Etery Sharon

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.

Optical, Electrical and Surface Plasmon Resonance Methods for Detecting Telomerase Activity

Three different sensing platforms for the analysis of telomerase activity in human cells are described. One sensing platform involves the label-free analysis of the telomerase activity by a field-effect-transistor (FET) device. The telomerase-induced extension of a primer associated with the gate of the FET device, in the presence of the nucleotide mixture dNTPs, alters the gate potential, and this allows the detection of telomerase extracted from 65 ± 10 293T (transformed human embryonic kidney) cells/μL. The second sensing platform involves the optical detection of telomerase using CdSe/ZnS quantum dots (QDs). The telomerase-stimulated telomerization of the primer-functionalized QDs in the presence of the nucleotide mixture dNTPs results in the synthesis of the G-rich telomeres. The stacking of hemin on the self-organized G-quadruplexes found on the telomers results in the electron transfer quenching of the QDs, thus providing an optical readout signal. This method enables the detection of telomerase originating from 270 ± 20 293T cells/μL. The third sensing method involves the amplified surface plasmon resonance (SPR) detection of telomerase activity. The telomerization of a primer associated with Au film-coated glass slides, in the presence of telomerase and the nucleotide mixture (dNTPs), results in the formation of telomeres on the surface, and these alter the dielectric properties of the surface resulting in a shift in the SPR spectrum. The hybridization of Au NPs functionalized with nucleic acids complementary to the telomere repeat units with the telomeres amplifies the SPR shifts due to the coupling between the local plasmon of the NPs and the surface plasmon wave. This method enables the detection of telomerase extracted from 18 ± 3 293T cells/μL.

Ag Nanocluster/DNA Hybrids: Functional Modules for the Detection of Nitroaromatic and RDX Explosives

Luminescent Ag nanoclusters (NCs) stabilized by nucleic acids are implemented as optical labels for the detection of the explosives picric acid, trinitrotoluene (TNT), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The sensing modules consist of two parts, a nucleic acid with the nucleic acid-stabilized Ag NCs and a nucleic acid functionalized with electron-donating units, including L-DOPA, L-tyrosine and 6-hydroxy-L-DOPA, self-assembled on a nucleic acid scaffold. The formation of donor-acceptor complexes between the nitro-substituted explosives, exhibiting electron-acceptor properties, and the electron-donating sites, associated with the sensing modules, concentrates the explosives in close proximity to the Ag NCs. This leads to the electron-transfer quenching of the luminescence of the Ag NCs by the explosive molecule. The quenching of the luminescence of the Ag NCs provides a readout signal for the sensing process. The sensitivities of the analytical platforms are controlled by the electron-donating properties of the donor substituents, and 6-hydroxy-L-DOPA was found to be the most sensitive donor. Picric acid, TNT, and RDX are analyzed with detection limits corresponding to 5.2 × 10(-12) M, 1.0 × 10(-12) M, and 3.0 × 10(-12) M, respectively, using the 6-hydroxy-L-DOPA-modified Ag NCs sensing module.

Multiplexed Analysis of Genes Using Nucleic Acid-Stabilized Silver-Nanocluster Quantum Dots

Luminescent nucleic acid-stabilized Ag nanoclusters (Ag NCs) are applied for the optical detection of DNA and for the multiplexed analysis of genes. Two different sensing modules including Ag NCs as luminescence labels are described. One sensing module involves the assembly of a three-component sensing module composed of a nucleic acid-stabilized Ag NC and a quencher-modified nucleic acid hybridized with a nucleic acid scaffold that is complementary to the target DNA. The luminescence of the Ag NCs is quenched in the sensing module nanostructure. The strand displacement of the scaffold by the target DNA separates the nucleic acid-functionalized Ag NCs, leading to the turned-on luminescence of the NCs and to the optical readout of the sensing process. By implementing two different-sized Ag NC-modified sensing modules, the parallel multiplexed analysis of two genes (the Werner Syndrome gene and the HIV, human immunodeficiency, gene), using 615 and 560 nm luminescent Ag NCs, is demonstrated. The second sensing module includes the nucleic acid functionalized Ag NCs and the quencher-modified nucleic acid hybridized with a hairpin DNA scaffold. The luminescence of the Ag NCs is quenched in the sensing module. Opening of the hairpin by the target DNA triggers the luminescence of the Ag NCs, due to the spatial separation of the Ag NCs/quencher units. The system is applied for the optical detection of the BRAC1 gene. In addition, by implementing two-sized Ag NCs, the multiplexed analysis of two genes by the hairpin sensing module approach is demonstrated.

DNAzyme-like activity of hemin-telomeric G-quadruplexes for the optical analysis of telomerase and its inhibitors.

The telomeres cap and protect the eukaryotic chromosome ends from undesired degradation and end-to-end fusion. They consist of short tandem DNA repeats (TTAGGG in vertebrates). In human somatic cells, the telomeres undergo progressive shortening during cell proliferation, and at a certain length they signal the cell to terminate its life cycle and undergo apoptosis. Telomerase is a ribonucleoprotein reverse transcriptase. It binds to the telomere ends and elongates them by copying telomeric repeats from its endogenous RNA template. This telomerase-mediated elongation of the telomeres compensates for the natural shortening of the telomeres, thus prolonging cellular lifespan and potentially transforming the cell into a malignant form. Indeed, over 85 % of all cancer cells exhibit elevated amounts of telomerase. Accordingly, the rapid, cost-effective, easy, and sensitive analysis of telomerase activity is important for diagnosis and for the development of anticancer drugs (telomerase inhibitors). Various analytical methods have been developed to analyze the activity of telomerase. The most frequently used method is the telomeric repeat amplification protocol (TRAP), which is PCR-based. However, this method is susceptible to inhibition of the PCR by the cell extract, requires costly instrumentation and reagents, and is time consuming. Moreover, the exponential amplification of the telomerase products increases the risk of false-positive results, and makes the quantitative analysis of telomerase products difficult. Various electrochemical and optical methods to monitor telomerase activity have been reported. Electrochemical methods include the electrochemical detection of ferrocenyl naphthalene diimide, which binds to the telomeric tetraplex structure, and the coulometric analysis of the Ru(NH3)6 3+ label, which binds to duplex DNA formed between the telomeric repeats and a nucleic acid complementary to the telomere repeat units. Optical methods for detection of telomerase activity include the fluorescence resonance energy transfer (FRET) process between quantum dots and an acceptor dye incorporated into telomeres synthesized by telomerase on the quantum dots. The detection of telomerase activity has also been carried out by designing DNA hairpin structures that include a protected horseradish peroxidase mimicking DNAzyme structure in the stem region. Activation of the DNAzyme stimulates a colored reaction upon opening of the hairpins by the telomeres. Nanotechnology-based detection of telomeres by magnetomechanical deflection of cantilevers has been reported. Activation of telomerase activity on the cantilevers in combination with specific hybridization of magnetic particles functionalized with nucleic acids complementary to the telomere repeat units enables forced deflection of the cantilever under an applied magnetic field. Although substantial progress has been achieved in the analysis of telomerase activity, the sensitivity of the various platforms is usually insufficient and requires a preceding PCR amplification step, which is accompanied by the aforementioned drawbacks. In the presence of hemin (1), G-rich DNA sequences are known to form catalytic hemin–G-quadruplex nanostructures. The best known system is the horseradish peroxidase mimicking hemin–G-quadruplex that was found to catalyze the H2O2-mediated oxidation of 2,2’-azinobis(3-ethylbenzthiazoline6-sulfonic acid), ABTS , to the colored product, ABTSC , or to catalyze the generation of chemiluminescence in the presence of luminol/H2O2. In fact, many sensing platforms have used the hemin–G-quadruplex DNAzyme as a catalytic label. Further studies have demonstrated that various G-rich DNA sequences which self-assemble into parallel or antiparallel G-quadruplexes reveal horseradish-peroxidase-like catalytic activities in the presence of hemin. The relative activities of the hemin–Gquadruplexes are dominated by the specific configuration of the nanostructures. The G-rich telomeric repeat units are known to adopt a G-quadruplex conformation, and are therefore anticipated to incorporate hemin in catalytically active structures. These could then serve as biocatalytic amplifying labels that follow telomerase activity. In fact, several studies have reported the catalytic activities of the complex generated between hemin and a synthetic DNA strand that includes the telomeric repeat sequence, but this phenomenon was never implemented to detect telomerase activities in biological samples. Herein we report the development of a colorimetric assay that allows quantitative monitoring of telomerase activity originating from transformed human embryonic kidney (293T) cells, and to probe inhibitors of telomerase. Hemin inhibits the activity of telomerase through stabilization of the G-quadruplex structure of the telomere ends, and we found that the cell extract perturbs the hemin–G-quadruplex DNAzyme functions. Nonetheless, these difficulties were resolved by the appropriate design of the analytical steps, and a colorimetric [a] R. Freeman, E. Sharon, Dr. C. Teller, A. Henning , Prof. I. Willner Institute of Chemistry, The Center for Nanoscience and Nanotechnology The Hebrew University of Jerusalem, Jerusalem 91904 (Israel) Fax: (+ 972) 2-6527715 E-mail : willnea@vms.huji.ac.il [b] Dr. C. Teller Fraunhofer Institute for Biomedical Engineering IBMT Am M hlenberg 13, 14476 Potsdam-Golm (Germany) [c] A. Henning Physikalische Chemie, Messund Sensortechnik Technische Universit t Dresden, 01062 Dresden (Germany) [d] Dr. Y. Tzfati Department of Genetics, The Silberman Institute of Life Sciences The Hebrew University of Jerusalem, Jerusalem 91904 (Israel) Fax: (+ 972) 2-6584902 Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000512.

Analysis of Telomerase by the Telomeric Hemin/G-Quadruplex-Controlled Aggregation of Au Nanoparticles in the Presence of Cysteine

Telomeres are guanosine-rich nucleic-acid chains that fold, in the presence of K(+) ions and hemin, into the telomeric hemin/G-quadruplex structure, exhibiting horseradish peroxidase mimicking functions. The telomeric hemin/G-quadruplex structures catalyze the oxidation of thiols (e.g., l-cysteine) into disulfides (e.g., cystine). As l-cysteine stimulates the aggregation of Au nanoparticles (NPs), accompanied by absorbance changes from red (individual Au NPs) to blue (aggregated Au NPs), the process is implemented to quantitatively analyze the activity (content) of telomerase, a versatile biomarker for cancer cells. Telomerase extracted from 293T cancer cells catalyzes, in the presence of a dNTPs mixture and an appropriate primer probe, the telomerization process, leading to the generation of catalytic telomeric hemin/G-quadruplex chains that control the l-cysteine-mediated aggregation of Au NPs. The extent of aggregation is thus controlled by the concentration of telomerase. The method enabled the detection of telomerase with a detection limit of 27 cells/μL. The spectral changes accompanying the aggregation of Au NPs are further supported by transmission electron microscopy imaging.

Electron-transfer quenching of nucleic acid-functionalized CdSe/ZnS quantum dots by doxorubicin: A versatile system for the optical detection of DNA, aptamer–substrate complexes and telomerase activity

The optical detection of DNA or the sensing of low-molecular-weight substrates or proteins by aptamer nucleic acids is a long term challenge in the design of biosensors. Similarly, the detection of the telomerase activity, a versatile biomarker of cancer cells, is important for rapid cancer diagnostics. We implement the luminescence quenching of the CdSe/ZnS quantum dots (QDs) as a versatile process to develop DNA sensors and aptasensors, and to design an analytical platform for the detection of telomerase activity. The formation of nucleic acid duplexes on QDs, or the assembly of aptamer-substrate complexes on the QDs (substrate=cocaine or thrombin) is accompanied by the intercalation of doxorubicin (DB) into the duplex domains of the resulting recognition complexes. The intercalated DB quenches the luminescence of the QDs, thus leading to the detection readout signal. Similarly, the telomerase-induced formation of the telomere chains on the QDs is followed by the hybridization of nucleic-acid units complementary to the telomere repeat units, and the intercalation of DB into the resulting duplex structure. The resulting luminescence quenching of the QDs provides an indicating signal for the activity of telomerase.

DNA Sensors and Aptasensors Based on the Hemin/G‐quadruplex‐Controlled Aggregation of Au NPs in the Presence of L‐Cysteine

L-cysteine induces the aggregation of Au nanoparticles (NPs), resulting in a color transition from red to blue due to interparticle plasmonic coupling in the aggregated structure. The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme catalyzes the aerobic oxidation of L-cysteine to cystine, a process that inhibits the aggregation of the NPs. The degree of inhibition of the aggregation process is controlled by the concentration of the DNAzyme in the system. These functions are implemented to develop sensing platforms for the detection of a target DNA, for the analysis of aptamer-substrate complexes, and for the analysis of L-cysteine in human urine samples. A hairpin DNA structure that includes a recognition site for the DNA analyte and a caged G-quadruplex sequence, is opened in the presence of the target DNA. The resulting self-assembled hemin/G-quadruplex acts as catalyst that controls the aggregation of the Au NPs. Also, the thrombin-binding aptamer folds into a G-quadruplex nanostructure upon binding to thrombin. The association of hemin to the resulting G-quadruplex aptamer-thrombin complex leads to a catalytic label that controls the L-cysteine-mediated aggregation of the Au NPs. The hemin/G-qaudruplex-controlled aggregation of Au NPs process is further implemented for visual and spectroscopic detection of L-cysteine concentration in urine samples.

Gossypol-Capped Mitoxantrone-Loaded Mesoporous SiO2 NPs for the Cooperative Controlled Release of Two Anti-Cancer Drugs

Mesoporous SiO2 nanoparticles, MP-SiO2 NPs, are functionalized with the boronic acid ligand units. The pores of the MP-SiO2 NPs are loaded with the anticancer drug mitoxantrone, and the pores are capped with the anticancer drug gossypol. The resulting two-drug-functionalized MP-SiO2 NPs provide a potential stimuli-responsive anticancer drug carrier for cooperative chemotherapeutic treatment. In vitro experiments reveal that the MP-SiO2 NPs are unlocked under environmental conditions present in cancer cells, e.g., acidic pH and lactic acid overexpressed in cancer cells. The effective unlocking of the capping units under these conditions is attributed to the acidic hydrolysis of the boronate ester capping units and to the cooperative separation of the boronate ester bridges by the lactate ligand. The gossypol-capped mitoxantrone-loaded MP-SiO2 NPs reveals preferential cytotoxicity toward cancer cells and cooperative chemotherapeutic activities toward the cancer cells. The MCF-10A epithelial breast cells and the malignant MDA-MB-231 breast cancer cells treated with the gossypol-capped mitoxantrone-loaded MP-SiO2 NPs revealed after a time-interval of 5 days a cell death of ca. 8% and 60%, respectively. Also, the gossypol-capped mitoxantrone-loaded MP-SiO2 NPs revealed superior cancer-cell death (ca. 60%) as compared to control carriers consisting of β-cyclodextrin-capped mitoxantrone-loaded (ca. 40%) under similar loading of the mitoxantrone drug. The drugs-loaded MP-SiO2 NPs reveal impressive long-term stabilities.