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.

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 1–2 nm localization precision with 2000–1000 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.

† These authors contributed equallyThe advent of highly sensitive photodetectors 1,2 and the development of photostabilization strategies 3 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 ".Early detection of disease biomarkers generally requires high sensitivity enabled by molecular amplification mechanisms 5-9 or physical signal enhancement of commonly used fluorescence signals. [10][11][12][13] Physical fluorescence signal enhancement could enable sensitivity improvement, detection of singlemolecules on cost-effective and mobile devices and therefore help to distinguish specific signals against an unavoidable background of impurities even in low-resource settings. Fluorescence from emitters such as fluorescent dyes can be enhanced using plasmonic nanoantennas, [14][15][16] and the challenge of placing quantum emitters in their hotspots was overcome using DNA origami as constructing material. 17,18 The immense requirements for small, defined and rigid gaps between the gold or silver nanoparticles forming the gap in the nanoantenna aggravated the usability of the space between the nanoparticles for a biosensing assay that could thus far only be realized with mono-particle antennas and low enhancement values. 19 In this work, we introduce NACHOS that enable high fluorescence signal amplification and use them for a single-molecule diagnostic assay on a portable and inexpensive smartphone microscope. A novel threedimensional DNA origami structure was designed ( Fig. 1a) and folded from an M13mp18-derived scaffold strand and complementary staple strands ( Supplementary Tables 1-3). The NACHOS origami design uses two pillars to attach silver nanoparticles and creates the plasmonic hotspot at the bifurcation in the gap between the two pillars and the nanoparticles (see DNA origami sketches in Fig. 1a and full NACHOS structure in Fig.