Quantum Dots DOI: 10.1002/ange.200704794 OpticalDetectionofGlucoseandAcetylcholineEsteraseInhibitorsby H 2 O 2 -SensitiveCdSe/ZnSQuantumDots** RonGill,LilyBahshi,RonitFreeman,andItamarWillner* There is a growing interest in using semiconductor quantum dots (QDs) as optical labels for biosensing events. [1] The size- controlled fluorescence properties of QDs, [2] the high fluo- rescence quantum yields of QDs, [3] and their stability against photobleaching [4] makes QDs superior optical labels for multiplexed analysis of antigen–antibody complexes, [5] nucleic acid–DNA hybrids, [6] and other biorecognition com- plexes. [7] QDs were also applied to monitor biocatalytic transformations using fluorescence resonance energy transfer (FRET) processes. FRET processes between CdSe/ZnS QDs and dye units incorporated into replicated DNA systems or into telomers were used to probe the activities of polymerase and telomerase, respectively. [8] Similarly, FRET reactions were used to monitor the biocatalytic cleavage of peptides by hydrolytic enzymes. [9] Alternatively, electron-transfer quenching of QDs by quinone-functionalized peptides was used to detect the activity of tyrosinase, and the hydrolytic cleavage of the quinone-modified peptide and the restoration of the fluorescence of the QDs were used to probe the activities of tyrosinase and thrombin, respectively. [10] In all of these QD assays for monitoring enzyme activities, it is mandatory to include a quencher (energy or electron-transfer quencher) in the analyzed samples as a reporter unit. Also, for each of the enzymes, a specific assay needs to be developed. Numerous oxidases generate hydrogen peroxide (H 2 O 2 ) as a product. Thus, controlling the photophysical properties of QDs by H 2 O 2 may provide a new and versatile method to develop QD-based sensors. In fact, the biocatalyzed gener- ation of H 2 O 2 by oxidases was used for the development of different electrochemical biosensors, [11] and recently for the development of optical biosensors using Au nanoparticles. [12] Herein we demonstrate that the fluorescence of CdSe/ZnS QDs is sensitive to H 2 O 2 . This sensitivity enables the use of the QDs as H 2 O 2 sensors and provides a versatile fluorescent reporter for the activities of oxidases and for the detection of their substrates. This utility is exemplified herein for the analysis of glucose in the presence of glucose oxidase. Furthermore, we apply the fluorescent QDs as sensors that monitor the inhibition of acetylcholine esterase (AChE). AChE hydrolyzes acetylcholine to choline and, subsequently, choline oxidase (ChOx) oxidizes choline to betaine while generating H 2 O 2 . In the presence of an inhibitor, the hydro- lytic cleavage of acetycholine by AChE is perturbed, and the inhibited formation of H 2 O 2 is reflected by the fluorescence of the QDs. In addition to the broad application of the CdSe/ ZnS for different sensing processes, we introduce the ratiometric fluorescent analysis of the different substrates. This analysis enables us to monitor the stability of the different sensors, and to correct for any precipitation events of the QDs that might cause an “apparent” decrease in the observed fluorescence intensities. We describe the use of the enzymes in solution or in immobilized forms on the QDs. Figure 1 a depicts the time-dependent luminescence changes upon the reaction of mercaptoundecanoic acid (MUA) capped CdSe/ZnS QDs with H 2 O 2 (0.4 mm). The fluorescence of the QDs decreases with time, and addition of catalase to the system, which includes H 2 O 2 , blocks the decrease in the fluorescence, implying that H 2 O 2 is, indeed, the component affecting the fluorescence. Figure 1b shows the fluorescence quenching of the QDs upon interaction with different concentrations of H 2 O 2 for a fixed time interval of 10 minutes. Although the precise mechanism that stimulates the decrease in the fluorescence of the QDs is not fully Figure 1. a) Time-dependent fluorescence changes of mercaptoundeca- noic acid capped CdSe/ZnS QDs in the presence of 0.4 mm H 2 O 2 : (1) 0 min, (2) 2 min, (3) 5 min, (4) 10 min, (5) 15 min; b) Quenched fluorescence in the presence of variable concentrations of H 2 O 2 upon interaction with the CdSe/ZnS QDs for a fixed time interval of 10 min. All measurements were performed in a 10 mm HEPES buffer solution (pH 7.2). [*] R. Gill, L. Bahshi, R. Freeman, Prof. I. Willner Institute of Chemistry and Center for Nanoscience The Hebrew University of Jerusalem Jerusalem 91904 (Israel) Fax: (+ 972)2-652-7715 E-mail: willnea@vms.huji.ac.il Homepage: http://chem.ch.huji.ac.il/willner [**] This research is supported by the James Franck Program, the VSN EC Project, and the Israel Science Foundation. Zuschriften 1700 # 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2008, 120, 1700 –1703