of a pair of thrombin aptamers. Here, binder spacing was optimised at 5.8 nm, attaching to either side of the thrombin protein. Larger separa- tions were shown to result in only single binder interactions which sig- ni ﬁ cantly reduced af ﬁ nity and therefore the ef ﬁ ciency of the assay. Recently, the energetics of this system have been probed independently by others . By simulating a dual aptamer platform the authors pro- vide evidence to suggest that parallel binder approaches provide the most robust routes to biosensing with improved measurement con ﬁ - dence and reduced crosstalk. This work highlights the advantage of the spatial addressability that DNA nanostructures can provide within bio-sensing and bio-labelling applications. In order to ensure detection con ﬁ dence and reduce non-speci ﬁ c binding producing false positives, it is preferable to have a two-pronged approach of binding multiple analytes or binding the same analyte with multiple probes simulta- neously. However, typically the latter approach is hampered by the ran- dom distribution of the binder pairs when attached to support surfaces using bulk chemistry. Here, DNA nanostructures are suitably placed to act as an adapter between the support surface whilst providing spatial precision to binding pair placements. This has been expertly applied to the development of platforms which have helped to de ﬁ ne the spatial binding tolerance of antibodies  and for fragment-based drug dis- covery .
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The ability to create DNA nanostructures containing precise spacing’s and shapes is an important requirement for bottom-up self assembly. 1-4 In this approach, DNA templates are used for the organization of secondary molecular components. To this end, DNA origami has been shown to be an ideal method for the creation of arbitrary 2- dimensional shapes. 5 As originally demonstrated by Paul Rothemund, DNA origami entails the folding of a long, single-stranded DNA scaffold strand into a variety of shapes by the addition of a large number (typically >200) of short oligonucleotide staple strands. The scaffold strand used is the 7,249 nt long M13mp18 viral DNA strand. The staple strands are typically 32 nt long and can be used without purification. A variety of different shapes have been generated using this method including squares, rectangles, five-point stars, smiley faces, and even a map of China. 5, 6 The origami itself can be decorated with DNA dumbbells by modifying the staple strands, allowing words such as “DNA” and even pictures to be drawn on the surface of the origami. 5 DNA origami can also be modified to display single stranded DNA sticky ends on the edges, as well as on the surface. 7, 8
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Combinatorial approaches for fabricating one-dimensional polymer chains have revolutionized chemical synthesis  and the selection of functional nucleic acids . This chapter expand these principles to random two-dimensional networks to open new opportunities for fabricating more complex molecular devices on DNA nanostructures. Nature adapts through an inherent stochasticity. One has only to look at human's long biologic struggle against viruses to see an example of this in action. Nobel prize winning  research showed that developing human immune cells undergo a combinatorial rearrangement of antibody related gene segments. The goal is the formation of novel amino acid sequences in the antigen-binding region of antibodies for targeting a wide range of ever-adapting foreign invaders such as bacteria, parasites, or viruses. Yet we still get sick. Viruses also take advantage of combinatorial diversity to find functional units that have not yet been targeted by the immune system. Every year, people get flu shots yet become sick with the flu. While immunity is developed to the flu, inevitably, illness reoccurs the following year. The resilience of the flu is due to massive combinatorial changes in its surface receptors through random mutations and random switching of receptors among virus strains . Applying these combinatorial methods, so successful in nature, to scientific research has yielded tremendous results.
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Trimerization of CD30 by multimeric C2NP aptamers could significantly enhance the downstream signaling pathway. To explore if multimeric SL2B aptamers had a similar biological function, possibly via oligomerization of VEGF165 proteins and/or its receptors, TD with 1–4 SL2B was designed in this work. However, results showed that single SL2B (TD-SL2B) showed ~ 84% HT-29 cell inhibition while TD-2SL2B and TD-3SL2B demonstrated only ~ 40% and ~ 20% HT-29 cell inhibition, respectively, at the same SL2B concentration (Figure 5A). One possibility was that only one SL2B on the TD could bind to VEGF165 and cause downstream signal. Or more than one SL2B on the TD could bind to more than one VEGF165, but only one SL2B bound VEGF165 could interact with the VEGF receptor to cause downstream signal change. In future, DNA nanostructures with varied lengths among aptamers will be designed and used to explore the issue.
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kinase-7 (PTK7) receptor on tumor cells and led to higher gene silencing ef ﬁ ciency with lower cytotoxicity when compared to the commercial cationic lipid transfection agents. 141 Another study demonstrated that the formulation of dendrimeric siRNA improves condensation, stability, and gene silencing ef ﬁ ciencies. 52 Moreover, branched DNA constructs – containing anti-miRNA – have been used for oncomiRNA targeting of cancer. In another report, a multifunctional dendrimeric-based system composing of tumor-targeting ligands – such as MUC1, AS1411, and ATP aptamers – was used for high loading and controlled delivery of an anthracycline drug called “ epirubicin ” to cancer cells. MUC1 and AS1411 aptamer were coated on the surface of dendrimer to assist crossing the cellular and nuclear membranes, respectively. Meanwhile, ATP aptamer incorporated in the building blocks directed dendrimer to the ATP-enriched lysozymes where more dendrimer disas- sembly can occur. 28 Furthermore, ligase-independent ef ﬁ - cient self-assembly of DNA dendrimer is reported by annealing DNA units with elongated adhesive ends. These structures were used for delivery of immunostimulatory CpG DNA encoding tumor necrosis factor- α to immune cells. 142 Programmable DNA dendrimers coated with CpG-containing hairpin-loops were found to trigger stron- ger immune responses than those conjugated with linear CpG. Further surface functionalization of DNA dendrimer with TAT – a classic cell-penetrating peptide – enhanced cell internalization and cytokines production (Figure 6B). 34
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tures are functionalized with different recognition sites, they can be used as probes for multiple targets at once [ 73 ]. Fluorescent dyes on a DNA structure can be used to realize energy transfer pathways by building a line of different dyes [ 74 ]. Transport efficiencies and dis- tances of light energy transport can be controlled and evaluated with this system. This could eventually lead to the development of new artificial light-harvesting systems. With an effective method to func- tionalize metal nanoparticles with DNA [ 75 ], it was an obvious step to use DNA origami to arrange particle in specific geometries. This is especially useful to construct optically active assemblies, by uti- lizing plasmonic effects in metal nanoparticles. One useful effect of nanoparticles is the enhanced electric fields in the area between two particles called "hot spot". With a dimer of gold nanoparticles con- nected by a DNA origami structure, the hot spot can be used to en- hance the signal of a fluorescent dye [ 76 ]. Fluorescent dyes attached to the structure in the hot spot showed an enhanced Raman signal. Multiple particles arranged in a helical structure can show circular dichroism (CD). Contrary to natural molecules that show a CD in the UV spectrum, artificial chiral arrangements can be tuned for a CD in the visible spectrum opening up opportunities for new optical ma- terials [ 77 ]. Those CD signals have been theoretically predicted with an approach of interacting dipoles. Another chiral structure can be constructed with two nanorods [ 78 ]. Plasmonic nanostructures and plasmonic chirality will be discussed in the next chapter.
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Here, we report the effect of the introduction of basepair mismatches and single-strand nicks on the yield of RecA-based nucleoprotein filament assembly on a dsDNA scaffold. Significant increases in yield were observed upon the introduction of unpaired regions – reminiscent of permanent DNA bubbles – directly adjacent to the assembly region. However, a more complex behaviour was found when short unpaired regions were intro- duced away from the assembly site, where, as the length of the unpaired region was increased, the yield initially decreased, and then increased. These results suggest that an unpaired region in general interferes with the search mechanism of the RecA nucleoprotein filaments, although when located directly adjacent to the assembly site, it conversely enhances access to the homologous region and thus binding of the RecA filament. We propose that this enhanced access is enabled by increased breathing of the homologous site resulting from being positioned directly adjacent to the unpaired region. This hypothesis is corroborated by the introduction of single-strand nicks in the dsDNA adjacent to the assembly site, which also enhances breathing in the vicinity of the nick and thus also provides greater access to the DNA scaffold for the RecA nucleoprotein filament.
Figure 1. Design and modelling of DNA nanotubes. (A) Top: a single tile REs, based on the core RE, bears 4 sticky ends. Bottom: Complementarity between sticky ends directs the tiles to form a regular lattice. (B) A single tile SEs, based on a difference core SE, and its lattice. (C) Two tiles, REd and SEd, can assemble into a lattice with diagonal stripes; alone each tile could assemble into a linear strip. (D) Another pair of tiles, REp and SEp, cannot assemble independently but together can form a lattice with stripes perpendicular to the long axis of the tiles. (E) Structure of a DAE-E molecule. Each tile is assembled from five single strands: two of 37 nucleotides (nt) (top and bottom, no. 1 and no. 5, red and magenta), two of 26 nt (left and right, no. 2 and no. 4, yellow and green) and one of 42 nt (central, no. 3, blue). Triangles mark two crossover points, separated by two helical turns (21 nt). Arrowheads point from 5" to 3". Sticky ends (5 nt) are at the ends of the no. 2 and no. 4 strands. (F) Tile structure with hairpins (8 nt stem, 4 nt loop) on the no. 1 and no. 5 strands between the 14th and 15th nt from their 5" ends. Molecular models suggest that these hairpins attach underneath the molecule, as depicted here; in a tube they would be on the outside. (G and H) Two in-plane rotational symmetries that, if satisfied by a patch of tiles, encourage molecular strain to balance, resulting in a flat sheet. (I) A rotational symmetry, satisfied by DAE-E molecules, that permits curvature. (J) Heptagonal tube of radius R. In each tile, two cylinders of radius r represent the double-helices. Black circles mark crossover points. Blue and orange lines connect the position of phosphate backbones to the center of a helix. The smaller angle between the blue and orange lines defines the minor groove. Tiles from (A), (B), or (D) may form tubes of any number of tiles in circumference; tiles from (C) only tubes of an even number. (K) Cross-section of the red tile from (J) at a crossover point.
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TDN: tetrahedral DNA nanostructure; TDNN: ﬂuorescence resonance energy transfer based nanosensor in this experiment; Au-NP: gold nano- particles; DF: discrimination factor; miRNAs: micro- RNAs; FRET: ﬂuorescence resonance energy transfer; TCEP: tri (2-carboxyethyl) phosphine hydrochloride; PBS: phosphate buffer solution; TEM: transmission electron microscopy; PAGE: polyacrylamide gel electrophoresis; DLS: dynamic light scattering; DMEM: Dulbecco's modified Eagle's media; FBS: fetal bovine serum; ExP1: Expected detection probe1; ExP2: expected detection probe2; OrP: ordinary detection probe; SSC: saline-sodium citrate. RFI: relative fluorescence intensity.
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In summary, two FRET-based waveguides and a systematic approach to fabricate four different nanoparticle arrays have been demonstrated. FRET-based waveguides have been successfully designed, fabricated, and characterized using DNA tiles, DNA origami nanotubes, and fluorophore-labeled DNA. It has been established that the photonic energy was diffusively transferred from one end of the devices to the other through the FRET processes. The limitation of the persistent length of a single duplex DNA has been overcome by using larger DNA scaffolds (e.g., branched DNA junctions and DNA origami). The strengths of these approaches are (1) DNA materials are capable of self- assembly, molecular recognition, and programmability, (2) the flexibility of choosing fluorophores to form the energy cascade as the driving force in order to transfer photon energy through FRET, (3) the synthetic simplicity, and (4) the design flexibility of the structure for future enhancement for more complex circuitries. However, the current fluorescence measurements only provided the average representation of all FRET-based waveguides contained in the tested solution. To gain more insight on the complex photophysical behavior of the FRET-based waveguides, time-resolved fluorescence spectroscopy, single-molecule fluorescence spectroscopy, or total internal reflection fluorescence microscopy is needed.
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also observed at 123 nm, 195 nm, and 275 nm, corresponding to the 2nd, 4th, and 7th nearest neighbor distances, respectively (Fig. 2.12). For reference, from AFM images the dimensions of individual tiles on mica were approximately 100 nm per side, 13 nm longer than the center-to-center spacing of tiles imaged by Xtal-PAINT in solution. This 13% difference is likely caused by out-of-plane curvature of tiles immobilized by protein- binding in the fluid cell compared to lying flat on mica; Cando analysis of the tile indicates a ~12% reduction of tile dimensions from curvature and twist, consistent with the dimensions observed in Xtal-PAINT (Fig. 2.12). 33 The correlation length (g(r)→1) of the distribution indicates that order persists until nearly 1.6 μm, approximately equal to the largest dimension of array iv from Figure 2.4. The lack of distinct peaks beyond 300 nm suggests that large arrays were typically polycrystalline, consistent with the array analyzed in Figure 2.3. Thus, the results of Figure 2.4 validate the use of Xtal-PAINT in characterizing 2D crystalline DNA origami arrays. In comparison to AFM imaging, two- color Xtal-PAINT images were typically captured in ~75 minutes under conservative imaging conditions and could be expanded to capture over 10 5 μm 2 without increasing capture time, while AFM imaging of an equal area would be impractical.
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the fabrication processes required to realize such nano- structures are complex and expensive [9,10]. Thus, based on theoretical calculations, it is necessary to deter- mine the period and height of the nanostructure that can be fabricated at ease using the proposed technique to achieve desirable antireflection properties. For prac- tical applications such as solar cells, it is important that the nanostructures have a low reflectance over a broad wavelength range. To determine the desirable geometric features (i.e., period and height) for Si nanostructures that can achieve broadband antireflection for practical applications, we conducted a theoretical investigation of the reflectance behavior using the RCWA method . To calculate the reflectance, a truncated cone-shaped Si nanostructure with a bottom diameter to period ratio of 0.8 and a top diameter to period ratio of 0.15 was assumed in order to simplify the calculations. The simulation model was constructed based on previous experimental results which used metal nanoparticles as a dry etching mask [8,11,12]. Figure 1a shows the calculated reflectance of the Si nanostructures for various periods for a fixed height of 300 nm. The overall reflectance at first somewhat de- creased with an increasing period and then began to increase as the period was further increased. We also ob- served that there were regions with low reflectance (<3%) over a broad wavelength range, when the period was around 200 to 400 nm. This indicates that the selection of proper period is essential to obtain nanostructures with broadband antireflection properties. Figure 1b shows the calculated height-dependent reflectance of the Si nano- structures when their period was fixed at 300 nm. It is clear that the reflectance decreased considerably with an
Our quantitative analysis of the cell density shows that even after 4 h culturing period, cells seem well to prolif- erate on 0D nanostructures region. At a time point of 48 h, one can see a clear difference in the cell densities. While the density of cells on 0D nanostructures is around 189.8% ± 3.7%, this value drops down to 54.3% ± 0.6% in case of transitional nanostructures (see Figure 3). Al- though the cell density of 1D nanostructures is much lower than that of 0D nanostructures, these surfaces en- hance the cell adhesion and proliferation with respect to the glass substrate (control). This is a clear indication of cytocompatibility of our surfaces.
It is tempting to believe that modelling in nanotechnology is much the same as that for conventional solid-state physics. However, important areas of nanotechnology address different systems. The mechanics of DNA (for instance) resembles spaghetti more than silicon, the statistical physics needed is often not carrier statistics, and the role of viscosity (the low Reynolds number limit) is not always the familiar one. The idea of equilibrium may be irrelevant, as the kinetics of nonequilibrium (perhaps quasi-steady state) can be crucial. Even when the issues are limited to nanoscale structures (rather than functions), there is a complex range of ideas. Some features, like elasticity and electrostatic energies, have clear macroscopic analogies, but different questions emerge, such as the accuracy of self-organisation. Others concepts like epitaxy and templating are usually micro- or mesostructural. Some of the ideas, which emerge in modelling for the nanoscale, suggest parallels between molecular motors and recombination enhanced diffusion in semiconductors.
III. C HARACTERIZATION O F ZNO N ANOSTRUCTURES The structural and phase formation of the samples were identified by Reich Seifert XRD 3000 diffractometer using Cu-Kα (λ=1.5406 Å) radiation. The morphology and size of the ZnO nanostructures were evaluated by scanning electron microscopy (SEM, FEI- Quanta 250) and transmission electron microscopy (TEM, FEI-Technai Sprit). The presence of functional groups is analyzed by FTIR spectroscopy (JSO DEBYEFLEX 2002). UV-vis absorption / reflectance measurements were made by Lamda 650 UV-vis diffuse reflectance spectrometer (PerkinElmer) and room temperature PL spectral study was performed by a luminescence spectrometer (LS/55, PerkinElmer).
energy dispersive spectroscopy (EDS). Figure 3a–c show representative TEM images of the BSA–CuSe nanosnakes at the different reaction time such as 24, 48, and 96 h, respectively. We can clearly observe that the well-dis- persed nanostructures displayed different sizes, represent- ing the different growth stages. Within the 24-h reaction time, BSA–CuSe nanostructures mainly exhibited cubic structure with average size of 30 nm. After 24 h, the BSA– CuSe nanosnakes formed gradually, their sizes were about 130 nm in length and 12 nm in width. After 48 h, the cubic nanostructures had little change, short rods appeared, and the nanosnakes grew wide and long (Fig. 3b). When the reaction time reached to 96 h, the cubic nanostructures almost completely disappeared, and the nanosnakes grew homogeneously up to about 200 nm in length, and 14 nm in width (Fig. 3c, f). When the reaction time was over 96 h, the sizes of nanosnakes were almost unchanged. As shown in Fig. 3d, the single nanosnake exhibits good crystalline and clear lattice fringes. The lattice fringe spacing was 0.172 nm, consistent with the interplanar spacing of the (113) plane of cubic berzelianite (Cu 2-x Se) crystallites.
Different transducers are in place ranging from electrochemical, electromechanical, and fluorescent based biosensors. Some of these biosensors are a great application in the biomedical field in the prospects of disease diagnosis, particularly in the Cancer area. The demand and need for using a biosensor for rapid analysis with cost effectiveness is still being worked upon by scientists and engineers. In that situation, both 2D and 3D detection are required with sophisticated transducers for targeting and quantifying small analytes. The level of development in the area of biosensors should take a notch in discovering more robust regenerative biosensors for long term use. If the technology can improve to this level, new diagnostic biosensors can be developed for many biomedical applications and help clinicians and patients understand the integrative understanding of diseases and therapy. In relation to this, the fluorescent biosensor is excellent for assessing the efficacy of the tumor cell concentration and see how early Cancer could be detected. Currently, the use of aptamers, peptides, antibodies, and other materials are examples for the prospective approach in delving into this research. In this thesis, the engineered design of the nanomaterials to see the sensitivity of the target DNA by two different approaches were studied. This approach could be the materials used to view the Cancer Diagnosis as an in- vitro analysis in the future. Different biological agents can be used in the detection, ranging from other various nanomaterials, polymers that can provide hybrid devices for better usage in the earlier detection. Looking out in the horizon, potential application and characteristics like analyte detection ability, analysis time, portability, cost and customization have to be taken into account and be improved upon in this field.
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with yellow-orange emissions [24,27]. By contrast, oxygen vacancies have been attributed to the green emission band . XPS and TEM-EDS analyses indicated that the Sn content of the nanostructures of sample 1 (2.0 at.%) was slightly lower than those of sample 2 (2.4 at.%) and sample 3 (2.3 at.%). Moreover, the density of oxygen vacancies at the surface of the nanostructures was relatively high in sample 1 (39%) compared with those in sample 2 (28%) and sample 3 (21%). Comparatively, the ratio of yellow- orange emission band to total visible emission band for sample 1 (72.2%) was larger than those of sample 2 (32.3%) and sample 3 (32.0%). Our results suggested that the oxygen vacancies near the surface of the nanostructures might dominate the yellow-orange emission band. Recent work on the PL spectra of In-Sn-O nanostructures has shown that a relatively high Sn content (3.8 at.%) in the nanostructures causes a clear blueshift (590 to 430 nm) in the visible emission band . Kar et al. reported that the blue-green emission band of In 2 O 3 can be attributed
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washed and resuspended in water (solid, blue line). The AuNR longitudinal peak is blue- shifted, indicating partial aggregation..................................................................................... 72 Figure 34. UV-vis absorbance spectroscopy of AuNRs and single-thiol-DNA (solid, blue line) and later, those same AuNRs, fully functionalized with DNA, after NaCl and TAEMg addition to show that the peak position is maintained (green, short dashes). Also shown are AuNRs with dithiol-DNA (red, long dashes), which shows a peak shift and peak broadening, indicating aggregation. ............................................................................................................ 73 Figure 35. Both native CTAB-AuNRs and lipoic-acid AuNRs are aggregated and cannot enter the gel. The thiol-AuNRs migrate into the gel and remain red in color. ....................... 75 Figure 36. EtBr stained agarose gel electrophoresis (1% agarose, 1xTAEMg) of A) DNA- AuNSs, B) Design 1 tetrahedra with excess ssDNA, C) filtered Design 1 tetrahedra, and D) tetrahedra hybridized with AuNSs, in which the AuNSs are in 5x excess of the origami. (Top) White light illuminated image of the gel and (bottom) UV illuminated. ..................... 77 Figure 37. Tall Rectangle origami with AuNSs A) along one edge, and B) along two edges, or C) at each corner. For each of A and B, i) shows a schematic of the desired product, ii) shows AFM images of the samples, and iii) shows SEM images of the samples. For C, i) shows the schematic, ii) shows SEM images, and iii) shows TEM images using the method from Chapter 2....................................................................................................................... 79 Figure 38. A) Tall Rectangle origami with a AuNR on one edge. i) Schematic and ii) SEM images. B) Tall Rectangle origami with a AuNR on each edge. C) Design 1 tetrahedra with a AuNR on an arm, and D) the same but with AuNSs at the vertices. E) A core-satellite
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Fig (3) depicts the simulated result of eqn (2) which shows the dependence of EL intensity on the temperature for GaN nanostructures. As temperature increases EL intensity decreases. This is due to high absorption and the shrinkage of band gap with increasing temperature. The EL quenching at increased temperature is a combination of a reduced confinement and thermally activated non-radiative recombination processes within "active" crystallites.