Fluorophore photostability and saturation in the hotspot of DNA origami nanoantennas

Grabenhorst, Lennart; Trofymchuk, Kateryna; Steiner, Florian; Glembockyte, Viktorija; Tinnefeld, Philip

doi:10.1088/2050-6120/ab6ac8

Cited by 25 publications

(58 citation statements)

References 69 publications

(108 reference statements)

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“…The excitation powers at 639 nm were set to 200 nW or for 500 nW for the reference samples and to 50 nW for the NACHOS for the recording of the fluorescence transients. These powers were chosen to ensure that the samples are excited in the linear regime and to avoid saturation in the nanoantenna hotspot 29 . For the confocal scans, 2 µW at 532 nm and 2 µW and 500 nW at 639 nm were used for the reference and NACHOS samples, respectively.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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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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Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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“…These powers were chosen to avoid the saturation in the hotspot. 33 For the confocal scans, 2 µW at 532 nm and 2 µW and 500 nW at 639 nm were used for the reference and NACHOS samples, respectively. Emitted light was then collected using the same objective and filtered from the excitation light by the dichroic beam splitter.…”

Section: Confocal Measurements and Data Analysismentioning

confidence: 99%

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

Trofymchuk

Glembockyte

Grabenhorst

et al. 2020

Preprint

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† 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.

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“…When plotted as a function of normalized excitation intensity, Cy5B-disulfo is significantly brighter than AF647 at all excitation intensities. While both samples show a degree of saturation, [24] Cy5B-disulfo starts saturating only at much higher excitation intensities (Fig. 1B).…”

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confidence: 92%

Targetable Conformationally Restricted Cyanines Enable Photon-Count Limited Applications

Eiring¹,

McLaughlin²,

Matikonda³

et al. 2021

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Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications. 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 parent species in a range of contexts including their use on a DNA-nano-antenna, in single-molecule Förster resonance energy transfer (FRET) applications, far-red fluorescence lifetime imaging microscopy (FLIM), and single-molecule localization microscopy. These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.

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Fluorophore photostability and saturation in the hotspot of DNA origami nanoantennas

Cited by 25 publications

References 69 publications

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

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

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

Targetable Conformationally Restricted Cyanines Enable Photon-Count Limited Applications

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