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@article{nokey,

title = {Bringing Attomolar Detection to the Point-of-Care with Nanopatterned DNA Origami Nanoantennas},

author = {R Yaadav and K Trofymchuk and M Dass and V Behrendt and B Hauer and J Sch\"{u}tz and C Close and M Scheckenbach and G Ferrari and L M\"{a}urer and S Sebina and V Glembockyte and T Liedl and P Tinnefeld},

url = {\<Go to ISI\>://WOS:001536107000001},

doi = {10.1002/adma.202507407},

issn = {0935-9648},

year  = {2025},

date = {2025-07-26},

journal = {Advanced Materials},

abstract = {Creating increasingly sensitive and cost-effective nucleic acid detection methods is critical for enhancing point-of-care (POC) applications. This requires highly specific capture of biomarkers and efficient transduction of capture events. However, the signal from biomarkers present at extremely low amounts often falls below the detection limit of typical fluorescence-based methods, necessitating molecular amplification. Here, we present single-molecule detection of a non-amplified, 151-nucleotide sequence specific to antibiotics-resistant Klebsiella pneumoniae down to attomolar concentrations, using Trident NanoAntennas with Cleared HOtSpots (NACHOS). This NACHOS-diagnostics assay leverages a compact microscope with a large field-of-view, including microfluidic flow to enhance capturing efficiency. Fluorescence enhancement is provided by NanoAntennas, arranged using a combination of nanosphere lithography and site-specific DNA origami placement. This method can detect 200 +/- 50 out of 600 molecules in a 100 mu L sample volume within an hour. This represents a typical number of pathogens in clinical samples commonly detected by Polymerase Chain Reaction. We achieve similar sensitivity in untreated human plasma, enhancing the practical applicability of the system. This platform can be adapted to detect shorter nucleic acid fragments that are not compatible with traditional amplification-based technologies. This provides a robust and scalable solution for sensitive nucleic acid detection in diverse clinical settings.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Single-molecule dynamic structural biology with vertically arranged DNA on a fluorescence microscope},

author = {A M Szalai and G Ferrari and L Richter and J Hartmann and M-Z Kesici and B Ji and K Coshic and M R J Dagleish and A Jaeger and A Aksimentiev and I Tessmer and I Kami\'{n}ska and A M Vera and P Tinnefeld},

url = {https://doi.org/10.1038/s41592-024-02498-x},

doi = {10.1038/s41592-024-02498-x},

issn = {1548-7105},

year  = {2024},

date = {2024-11-08},

journal = {Nature Methods},

abstract = {The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription and repair. Dynamic nanoscale observations of DNA structural features are necessary for understanding these interactions. Here we introduce graphene energy transfer with vertical nucleic acids (GETvNA), a method to investigate DNA\textendashprotein interactions that exploits the vertical orientation adopted by double-stranded DNA on graphene. This approach enables the dynamic study of DNA conformational changes via energy transfer from a probe dye to graphene, achieving spatial resolution down to the r{A}ngstr\"{o}m scale at subsecond temporal resolution. We measured DNA bending induced by adenine tracts, bulges, abasic sites and the binding of endonuclease IV. In addition, we observed the translocation of the O6-alkylguanine DNA alkyltransferase on DNA, reaching single base-pair resolution and detecting preferential binding to adenine tracts. This method promises widespread use for dynamical studies of nucleic acids and nucleic acid\textendashprotein interactions with resolution so far reserved for traditional structural biology techniques.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {2D Titanium Carbide MXene and Single-Molecule Fluorescence: Distance-Dependent Nonradiative Energy Transfer and Leaflet-Resolved Dye Sensing in Lipid Bilayers},

author = {L Manzanares and D Spurling and A M Szalai and T Schr\"{o}der and E B\"{u}ber and G Ferrari and M R J Dagleish and V Nicolosi and P Tinnefeld},

doi = {10.1002/adma.202411724},

issn = {0935-9648 (Print) 

0935-9648},

year  = {2024},

date = {2024-10-24},

journal = {Adv Mater},

volume = {36},

number = {49},

pages = {e2411724},

abstract = {Despite their growing popularity, many fundamental properties and applications of MXene materials remain underexplored. Here, the nonradiative energy transfer properties of 2D titanium carbide MXene are investigated and their application in single-molecule biosensing is explored for the first time. DNA origami positioners are used for single dye placement immobilized by a specific chemistry based on glycine-MXene interactions, allowing precise control of their orientation on the surface. Each DNA origami structure carries a single dye molecule at predetermined heights. Single-molecule fluorescence confocal microscopy reveals that energy transfer of an organic emitter (ATTO 542) on transparent thin films made of spincast Ti(3)C(2)T(x) flakes follows a cubic distance dependence, where 50% of energy transfer efficiency is reached at 2.7 nm (d(0)). MXenes are applied as short-distance spectroscopic nanorulers, determining z distances of dye-labeled supported lipid bilayers fused on MXene's hydrophilic surface. Hydration layer (2.1 nm) and lipid bilayer thickness (4.5 nm) values that agree with the literature are obtained. These results highlight titanium carbide MXenes as promising substrates for single-molecule biosensing of ultrathin assemblies, owing to their sensitivity near the interface, a distance regime that is typically inaccessible to other energy transfer tools.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Broad-Band Fluorescence Enhancement of QDs Captured in the Hotspot of DNA Origami Nanonantennas},

author = {R Yaadav and K Trofymchuk and F Gong and X Ji and F Steiner and P Tinnefeld and Z He},

url = {https://doi.org/10.1021/acs.jpcc.4c01797},

doi = {10.1021/acs.jpcc.4c01797},

issn = {1932-7447},

year  = {2024},

date = {2024-06-06},

journal = {The Journal of Physical Chemistry C},

volume = {128},

number = {22},

pages = {9154-9160},

abstract = {The integration of the DNA origami technique with plasmonic nanostructures has led to the development of optical antennas capable of significantly enhancing the excitation and emission rates of proximal fluorophores by offering precise control over their spatial positioning. However, despite these advancements, conventional fluorophores still face challenges due to their susceptibility to photobleaching. This limitation highlights the need for more stable alternatives. Quantum dots (QDs), also known as “artificial atoms”, emerge as a promising candidate, offering an array of distinctive size-dependent spectral qualities, encompassing broad absorption ranges, tight emission bands, adjustable fluorescence, and better photostability. The pivotal challenge is ensuring the QDs’ precise placement within plasmonic hotspots to unlock maximal signal enhancement. To tackle this, we used DNA origami-based NanoAntennas with Cleared HOtSpots (NACHOS) to successfully incorporate divalent DNA-tagged QDs of different sizes within the hotspot of two 100 nm silver nanoparticles. Known for their broad-band optical properties, silver nanoparticles enabled fluorescence enhancement for three spectrally different QDs, with more than 100-fold enhancement for the smallest QDs, alongside a reduction in their fluorescence lifetime. This integration deepens our understanding of nanoscale interactions and charts a path for the development of advanced plasmonic devices, establishing a robust framework for tailored bioassays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Super-resolved FRET and co-tracking in pMINFLUX},

author = {F Cole and J Z\"{a}hringer and J Bohlen and T Schr\"{o}der and F Steiner and M Pfeiffer and P Sch\"{u}ler and F D Stefani and P Tinnefeld},

url = {https://doi.org/10.1038/s41566-024-01384-4},

doi = {10.1038/s41566-024-01384-4},

issn = {1749-4893},

year  = {2024},

date = {2024-05-01},

journal = {Nature Photonics},

volume = {18},

number = {5},

pages = {478-484},

abstract = {Single-molecule fluorescence resonance energy transfer (smFRET) is widely used to investigate dynamic (bio)molecular interactions occurring over distances of up to 10 nm. Recent advances in super-resolution methods have brought their spatiotemporal resolution closer towards the smFRET regime. Although these methods do not suffer from the spatial restrictions of FRET, they only visualize one emitter at a time, thus making it difficult to capture fast dynamics of the interactions. Here we describe two approaches to overcome this limitation in pulsed-interleaved MINFLUX (pMINFLUX) microscopy by using its intrinsic fluorescence lifetime information. First we combine pMINFLUX with smFRET, which enables tracking a FRET donor with nanometre precision while simultaneously determining its distance to a FRET acceptor, yielding the acceptor position by multilateration. Second, we developed pMINFLUX lifetime multiplexing\textemdasha method that simultaneously tracks two fluorophores with similar spectral properties but distinct fluorescence lifetimes\textemdashto extend co-localized tracking beyond the FRET range. We demonstrate applications on DNA origami systems as well as by imaging the paratopes of an antibody with precision better than 2 nm, paving the way for nanometre precise co-localized tracking for inter-dye distances between 4 nm and 100 nm, and closing the resolution gap between smFRET and co-tracking.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Single-Molecule FRET at 10 MHz Count Rates},

author = {L Grabenhorst and F Sturzenegger and M Hasler and B Schuler and P Tinnefeld},

url = {https://doi.org/10.1021/jacs.3c13757},

doi = {10.1021/jacs.3c13757},

issn = {0002-7863},

year  = {2024},

date = {2024-02-07},

journal = {Journal of the American Chemical Society},

volume = {146},

number = {5},

pages = {3539-3544},

abstract = {A bottleneck in many studies utilizing single-molecule F\"{o}rster resonance energy transfer is the attainable photon count rate, as it determines the temporal resolution of the experiment. As many biologically relevant processes occur on time scales that are hardly accessible with currently achievable photon count rates, there has been considerable effort to find strategies to increase the stability and brightness of fluorescent dyes. Here, we use DNA nanoantennas to drastically increase the achievable photon count rates and observe fast biomolecular dynamics in the small volume between two plasmonic nanoparticles. As a proof of concept, we observe the coupled folding and binding of two intrinsically disordered proteins, which form transient encounter complexes with lifetimes on the order of 100 μs. To test the limits of our approach, we also investigated the hybridization of a short single-stranded DNA to its complementary counterpart, revealing a transition path time of 17 μs at photon count rates of around 10 MHz, which is an order-of-magnitude improvement compared to the state of the art. Concomitantly, the photostability was increased, enabling many seconds long megahertz fluorescence time traces. Due to the modular nature of the DNA origami method, this platform can be adapted to a broad range of biomolecules, providing a promising approach to study previously unobservable ultrafast biophysical processes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {MXene-induced nonradiative energy transfer},

author = {C L M Palenzuela and D Spurling and A Szalai and T Schr\"{o}der and V Nicolosi and P Tinnefeld},

url = {https://chemrxiv.org/engage/chemrxiv/article-details/6544ea8948dad23120fe8d2a},

doi = {10.26434/chemrxiv-2023-r54g8},

year  = {2023},

date = {2023-11-06},

abstract = {Since their discovery in 2011, MXenes have risen to prominence for energy storage, electromagnetic shielding, and optoelectronics. Yet, the nonradiative energy transfer properties of this family of 2D materials remain elusive, which may have implications in optoelectronics, photovoltaics and biosensing. Here, we use single-molecule fluorescence confocal microscopy and DNA origami nanopositioners to investigate, for the first time, the distance-dependent energy transfer of an organic emitter (ATTO 542) placed on transparent thin films made of spincast Ti3C2Tx flakes. We propose a specific immobilization chemistry for DNA origami nanostructures based on glycine-MXene interaction, allowing us to precisely control their orientation on the surface. Each DNA origami structure is designed to carry a single dye molecule at predetermined heights. Our findings reveal that when the dye is located at distances of 1 nm \< d \< 8 nm from the surface, the fluorescence is quenched following a distance dependence of d-3. This is in agreement with the F\"{o}rster-type mechanism of energy transfer in transparent conductors at the bulk level. 50% of energy transfer efficiency is reached at 2.7 nm (d0). MXenes could therefore be used as short-distance spectroscopic nanorulers, sensitive at a distance regime that common energy transfer tools cannot access.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Deep-LASI: deep-learning assisted, single-molecule imaging analysis of multi-color DNA origami structures},

author = {S Wanninger and P Asadiatouei and J Bohlen and C-B Salem and P Tinnefeld and E Ploetz and D C Lamb},

url = {https://doi.org/10.1038/s41467-023-42272-9},

doi = {10.1038/s41467-023-42272-9},

issn = {2041-1723},

year  = {2023},

date = {2023-10-17},

journal = {Nature Communications},

volume = {14},

number = {1},

pages = {6564},

abstract = {Single-molecule experiments have changed the way we explore the physical world, yet data analysis remains time-consuming and prone to human bias. Here, we introduce Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software suite powered by deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, especially from single-molecule F\"{o}rster Resonance Energy Transfer (smFRET) experiments. Deep-LASI automatically sorts recorded traces, determines FRET correction factors and classifies the state transitions of dynamic traces all in ~20\textendash100 ms per trajectory. We benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Exploring the Synergies of Single-Molecule Fluorescence and 2D Materials Coupled by DNA},

author = {L Richter and A M Szalai and C L Manzanares-Palenzuela and I Kami\'{n}ska and P Tinnefeld},

url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/adma.202303152},

doi = {https://doi.org/10.1002/adma.202303152},

issn = {0935-9648},

year  = {2023},

date = {2023-09-05},

journal = {Advanced Materials},

volume = {35},

number = {41},

pages = {2303152},

abstract = {Abstract The world of 2D materials is steadily growing, with numerous researchers attempting to discover, elucidate, and exploit their properties. Approaches relying on the detection of single fluorescent molecules offer a set of advantages, for instance, high sensitivity and specificity, that allow the drawing of conclusions with unprecedented precision. Herein, it is argued how the study of 2D materials benefits from fluorescence-based single-molecule modalities, and vice versa. A special focus is placed on DNA, serving as a versatile adaptor when anchoring single dye molecules to 2D materials. The existing literature on the fruitful combination of the two fields is reviewed, and an outlook on the additional synergies that can be created between them provided.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy},

author = {J Z\"{a}hringer and F Cole and J Bohlen and F Steiner and I Kami\'{n}ska and P Tinnefeld},

url = {https://doi.org/10.1038/s41377-023-01111-8},

doi = {10.1038/s41377-023-01111-8},

issn = {2047-7538},

year  = {2023},

date = {2023-03-10},

journal = {Light: Science \& Applications},

volume = {12},

number = {1},

pages = {70},

abstract = {3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques with fluorescence imaging. Here, we achieve 3D super-resolution by combining the 2D localization of pMINFLUX with the axial information of graphene energy transfer (GET) and the single-molecule switching by DNA-PAINT. We demonstrate \<2 nm localization precision in all 3 dimension with axial precision reaching below 0.3 nm. In 3D DNA-PAINT measurements, structural features, i.e., individual docking strands at distances of 3 nm, are directly resolved on DNA origami structures. pMINFLUX and GET represent a particular synergetic combination for super-resolution imaging near the surface such as for cell adhesion and membrane complexes as the information of each photon is used for both 2D and axial localization information. Furthermore, we introduce local PAINT (L-PAINT), in which DNA-PAINT imager strands are equipped with an additional binding sequence for local upconcentration improving signal-to-background ratio and imaging speed of local clusters. L-PAINT is demonstrated by imaging a triangular structure with 6 nm side lengths within seconds.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics},

author = {T Schr\"{o}der and J Bohlen and S E Ochmann and P Sch\"{u}ler and S Krause and D C Lamb and P Tinnefeld},

url = {https://www.pnas.org/doi/abs/10.1073/pnas.2211896120},

doi = {doi:10.1073/pnas.2211896120},

year  = {2023},

date = {2023-01-18},

journal = {Proceedings of the National Academy of Sciences},

volume = {120},

number = {4},

pages = {e2211896120},

abstract = {Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with F\"{o}rster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Gold Nanorod DNA Origami Antennas for 3 Orders of Magnitude Fluorescence Enhancement in NIR},

author = {K Trofymchuk and K Ko\l\k{a}taj and V Glembockyte and F Zhu and G P Acuna and T Liedl and P Tinnefeld},

url = {https://doi.org/10.1021/acsnano.2c09577},

doi = {10.1021/acsnano.2c09577},

issn = {1936-0851},

year  = {2023},

date = {2023-01-03},

urldate = {2023-01-03},

journal = {ACS Nano},

volume = {17},

issue = {2},

pages = {1327-1334},

abstract = {DNA origami has taken a leading position in organizing materials at the nanoscale for various applications such as manipulation of light by exploiting plasmonic nanoparticles. We here present the arrangement of gold nanorods in a plasmonic nanoantenna dimer enabling up to 1600-fold fluorescence enhancement of a conventional near-infrared (NIR) dye positioned at the plasmonic hotspot between the nanorods. Transmission electron microscopy, dark-field spectroscopy, and fluorescence analysis together with numerical simulations give us insights on the heterogeneity of the observed enhancement values. The size of our hotspot region is ∼12 nm, granted by using the recently introduced design of NAnoantenna with Cleared HotSpot (NACHOS), which provides enough space for placing of tailored bioassays. Additionally, the possibility to synthesize nanoantennas in solution might allow for production upscaling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Gold Nanorod DNA Origami Antennas for 3 Orders of Magnitude Fluorescence Enhancement in NIR},

author = {K Trofymchuk and K Ko\l\k{a}taj and V Glembockyte and F Zhu and G P Acuna and T Liedl and P Tinnefeld},

url = {https://doi.org/10.1021/acsnano.2c09577},

doi = {10.1021/acsnano.2c09577},

issn = {1936-0851},

year  = {2023},

date = {2023-01-03},

journal = {ACS Nano},

volume = {17},

number = {2},

pages = {1327-1334},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Maximizing the Accessibility in DNA Origami Nanoantenna Plasmonic Hotspots},

author = {C Close and K Trofymchuk and L Grabenhorst and B Lalkens and V Glembockyte and P Tinnefeld},

url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/admi.202200255},

doi = {https://doi.org/10.1002/admi.202200255},

issn = {2196-7350},

year  = {2022},

date = {2022-07-01},

journal = {Advanced Materials Interfaces},

volume = {n/a},

number = {n/a},

pages = {2200255},

abstract = {Abstract DNA nanotechnology has conquered the challenge of positioning quantum emitters in the hotspot of optical antenna structures for fluorescence enhancement. Therefore, DNA origami serves as the scaffold to arrange nanoparticles and emitters, such as fluorescent dyes. For the next challenge of optimizing the applicability of plasmonic hotspots for molecular assays, a Trident DNA origami structure that increases the accessibility of the hotspot is introduced, thereby improving the kinetics of target molecule binding. This Trident NanoAntenna with Cleared HOtSpot (NACHOS) is compared with previous DNA origami nanoantennas and improved hotspot accessibility is demonstrated without compromising fluorescence enhancement. The approach taps into the potential of Trident NACHOS for single-molecule-based plasmonic biosensing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Salt-induced conformational switching of a flat rectangular DNA origami structure},

author = {K H\"{u}bner and M Raab and J Bohlen and J Bauer and P Tinnefeld},

url = {http://dx.doi.org/10.1039/D1NR07793G},

doi = {10.1039/D1NR07793G},

issn = {2040-3364},

year  = {2022},

date = {2022-05-11},

journal = {Nanoscale},

volume = {14},

number = {21},

pages = {7898-7905},

abstract = {A rectangular DNA origami structure is one of the most studied and often used motif for applications in DNA nanotechnology. Here, we present two assays to study structural changes in DNA nanostructures and reveal a reversible rolling-up of the rectangular DNA origami structure induced by bivalent cations such as magnesium or calcium. First, we applied one-color and two-color superresolution DNA-PAINT with protruding strands along the long edges of the DNA origami rectangle. At increasing salt concentration, a single line instead of two lines is observed as a first indicator of rolling-up. Two-color measurements also revealed different conformations with parallel and angled edges. Second, we placed a gold nanoparticle and a dye molecule at different positions on the DNA origami structure. Distance dependent fluorescence quenching by the nanoparticle reports on dynamic transitions as well as it provides evidence that the rolling-up occurs preferentially along the diagonal of the DNA origami rectangle. The results will be helpful to test DNA structural models and the assays presented will be useful to study further structural transitions in DNA nanotechnology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Quantitative Single-Molecule Measurements of Membrane Charges with DNA Origami Sensors},

author = {S E Ochmann and T Schr\"{o}der and C M Schulz and P Tinnefeld},

url = {https://doi.org/10.1021/acs.analchem.1c05092},

doi = {10.1021/acs.analchem.1c05092},

issn = {0003-2700},

year  = {2022},

date = {2022-01-28},

journal = {Analytical Chemistry},

abstract = {Charges in lipid head groups generate electrical surface potentials at cell membranes, and changes in their composition are involved in various signaling pathways, such as T-cell activation or apoptosis. Here, we present a DNA origami-based sensor for membrane surface charges with a quantitative fluorescence read-out of single molecules. A DNA origami plate is equipped with modifications for specific membrane targeting, surface immobilization, and an anionic sensing unit consisting of single-stranded DNA and the dye ATTO542. This unit is anchored to a lipid membrane by the dye ATTO647N, and conformational changes of the sensing unit in response to surface charges are read out by fluorescence resonance energy transfer between the two dyes. We test the performance of our sensor with single-molecule fluorescence microscopy by exposing it to differently charged large unilamellar vesicles. We achieve a change in energy transfer of ∼10% points between uncharged and highly charged membranes and demonstrate a quantitative relation between the surface charge and the energy transfer. Further, with autocorrelation analyses of confocal data, we unravel the working principle of our sensor that is switching dynamically between a membrane-bound state and an unbound state on the timescale of 1\textendash10 ms. Our study introduces a complementary sensing system for membrane surface charges to previously published genetically encoded sensors. Additionally, the single-molecule read-out enables investigations of lipid membranes on the nanoscale with a high spatial resolution circumventing ensemble averaging.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {How Blinking Affects Photon Correlations in Multichromophoric Nanoparticles},

author = {T Schr\"{o}der and S Bange and J Schedlbauer and F Steiner and J M Lupton and P Tinnefeld and J Vogelsang},

url = {https://doi.org/10.1021/acsnano.1c06649},

doi = {10.1021/acsnano.1c06649},

issn = {1936-0851},

year  = {2021},

date = {2021-11-04},

urldate = {2021-11-04},

journal = {ACS Nano},

abstract = {A single chromophore can only emit a maximum of one single photon per excitation cycle. This limitation results in a phenomenon commonly referred to as photon antibunching (pAB). When multiple chromophores contribute to the fluorescence measured, the degree of pAB has been used as a metric to “count” the number of chromophores. But the fact that chromophores can switch randomly between bright and dark states also impacts pAB and can lead to incorrect chromophore numbers being determined from pAB measurements. By both simulations and experiment, we demonstrate how pAB is affected by independent and collective chromophore blinking, enabling us to formulate universal guidelines for correct interpretation of pAB measurements. We use DNA-origami nanostructures to design multichromophoric model systems that exhibit either independent or collective chromophore blinking. Two approaches are presented that can distinguish experimentally between these two blinking mechanisms. The first one utilizes the different excitation intensity dependence on the blinking mechanisms. The second approach exploits the fact that collective blinking implies energy transfer to a quenching moiety, which is a time-dependent process. In pulsed-excitation experiments, the degree of collective blinking can therefore be altered by time gating the fluorescence photon stream, enabling us to extract the energy-transfer rate to a quencher. The ability to distinguish between different blinking mechanisms is valuable in materials science, such as for multichromophoric nanoparticles like conjugated-polymer chains as well as in biophysics, for example, for quantitative analysis of protein assemblies by counting chromophores.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Targetable Conformationally Restricted Cyanines Enable Photon-Count-Limited Applications**},

author = {P Eiring and R Mclaughlin and S S Matikonda and Z Han and L Grabenhorst and D A Helmerich and M Meub and G Beliu and M Luciano and V Bandi and N Zijlstra and Z-D Shi and S G Tarasov and R Swenson and P Tinnefeld and V Glembockyte and T Cordes and M Sauer and M J Schnermann},

url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202109749},

doi = {https://doi.org/10.1002/anie.202109749},

issn = {1433-7851},

year  = {2021},

date = {2021-10-04},

urldate = {2021-10-04},

journal = {Angewandte Chemie International Edition},

volume = {60},

number = {51},

pages = {26685-26693},

abstract = {Abstract Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule F\"{o}rster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Single Antibody Detection in a DNA Origami Nanoantenna},

author = {M Pfeiffer and K Trofymchuk and S Ranallo and F Ricci and F Steiner and F Cole and V Glembockyte and P Tinnefeld},

url = {https://www.sciencedirect.com/science/article/pii/S2589004221010403},

doi = {https://doi.org/10.1016/j.isci.2021.103072},

issn = {2589-0042},

year  = {2021},

date = {2021-09-01},

urldate = {2021-09-01},

journal = {iScience},

pages = {103072},

abstract = {Summary 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {DNA Origami Nanoantennas for Fluorescence Enhancement},

author = {V Glembockyte and L Grabenhorst and K Trofymchuk and P Tinnefeld},

url = {https://doi.org/10.1021/acs.accounts.1c00307},

doi = {10.1021/acs.accounts.1c00307},

issn = {0001-4842},

year  = {2021},

date = {2021-08-26},

urldate = {2021-08-26},

journal = {Accounts of Chemical Research},

volume = {54},

number = {17},

pages = {3338-3348},

abstract = {ConspectusThe 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Graphene Energy Transfer for Single-Molecule Biophysics, Biosensing, and Super-Resolution Microscopy},

author = {I Kami\'{n}ska and J Bohlen and R Yaadav and P Sch\"{u}ler and M Raab and T Schr\"{o}der and J Z\"{a}hringer and K Zielonka and S Krause and P Tinnefeld},

url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/adma.202101099},

doi = {https://doi.org/10.1002/adma.202101099},

issn = {0935-9648},

year  = {2021},

date = {2021-05-03},

urldate = {2021-05-03},

journal = {Advanced Materials},

volume = {33},

issue = {24},

pages = {2101099},

abstract = {Abstract Graphene is considered a game-changing material, especially for its mechanical and electrical properties. This work exploits that graphene is almost transparent but quenches fluorescence in a range up to ≈40 nm. Graphene as a broadband and unbleachable energy-transfer acceptor without labeling, is used to precisely determine the height of molecules with respect to graphene, to visualize the dynamics of DNA nanostructures, and to determine the orientation of F\"{o}rster-type resonance energy transfer (FRET) pairs. Using DNA origami nanopositioners, biosensing, single-molecule tracking, and DNA PAINT super-resolution with \<3 nm z-resolution are demonstrated. The range of examples shows the potential of graphene-on-glass coverslips as a versatile platform for single-molecule biophysics, biosensing, and super-resolution microscopy.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Graphene-on-Glass Preparation and Cleaning Methods Characterized by Single-Molecule DNA Origami Fluorescent Probes and Raman Spectroscopy},

author = {S Krause and E Ploetz and J Bohlen and P Sch\"{u}ler and R Yaadav and F Selbach and F Steiner and I Kami\'{n}ska and P Tinnefeld},

url = {https://pubs.acs.org/doi/abs/10.1021/acsnano.0c08383},

doi = {10.1021/acsnano.0c08383},

issn = {1936-0851},

year  = {2021},

date = {2021-04-09},

urldate = {2021-04-09},

journal = {ACS Nano},

abstract = {Graphene exhibits outstanding fluorescence quenching properties that can become useful for biophysics and biosensing applications, but it remains challenging to harness these advantages due to the complex transfer procedure of chemical vapor deposition-grown graphene to glass coverslips and the low yield of usable samples. Here, we screen 10 graphene-on-glass preparation methods and present an optimized protocol. To obtain the required quality for single-molecule and super-resolution imaging on graphene, we introduce a graphene screening method that avoids consuming the investigated sample. We apply DNA origami nanostructures to place fluorescent probes at a defined distance on top of graphene-on-glass coverslips. Subsequent fluorescence lifetime imaging directly reports on the graphene quality, as deviations from the expected fluorescence lifetime indicate imperfections. We compare the DNA origami probes with conventional techniques for graphene characterization, including light microscopy, atomic force microscopy, and Raman spectroscopy. For the latter, we observe a discrepancy between the graphene quality implied by Raman spectra in comparison to the quality probed by fluorescence lifetime quenching measured at the same position. We attribute this discrepancy to the difference in the effective area that is probed by Raman spectroscopy and fluorescence quenching. Moreover, we demonstrate the applicability of already screened and positively evaluated graphene for studying single-molecule conformational dynamics on a second DNA origami structure. Our results constitute the basis for graphene-based biophysics and super-resolution microscopy.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Determining the In-Plane Orientation and Binding Mode of Single Fluorescent Dyes in DNA Origami Structures},

author = {K H\"{u}bner and H Joshi and A Aksimentiev and F D Stefani and P Tinnefeld and G P Acuna},

url = {https://doi.org/10.1021/acsnano.0c10259},

doi = {10.1021/acsnano.0c10259},

issn = {1936-0851},

year  = {2021},

date = {2021-03-04},

journal = {ACS Nano},

abstract = {We present a technique to determine the orientation of single fluorophores attached to DNA origami structures based on two measurements. First, the orientation of the absorption transition dipole of the molecule is determined through a polarization-resolved excitation measurement. Second, the orientation of the DNA origami structure is obtained from a DNA-PAINT nanoscopy measurement. Both measurements are performed consecutively on a fluorescence wide-field microscope. We employed this approach to study the orientation of single ATTO 647N, ATTO 643, and Cy5 fluorophores covalently attached to a 2D rectangular DNA origami structure with different nanoenvironments, achieved by changing both the fluorophores’ binding position and immediate vicinity. Our results show that when fluorophores are incorporated with additional space, for example, by omitting nucleotides in an elsewise double-stranded environment, they tend to stick to the DNA and to adopt a preferred orientation that depends more on the specific molecular environment than on the fluorophore type. With the aid of all-atom molecular dynamics simulations, we rationalized our observations and provide insight into the fluorophores’ probable binding modes. We believe this work constitutes an important step toward manipulating the orientation of single fluorophores in DNA origami structures, which is vital for the development of more efficient and reproducible self-assembled nanophotonic devices.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Picosecond time-resolved photon antibunching measures nanoscale exciton motion and the true number of chromophores},

author = {G J Hedley and T Schr\"{o}der and F Steiner and T Eder and F J Hofmann and S Bange and D Laux and S H\"{o}ger and P Tinnefeld and J M Lupton and J Vogelsang},

url = {https://doi.org/10.1038/s41467-021-21474-z},

doi = {10.1038/s41467-021-21474-z},

issn = {2041-1723},

year  = {2021},

date = {2021-02-26},

journal = {Nature Communications},

volume = {12},

number = {1},

pages = {1327},

abstract = {The particle-like nature of light becomes evident in the photon statistics of fluorescence from single quantum systems as photon antibunching. In multichromophoric systems, exciton diffusion and subsequent annihilation occurs. These processes also yield photon antibunching but cannot be interpreted reliably. Here we develop picosecond time-resolved antibunching to identify and decode such processes. We use this method to measure the true number of chromophores on well-defined multichromophoric DNA-origami structures, and precisely determine the distance-dependent rates of annihilation between excitons. Further, this allows us to measure exciton diffusion in mesoscopic H- and J-type conjugated-polymer aggregates. We distinguish between one-dimensional intra-chain and three-dimensional inter-chain exciton diffusion at different times after excitation and determine the disorder-dependent diffusion lengths. Our method provides a powerful lens through which excitons can be studied at the single-particle level, enabling the rational design of improved excitonic probes such as ultra-bright fluorescent nanoparticles and materials for optoelectronic devices.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET},

author = {A M Molszalai and B Siarry and J Lukin and S Giusti and N Unsain and A C\'{a}ceres and F Steiner and P Tinnefeld and D Refojo and T M Jovin and F D Stefani},

url = {https://doi.org/10.1021/acs.nanolett.1c00158},

doi = {10.1021/acs.nanolett.1c00158},

issn = {1530-6984},

year  = {2021},

date = {2021-02-23},

journal = {Nano Letters},

abstract = {F\"{o}rster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope},

author = {K Trofymchuk and V Glembockyte and L Grabenhorst and F Steiner and C Vietz and C Close and M Pfeiffer and L Richter and M L Sch\"{u}tte and F Selbach and R Yaadav and J Z\"{a}hringer and Q Wei and A Ozcan and B Lalkens and G P Acuna and P Tinnefeld},

url = {https://doi.org/10.1038/s41467-021-21238-9},

doi = {10.1038/s41467-021-21238-9},

issn = {2041-1723},

year  = {2021},

date = {2021-02-11},

urldate = {2021-02-11},

journal = {Nature Communications},

volume = {12},

number = {1},

pages = {950},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Pulsed Interleaved MINFLUX},

author = {L A Masullo and F Steiner and J Z\"{a}hringer and L F Lopez and J Bohlen and L Richter and F Cole and P Tinnefeld and F D Stefani},

url = {https://doi.org/10.1021/acs.nanolett.0c04600},

doi = {10.1021/acs.nanolett.0c04600},

issn = {1530-6984},

year  = {2021},

date = {2021-01-13},

urldate = {2021-01-13},

journal = {Nano Letters},

volume = {21},

number = {1},

pages = {840-846},

abstract = {We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1\textendash2 nm localization precision with 2000\textendash1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {DNA origami based superconducting nanowires},

author = {L Shani and P Tinnefeld and Y Fleger and A Sharoni and B Y Shapiro and A Shaulov and O Gang and Y Yeshurun},

url = {https://doi.org/10.1063/5.0029781},

doi = {10.1063/5.0029781},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {AIP Advances},

volume = {11},

number = {1},

pages = {015130},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Self-Regeneration and Self-Healing in DNA Origami Nanostructures},

author = {J M Scheckenbach and P Tinnefeld and V Glembockyte and T Schubert and C Forthmann},

url = {https://doi.org/10.1002/anie.202012986},

doi = {https://doi.org/10.1002/anie.202012986},

issn = {1433-7851},

year  = {2020},

date = {2020-11-23},

journal = {Angewandte Chemie International Edition},

volume = {n/a},

number = {n/a},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {DNA origami nanorulers and emerging reference structures},

author = {M Scheckenbach and J Bauer and J Z\"{a}hringer and F Selbach and P Tinnefeld},

url = {https://doi.org/10.1063/5.0022885},

doi = {10.1063/5.0022885},

year  = {2020},

date = {2020-11-01},

journal = {APL Materials},

volume = {8},

number = {11},

pages = {110902},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Self-Healing Dyes\textemdashKeeping the Promise?},

author = {M Isselstein and L Zhang and V Glembockyte and O Brix and G Cosa and P Tinnefeld and T Cordes},

url = {https://doi.org/10.1021/acs.jpclett.9b03833},

doi = {10.1021/acs.jpclett.9b03833},

year  = {2020},

date = {2020-06-04},

journal = {The Journal of Physical Chemistry Letters},

volume = {11},

number = {11},

pages = {4462-4480},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Impact of Cyanine Conformational Restraint in the Near-Infrared Range},

author = {S S Matikonda and G Hammersley and N Kumari and L Grabenhorst and V Glembockyte and P Tinnefeld and J Ivanic and M Levitus and M J Schnermann},

url = {https://doi.org/10.1021/acs.joc.0c00236},

doi = {10.1021/acs.joc.0c00236},

issn = {0022-3263},

year  = {2020},

date = {2020-04-10},

urldate = {2020-04-10},

journal = {The Journal of Organic Chemistry},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Addressable Nanoantennas with Cleared Hotspots for Single-Molecule Detection on a Portable Smartphone Microscope},

author = {K Trofymchuk and V Glembockyte and L Grabenhorst and F Steiner and C Vietz and C Close and M Pfeiffer and L Richter and M L Sch\"{u}tte and F Selbach and R Yaadav and J Z\"{a}hringer and Q Wei and A Ozcan and B Lalkens and G P Acuna and P Tinnefeld},

url = {http://biorxiv.org/content/early/2020/04/09/2020.04.09.032037.abstract},

doi = {10.1101/2020.04.09.032037},

year  = {2020},

date = {2020-04-09},

urldate = {2020-04-09},

journal = {bioRxiv},

pages = {2020.04.09.032037},

abstract = {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 “.Competing Interest StatementPT and GPA are inventors on a patent of the described Bottom-up method for fluorescence enhancement in molecular assays, EP1260316.1, 2012, US20130252825 A1.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Fluorophore photostability and saturation in the hotspot of DNA origami nanoantennas},

author = {L Grabenhorst and K Trofymchuk and F Steiner and V Glembockyte and P Tinnefeld},

url = {http://dx.doi.org/10.1088/2050-6120/ab6ac8},

doi = {10.1088/2050-6120/ab6ac8},

issn = {2050-6120},

year  = {2020},

date = {2020-02-05},

urldate = {2020-02-05},

journal = {Methods and Applications in Fluorescence},

volume = {8},

number = {2},

pages = {024003},

abstract = {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 quencher.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Ultrafast Single-Molecule Fluorescence Measured by Femtosecond Double-Pulse Excitation Photon Antibunching},

author = {J Schedlbauer and P Wilhelm and L Grabenhorst and M E Federl and B Lalkens and F Hinderer and U Scherf and S H\"{o}ger and P Tinnefeld and S Bange and J Vogelsang and J M Lupton},

url = {https://doi.org/10.1021/acs.nanolett.9b04354},

doi = {10.1021/acs.nanolett.9b04354},

issn = {1530-6984},

year  = {2019},

date = {2019-12-23},

journal = {Nano Letters},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Directing Single-Molecule Emission with DNA Origami-Assembled Optical Antennas},

author = {K H\"{u}bner and M Pilo-Pais and F Selbach and T Liedl and P Tinnefeld and F D Stefani and G P Acuna},

url = {https://doi.org/10.1021/acs.nanolett.9b02886},

doi = {10.1021/acs.nanolett.9b02886},

issn = {1530-6984},

year  = {2019},

date = {2019-09-11},

urldate = {2019-09-11},

journal = {Nano Letters},

volume = {19},

number = {9},

pages = {6629-6634},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Distance Dependence of Single-Molecule Energy Transfer to Graphene Measured with DNA Origami Nanopositioners},

author = {I Kaminska and J Bohlen and S Rocchetti and F Selbach and G P Acuna and P Tinnefeld},

url = {https://doi.org/10.1021/acs.nanolett.9b00172},

doi = {10.1021/acs.nanolett.9b00172},

issn = {1530-6984},

year  = {2019},

date = {2019-07-10},

journal = {Nano Letters},

volume = {19},

number = {7},

pages = {4257-4262},

abstract = {Despite the thorough investigation of graphene since 2004, altering its surface chemistry and reproducible functionalization remain challenging. This hinders fabrication of more complex hybrid materials with controlled architectures, and as a consequence the development of sensitive and reliable sensors and biological assays. In this contribution, we introduce DNA origami structures as nanopositioners for placing single dye molecules at controlled distances from graphene. The measurements of fluorescence intensity and lifetime of single emitters carried out for distances ranging from 3 to 58 nm confirmed the d\textendash4 dependence of the excitation energy transfer to graphene. Moreover, we determined the characteristic distance for 50% efficiency of the energy transfer from single dyes to graphene to be 17.7 nm. Using pyrene molecules as a glue to immobilize DNA origami nanostructures of various shape on graphene opens new possibilities to develop graphene-based biophysics and biosensing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Interchromophoric Interactions Determine the Maximum Brightness Density in DNA Origami Structures},

author = {T Schr\"{o}der and M B Scheible and F Steiner and J Vogelsang and P Tinnefeld},

url = {https://doi.org/10.1021/acs.nanolett.8b04845},

doi = {10.1021/acs.nanolett.8b04845},

issn = {1530-6984},

year  = {2019},

date = {2019-02-13},

urldate = {2019-02-13},

journal = {Nano Letters},

volume = {19},

number = {2},

pages = {1275-1281},

abstract = {An ideal point light source is as small and as bright as possible. For fluorescent point light sources, homogeneity of the light sources is important as well as that the fluorescent units inside the light source maintain their photophysical properties, which is compromised by dye aggregation. Here we propose DNA origami as a rigid scaffold to arrange dye molecules in a dense pixel array with high control of stoichiometry and dye\textendashdye interactions. In order to find the highest labeling density in a DNA origami structure without influencing dye photophysics, we alter the distance of two ATTO647N dyes in single base pair steps and probe the dye\textendashdye interactions on the single-molecule level. For small distances strong quenching in terms of intensity and fluorescence lifetime is observed. With increasing distance, we observe reduced quenching and molecular dynamics. However, energy transfer processes in the weak coupling regime still have a significant impact and can lead to quenching by singlet-dark-state-annihilation. Our study fills a gap of studying the interactions of dyes relevant for superresolution microscopy with dense labeling and for single-molecule biophysics. Incorporating these findings in a 3D DNA origami object will pave the way to bright and homogeneous DNA origami nanobeads.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}