DNA Origami Nanoantennas for Fluorescence Enhancement

[Image: see text] The possibility to increase fluorescence by plasmonic effects in the near-field of metal nanostructures was recognized more than half a century ago. A major challenge, however, was to use this effect because placing single quantum emitters in the nanoscale plasmonic hotspot remained unsolved for a long time. This not only presents a chemical problem but also requires the nanostructure itself to be coaligned with the polarization of the excitation light. Additional difficulties arise from the complex distance dependence of fluorescence emission: in contrast to other surface-enhanced spectroscopies (such as Raman spectroscopy), the emitter should not be placed as close as possible to the metallic nanostructure but rather needs to be at an optimal distance on the order of a few nanometers to avoid undesired quenching effects. Our group addressed these challenges almost a decade ago by exploiting the unique positioning ability of DNA nanotechnology and reported the first self-assembled DNA origami nanoantennas. This Account summarizes our work spanning from this first proof-of-principle study to recent advances in utilizing DNA origami nanoantennas for single DNA molecule detection on a portable smartphone microscope. We summarize different aspects of DNA origami nanoantennas that are essential for achieving strong fluorescence enhancement and discuss how single-molecule fluorescence studies helped us to gain a better understanding of the interplay between fluorophores and plasmonic hotspots. Practical aspects of preparing the DNA origami nanoantennas and extending their utility are also discussed. Fluorescence enhancement in DNA origami nanoantennas is especially exciting for signal amplification in molecular diagnostic assays or in single-molecule biophysics, which could strongly benefit from higher time resolution. Additionally, biophysics can greatly profit from the ultrasmall effective detection volumes provided by DNA nanoantennas that allow single-molecule detection at drastically elevated concentrations as is required, e.g., in single-molecule DNA sequencing approaches. Finally, we describe our most recent progress in developing DNA NanoAntennas with Cleared HOtSpots (NACHOS) that are fully compatible with biomolecular assays. The developed DNA origami nanoantennas have proven robustness and remain functional after months of storage. As an example, we demonstrated for the first time the single-molecule detection of DNA specific to antibiotic-resistant bacteria on a portable and battery-driven smartphone microscope enabled by DNA origami nanoantennas. These recent developments mark a perfect moment to summarize the principles and the synthesis of DNA origami nanoantennas and give an outlook of new exciting directions toward using different nanomaterials for the construction of nanoantennas as well as for their emerging applications

Fluorophore photostability and saturation in the hotspot of DNA origami nanoantennas

Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the conditions for fluorescent dyes are even more demanding in DNA origami nanoantennas. Here, we briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal additional factors relevant in the context of plasmonic fluorescence enhancement. We show that despite the improved photostability of single-molecule fluorophores by DNA origami nanoantennas, their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying photophysical processes, such as formation of dim states and photoisomerization. These photophysical processes limit the photon count rates, increase heterogeneity and aggravate quantification of fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved in biophysical single-molecule experiments. Finally, we show how the photophysics of a DNA hairpin assay with a fluorophore-quencher pair can be influenced by plasmonic DNA origami nanoantennas leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that such assays can produce false positive results by premature photobleaching of the dark quenche

ChipScope Symposium: Novel Approaches for a Chip-Sized Optical Microscope

In the Chipscope project funded by the EU, a completely new strategy towards optical microscopy is explored by a team of researchers from different European institutions. In this workshop, the different researchers of the project will explain the last advances obtained in the project, presenting the microscopes, how light emission is produced, and the detection principles and simulations

Single antibody detection in a DNA origami nanoantenna

DNA nanotechnology offers new biosensing approaches by templating different sensor and transducer components. Here, we combine DNA origami nanoantennas with label-free antibody detection by incorporating a nanoswitch in the plasmonic hotspot of the nanoantenna. The nanoswitch contains two antigens that are displaced by antibody binding, thereby eliciting a fluorescent signal. Single-antibody detection is demonstrated with a DNA origami integrated anti-digoxigenin antibody nanoswitch. In combination with the nanoantenna, the signal generated by the antibody is additionally amplified. This allows the detection of single antibodies on a portable smartphone microscope. Overall, fluorescence-enhanced antibody detection in DNA origami nanoantennas shows that fluorescence-enhanced biosensing can be expanded beyond the scope of the nucleic acids realm

Single antibody detection in a DNA origami nanoantenna

DNA nanotechnology offers new biosensing approaches by templating different sensor and transducer components. Here, we combine DNA origami nanoantennas with label-free antibody detection by incorporating a nanoswitch in the plasmonic hotspot of the nanoantenna. The nanoswitch contains two antigens that are displaced by antibody binding, thereby eliciting a fluorescent signal. Single-antibody detection is demonstrated with a DNA origami integrated anti-digoxigenin antibody nanoswitch. In combination with the nanoantenna, the signal generated by the antibody is additionally amplified. This allows the detection of single antibodies on a portable smartphone microscope. Overall, fluorescence-enhanced antibody detection in DNA origami nanoantennas shows that fluorescence-enhanced biosensing can be expanded beyond the scope of the nucleic acids realm

Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope

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

Addressable Nanoantennas with Cleared Hotspots for Single-Molecule Detection on a Portable Smartphone Microscope

The advent of highly sensitive photodetectors1,2 and the development of photostabilization strategies3 made detecting the fluorescence of a single molecule 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.4 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 the NACHOS emit up to 461-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 “

Photoprotection of Fluorophores used for single-molecule imaging: a battle for more photons

While fluorescence imaging techniques have seen revolutionary advancements in both sensitivity and resolution in the past decade, the poor inherent photostability of organic dyes used for single molecule detection remains one of the major limitations. The rapid photobleaching of fluorophores is a vexing problem in single-molecule imaging applications and novel super-resolution imaging studies, where achieving high signal-to-background ratios and extended imaging times are crucial. In this work, we explore new methods and revise existing mechanisms utilized toward increasing the photostability of fluorophores used for single-molecule imaging. Our approach relies on the quenching of dye triplet excited states, thereby eliminating one of the key intermediates in the photobleaching pathway. Using single-molecule fluorescence as well as mechanistic ensemble fluorescence and laser flash photolysis studies, we first demonstrate that Ni(II) ions can be used as a photostabilizing agent for Cy3 in single-molecule fluorescence imaging. Importantly, this work provides a much-desired physical quenching route (chemically inert) to increase the photostability of Cy3. We then explore the scope of this photostabilization approach and show that it can be extended to a wide range of fluorophores used for single-molecule imaging including: Cy5, ATTO647N, Alexa647, ATTO532, and Alexa532. Here, a 10- to 40-fold increase in photon output was observed in the presence of Ni (II) ions. Moreover, we show that in contrast to other commonly used triplet state quenchers, Ni(II) is effective in photostabilizing both green- and red-absorbing dyes, which is essential for multicolor imaging applications. We also demonstrate that photostabilization of fluorophores by Ni(II) ions can be achieved via an intramolecular approach relying on tris-NTA-fluorophores where we exploit the high effective Ni(II) concentration in the tris-NTA-fluorophore construct. This strategy precludes the use of solution additives and is of great interest for live-cell imaging applications. Our results confirm that the tris-NTA group can indeed serve a dual role – allowing both for the specific labelling of biomolecules and also for increased photostability via a 'self-healing' approach.Finally, we address the mechanistic aspects of fluorophore photostabilization by studying the direct triplet excited state recovery pathways when using redox based triplet state quenchers. We designed transient-absorption studies to probe the key transient intermediates formed with commonly used reducing agents/photostabilizers. To rationalize varying degrees of direct recovery, we revisit the factors that influence the ISC step in geminate radical ion pairs and biradicals and highlight mechanistic aspects to consider when designing new photostabilizers or 'self-healing' dyes. Altogether, our work provides new mechanistic insights and guiding principles that should result in improved fluorophore properties and, thus, long-lasting fluorescence imaging studies. The work presented here holds promise to enhance the potential of fluorescence methodologies, including single-molecule fluorescence and fluorescence superresolution imaging, and, given the emerging role of these methods in materials, biological and chemical sciences, to advance our understanding of complex biological and chemical systems.Alors que les techniques d'imagerie par fluorescence ont connu des progrès révolutionnaires en sensibilité et en résolution au cours de la dernière décennie, la faible photostabilité inhérente des colorants organiques utilisés pour la détection d'une seule molécule demeure l'une des principales barrières. Le photoblanchiment rapide des molécules fluorescentes est un problème énorme dans les applications d'imagerie à l'échelle de la molécule unique et de nouvelles études d'imagerie de super résolution, où l'obtention de rapports élevés de signal/bruit de fond et les temps d'imagerie prolongés sont cruciaux. Dans ce travail, nous explorons des nouvelles méthodes et révisons les mécanismes existants utilisés afin d'augmenter la photostabilité des molécules fluorescentes utilisées pour l'imagerie à l'échelle de la molécule unique. Notre approche repose sur la trempe des états excités de triplet, éliminant ainsi l'un des intermédiaires clés du photoblanchiment. En utilisant la technique d'imagerie par fluorescence à l'échelle de la molécule unique ainsi qu'à l'échelle de l'ensemble des molécules fluorescentes et la photolyse à flash laser, nous démontrons d'abord que Ni(II) peut être utilisé comme agent photostabilisant pour Cy3 dans l'imagerie à fluorescence à l'échelle de la molécule unique. Il est important de noter que ce travail fournit une voie de trempe physique très exigée (chimiquement inerte) pour augmenter la photostabilité de Cy3. Nous explorons ensuite la portée de cette approche de la photostabilisation et démontrons qu'elle peut être étendue à une large gamme de molécules fluorescentes utilisées pour l'imagerie à l'échelle de la molécule unique, y compris : Cy5, ATTO647N, Alexa647, ATTO532 et Alexa532. Dans ce cas, une augmentation de 10 à 40 fois de la production de photons a été observée en présence de Ni(II). En outre, nous montrons que, contrairement aux autres inhibiteurs d'état triplet couramment utilisés, Ni(II) est efficace pour la photostabilisation des colorants absorbant le rouge et le vert, ce qui est essentiel pour les applications d'imagerie multicolores. Nous démontrons également que la photostabilisation des molécules fluorescentes par Ni(II) peut être réalisée par une approche intramoléculaire en s'appuyant sur les molécules fluorescentes trisNTA, où nous exploitons la concentration élevée en Ni(II) efficace pour la génération du complexe fluorescent trisNTA. Cette stratégie ne nécessite pas l'utilisation de solutions d'additifs et est très intéressante pour les applications d'imagerie de cellules vivantes. Nos résultats confirment que le groupe tris-NTA peut effectivement jouer un double rôle, permettant à la fois l'étiquetage spécifique des biomolécules ainsi qu'une augmentation de la photostabilité par une approche d'« auto-rétablissement». Enfin, nous abordons les aspects mécanistiques de la photostabilisation des molécules fluorescentes en étudiant les voies directes de récupération de l'état triplet excité lors de l'utilisation d'extincteurs à l'état triplet à base de redox. Nous avons conçu des études d'absorption transitoire pour examiner les intermédiaires transitoires clés formés avec des agents réducteurs / photostabilisants couramment utilisés. Pour rationaliser les différents degrés de variabilité de la récupération directe, nous étudions les facteurs qui influent l'étape ISC dans les paires d'ions radicaux géminés et les bi radicaux et nous soulignons en plus les aspects mécanistiques à prendre en considération lors de la conception de nouveaux photostabilisants ou des colorants capables d'auto-rétablissements.Dans l'ensemble, notre travail fournit de nouvelles idées mécaniques et des principes généraux qui devraient aboutir à des propriétés améliorées de molécules fluorescentes et, par conséquent, à des études d'imagerie par fluorescence durable

Self-Regeneration and Self-Healing in DNA Origami Nanostructures

DNA nanotechnology and advances in the DNA origami technique have enabled facile design and synthesis of complex and functional nanostructures. Molecular devices are, however, prone to rapid functional and structural degradation due to the high proportion of surface atoms at the nanoscale and due to complex working environments. Besides stabilizing mechanisms, approach for the self‐repair of functional molecular devices are desirable. Here we exploit the self‐assembly and reconfigurability of DNA origami nanostructures to induce the self‐repair of defects of photoinduced and enzymatic damage. With different examples of repair in DNA nanostructures, we distinguish between unspecific self‐regeneration and damage specific self‐healing mechanisms. Using DNA origami nanorulers studied by atomic force and superresolution DNA PAINT microscopy, quantitative preservation of fluorescence properties is demonstrated with direct potential for improving nanoscale calibration samples