Ratiometric fluorescence sensor for protamine and heparin based on N,P-doped carbon dots and Eosin Y

A novel ratiometric fluorescent assay was proposed for protamine and heparin based on blue-emitting N,P-doped carbon dots (N,P-CDs) and green-emitting Eosin Y. The N,P-CDs were prepared using arginine as carbon and nitrogen sources according to hydrothermal method with favorable luminescence intensity, photostability, and good reproducibility. And the N,P-CDs were used as internal reference fluorescence molecules. The anionic Eosin Y was used as fluorescence signal molecule. Protamine with positive group could interact with Eosin Y through electrostatic effect, leading to the fluorescence quenching of Eosin Y. Upon the addition of heparin, heparin was combined with protamine with stronger affinity through electrostatic interactions and hydrogen bonds, which preventing the bonding of protamine with Eosin Y and obvious fluorescence emissions of Eosin Y at 538 nm could be observed. Meanwhile, the fluorescence signal of N,P-CDs at 420 nm remained stable during the whole process. A good linear range between the fluorescence emission intensity ratio of I₅₃₈/I₄₂₀ was obtained for protamine from 0.01 to 40 μg/mL with a low detection limit of 5.6 ng/mL, and heparin from 0 to 35 μg/mL with a low detection limit of 2.5 ng/mL. Moreover, the method was further applied to detection of heparin with satisfactory results in fetal bovine serum samples.