²⁺ complexes provides the response, and the presence of His binds to Ni²⁺ via cooperative chelation and produces free calcein causing fluorescence to be recovered. CDs provide a self-calibration fluorescence signal, the intensity of which remains almost unchanged in the presence of His. The ratio of the fluorescence intensities at 512 and 617 nm (I₅₁₂/I₆₁₇) was strongly related to the His concentration in the range 0.5-22 μM, and the detection limit was 0.16 μM. The specificity of Ni²⁺/His interactions allows His to be determined without interference from other species. The method was successfully used to determine His in diluted human urine. The recovery was acceptable, suggesting that the biosensor can be used to determine His in real samples.