Biosensors and Bioelectronics 21 (2006) 1302–1308 DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu + and Cu 2+ ions James P. Sumner a,1 , Nissa M. Westerberg a , Andrea K. Stoddard a , Tamiika K. Hurst a , Michele Cramer b,2 , Richard B. Thompson b,2 , Carol A. Fierke a,∗ , Raoul Kopelman a,∗ a Department of Chemistry, The University of Michigan, 930N University AVE, Ann Arbor, MI 48109, USA b Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108N Greene St., Baltimore, MD 21201, USA Received 24 November 2004; received in revised form 31 March 2005; accepted 1 April 2005 Available online 18 July 2005 Abstract The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu + and Cu 2+ , with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K d values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu 2+ (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling. © 2005 Elsevier B.V. All rights reserved. 1. Introduction Fluorophores that are both sensitive and selective are a prerequisite for luminescent sensor development (Aylott, 2003; Cullum and Vo-Dinh, 2000; Wolfbeis, 2002; Brooks et al., 1991; Buck et al., 2004). Very few fluorophores have both of these qualities for measuring transition metals. Transition metals are essential components in all classes of enzymes, participating in functions as diverse as electron transport, maintenance of structural integrity, enzymatic catalysis, and neuronal communication (Silvia and Williams, 1991). Copper is an integral metal ion in several biological path- ways with major functions inside organisms, including both electron transfer and O 2 metabolism and transport (Sigel and Editor, 1981). Copper-containing proteins have a prominent ∗ Corresponding authors. Tel.: +1 734 764 7541; fax: +1 734 647 4865. E-mail addresses: sumnerj@umich.edu (J.P. Sumner), rthompso@umaryland.edu (R.B. Thompson), fierke@umich.edu (C.A. Fierke), kopelman@umich.edu (R. Kopelman). 1 Tel.: +1 7346472170; fax: +1 7349362778. 2 Tel.: +1 410 706 7142; fax: +1 410 706 7122. role in the human nervous system, and important neurological ailments have been linked to defects in copper homoeostasis (Waggoner et al., 1999). Menkes’ and Wilson’s diseases, both neurological disorders, are connected with the body’s inabil- ity to metabolize copper effectively. At normal biological conditions, free Cu + is believed to be at or below femtomolar quantities (Finney and O’Halloran, 2003), while total concentration in blood is micromolar (Hartter and Barnea, 1988). However, free copper concentrations can reach ele- vated concentrations under aggravated conditions (Hartter and Barnea, 1988; Bush, 2000). Copper is also widely used in industrial processes, such as anti-fouling agents for com- mercial shipping vessels, and is a source of pollution in the environment (High et al., 2001). This could result in toxicity in the biological environment (Tapia et al., 2003) considering that micromolar amounts of Cu 2+ are toxic to commercial shellfish. Current methods for determining copper levels in biolog- ical and environmental samples include atomic absorption spectroscopy and ICP–MS, both of which measure total copper concentrations and destroy the sample in the process. In keeping with trends in cell biology and environmental sci- 0956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2005.04.023