Abstract
We herein developed a highly selective replacement-type fluorescence sensor using Cu-Calcein probe, which exhibits “turn on” fluorescence detection for sensing zinc ion. When zinc ion replaced cupper ion in Cu-Calcein complex, the recovery of fluorescence to the original intensity of calcein was observed. Since the equilibrium between dissolved metal specie, zinc ion, and complexing agents, Cu-Calcein, could be attained in 20 minutes, it is well-suited for the rapid detection and quantitation of zinc ion. After optimization on pH of reaction solution and calcein concentration, the linear range of such sensor for the detection of zinc ion was found to be 1-16 □M (y = 3.7811x-0.0262, R2 = 0.9993), providing a limit of detection (LOD) of 900 nM, defined by adding 3 times the standard deviation of the control (free of target Zn2+) from its average value. Comparing the results for the analysis of real samples (commercial available nutrient supplement) obtained by the proposed sensor and ICP-MS, 16.8 % of relative error was calculated. Our study verified that Hg2+, Cd2+ or most of the transitional metal ions were unable to trigger the turn-on mechanism of fluorescence, though some of them were still capable of replacing Cu2+ in Cu-Calcein complex. We thus confirmed that the proposed Cu-Calcein sensor demonstrates enough sensitivity and high selectivity, which is capable of distinguishing Zn2+ and Hg2+, Cd2+ or most of the transitional metal ions presented in biological system. Furthermore, it was also found that the cations, such as sodium, potassium, magnesium, calcium, commonly seen in biological system, or various anions, such as chloride, acetate, nitrate, sulfate did not exert interference on the detection of Zn2+. In addition, to examine the reversibility of sensor, EDTA titration was conducted. It was found that the regeneration of the sensor could be accomplished. Due to the significant difference in the as-obtained quantum yields between Cu-Caclein (0.0033) and Zn-Calcein (0.79), the sensor described in this study has achieved high performance in terms of signal to noise ratio (S/N ratio). Moreover the phenomenon of blue-shift for emission and absorption peaks of Zn-Calcein was observed, we suspected that the new coordination bonding between zinc ion and fluorophore (calcein) was formed, and subsequently triggered the Photoinduced Charge Transfer (PCT) mechanism. NMR analysis was therefore adopted to investigate our hypothesis. Compared to the 1H NMR spectrum of the free ligand calcein, the Zn2+ binding caused the small but significant downfield shifts, especially for the protons in the 3-hydroxyxanthen-6-one ring, 6.45 ppm and 7.06 ppm and so forth, strongly suggested the participation of the 3-hydroxyxanthen-6-one rings in the coordination. Owing to the neutral molecular structure of Cu-Calcein, it is easier to diffuse passively across the cell membrane. In this study, we confirmed its applicability of being used in monitoring zinc influx in RAW 264.7 cells. Finally, MTT assay was utilized to examine the cytotoxicity of Cu-Calcein probe. After co-incubation with RAW 264.7 for 24 hours, the cell viability remained above 90 %, validating the biocompatibility of such probe and its feasibility of being further developed as a device in monitoring the trafficking and regulation of mobile zinc pools, which are critically important in cellular functions and pathological conditions in multiple organs, such as brain, pancreas, and prostate.