Abstract: Background and purpose: Tracer is the key to sentinel lymph node biopsy, but the traditional fluorescence tracer could not target at sentinel lymph node with weak intensity. A new near-infrared fluorescence tracer polymethylene cyanine dye Cy754 with stronger permeability and intensity was synthetized and combined with rituximab (Rit) to produce a new near-infrared fluorescence-target tracer Cy754-Rit. Methods: The new near-infrared fluorescence tracer Cy754 was synthetized by chemical method, and the basic parameters including molecular weight, absorption and emission spectra were tested. The molecule of Cy754 was combined directly with Rit to produce Cy754-Rit. The Cy754-Rit was analyzed for labeled rate, molecular integrity, and molecular immune activity. The fluorescence intensity of Cy754-Rit was compared with indocyanine green (ICG)-Rit by using near-infrared fluorescence detector. The safety limitation was tested. The localization ability of Cy754-Rit was tested in animal model of sentinel lymph node and compared with Cy754, ICG-Rit and radiotracer. Results: The molecular weight of Cy754 was 818, and the absorption and emission spectra were 740 nm and 760 nm. The Cy754-Rit was intact and kept the immune activity of Rit. The Cy754 labeled rate of Rit was 100%. The fluorescence intensity of Cy754-Rit was stronger than that of ICG-Rit (3.08×10 ¹⁰ vs 6.56×10 ⁸ ). The Cy754-Rit was bacteria- and pyrogen-free, and was safe to body with location injection. The best molarity ratio of Cy754-Rit was 80∶1. The Cy754-Rit could identify the location of sentinel lymph node which was accorded with the radiotracer and had a stronger permeability than ICG-Rit (14.3 mm vs 13.2 mm). Conclusion: The method of producing Cy754-Rit is simple, effective and non-radioactive. The fluorescence intensity of Cy754-Rit is stronger than that of traditional tracer. The Cy754-Rit could identify the location of sentinel lymph node in the animal model of sentinel lymph node but should be validated by clinical trial before its use in clinical practice.

Key words: Sentinel lymph node, Target tracer, Rituximab, Polymethylene cyanine dye, Lymphoscintigraphy