ARTICLES PUBLISHED ONLINE: 23 JULY 2010 | DOI: 10.1038/NMAT2811 Quantum-dot/dopamine bioconjugates function as redox coupled assemblies for in vitro and intracellular pH sensing Igor L. Medintz 1 * , Michael H. Stewart 2 , Scott A. Trammell 1 , Kimihiro Susumu 2 , James B. Delehanty 1 , Bing C. Mei 2,3 , Joseph S. Melinger 4 , Juan B. Blanco-Canosa 5 , Philip E. Dawson 5 and Hedi Mattoussi 2† The use of semiconductor quantum dots (QDs) for bioimaging and sensing has progressively matured over the past decade. QDs are highly sensitive to charge-transfer processes, which can alter their optical properties. Here, we demonstrate that QD–dopamine–peptide bioconjugates can function as charge-transfer coupled pH sensors. Dopamine is normally characterized by two intrinsic redox properties: a Nernstian dependence of formal potential on pH and oxidation of hydroquinone to quinone by O 2 at basic pH. We show that the latter quinone can function as an electron acceptor quenching QD photoluminescence in a manner that depends directly on pH. We characterize the pH-dependent QD quenching using both electrochemistry and spectroscopy. QD–dopamine conjugates were also used as pH sensors that measured changes in cytoplasmic pH as cells underwent drug-induced alkalosis. A detailed mechanism describing the QD quenching processes that is consistent with dopamine’s inherent redox chemistry is presented. S emiconductor QDs have become well-established photolumi- nescent (PL) platforms for biological applications 1,2 . Unlike most organic dyes, QDs are also highly sensitive to charge transfer, which can alter their optical properties 3,4 , thus gener- ating interest in charge-transfer-based biosensing 5 . Redox-active compounds including metal complexes, ions and dyes have al- ready been investigated for use in photoinduced electron-transfer QD biosensing 6–12 . Catechols have also undergone extensive testing with QDs owing to their interesting electrochemistry, and differing interpretations have been used to explain the disparate results. Progressive quenching of CdSe/ZnS (ref. 13), CdS:Mn/ZnS (ref. 14), CdSe (ref. 15), CdTe (refs 16,17) and CdS (ref. 18) QDs in the presence of increasing benzoquinones and dopamine has been most commonly reported where the quinone is suggested as an electron acceptor. A mechanism whereby dopamine increases the rate of quenching through Förster resonance energy transfer (FRET) has also been postulated 18 . In contrast, PL increases of CdSe/CdS QDs in the presence of benzoquinone 19 along with both CdSe/ZnS (ref. 20) and CdTe (ref. 21) QDs by dopamine have also been found. Nadeau and co-workers reported that QD–dopamine bioconjugates can stain dopamine-receptor-expressing cells in redox-sensitive patterns where increased fluorescence was noted under oxidizing conditions 20 . Dopamine was suggested here as an electron donor that could quench or sensitize QDs through different mechanisms involving reactive oxygen 20,21 . Cumulatively, this confirms a role for quinones and especially dopamine in redox interactions with QDs; however, a full understanding of this system and how to exploit it for biosensing is lacking. Using peptides covalently displaying dopamine-hydroquinone controllably self-assembled onto QDs, we demonstrate that PL 1 Center for Bio/Molecular Science and Engineering Code 6900, US Naval Research Laboratory, Washington, District of Columbia 20375, USA, 2 Optical Sciences Division Code 5611, US Naval Research Laboratory, Washington, District of Columbia 20375, USA, 3 Department of Chemical Engineering, University of Massachusetts, Amherst, Massachusetts 01003, USA, 4 Electronic Science and Technology Code 6812, US Naval Research Laboratory, Washington, District of Columbia 20375, USA, 5 Departments of Cell Biology & Chemistry, The Scripps Research Institute, La Jolla, California 92037, USA. † Present address: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA. *e-mail: igor.medintz@nrl.navy.mil. quenching arises from a pH-dependent electron-transfer process from QDs to oxidized dopamine-quinone functioning as an electron acceptor (see Fig. 1a,b). Following photoexcitation, the QD conduction-band electron is transferred to the lowest unoccupied molecular orbital of a quinone acceptor, resulting in PL quenching, and the electron is then shuttled back to the QD valence band (see below). The pH-dependent concentration of oxidized dopamine in the QD conjugate at any point determines the magnitude of electron transfer and concomitant QD PL quenching. At low pH, the concentration of oxidized dopamine is small producing only marginal quenching. As pH increases, dopamine undergoes a proportional increase in oxidation to quinone by ambient O 2 and the appearance of this electron acceptor near the QD provides a favourable non-radiative channel for increased QD quenching as verified by shortening of the QD exciton lifetime. We show PL quenching efficiency to be dependent on QD size, as more pronounced quenching was observed for smaller-sized QDs. This arises as decreasing nanocrystal size provides better spatial overlap of carriers and a larger driving force, enhancing the probability of electron transfer. This mechanism is consistent with the electrochemistry of dopamine and structurally related quinone molecules, which are known potent electron acceptors in biological and abiotic formats 22–24 . As with other hydroquinones, dopamine undergoes autoxidation and is also oxidized by molecular O 2 , generating a concomitant H 2 O 2 species. Such coupled electron–proton systems exhibit slow redox kinetics with rate constants in aqueous solution that directly depend on pH (refs 22,25). Rates of oxidation to quinone increase markedly in buffers by >1,000-fold as pH increases from 6 to 12 (refs 22,24,26). 676 NATURE MATERIALS | VOL 9 | AUGUST 2010 | www.nature.com/naturematerials © 2010 Macmillan Publishers Limited. All rights reserved.