Yossi Weizmann

Au-Nanoparticle Nanowires Based on DNA and Polylysine Templates

The assembly of ordered nanoparticle architectures is a challenging topic in nanotechnology directed to the construction of nanoscale devices.[1] Within this broad subject, the conjugation of biomaterials and nanoparticles to yield ordered architectures is a promising route to tailor future sensing and catalytic devices, nanocircuitry, or nanodevices, for example transistors, and computing devices.[2] DNA is an attractive biomaterial for use as a template in programmed nanoparticle structures. The ability to synthesize nucleic acids of predesigned shapes and composition, the versatile biocatalytic transformations that can be performed on DNA, for example, ligation, scission, or polymerization, enable TMcut and paste∫ procedures to be carried out on the template DNA, thus enabling us to design and manipulate the DNA ∫mold∫. Furthermore, the association of metal ions to the DNA phosphate units, or the intercalation of transition-metal complexes or molecular substrates into the DNA provide a means to functionalize the DNA-template and to initiate further chemical transformations on the mold. Nanoparticle ± DNA assemblies were organized by the hybridization of nucleic-acid-functionalized metal[3] or semiconductor nanoThe fluorescence data in our experiments indicated that the surface coverage of the final printed layer for each of the three patterning methods presented here is nearly equivalent and reaches about 60% of the surface coverage obtained by direct deposition of the antibodies from solution. As already described for CP and CP of proteins, the printing process does not compromise the binding efficiency of the printed antibody. This strategy might not be suitable for patterning a large number of different proteins on a surface. However, it can place a few different proteins as adjacent high-density arrays on a surface. Such arrays could find an application for high-throughput screening in which a large number of analytes could be spotted using a subset of the patterned areas. Another possibility for creating high-density immunoassays on planar surfaces is by performing surface immunoassays using many different analytes and capture sites, such as shown in Figure 5. The main limiting factor in using the prepared microarrays for diagnostic purposes could be misplacement of target molecules during the inking of the stamp. Such a misplacement, which may induce false positive reactions, can arise from cross-reactions of the target molecules with different capture proteins and/or from nonspecific adsorption on the -stamp. The former is limited by biological specificity of affinity extraction. The latter can be limited by the systematic use of blocking agents such as BSA. Indeed, for the recognition of goat antigen by the printed array shown in Figure 5a, the recognition signal in the areas with printed anti-chicken antibodies was only 5% of that in the areas with printed anti-goat antibodies. In summary, we have illustrated how CP can complement different patterning methods to produce repeatedly, and in parallel, high resolution arrays of proteins in three simple steps: 1) TMinking∫, 2) rinsing, and 3) printing the stamp on the substrate. Since -stamps carry the complementary pattern of binding partners specific to the target proteins on their surface, the proteins self-assemble into the predefined array on the stamp surface during inking in solution, and dissociate upon printing. Hence, the (re)production of the target protein arrays is fast and easy. The initial production of the -stamp is a one-time burden only. We thus believe that the methodology presented is powerful and versatile, and should be useful in detection and fabrication strategies that are based on arrays of proteins.

Diagnosing viruses by the rolling circle amplified synthesis of DNAzymes

Circular DNA is used as a template for the amplified detection of M13 phage ssDNA by a rolling circle amplification (RCA) process that synthesizes DNAzyme chains, thus enabling the colorimetric or chemiluminescent detection of the analyte.

Amplified detection of single-base mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction

Three different methods for the amplified detection of a single-base mismatch in DNA are described using microgravimetric quartz-crystal-microbalance as transduction means. All methods involve the primary incorporation of a biotinylated base complementary to the mutation site in the analyzed double-stranded primer/DNA assembly. The double-stranded assembly is formed between 25 complementary bases of the probe DNA assembled on the Au-quartz crystal and the target DNA. One method of amplification includes the association of avidin- and biotin-labeled liposomes to the sensing interface. The second method of amplified detection of the base mismatch includes the association of an Au-nanoparticle-avidin conjugate to the sensing interface, and the secondary Au-nanoparticle-catalyzed deposition of gold on the particles. The third amplification route includes the binding of the avidin-alkaline phosphatase biocatalytic conjugate to the double-stranded surface followed by the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate to the insoluble product indigo derivative that precipitates on the transducer. Comparison of the three amplification routes reveals that the catalytic deposition of gold on the Au-nanoparticle/avidin conjugate is the most sensitive method, and the single-base mismatch in the analyzed DNA is detected with a sensitivity that corresponds to 3x10(-16) M.

A polycatenated DNA scaffold for the one-step assembly of hierarchical nanostructures

A unique DNA scaffold was prepared for the one-step self-assembly of hierarchical nanostructures onto which multiple proteins or nanoparticles are positioned on a single template with precise relative spatial orientation. The architecture is a topologically complex ladder-shaped polycatenane in which the “rungs” of the ladder are used to bring together the individual rings of the mechanically interlocked structure, and the “rails” are available for hierarchical assembly, whose effectiveness has been demonstrated with proteins, complementary DNA, and gold nanoparticles. The ability of this template to form from linear monomers and simultaneously bind two proteins was demonstrated by chemical force microscopy, transmission electron microscopy, and confocal fluorescence microscopy. Finally, fluorescence resonance energy transfer between adjacent fluorophores confirmed the programmed spatial arrangement between two different nanomaterials. DNA templates that bring together multiple nanostructures with precise spatial control have applications in catalysis, biosensing, and nanomaterials design.

Live Cell MicroRNA Imaging Using Cascade Hybridization Reaction

Recent advances in RNA research have posed new directives in biology and chemistry to uncover the complex roles of ribonucleic acids in cellular processes. Innovative techniques to visualize native RNAs, particularly, short, low-abundance RNAs in live cells, can dramatically impact current research on the roles of RNAs in biology. Herein, we report a novel method for real-time, microRNA imaging inside live cells based on programmable oligonucleotide probes, which self-assemble through the Cascade Hybridization Reaction (CHR).

Real-time detection of telomerase activity using the exponential isothermal amplification of telomere repeat assay.

As crucial pieces in the puzzle of cancer and human aging, telomeres and telomerase are indispensable in modern biology. Here we describe a novel exponential isothermal amplification of telomere repeat (EXPIATR) assay--a sensitive, simple, and reliable in vitro method for measuring telomerase activity in cell extracts. Through a strategically designed path of nucleic acid isothermal amplifications, EXPIATR abandons the expensive thermal cycling protocol and achieves ultrafast detection: telomerase activity equivalent to a single HeLa cancer cell can be detected in ∼25 min.

DNA-CNT Nanowire Networks for DNA Detection

The ability to detect biological analytes in a rapid, sensitive, operationally simple, and cost-effective manner will impact human health and safety. Hybrid biocatalyzed-carbon nanotube (CNT) nanowire-based detection methods offer a highly sensitive and specific platform for the fabrication of simple and effective conductometric devices. Here, we report a conductivity-based DNA detection method utilizing carbon nanotube-DNA nanowire devices and oligonucleotide-functionalized enzyme probes. Key to our sensor design is a DNA-linked-CNT wire motif, which forms a network of interrupted carbon nanotube wires connecting two electrodes. Sensing occurs at the DNA junctions linking CNTs, followed by amplification using enzymatic metalization leading to a conductimetric response. The DNA analyte detection limit is 10 fM with the ability to discriminate single, double, and triple base pair mismatches. DNA-CNT nanowires and device sensing gaps were characterized by scanning electron microscopy (SEM) and confocal Raman microscopy, supporting the enhanced conductometric response resulting from nanowire metallization.

Enzymatic Synthesis of Periodic DNA Nanoribbons for Intracellular pH Sensing and Gene Silencing

We report the construction of periodic DNA nanoribbons (DNRs) by a modified DNA origami method. Unlike the conventional DNA origami, the DNR scaffold is a long, single-stranded DNA of tandem repeats, originating from the rolling circular amplification (RCA). Consequently, the number of folding staple strands tremendously decreases from hundreds to a few, which makes the DNR production scalable and cost-effective, thus potentially removing the barrier for practical applications of DNA nanostructures. Moreover, the co-replicational synthesis of scaffold and staple strands by RCA-based enzymatic reactions allows the generation of DNRs in one pot, further reducing the cost. Due to their unique periodicity, rigidity, and high aspect ratio, DNRs are efficiently internalized into cells and escape from endosomal entrapment, making them potential nanocarriers for imaging agents and biological therapeutics. We demonstrated proof-of-concept applications of DNRs as an intracellular pH sensor and an efficient small interfering RNA delivery vehicle in human cancer cells.

Bipyramid-templated synthesis of monodisperse anisotropic gold nanocrystals

Much of the interest in noble metal nanoparticles is due to their plasmonic resonance responses and local field enhancement, both of which can be tuned through the size and shape of the particles. However, both properties suffer from the loss of monodispersity that is frequently associated with various morphologies of nanoparticles. Here we show a method to generate diverse and monodisperse anisotropic gold nanoparticle shapes with various tip geometries as well as highly tunable size augmentations through either oxidative etching or seed-mediated growth of purified, monodisperse gold bipyramids. The conditions employed in the etching and growth processes also offer valuable insights into the growth mechanism difficult to realize with other gold nanostructures. The high-index facets and more complicated structure of the bipyramid lead to a wider variety of intriguing regrowth structures than in previously studied nanoparticles. Our results introduce a class of gold bipyramid-based nanoparticles with interesting and potentially useful features to the toolbox of gold nanoparticles.

Amplified Telomerase Analysis by Using Rotating Magnetic Particles: The Rapid and Sensitive Detection of Cancer Cells

A highly sensitive telomerase detection method that involves amplified telomerase analysis and the use of rotating magnetic particles has been developed. Magnetic particles, functionalized with a primer (1) that is recognized by telomerase, are mixed with a nucleotide mixture that includes biotinylated‐dUTP, and telomerase‐induced elongation of the primers proceeds with simultaneous biotin incorporation. Avidin–Horseradish peroxidase conjugate, coupled to biotin labels, yields the biocatalytic functional particles. Mixing the resulting particles with naphthoquinone‐modified magnetic particles enables the optoelectronic detection of telomerase. Attraction of the magnetic particles to an electrode, followed by rotation of the particles, causes the electrocatalytic reduction of O2 to H2O2 and HRP‐catalyzed oxidation of luminol (3); this results in chemilumunescence. The intensity of the emitted light depends on the telomerase content of the sample and the rotation speed of the particles. A minimum number of 10 cancer cells could be detected.