Near infrared fluorescence quenching properties of copper (II) ions for potential applications in biological imaging Dolonchampa Maji 1, 2 , Mingzhou Zhou 2 , Pinaki Sarder 2 , Samuel Achilefu 1, 2, 3,* 1 Department of Biomedical Engineering, Washington University in St. Louis, Missouri, USA; 2 Department of Radiology, Washington University School of Medicine, St. Louis, Missouri, USA. 3 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA. *Correspondence: achilefus@mir.wustl.edu ABSTRACT Fluorescence quenching properties of copper(II) ions have been used for designing Cu(II) sensitive fluorescent molecular probes. In this paper, we demonstrate that static quenching plays a key role in free Cu(II)-mediated fluorescence quenching of a near infrared (NIR) fluorescent dye cypate. The Stern-Volmer quenching constant was calculated to be K SV = 970,000 M -1 in 25 mM MES buffer, pH 6.5 at room temperature. We synthesized LS835, a compound containing cypate attached covalently to chelated Cu(II) to study fluorescence quenching by chelated Cu(II). The fluorescence quenching mechanism of chelated Cu(II) is predominantly dynamic or collisional quenching. The quenching efficiency of chelated Cu(II) was calculated to be 58% ± 6% in dimethylsulfoxide at room temperature. Future work will involve further characterization of the mechanism of NIR fluorescence quenching by Cu(II) and testing its reversibility for potential applications in designing fluorophore-quencher based molecular probes for biological imaging. Keywords: Copper, cypate, fluorescence quenching, near infrared fluorescence, Stern-Volmer quenching constant. 1. INTRODUCTION Metal ions have been shown to interact with and alter the spectral properties of fluorescent molecules. This feature is widely used to design metal sensitive fluorescent molecular probes to detect the presence of metal ions 1,2 . Such interactions result in effects ranging from quenching of fluorescence by metal ions such as iron (Fe (III)) 3 and copper (Cu(II)) 4 to fluorescence enhancement by zinc (Zn(II)) 5 . Cu(II) ions have been shown to quench fluorescence of organic dyes, fluorescent proteins and quantum dots 6-10 . Fluorescence quenching properties of Cu(II) are very prominent in the near infrared (NIR) fluorescence range (700-900 nm). In this study we studied and quantified the fluorescence quenching properties Cu(II) ions for a NIR dye cypate. We used both free Cu(II) as well as chelated Cu(II) attached to the dye molecule for this study. The ultimate goal is to demonstrate the feasibility of exploiting this NIR fluorescence quenching properties of Cu(II) to design fluorophore-quencher pair based molecular probes for potential applications in biological imaging. 2. MATERIALS AND METHODS 2.1 Materials Dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethylenediamine, N,N-Diisopropylethylamine (DIEA), MES hydrate, hydrion (sodium carbonate and sodium bicarbonate) buffer powder, ammonium acetate, and copper (II) chloride Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VI, edited by Samuel Achilefu, Ramesh Raghavachari, Proc. of SPIE Vol. 8956, 89560K · © 2014 SPIE · CCC code: 1605-7422/14/$18 · doi: 10.1117/12.2053746 Proc. of SPIE Vol. 8956 89560K-1 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 04/26/2015 Terms of Use: http://spiedl.org/terms