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DNA Nanotechnology is being applied to multiple research fields. The functionality of DNA nanostructures is significantly enhanced by decorating them with nanoscale moieties including: proteins, metallic nanoparticles, quantum dots, and chromophores. Decoration is a complex process and developing protocols for reliable attachment routinely requires extensive trial and error. Additionally, the granular nature of scientific communication makes it difficult to discern general principles in DNA nanostructure decoration. This tutorial is a guidebook designed to minimize experimental bottlenecks and avoid dead-ends for those wishing to decorate DNA nanostructures. We supplement the reference material on available technical tools and procedures with a conceptual framework required to make efficient and effective decisions in the lab. Together these resources should aid both the novice and the expert to develop and execute a rapid, reliable decoration protocols.
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DNA , Nanoestruturas , Nanotecnologia , DNA/química , Nanoestruturas/química , Nanotecnologia/métodos , Pontos Quânticos/química , Nanopartículas Metálicas/químicaRESUMO
We present a method for extracting temperature-dependent thermodynamic and photophysical properties of SYTO-13 dye bound to DNA from fluorescence measurements. Together, mathematical modeling, control experiments, and numerical optimization enable dye binding strength, dye brightness, and experimental noise (or error) to be discriminated from one another. By focusing on the low-dye-coverage regime, the model avoids bias and can simplify quantification. Utilizing the temperature-cycling capabilities and multi-reaction chambers of a real-time PCR machine increases throughput. Significant well-to-well and plate-to-plate variation is quantified by using total least squares to account for error in both fluorescence and nominal dye concentration. Properties computed independently for single-stranded DNA and double-stranded DNA by numerical optimization are consistent with intuition and explain the advantageous performance of SYTO-13 in high-resolution melting and real-time PCR assays. Distinguishing between binding, brightness, and noise also clarifies the mechanism for the increased fluorescence of dye in a solution of double-stranded DNA compared to single-stranded DNA; in fact, the explanation changes with temperature.
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DNA de Cadeia Simples , DNA , Temperatura , DNA/química , Compostos Orgânicos , Corantes Fluorescentes/químicaRESUMO
Biomolecular thermodynamics, particularly for DNA, are frequently determined via van't Hoff analysis of optically measured melt curves. Accurate and precise values of thermodynamic parameters are essential for the modeling of complex systems involving cooperative effects, such as RNA tertiary structure and DNA origami, because the uncertainties associated with each motif in a folding energy landscape can compound, significantly reducing the power of predictive models. We follow the sources of uncertainty as they propagate through a typical van't Hoff analysis to derive best practices for melt experiments and subsequent data analysis, assuming perfect signal baseline correction. With appropriately designed experiments and analysis, a van't Hoff approach can provide surprisingly high precision, e.g., enthalpies may be determined with a precision as low as 10-2 kJ â mol-1 for an 8-base DNA oligomer.
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TermodinâmicaRESUMO
Focused-ion-beam machining is a powerful process to fabricate complex nanostructures, often through a sacrificial mask that enables milling beyond the resolution limit of the ion beam. However, current understanding of this super-resolution effect is empirical in the spatial domain and nonexistent in the temporal domain. This article reports the primary study of this fundamental tradespace of resolution and throughput. Chromia functions well as a masking material due to its smooth, uniform, and amorphous structure. An efficient method of in-line metrology enables characterization of ion-beam focus by scanning electron microscopy. Fabrication and characterization of complex test structures through chromia and into silica probe the response of the bilayer to a focused beam of gallium cations, demonstrating super-resolution factors of up to 6 ± 2 and improvements to volume throughput of at least factors of 42 ± 2, with uncertainties denoting 95% coverage intervals. Tractable theory models the essential aspects of the super-resolution effect for various nanostructures. Application of the new tradespace increases the volume throughput of machining Fresnel lenses by a factor of 75, enabling the introduction of projection standards for optical microscopy. These results enable paradigm shifts of sacrificial masking from empirical to engineering design and from prototyping to manufacturing.
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Structural DNA nanotechnology, as exemplified by DNA origami, has enabled the design and construction of molecularly-precise objects for a myriad of applications. However, limitations in imaging, and other characterization approaches, make a quantitative understanding of the folding process challenging. Such an understanding is necessary to determine the origins of structural defects, which constrain the practical use of these nanostructures. Here, we combine careful fluorescent reporter design with a novel affine transformation technique that, together, permit the rigorous measurement of folding thermodynamics. This method removes sources of systematic uncertainty and resolves problems with typical background-correction schemes. This in turn allows us to examine entropic corrections associated with folding and potential secondary and tertiary structure of the scaffold. Our approach also highlights the importance of heat-capacity changes during DNA melting. In addition to yielding insight into DNA origami folding, it is well-suited to probing fundamental processes in related self-assembling systems.
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DNA/química , Termodinâmica , Varredura Diferencial de Calorimetria , Entropia , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Nanoestruturas/química , Conformação de Ácido Nucleico , Desnaturação de Ácido NucleicoRESUMO
Fluorescence-based measurements are a standard tool for characterizing the thermodynamic properties of DNA systems. Nonetheless, experimental melt data obtained from polymerase chain-reaction (PCR) machines (for example) often leads to signals that vary significantly between datasets. In many cases, this lack of reproducibility has led to difficulties in analyzing results and computing reasonable uncertainty estimates. To address this problem, we propose a data analysis procedure based on constrained, convex optimization of affine transformations, which can determine when and how melt curves collapse onto one another. A key aspect of this approach is its ability to provide a reproducible and more objective measure of whether a collection of datasets yields a consistent "universal" signal according to an appropriate model of the raw signals. Importantly, integrating this validation step into the analysis hardens the measurement protocol by allowing one to identify experimental conditions and/or modeling assumptions that may corrupt a measurement. Moreover, this robustness facilitates extraction of thermodynamic information at no additional cost in experimental time. We illustrate and test our approach on experiments of Förster resonance energy transfer (FRET) pairs used study the thermodynamics of DNA loops.
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DNA/análise , Bases de Dados Factuais , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Conformação de Ácido Nucleico , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Termodinâmica , Temperatura de TransiçãoRESUMO
Nanofabrication/characterization facilities enable research and development activities across a host of science and engineering disciplines. The collection of tools and supporting infrastructure necessary to construct, image, and measure micro- and nanoscale materials, devices, and systems is complex and expensive to establish, and it is costly to maintain and optimize. As a result, these facilities are typically operated in a shared-use mode. We discuss the key factors that must be considered to successfully create and sustain such facilities. These include the need for long-term vision and institutional commitment, and the hands-on involvement of managers in facility operations. We consider startup, operating, and recapitalization costs, together with algorithms for cost recovery and tool-time allocation. The acquisition of detailed and comprehensive project and tool-utilization data is essential for understanding and optimizing facility operations. Only such a data-driven decision-making approach can maximize facility impact on institutional goals. We illustrate these concepts using the National Institute of Standards and Technology (NIST) NanoFab as our test case, but the methodologies and resources presented here should be useful to all those faced with this challenging task.
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Synthesis of low-dimensional carbon nanomaterials such as carbon nanotubes (CNTs) is a key driver for achieving advances in energy storage, computing, and multifunctional composites, among other applications. Here, we report high-yield thermal chemical vapor deposition (CVD) synthesis of CNTs catalyzed by reagent-grade common sodium-containing compounds, including NaCl, NaHCO3 , Na2 CO3 , and NaOH, found in table salt, baking soda, and detergents, respectively. Coupled with an oxidative dehydrogenation reaction to crack acetylene at reduced temperatures, Na-based nanoparticles have been observed to catalyze CNT growth at temperatures below 400 °C. Ex situ and in situ transmission electron microscopy (TEM) reveal unique CNT morphologies and growth characteristics, including a vaporizing Na catalyst phenomenon that we leverage to create CNTs without residual catalyst particles for applications that require metal-free CNTs. Na is shown to synthesize CNTs on numerous substrates, and as the first alkali group metal catalyst demonstrated for CNT growth, holds great promise for expanding the understanding of nanocarbon synthesis.
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As carbon nanotube (CNT) infused hybrid composites are increasingly identified as next-generation aerospace materials, it is vital to evaluate their long-term structural performance under aging environments. In this work, the durability of hierarchical, aligned CNT grafted aluminoborosilicate microfiber-epoxy composites (CNT composites) are compared against baseline aluminoborosilicate composites (baseline composites), before and after immersion in water at 25 °C (hydro) and 60 °C (hydrothermal), for extended durations (90 d and 180 d). The addition of CNTs is found to reduce water diffusivities by approximately 1.5 times. The mechanical properties (bending strength and modulus) and the damage sensing capabilities (DC conductivity) of CNT composites remain intact regardless of exposure conditions. The baseline composites show significant loss of strength (44 %) after only 15 d of hydrothermal aging. This loss of mechanical strength is attributed to fiber-polymer interfacial debonding caused by accumulation of water at high temperatures. In situ acoustic and DC electrical measurements of hydrothermally aged CNT composites identify extensive stress-relieving micro-cracking and crack deflections that are absent in the aged baseline composites. These observations are supported by SEM images of the failed composite cross-sections that highlight secondary matrix toughening mechanisms in the form of CNT pullouts and fractures which enhance the service life of composites and maintain their properties under accelerated aging environments.
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This article introduces in archival form the Nanolithography Toolbox, a platform-independent software package for scripted lithography pattern layout generation. The Center for Nanoscale Science and Technology (CNST) at the National Institute of Standards and Technology (NIST) developed the Nanolithography Toolbox to help users of the CNST NanoFab design devices with complex curves and aggressive critical dimensions. Using parameterized shapes as building blocks, the Nanolithography Toolbox allows users to rapidly design and layout nanoscale devices of arbitrary complexity through scripting and programming. The Toolbox offers many parameterized shapes, including structure libraries for micro- and nanoelectromechanical systems (MEMS and NEMS) and nanophotonic devices. Furthermore, the Toolbox allows users to precisely define the number of vertices for each shape or create vectorized shapes using Bezier curves. Parameterized control allows users to design smooth curves with complex shapes. The Toolbox is applicable to a broad range of design tasks in the fabrication of microscale and nanoscale devices.
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This article reports a process for rapidly prototyping nanofluidic devices, particularly those comprising slits with microscale widths and nanoscale depths, in silicone. This process consists of designing a nanofluidic device, fabricating a photomask, fabricating a device mold in epoxy photoresist, molding a device in silicone, cutting and punching a molded silicone device, bonding a silicone device to a glass substrate, and filling the device with aqueous solution. By using a bilayer of hard and soft silicone, we have formed and filled nanofluidic slits with depths of less than 400 nm and aspect ratios of width to depth exceeding 250 without collapse of the slits. An important attribute of this article is that the description of this rapid prototyping process is very comprehensive, presenting context and details which are highly relevant to the rational implementation and reliable repetition of the process. Moreover, this process makes use of equipment commonly found in nanofabrication facilities and research laboratories, facilitating the broad adaptation and application of the process. Therefore, while this article specifically informs users of the Center for Nanoscale Science and Technology (CNST) at the National Institute of Standards and Technology (NIST), we anticipate that this information will be generally useful for the nanofabrication and nanofluidics research communities at large, and particularly useful for neophyte nanofabricators and nanofluidicists.
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DNA origami is a powerful platform for assembling gold nanoparticle constructs, an important class of nanostructure with numerous applications. Such constructs are assembled by the association of complementary DNA oligomers. These association reactions have yields of <100%, requiring the development of methods to purify the desired product. We study the performance of centrifugation as a separation approach by combining optical and hydrodynamic measurements and computations. We demonstrate that bench-top microcentrifugation is a simple and efficient method of separating the reaction products, readily achieving purities of >90%. The gold nanoparticles play a number of critical roles in our system, functioning not only as integral components of the purified products, but also as hydrodynamic separators and optical indicators of the reaction products during the purification process. We find that separation resolution is ultimately limited by the polydispersity in the mass of the gold nanoparticles and by structural distortions of DNA origami induced by the gold nanoparticles. Our study establishes a methodology for determining the design rules for nanomanufacturing DNA origami-nanoparticle constructs.
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Centrifugação/métodos , DNA/química , Ouro/química , Nanopartículas Metálicas/química , Simulação por Computador , DNA de Cadeia Simples/química , Difusão , Hidrodinâmica , Luz , Nanocompostos/química , Nanotecnologia , Tamanho da Partícula , Pressão , Espalhamento de Radiação , Temperatura , ViscosidadeRESUMO
DNA-based Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is an effective super resolution microscopy technique, and its optimization is key to improve nanoscale detection. The state-of-the-art improvements that are at the base of this optimization have been first routinely validated on DNA nanostructure devices before being tested on biological samples. This allows researchers to finely tune DNA-PAINT imaging features in a more controllable in vitro environment. Dye-labeled oligonucleotide probes with short hybridization domains can expand DNA-PAINT's detection by targeting short nucleotide sequences and improving resolution, speed, and multiplexing. However, developing these probes is challenging as their brief bound state makes them difficult to capture under routine imaging conditions. To extend dwell binding times and promote duplex stability, we introduced structural and chemical modifications to our imager probes. The modifications included mini-hairpins and/or Bridged Nucleic Acids (BNA); both of which increase the thermomechanical stability of a DNA duplex. Using this approach we demonstrate DNA-PAINT imaging with approximately 5 nm resolution using a 4-nucleotide hybridization domain that is 43% shorter than previously reported probes. Imager probes with such short hybridization domains are key for improving detection on DNA nanostructure devices because they have the capability to target a larger number of binding domains per localization unit. This is essential for metrology applications such as Nucleic Acid Memory (NAM) where the information density is dependent on the binding site length. The selected imager probes reported here present imaging resolution equivalent to current state-of-the-art DNA-PAINT probes, creating a strategy to image shorter DNA domains for nanoscience and nanotechnology alike.
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DNA , DNA/química , Sondas de DNA/química , Nanoestruturas/química , Hibridização de Ácido NucleicoRESUMO
We observe a resonant transition in the microwave absorption of thin thermally deposited Au nanoparticle films near the geometrical percolation transition pc where the films exhibit a 'fractal' heterogeneous geometry. Absorption of incident microwave radiation increases sharply near pc, consistent with effective medium theory predictions. Both the theory and our experiments indicate that the hierarchical structure of these films makes their absorption insensitive to the microwave radiation wavelength λ, so that this singular absorption of microwave radiation is observed over a broad frequency range between 100 MHz and 20 GHz. The interaction of electromagnetic radiation with randomly distributed conductive scattering particles gives rise to localized resonant modes, and our measurements indicate that this adsorption process is significantly enhanced for microwaves in comparison to ordinary light. In particular, above the percolation transition a portion of the injected microwave power is stored within the film until dissipated. Finally, we find that the measured surface conductivity can be quantitatively described at all Au concentrations by generalized effective medium theory, where the fitted conductivity percolation exponents and pc itself are consistent with known two-dimensional estimates. Our results demonstrate that microwave measurements provide a powerful means of remotely measuring the electromagnetic properties of highly heterogeneous conducting films, enabling purposeful engineering of the electromagnetic properties of thin films in the microwave frequency range through fabrication of 'disordered' films of conducting particles such as metal nanoparticles or carbon nanotubes.
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Single-particle tracking with real-time feedback control can be used to study three-dimensional nanoparticle transport dynamics. We apply the method to study the behavior of adsorbed nanoparticles at a silicone oil-water interface in a microemulsion system over a range of particles sizes from 24 nm to 2000 nm. The diffusion coefficient of large particles (>200 nm) scales inversely with particle size, while smaller particles exhibit an unexpected increase in drag force at the interface. The technique can be applied in the future to study three-dimensional dynamics in a range of systems, including complex fluids, gels, biological cells, and geological media.
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Nanopartículas/química , Óleos/química , Água/química , Difusão , Emulsões , Corantes Fluorescentes/química , Movimento (Física) , Poliestirenos/química , Fatores de TempoRESUMO
Since its inception nearly 40 years ago [Kallenbach, et al., Nature, 1983, 305, 829; N. C. Seeman, J. Theoretical Biology, 1982, 99, 237], Nucleic Acid Nanotechnology (NAN) has matured and is beginning to find commercial applications. For the last 20 years, it has been suggested that NAN might be an effective replacement for parts of the semiconductor lithography or protein engineering workflows. However, in that time, these incumbent technologies have made significant advances, and our understanding of NAN's strengths and weaknesses has progressed, suggesting that the greatest opportunities in fact lie elsewhere. Given the commitment of resources necessary to bring new technologies to the market and the desire to use those resources as wisely as possible, we conduct a critical examination of where NAN may benefit from, and provide benefit to, adjacent technologies and compete least with market incumbents. While the accuracy of our conclusions may be limited by our ability to extrapolate from the current state of NAN to its future commercial success, we conclude that the next promising direction is to create a bridge between biology and semiconductor technology. We also hope to stimulate a robust conversation around this technology's capabilities with the goal of building consensus on those research and development efforts that would advance NAN to the greatest effect in real-world applications.
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Ácidos Nucleicos , Nanotecnologia , SemicondutoresRESUMO
Analytical tools that have spatial resolution at the nanometre scale are indispensable for the life and physical sciences. It is desirable that these tools also permit elemental and chemical identification on a scale of 10 nm or less, with large penetration depths. A variety of techniques in X-ray imaging are currently being developed that may provide these combined capabilities. Here we report the achievement of sub-15-nm spatial resolution with a soft X-ray microscope--and a clear path to below 10 nm--using an overlay technique for zone plate fabrication. The microscope covers a spectral range from a photon energy of 250 eV (approximately 5 nm wavelength) to 1.8 keV (approximately 0.7 nm), so that primary K and L atomic resonances of elements such as C, N, O, Al, Ti, Fe, Co and Ni can be probed. This X-ray microscopy technique is therefore suitable for a wide range of studies: biological imaging in the water window; studies of wet environmental samples; studies of magnetic nanostructures with both elemental and spin-orbit sensitivity; studies that require viewing through thin windows, coatings or substrates (such as buried electronic devices in a silicon chip); and three-dimensional imaging of cryogenically fixed biological cells.
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Microscopia/instrumentação , Microscopia/métodos , Raios X , Disciplinas das Ciências Biológicas/instrumentação , Disciplinas das Ciências Biológicas/métodos , Magnetismo , Procedimentos Analíticos em Microchip , Microscopia Eletrônica de Varredura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fótons , Sensibilidade e Especificidade , Silício , Água/químicaRESUMO
Understanding the folding process of DNA origami is a critical stepping stone to the broader implementation of nucleic acid nanofabrication technology but is notably nontrivial. Origami are formed by several hundred cooperative hybridization events-folds-between spatially separate domains of a scaffold, derived from a viral genome, and oligomeric staples. Individual events are difficult to detect. Here, we present a real-time probe of the unit operation of origami assembly, a single fold, across the scaffold as a function of hybridization domain separation-fold distance-and staple/scaffold ratio. This approach to the folding problem elucidates a predicted but previously unobserved blocked state that acts as a limit on yield for single folds, which may manifest as a barrier in whole origami assembly.
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DNA , Nanoestruturas , Nanotecnologia , Conformação de Ácido NucleicoRESUMO
Several recently developed particle-tracking and imaging methods have achieved three-dimensional sensitivity through the introduction of angled micromirrors into the observation volume of an optical microscope. We model the imaging response of such devices and show how the direct and reflected images of a fluorescent particle are affected. In particle-tracking applications, asymmetric image degradation manifests itself as systematic tracking errors. Based on our results, we identify strategies for reducing systematic errors to the 10nm level in practical applications.
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Biopolímeros/análise , Imageamento Tridimensional/métodos , Lentes , Microscopia/instrumentação , Modelos Teóricos , Simulação por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Tamanho da Partícula , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Carbon nanostructure (CNS) based polymer nanocomposites (PNCs) are of interest due to the superior properties of the CNS themselves, scale effects, and the ability to transfer these properties anisotropically to the bulk material. However, measurements of physical properties of such materials are not in agreement with theoretical predictions. Recently, the ability to characterize the 3D morphology of such PNCs at the nanoscale has been significantly improved, with rich, quantitative data extracted from tomographic transmission electron microscopy (TEM). In this work, we use new, nanoscale quantitative 3D morphological information and stochastic modeling to re-interpret experimental measurements of continuous aligned carbon nanotube (A-CNT) PNC properties as a function of A-CNT packing/volume fraction. The 3D tortuosity calculated from tomographic reconstructions and its evolution with volume fraction is used to develop a novel definition of waviness that incorporates the stochastic nature of CNT growth. The importance of using randomly wavy CNTs to model these materials is validated by agreement between simulated and previously-measured PNC elastic moduli. Secondary morphological descriptors such as CNT-CNT junction density and inter-junction distances are measured for transport property predictions. The scaling of the junction density with CNT volume fraction is observed to be non-linear, and this non-linearity is identified as the primary reason behind the previously unexplained scaling of aligned-CNT PNC longitudinal thermal conductivity. By contrast, the measured electrical conductivity scales linearly with volume fraction as it is relatively insensitive to junction density beyond percolation. This result verifies prior hypotheses that electrical conduction in such fully percolated and continuous CNT systems is dominated by the bulk resistivity of the CNTs themselves. This combination of electron tomographic data and stochastic simulations is a powerful method for establishing a predictive capability for nanocomposite structure-property relations, making it an essential aid in understanding and tailoring the next-generation of advanced composites.