Ronit Freeman

Multiplexed Analysis of Hg2+ and Ag+ Ions by Nucleic Acid Functionalized CdSe/ZnS Quantum Dots and Their Use for Logic Gate Operations†

Semiconductor quantum dots (QDs) attract extensive research interest as optical labels for sensing events. The unique photophysical properties of QDs, such as high fluorescence quantum yields, narrow emission bands, high Stokes shifts, and stability against photobleaching, make them a superior sensing material. Specifically, the size-controlled luminescence features of QDs should enable the multiplexed detection of different analytes in solution or on-chip formats. Indeed, substantial progress was accomplished in the past few years in the application of QDs as optical labels for sensing and biosensing. Various QD-based biosensors were developed, including sensors that follow the activities of enzymes or their substrates, immunosensors, and sensors that follow DNA hybridizations or the formation of aptamer–substrate complexes. The development of multiplexed analytical assays is a major goal in sensing. The sizecontrolled emission properties of QDs enable the multiplexed analysis of different targets by QDs exhibiting controlled sizes. Indeed, different-sized QDs were used as optical labels for the multiplexed labeling of different immunocomplexes on surfaces. Whereas the use of QDs for biosensing is well advanced, the use of QDs for chemical sensing is scarce. The sensing of pH values by QDs or the detection of alkali metal ions by crown-ether-modified QDs has been reported. Recently, modified QDs were used to detect the RDX explosive, and boronic acid functionalized QDs were used for the competitive luminescence detection of saccharides and neurotransmitters. Also, b-cyclodextrin-modified QDs were applied to sense different substrates that bind to the cyclodextrin cavity using a competitive FRET assay. Metal ions, such as Hg and Ag, act as severe environmental pollutants and have serious medical effects on human health. Thus, the rapid and sensitive analysis of different ions in water or food resources is important. Various procedures for the analysis of Hg have been developed, including electrochemical and optical methods. Various ions form stable complexes by bridging specific nucleotide bases. For example, Hg ions bridge thymine bases, and Ag ions specifically bridge cytosine bases. These features were recently applied to develop oligo-Tor oligo-C-modified AuNPs for the optical detection of Hg or Ag through the ioninduced aggregation of the NPs. Herein we report on the use of T-richor C-rich-modified QDs for the selective analysis of Hg or Ag ions using an electron-transfer-quenching path. Specifically, we demonstrate the multiplexed analysis of Hg and Ag by different-sized QDs. Furthermore, the specific quenching of the QDs by the ions enabled us to use Hg and Ag ions as inputs that activate logic gates, and to implement QDs as optical readout signals for logic gate operations. CdSe/ZnS QDs (d = 3.8 nm, lem = 560 nm, QD ) were modified with the thymine-rich nucleic acid 1 using bis(sulfosuccinimidyl)suberate (BS) as a bifunctional coupling reagent. The average loading of the QDs with 1 was estimated to be three units per QD. Similarly, CdSe/ZnS QDs (d = 5.8 nm, lem = 620 nm, QD ) were modified with the cytosine-rich nucleic acid 2. The average loading of QD with 2 corresponded to five units per QD. Scheme 1 outlines the principle for the selective analysis of Hg or Ag ions by 1-QD or 2-QD, respectively. A rigid hairpin structure is formed in the presence of Hg or Ag ions, in which the T or C residues of the spatially separated nucleotides are linked by the ions. As the Hg–thymine or Ag–cytosine complexes lack any color that would enable energy transfer from the QDs, the decrease in their luminescence is attributed to electrontransfer quenching of the QDs by the ions bound to the thymine or cytosine bases. Figure 1A depicts the time-dependent luminescence changes upon interaction of 1-QD with 1 10 m Hg. The luminescence of the QDs decreased and reached (after 24 min) a steady-state value of 86% quenching of the initial luminescence of the QDs. The inset in Figure 1A shows the

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

Electrochemical, photoelectrochemical, and surface plasmon resonance detection of cocaine using supramolecular aptamer complexes and metallic or semiconductor nanoparticles.

Metallic or semiconductor nanoparticles (NPs) are used as labels for the electrochemical, photoelectrochemical, or surface plasmon resonance (SPR) detection of cocaine using a common aptasensor configuration. The aptasensors are based on the use of two anticocaine aptamer subunits, where one subunit is assembled on a Au support, acting as an electrode or a SPR-active surface, and the second aptamer subunit is labeled with Pt-NPs, CdS-NPs, or Au-NPs. In the different aptasensor configurations, the addition of cocaine results in the formation of supramolecular complexes between the NPs-labeled aptamer subunits and cocaine on the metallic surface, allowing the quantitative analysis of cocaine. The supramolecular Pt-NPs-aptamer subunits-cocaine complex allows the detection of cocaine by the electrocatalyzed reduction of H(2)O(2). The photocurrents generated by the CdS-NPs-labeled aptamer subunits-cocaine complex, in the presence of triethanol amine as a hole scavenger, allows the photoelectrochemical detection of cocaine. The supramolecular Au-NPs-aptamer subunits-cocaine complex generated on the Au support allows the SPR detection of cocaine through the reflectance changes stimulated by the electronic coupling between the localized plasmon of the Au-NPs and the surface plasmon wave. All aptasensor configurations enable the analysis of cocaine with a detection limit in the range of 10(-6) to 10(-5) M. The major advantage of the sensing platform is the lack of background interfering signals.

Chemiluminescence and Chemiluminescence Resonance Energy Transfer (CRET) Aptamer Sensors Using Catalytic Hemin/G-Quadruplexes

The incorporation of hemin into the thrombin/G-quadruplex aptamer assembly or into the ATP/G-quadruplex nanostructure yields active DNAzymes that catalyze the generation of chemiluminescence. These catalytic processes enable the detection of thrombin and ATP with detection limits corresponding to 200 pM and 10 μM, respectively. The conjugation of the antithrombin or anti-ATP aptamers to CdSe/ZnS semiconductor quantum dots (QDs) allowed the detection of thrombin or ATP through the luminescence of the QDs that is powered by a chemiluminescence resonance energy-transfer (CRET) process stimulated by the hemin/G-quadruplex/thrombin complex or the hemin/G-quadruplex/ATP nanostructure, in the presence of luminol/H(2)O(2). The advantages of applying the CRET process for the detection of thrombin or ATP, by the resulting hemin/G-quadruplex DNAzyme structures, are reflected by low background signals and the possibility to develop multiplexed aptasensor assays using different sized QDs.

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.

β-Cyclodextrin-Modified CdSe/ZnS Quantum Dots for Sensing and Chiroselective Analysis

Beta-cyclodextrin (beta-CD)-functionalized CdSe/ZnS quantum dots (QDs) are used for optical sensing and chiroselective sensing of different substrates using a fluorescence resonance energy transfer (FRET) or an electron transfer (ET) mechanisms. The FRET between the QDs and Rhodamine B incorporated in the beta-CD receptor sites is used for the competitive analysis of adamantanecarboxylic acid and of p-hydroxytoluene. Also, the dye-incorporated beta-CD-modified QDs are used for the chiroselective optical discrimination between D,L-phenylalanine and D,L-tyrosine. The receptor-functionalized QDs are also implemented for the optical detection of p-nitrophenol using an ET quenching route.

Amplified multiplexed analysis of DNA by the exonuclease III-catalyzed regeneration of the target DNA in the presence of functionalized semiconductor quantum dots.

Quantum dots (QDs) functionalized with a black-hole quencher are used as optical tracer for the detection of DNA using exonuclease as a biocatalyst. The binding of the target DNA or of a target/open hairpin complex to the functionalized QDs leads to the exonuclease-stimulated recycling of the target DNA or the target/hairpin complex. This results in the triggering of the luminescence of the QDs that provides a readout signal for the amplified sensing process. By using different-sized QDs, the multiplexed detection of DNAs is demonstrated.

CdSe/ZnS Quantum Dots-G-Quadruplex/Hemin Hybrids as Optical DNA Sensors and Aptasensors

The luminescence of CdSe/ZnS QDs is quenched via electron transfer by hemin/G-quadruplex associated with the particles. This phenomenon is implemented to develop DNA sensors or aptasensors by tailoring hairpin-functionalized QDs that generate the hemin/G-quadruplex quenchers upon sensing of the respective analytes.

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.

Amplified Fluorescence Aptamer‐Based Sensors Using Exonuclease III for the Regeneration of the Analyte

Quick and easy detection: The Exo III-stimulated regeneration of the analyte by the digestion of supramolecular aptamer-analyte complexes provides a means to develop amplified optical aptasensors (see figure).