Ron Gill

Semiconductor Quantum Dots for Bioanalysis

Semiconductor nanoparticles, or quantum dots (QDs), have unique photophysical properties, such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching. These properties enable the use of QDs as optical labels for the multiplexed analysis of immunocomplexes or DNA hybridization processes. Semiconductor QDs are also used to probe biocatalytic transformations. The time-dependent replication or telomerization of nucleic acids, the oxidation of phenol derivatives by tyrosinase, or the hydrolytic cleavage of peptides by proteases are probed by using fluorescence resonance energy transfer or photoinduced electron transfer. The photoexcitation of QD-biomolecule hybrids associated with electrodes enables the photoelectrochemical transduction of biorecognition events or biocatalytic transformations. Examples are the generation of photocurrents by duplex DNA assemblies bridging CdS NPs to electrodes, and by the formation of photocurrents as a result of biocatalyzed transformations. Semiconductor nanoparticles are also used as labels for the electrochemical detection of DNA or proteins: Semiconductor NPs functionalized with nucleic acids or proteins bind to biorecognition complexes, and the subsequent dissolution of the NPs allows the voltammetric detection of the related ions, and the tracing of the recognition events.

Optical Detection of Glucose and Acetylcholine Esterase Inhibitors by H2O2‐Sensitive CdSe/ZnS Quantum Dots

There is a growing interest in using semiconductor quantum dots (QDs) as optical labels for biosensing events. The sizecontrolled fluorescence properties of QDs, the high fluorescence quantum yields of QDs, and their stability against photobleaching makes QDs superior optical labels for multiplexed analysis of antigen–antibody complexes, nucleic acid–DNA hybrids, and other biorecognition complexes. 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. Similarly, FRET reactions were used to monitor the biocatalytic cleavage of peptides by hydrolytic enzymes. 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. 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 (H2O2) as a product. Thus, controlling the photophysical properties of QDs by H2O2 may provide a new and versatile method to develop QD-based sensors. In fact, the biocatalyzed generation of H2O2 by oxidases was used for the development of different electrochemical biosensors, and recently for the development of optical biosensors using Au nanoparticles. Herein we demonstrate that the fluorescence of CdSe/ZnS QDs is sensitive to H2O2. This sensitivity enables the use of the QDs as H2O2 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 H2O2. In the presence of an inhibitor, the hydrolytic cleavage of acetycholine by AChE is perturbed, and the inhibited formation of H2O2 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 1a depicts the time-dependent luminescence changes upon the reaction of mercaptoundecanoic acid (MUA) capped CdSe/ZnS QDs with H2O2 (0.4 mm). The fluorescence of the QDs decreases with time, and addition of catalase to the system, which includes H2O2, blocks the decrease in the fluorescence, implying that H2O2 is, indeed, the component affecting the fluorescence. Figure 1b shows the fluorescence quenching of the QDs upon interaction with different concentrations of H2O2 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

Probing protein kinase (CK2) and alkaline phosphatase with CdSe/ZnS quantum dots.

Semiconductor quantum dots (QDs) are used for the optical analysis of casein kinase (CK2) or the hydrolytic activity of alkaline phosphatase (ALP). Two schemes for the analysis of CK2 by a FRET-based mechanism are described. One approach involves the CK2-catalyzed phosphorylation of a serine-containing peptide (1), linked to CdSe/ZnS QDs, with Atto-590-functionalized ATP. The second analytical method involves the specific association of the Atto-590-functionalized antibody to the phosphorylated product. The hydrolytic activity of ALP is followed by the application of phosphotyrosine (4)-modified CdSe/ZnS QDs in the presence of tyrosinase as a secondary reporter biocatalyst. The hydrolysis of (4) yields the tyrosine units that are oxidized by O(2)/tyrosinase to the respective dopaquinone product. The latter quinone units quench the QDs via an electron transfer route, leading to the optical detection of the ALP activity.

Lighting up biochemiluminescence by the surface self-assembly of DNA-hemin complexes.

The discovery of catalytic RNAs (ribozymes) has sparked scientific activities directed to the preparation of new biocatalysts and raised the suggestion that these biomolecules participated in the evolutionary process as preprotein catalysts. 2] Analogously, deoxyribozymes, catalytic DNAzymes, are not found in nature but extensive research efforts have demonstrated the successful synthesis of catalytic deoxyribozymes for many chemical transformations. 4] One interesting example of a catalytic DNA that reveals peroxidase-like activity includes a supramolecular complex between hemin and a single-stranded guanine-rich nucleic acid (aptamer). This complex was reported to catalyze the oxidation of 2,2 -azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) by H2O2, a common reaction used as an assay for peroxidase activity. It was suggested that the supramolecular docking of the guanine-quadruplex layers facilitates the intercalation of hemin into the complex and the formation of the biocatalytically active hemin center. Enzymes and, specifically, horseradish peroxidase (HRP) 9] are used as biocatalytic labels for the amplified detection of DNA-sensing events. The electrochemical amplified detection of DNA has been accomplished in the presence of different enzymes 8] and the chemiluminescent analysis of DNA in the presence of HRP has been reported. The integration of a DNA biocatalyst into DNA-detection schemes could provide a new method for the detection of nucleic acids that might reveal important advantages: 1) The catalytic DNA may substitute the protein-based biocatalysts, and thus eliminate nonspecific binding phenomena; 2) Tailoring of the DNA biocatalyst as part of the labeled nucleic acid might reduce the number of analytical steps for DNA detection. Here we report that two separated nucleic acids that include the segments A and B–constituting the single-stranded peroxidase deoxyribozyme, which forms a layered G-quadruplex structure (see Scheme 1)–self-assemble in the presence of hemin to form a biocatalyst for the generation of chemiluminescence in the presence of H2O2 and luminol. The effect of hybridization with the DNAzyme compounds on the resulting biochemiluminescence is discussed. We also demonstrate the self-assembly of biocatalytic, supramolecular hemin ±nucleic acid complexes on gold electrodes in monolayer configurations, and describe the biocatalytic and bioelectrocatalytic formation of chemiluminescence at the Acknowledgements

Functionalized CdSe/ZnS QDs for the detection of nitroaromatic or RDX explosives.

Chemically modified CdSe/ZnS quantum dots (QDs) are used as fluorescent probes for the analysis of explosives, and specifically, the detection of trinitrotoluene (TNT) or trinitrotriazine (RDX). The QDs are functionalized with electron-donating ligands that bind nitro-containing explosives, exhibiting electron-acceptor properties, to the QD surface, via supramolecular donor-acceptor interactions leading to the quenching of the luminescence of the QDs.

Electrochemical, Photoelectrochemical, and Piezoelectric Analysis of Tyrosinase Activity by Functionalized Nanoparticles

The electrochemical and photoelectrochemical detection of tyrosinase (TR) activity (an indicative marker for melanoma cancer cells) is reported, using Pt nanoparticles (NPs) or CdS NPs as electrocatalytic labels or photoelectrochemical reporter units. The Pt NPs or CdS NPs are modified with a tyrosine methyl ester, (1), capping layer. Oxidation of the capping layer by TR/O2 yields the respective L-DOPA and dopaquinone products. The reduction of the resulting mixture of products with citric acid yields the L-DOPA derivative,(3), as a single product. The association of the (3)-functionalized Pt NPs or CdS NPs to a boronic acid monolayer-modified electrode enables the electrochemical transduction of TR activity by the Pt-NPs-electrocatalyzed reduction of H2O2 or the photoelectrochemical transduction of TR activity by the generation of photocurrents in the presence of triethanolamine as a sacrificial electron donor. The detection limits for analyzing TR corresponds to 1 U and 0.1 U by the electrochemical and photoelectrochemical methods, respectively. The association of the Pt NPs or CdS NPs to the functionalized monolayer electrode is followed by quartz crystal microbalance measurements.

Biosensing and Probing of Intracellular Metabolic Pathways by NADH-Sensitive Quantum Dots†

The use of semiconductor quantum dots (QDs) as optical labels for biorecognition events and biocatalytic processes attracts growing interest. 2] While numerous studies reported on the use of the QDs as fluorescent labels, applications of semiconductor QDs as optical probes of dynamic bioprocesses, such as enzymatic transformations, using fluorescence resonance energy transfer (FRET) or photoinduced electron transfer reactions are still scarce. The replication of DNA by polymerase or telomerization of a nucleic acid by telomerase were monitored by the incorporation of a dye into the replica/ telomers associated with QDs and the use of FRETas readout signal. The scission of duplex DNA linked to CdSe QDs by DNase and the hydrolytic cleavage of peptides bound to CdSe QDs were followed by FRET processes. Recently, the activities of tyrosinase and thrombin were analyzed by the tyrosinase-induced generation of quinone residues on amino acid or peptide capping layers associated with CdSe QDs. This resulted in electron-transfer quenching of the QDs. The subsequent hydrolytic cleavage of the peptide by thrombin removed the quencher and recovered the fluorescence of the QDs. We describe the synthesis of Nile-blue-functionalized CdSe/ZnS quantum dots as a hybrid material that optically senses 1,4-dihydronicotinamide adenine dinucleotide (phosphate) cofactors, NAD(P)H. The modified quantum dots enable the fluorescence imaging of 1,4-nicotinamide adenine dinucleotide (phosphate) {NAD(P)}-dependent biocatalytic transformations and allow the monitoring of the intracellular metabolism in HeLa cancer cells. This technique allows the application of the NAD(P)H-sensitive QDs to screen anticancer agents and to probe the effect of drugs on intracellular metabolism. Whereas previous applications of QDs to probe enzyme activities required the synthesis of specifically functionalized QDs, we sought generic functionalized QDs that could act as versatile probes to analyze different biocatalyzed transformations. Numerous redox enzymes use the common NAD(P) cofactor, and hence the use of appropriately functionalized QDs to analyze NAD(P)H could provide a generic method to analyze NAD(P)-dependent enzymes, as well as to detect their substrates. Indeed, substantial efforts have been directed to the development of biosensors based on NAD(P)-dependent biocatalysts. Different enzyme electrodes for the amperometric detection of the substrates of NAD(P)-dependent enzymes were designed, and molecular electron relays or redox polymers 15] were used to electrocatalyze the oxidation of NAD(P)H. Also, different integrated electrodes consisting of surface-confined relay–NAD(P)–enzyme assemblies for the electrochemical analysis of different substrates were developed. 17] Recently, the NAD(P)H-stimulated growth of Au nanoparticles was used to develop optical sensors that probe NAD-dependent enzymes and their substrates in solution or on surfaces. Similarly, the NADH-mediated growth of Cu nanoparticles was used for the electrochemical detection of NAD(P)-dependent enzymes and their substrates. Herein we report the design of functionalized semiconductor QDs for the detection of NADH and their use to follow NAD-dependent biocatalyzed transformations. Furthermore, we incorporated the NADH-sensing QDs into HeLa cancer cells and monitored the intracellular metabolism by the functionalized QDs and the effect of anticancer drugs on the cell metabolism. CdSe-core CdS(2 layers, Ls)/Cd0.5 Zn0.5S(3 Ls)/ZnS(2 Ls) multishell QDs with diameter 7.3 1.0 nm (core diameter 2.6 nm) were prepared according to a literature procedure. 20] These QDs were then transformed into watersoluble QDs by ligand exchange with 3-mercaptopropionic acid (MPA). The modified QDs were functionalized with bovine serum albumin (BSA), and then, Nile blue (1) was covalently linked to the BSA layer (see the Experimental Section). Spectroscopic analysis of the 1-functionalized QDs indicated that approximately seven units of 1 were associated with each particle. Nile blue acts as an electron mediator for the oxidation of the NAD(P)H cofactors. Accordingly, Figure 1a depicts the method to analyze NAD(P)H by the functionalized QDs. The fluorescence of the QDs is quenched by 1 through FRET quenching (QD emission: 635 nm, 1 absorbance: 630 nm). It should be noted that the quantum yield of emission of photoexcited 1 is very low at room temperature and cannot be detected. Thus, although 1 quenches effectively the luminescence of the QDs, it is nonemissive. In the presence of NADH, the reduced dye units (2) associated with the QDs lack absorbance in the visible spectral region, and thus do not quench the QDs. As a result, the reduction of the 1 capping layer by the NAD(P)H cofactors activates the fluorescence of the QDs, and provides a path for the optical detection of NADH. Figure 1b depicts the fluorescence intensity of the 1-functionalized QDs prior [*] R. Freeman, R. Gill, Dr. I. Shweky, Prof. M. Kotler, Prof. U. Banin, Prof. I. Willner Institute of Chemistry and Center for Nanoscience and Nanotechnology The Hebrew University of Jerusalem, Jerusalem 91904 (Israel) Fax: (+ 972)2-6527715 E-mail: willnea@vms.huji.ac.il Homepage: http://chem.ch.huji.ac.il/willner

Probing Kinase Activities by Electrochemistry, Contact‐Angle Measurements, and Molecular‐Force Interactions

Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistance in the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5 degrees , phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8 degrees. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3 degrees . The third method to characterize the CK2 system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6+/-2 pN), significant rupture forces (multiples of 120+/-20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region.

Probing of enzyme reactions by the biocatalyst-induced association or dissociation of redox labels linked to monolayer-functionalized electrodes

The activities of the enzymes tyrosinase and thrombin are probed by the association of the ferrocene boronic acid label to the enzyme-generated catechol ligand, and by the cleavage of the ligand-redox complex tethered to a peptide, respectively.