Abstract: Objective To screen a variety of high-affinity small molecule folate analogs as targeting ligands and different linkers, to summarize the rational molecular design of folate receptor-targeted fluorescent probes, and develop a new type of excellent folate receptor-targeted fluorescent probe. 
Methods The target fluorescent probe was obtained through various chemical synthesis reactions. Flow cytometry was used to investigate the selectivity of fluorescent probes for folate receptors, and fluorescence microscopy was conducted to evaluate the targeted imaging ability of fluorescent probes on tumor cells. 
Results Eight targeted fluorescent probe products were obtained, among which the fluorescent probe GT-FITC-1 could specifically bind to folate receptor and enter cells to achieve fluorescent imaging. The introduction of glutamic acid side chains reduced the relative average fluorescence intensity of cell uptake. GT-FITC-2 decreased by about 17% compared with GT-FITC-1, and GT-FITC-5 decreased by approximately 15% compared with GT-FITC-4. The fluorescence intensity of the fluorescent probes GT-FITC-7 and GT-FITC-8 containing rigid benzene ring linkers increased by 2-3 times compared with GT-FITC-4, and GT-FITC-8 showed the largest relative average fluorescence intensity. 
Conclusion A series of fluorescent probes designed and synthesized in this study all show high specificity for folate receptors, and the fluorescent probe GT-FITC-8 with excellent affinity for folate receptors is discovered. 

Key words: molecular probes, fluorescein, folate receptor