modification has been shown to adversely affect the reaction rate of the substrate [30]. It has also been reported that different quenchers affect the binding and conjugation of the substrate to the SNAPf differently [10]. IRDye QC1 (MW 1244) is a relatively large molecule [35], and may hinder the access of the substrate to the active site of the enzyme. A PEG linker between IRDye QC1 and guanine improved the reaction rate, possibly by alleviating the hindrance effect of IRDye QC1. However this PEG containing quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAPf-ADRb2 expressing tumors. Higher doses up to three times of that used for BG-800 were tried without any significant improvement (Gong et al., unpublished data). These results indicate that the reaction rate of CBG-800-PEG-QC1 is not fast enough to match the quick body clearance of the substrate in the system we used. 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Kobayashi H, Ogawa M, Alford R, Choyke PL, Urano Y (2010) New strategies for fluorescent probe design in medical diagnostic imaging. Chem Rev 110: 2620–2640. PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e34003