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This study explores important parameters for achieving a high-level positional control of DNA-nanoparticle hybrid structures by drop-casting onto a pre-structured silicon surface, in which the active adsorption sites were defined using electron beam lithography. By confining the adsorption sites to the scale of the DNA origami, we create multi-dimensional patterns and study the effect of diffusion and hybrid nanostructure concentration in the liquid on site occupation. We also propose a physical diffusion model that highlights the importance of surface diffusion in facilitating the adsorption of hybrid nanostructure onto active sites, particularly for two and one-dimensional adsorption sites. Our study shows prominent results of the hybrid nanostructure's selective adsorption, indicating high adsorption efficiency and precise control over the position, as well as the spatial orientation. We anticipate similar results in related systems, both in terms of different surfaces and similar DNA structures. Overall, our findings offer promising prospects for the development of large-scale nanoarrays on micrometer-scale surfaces with nanometer precision and orientation control.
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Nanopartículas , Nanoestructuras , Nanotecnología/métodos , Oro/química , Nanoestructuras/química , ADN/química , Conformación de Ácido NucleicoRESUMEN
The ongoing COVID-19 pandemic stresses the need for widely available diagnostic tests for the presence of SARS-CoV-2 in individuals. Due to the limited availability of vaccines, diagnostic assays which are cheap, easy-to-use at the point-of-need, reliable and fast, are currently the only way to control the pandemic situation. Here we present a diagnostic assay for the detection of pathogen-specific nucleic acids based on changes of the magnetic response of magnetic nanoparticles: The target-mediated hybridization of modified nanoparticles leads to an increase in the hydrodynamic radius. This resulting change in the magnetic behaviour in an ac magnetic field can be measured via magnetic particle spectroscopy (MPS), providing a viable tool for the accurate detection of target nucleic acids. In this work we show that single stranded DNA can be detected in a concentration-dependent manner by these means. In addition to detecting synthetic DNA with an arbitrary sequence in a concentration down to 500 pM, we show that RNA and SARS-CoV-2-specific DNA as well as saliva as a sample medium can be used for an accurate assay. These proof-of-principle experiments show the potential of MPS based assays for the reliable and fast diagnostics of pathogens like SARS-CoV-2 in a point-of-need fashion without the need of complex sample preparation.
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Técnicas Biosensibles , COVID-19 , Ácidos Nucleicos , Humanos , Fenómenos Magnéticos , Pandemias , ARN Viral , SARS-CoV-2 , Sensibilidad y Especificidad , Análisis EspectralRESUMEN
The advent of highly sensitive photodetectors and the development of photostabilization strategies made detecting the fluorescence of single molecules a routine task in many labs around the world. However, to this day, this process requires cost-intensive optical instruments due to the truly nanoscopic signal of a single emitter. Simplifying single-molecule detection would enable many exciting applications, e.g., in point-of-care diagnostic settings, where costly equipment would be prohibitive. Here, we introduce addressable NanoAntennas with Cleared HOtSpots (NACHOS) that are scaffolded by DNA origami nanostructures and can be specifically tailored for the incorporation of bioassays. Single emitters placed in NACHOS emit up to 461-fold (average of 89 ± 7-fold) brighter enabling their detection with a customary smartphone camera and an 8-US-dollar objective lens. To prove the applicability of our system, we built a portable, battery-powered smartphone microscope and successfully carried out an exemplary single-molecule detection assay for DNA specific to antibiotic-resistant Klebsiella pneumonia on the road.
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ADN/química , Microscopía , Nanotecnología , Teléfono Inteligente , Farmacorresistencia Bacteriana , Fluorescencia , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Masculino , Nanoestructuras , Pruebas en el Punto de Atención , Suero/químicaRESUMEN
Smartphone-based fluorescence microscopy has been rapidly developing over the last few years, enabling point-of-need detection of cells, bacteria, viruses, and biomarkers. These mobile microscopy devices are cost-effective, field-portable, and easy to use, and benefit from economies of scale. Recent developments in smartphone camera technology have improved their performance, getting closer to that of lab microscopes. Here, we report the use of DNA origami nanobeads with predefined numbers of fluorophores to quantify the sensitivity of a smartphone-based fluorescence microscope in terms of the minimum number of detectable molecules per diffraction-limited spot. With the brightness of a single dye molecule as a reference, we compare the performance of color and monochrome sensors embedded in state-of-the-art smartphones. Our results show that the monochrome sensor of a smartphone can achieve better sensitivity, with a detection limit of â¼10 fluorophores per spot. The use of DNA origami nanobeads to quantify the minimum number of detectable molecules of a sensor is broadly applicable to evaluate the sensitivity of various optical instruments.
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Because of the limited signal-to-background ratio, molecular diagnostics requires molecular amplification of the target molecules or molecular signal amplification after target recognition. For direct molecular detection, we demonstrate a purely physical fluorescence enhancement process which can elevate the fluorescence signal of single fluorescent dyes by several orders of magnitude. To this end, DNA origami-based optical antennas with a height of around 125 nm are used, which utilize metallic nanoparticles to create a hotspot where fluorescence signals are enhanced by plasmonic effects. By equipping the hotspot with a molecular beacon-like structure, we combine the plasmonic signal enhancement with a specific signal generation, leading to an enhanced and therefore easy to detect signal only in the presence of the specific target nucleic acid. Exemplified with Zika virus detection, we show the applicability of this approach by detecting Zika-specific artificial DNA and RNA both in buffer and in heat-inactivated human blood serum. We show the sensitivity against small nucleotide variations of this approach, allowing the discrimination of closely related pathogens. Furthermore, we show how the modularity offered by DNA nanotechnology enables multiplexing by incorporating orthogonal fluorescent labels for the simultaneous detection of different sequences. The achieved signal enhancement will allow technically simplified signal detection, paving the way for single molecule-based point-of-care diagnosis.
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ADN/sangre , Colorantes Fluorescentes/química , ARN/análisis , Virus Zika/genética , ADN/química , ADN/genética , Humanos , Secuencias Invertidas Repetidas , Mediciones Luminiscentes/métodos , Nanopartículas del Metal/química , Microscopía Confocal/métodos , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , ARN/genética , Plata/químicaRESUMEN
Fluorogenic nucleic acid hybridization probes are widely used for detecting and quantifying nucleic acids. The achieved sensitivity strongly depends on the contrast between a quenched closed form and an unquenched opened form with liberated fluorescence. So far, this contrast was improved by improving the quenching efficiency of the closed form. In this study, we modularly combine these probes with optical antennas used for plasmonic fluorescence enhancement and study the effect of the nanophotonic structure on the fluorescence of the quenched and the opened form. As quenched fluorescent dyes are usually enhanced more by fluorescence enhancement, a detrimental reduction of the contrast between closed and opened form was anticipated. In contrast, we could achieve a surprising increase of the contrast with full additivity of quenching of the dark form and fluorescence enhancement of the bright form. Using single-molecule experiments, we demonstrate that the additivity of the two mechanisms depends on the perfect quenching in the quenched form, and we delineate the rules for new nucleic acid probes for enhanced contrast and absolute brightness. Fluorogenic hybridization probes optimized not only for quenching but also for the brightness of the open form might find application in nucleic acid assays with PCR avoiding detection schemes.
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Sensing nucleic acids typically involves the recognition of a specific sequence and reporting by, for example, a fluorogenic reaction yielding one activated dye molecule per detected nucleic acid. Here, we show that after binding to a DNA origami track a bound DNA target (a "DNA walker") can release the fluorescence of many molecules by acting as the catalyst of an enzymatic nicking reaction. As the walking kinetics sensitively depends on the walker sequence, the resulting brightness distribution of DNA origamis is a sequence fingerprint with single-nucleotide sensitivity. Using Monte Carlo simulations, we rationalize that the random self-avoiding walk is mainly terminated when steps to nearest neighbors are exhausted. Finally, we demonstrate that the DNA walker is also active in a plasmonic hotspot for fluorescence enhancement, indicating the potential of combining different amplification mechanisms enabled by the modularity of DNA nanotechnology.
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ADN/química , ADN/genética , Nanoestructuras/química , Polimorfismo de Nucleótido Simple , Disparidad de Par Base , Técnicas Biosensibles/métodos , Fluorescencia , Cinética , Nanotecnología/métodosRESUMEN
Most fluorescence microscopy experiments today require a multicolor-capable setup, e.g. to study the interaction between different proteins. Multicolor capabilities are also well desirable for superresolution images. However, especially for STED (Stimulated Emission Depletion) microscopy, which requires two laser lines for a single color, multicolor imaging is technically challenging. Here we present a straightforward, easy-to-implement method to extend a single-color fluorescence (STED) microscope to a multichannel microscope without the need of modifying the optical setup. Therefore, we use a labeling technique based on complementary DNA sequences: a single-stranded short DNA sequence is attached to each structure to be imaged, different colors for labeling different features are represented by different sequences. Within the imaging process, the corresponding complementary sequence labeled with an organic fluorophore is added and transiently binds to the corresponding structure. After imaging, the labeled sequence is washed away and replaced by a second fluorescently labeled DNA strand complementary to the sequence bound to another feature. This way, multiplexing is achieved using only one arbitrary fluorophore, therefore aberrations are avoided.
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Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.
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Investigación , Espectrometría de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Espectrometría de Fluorescencia/métodosRESUMEN
Nanotechnology is challenged by the need to connect top-down produced nanostructures with the bottom-up world of chemistry. A nanobiotechnological prime example is the positioning of single polymerase molecules in small holes in metal films, so-called zeromode waveguides (ZMWs), which is required for single-molecule real-time DNA sequencing. In this work, we present nanoadapters made of DNA (DNA origami) that match the size of the holes so that exactly one nanoadapter fits in each hole. By site-selective functionalization of the DNA origami nanoadapters, we placed single dye molecules in the ZMWs, thus optimizing the hole usage and improving the photophysical properties of dyes compared to stochastically immobilized molecules.
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ADN , Nanoestructuras , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/instrumentaciónRESUMEN
The amount of information obtainable from a fluorescence-based measurement is limited by photobleaching: Irreversible photochemical reactions either render the molecules nonfluorescent or shift their absorption and/or emission spectra outside the working range. Photobleaching is evidenced as a decrease of fluorescence intensity with time, or in the case of single molecule measurements, as an abrupt, single-step interruption of the fluorescence emission that determines the end of the experiment. Reducing photobleaching is central for improving fluorescence (functional) imaging, single molecule tracking, and fluorescence-based biosensors and assays. In this single molecule study, we use DNA self-assembly to produce hybrid nanostructures containing individual fluorophores and gold nanoparticles at a controlled separation distance of 8.5 nm. By changing the nanoparticles' size we are able to systematically increase the mean number of photons emitted by the fluorophores before photobleaching.
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Técnicas Biosensibles , Oro/química , Nanopartículas del Metal/química , Fotoblanqueo , ADN/química , FotonesRESUMEN
Superresolution microscopy is currently revolutionizing optical imaging. A key factor for getting images of highest quality is - besides a well-performing imaging system - the labeling of the sample. We compared the fluorescent dyes Abberior Star 488, Alexa 488, Chromeo 488 and Oregon Green 488 for use in continuous wave (cw-)STED microscopy in aqueous buffer and in a durable polymer matrix. To optimize comparability, we designed DNA origami standards labeled with the fluorescent dyes including a bead-like DNA origami with dyes focused on one spot and a DNA origami with two marks at a designed distance of â¼100 nm. Our data show that all dyes are well suited for cw-STED microscopy but that the optimal dye depends on the embedding medium. The precise comparison enabled by DNA origami nanorulers indicates that these structures have matured from the proof-of-concept to easily applicable tools in fluorescence microscopy.
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ADN/química , Colorantes Fluorescentes/química , Nanoestructuras/química , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Propiedades de SuperficieRESUMEN
The dynamic concentration range is one of the major limitations of single-molecule fluorescence techniques. We show how bottom-up nanoantennas enhance the fluorescence intensity in a reduced hotspot, ready for biological applications. We use self-assembled DNA origami structures as a breadboard where gold nanoparticle (NP) dimers are positioned with nanometer precision. A maximum of almost 100-fold intensity enhancement is obtained using 100-nm gold NPs within a gap of 23 nm between the particles. The results obtained are in good agreement with numerical simulations. Due to the intensity enhancement introduced by the nanoantenna, we are able to perform single-molecule measurements at concentrations as high as 500 nM, which represents an increment of 2 orders of magnitude compared to conventional measurements. The combination of metallic NPs with DNA origami structures with docking points for biological assays paves the way for the development of bottom-up inexpensive enhancement chambers for single-molecule measurements at high concentrations where processes like DNA sequencing occur.
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ADN/química , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Avidina/química , Biotina/química , Dimerización , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Oro/química , Procesamiento de Imagen Asistido por Computador , Nanopartículas del Metal/química , Microscopía Confocal , Modelos Teóricos , Tamaño de la Partícula , Espectrometría de FluorescenciaRESUMEN
Nanopillars are promising nanostructures composed of various materials that bring new functionalities for applications ranging from photovoltaics to analytics. We developed DNA nanopillars with a height of 220 nm and a diameter of ~14 nm using the DNA origami technique. Modifying the base of the nanopillars with biotins allowed selective, upright, and rigid immobilization on solid substrates. With the help of site-selective dye labels, we visualized the structure and determined the orientation of the nanopillars by three-dimensional fluorescence superresolution microscopy. Because of their rigidity and nanometer-precise addressability, DNA origami nanopillars qualify as scaffold for the assembly of plasmonic devices as well as for three-dimensional superresolution standards.
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ADN/química , Imagenología Tridimensional , Nanoestructuras/química , Microscopía Fluorescente , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
We report the use of superresolution fluorescence microscopy for studying the nanoscale distribution of protein colocalization in living mammalian cells. Nanoscale imaging is attained both by a targeted and a stochastic fluorescence on-off switching superresolution method, namely by stimulated emission depletion (STED) and ground state depletion microscopy followed by individual molecular return (GSDIM), respectively. Analysis of protein colocalization is performed by bimolecular fluorescence complementation (BiFC). Specifically, a nonfluorescent fragment of the yellow fluorescent protein Citrine is fused to tubulin while a counterpart nonfluorescent fragment is fused to the microtubulin-associated protein MAP2 such that fluorescence is reconstituted on contact of the fragment-carrying proteins. Images with resolution down to 65 nm prove a powerful new way for studying protein colocalization in living cells at the nanoscale.