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In recent years, DNA nanotechnology has matured to enable robust production of complex nanostructures and hybrid materials. We have combined DNA nanotechnology with sensitive optical detection to create functional single-molecule devices that enable new applications in single-molecule biosensing and superresolution microscopy. Starting with superresolution nanorulers and brightness reference samples we determined the resolving power of superresolution microscopes and evaluated the sensitivity of smartphone cameras. To improve the sensitivity we created DNA origami optical antennas for metal enhanced fluorescence. The unique ability of our DNA origami nanoantennas to place molecular assays specifically in the plasmonic hotspot is used for detecting Zika-virus nucleic acids. The fluorescence enhancement is further exploited to achieve ultra-sensitive detection on low-cost devices such as a modified smartphone leading to the democratization of single-molecule detection.

We developed a handheld and cost-effective surface-enhanced smartphone fluorescence microscope that showed ~10-fold improvement in signal by using a thin silver film as a plasmonic substrate, enabling the detection of ~80 fluorophores per diffraction-limited spot.

In this paper we show that the emission of ordinary organic dyes can be controlled in order to increase photostability or to induce long off-states for superresolution microscopy. We therefore extend a recently introduced concept that utilizes triplet-state quenching via redox-reactions and recovery of the electronic ground-state by complementary redox-reactions: it is shown that different reagents in an oxidizing and reducing system (ROXS) can positively influence the fluorescence properties of organic dyes. In more detail, the effects of Trolox, a ferrocene-based compound, an oxidized quinone derivative of Trolox and nitrobenzoic acid are investigated and compared to the prototypical compounds ascorbic acid and N,N methylviologen. While the redox potential is the most important parameter for the realization of the ROXS concept it is demonstrated that also kinetic aspects have to be taken into account to explain the properties of the specific redox agents. Photostabilization and the induction of off-states are of paramount importance for fluorescence microscopy in general and especially for superresolution microscopy based on “blinking” molecules.

In recent years Fluorescence Resonance Energy Transfer (FRET) has been widely used to determine distances, observe distance dynamics, and monitor molecular binding at the single-molecule level. A basic constraint of single-molecule FRET studies is the limited distance resolution owing to low photon statistics. We demonstrate that by confining molecules in nanopipettes (50-100 nm diameter) smFRET can be measured with improved photon statistics reducing the width of FRET proximity ratio distributions (PRD). This increase in distance resolution makes it possible to reveal subpopulations and dynamics in biomolecular complexes. Our data indicate that the width of PRD is not only determined by photon statistics (shot noise) and distance distributions between the chromophores but that photoinduced dark states of the acceptor also contribute to the PRD width. Furthermore, acceptor dark states such as triplet states influence the accuracy of determined mean FRET values. In this context, we present a strategy for the correction of the shift of the mean PR that is related to triplet induced blinking of the acceptor using reference FCS measurements.

Most biological processes are governed by assemblies of several dynamically interacting molecules. We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. By comparing energy transfer of the different subpopulations, we can disentangle the reasons that lead to the occurrence of three-way junctions lacking one chromophore. A merit of the method is the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously.

Fluorescence based enzyme analysis is commonly done by FRET-probes, natural enzyme substrates flanked by two corresponding fluorophores, showing spectral changes upon distance variations between the fluorophores. However, the use of double labeled substrates displays several limitations such as reduction of sensitivity and high background signal accompanied by high costs for synthesis. Therefore, the development of new probes avoiding these factors is of general interest in enzyme research. A promising approach represents smart probes, i.e. singly labeled quenched enzyme substrates that increase fluorescence intensity upon enzymatic cleavage. Smart probes use the fact that certain rhodamine and oxazine dyes are selectively quenched upon contact formation with guanine or tryptophan residues via photoinduced electron transfer (PET). The rapid response time of the probes enables real-time monitoring of enzyme activity in ensemble as well as in single molecule measurements, which is an important prerequisite for the improved understanding of enzyme mechanisms. We present the design of smart probes for the detection of the two hydrolases, DNasel and Carboxypeptidase A (CPA) with respect to stability and substrate specificity in ensemble measurements. Furthermore, we investigate the influence of the attached fluorophore on hydrolysis efficiency in case of CPA and demonstrate first applications of smart probes in single enzyme experiments.