Thermometries for Single Nanoparticles Heated with Light.

The development of efficient nanoscale photon absorbers, such as plasmonic or high-index dielectric nanostructures, allows the remotely controlled release of heat on the nanoscale using light. These photothermal nanomaterials have found applications in various research and technological fields, ranging from materials science to biology. However, measuring the nanoscale thermal fields remains an open challenge, hindering full comprehension and control of nanoscale photothermal phenomena. Here, we review and discuss existent thermometries suitable for single nanoparticles heated under illumination. These methods are classified in four categories according to the region where they assess temperature: (1) the average temperature within a diffraction-limited volume, (2) the average temperature at the immediate vicinity of the nanoparticle surface, (3) the temperature of the nanoparticle itself, and (4) a map of the temperature around the nanoparticle with nanoscale spatial resolution. In the latter, because it is the most challenging and informative type of method, we also envisage new combinations of technologies that could be helpful in retrieving nanoscale temperature maps. Finally, we analyze and provide examples of strategies to validate the results obtained using different thermometry methods.

Unidirectional Meta-Emitters Based on the Kerker Condition Assembled by DNA Origami.

Optical quantum emitters near nanostructures have access to additional relaxation channels and thus exhibit structure-dependent emission properties, including quantum yield and emission directionality. A well-engineered quantum emitter-plasmonic nanostructure hybrid can be considered as an optical meta-emitter consisting of a transmitting nanoantenna driven by an optical-frequency generator. In this work, the DNA origami fabrication method is used to construct ultracompact unidirectional meta-emitters composed of a plasmonic trimer nanoantenna driven by a single dye molecule. The origami is designed to bring the dye to the gap to simultaneously excite the electric and magnetic dipole modes of the trimer nanoantenna. The interference of these modes fulfills the Kerker condition at the fluorophore's emission band, enabling unidirectional emission. We report unidirectional emission from a single molecule with a front-to-back ratio of up to 10.7 dB accompanied by a maximum emission enhancement of 23-fold.

DNA Origami Assembled Nanoantennas for Manipulating Single-Molecule Spectral Emission.

The emission spectrum of a dye is given by the energy of all of the possible radiative transitions weighted by their probability. This spectrum can be altered with optical nanoantennas that are able to manipulate the decay rate of nearby emitters by modifying the local density of photonic states. Here, we make use of DNA origami to precisely place an individual dye at different positions around a gold nanorod and show how this affects the emission spectrum of the dye. In particular, we observe a strong suppression or enhancement of the transitions to different vibrational levels of the excitonic ground state, depending on the spectral overlap with the nanorod resonance. This reshaping can be used to experimentally extract the spectral dependence of the radiative decay rate enhancement. Furthermore, for some cases, we argue that the drastic alteration of the fluorescence spectrum could arise from the violation of Kasha's rule.

Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.

Fabrication of 3D Oriented MOF Micropatterns with Anisotropic Fluorescent Properties.

Micropatterning crystalline materials with oriented pores is necessary for the fabrication of devices with anisotropic properties. Crystalline and porous metal-organic frameworks (MOFs) are ideal materials as their chemical and structural mutability enables precise tuning of functional properties for applications ranging from microelectronics to photonics. Herein, a patternable oriented MOF film is designed: by using a photomask under X-ray exposure, the MOF film decomposes in the irradiated areas, remaining intact in the unexposed regions. The MOF film acts simultaneously as a resist and as functional porous material. While the heteroepitaxial growth from aligned Cu(OH)2 nanobelts is used to deposit oriented MOF films, the sensitivity to radiation is achieved by integrating a brominated dicarboxylate ligand (Br2 BDC) into a copper-based MOF Cu2 L2 DABCO (DABCO = 1,4-diazabicyclo[2.2.2]octane; L = BDC/Br2 BDC). The lithographed samples act as diffraction gratings upon irradiation with a laser, thus confirming the quality of the extended MOF micropattern. Furthermore, the oriented MOF patterns are functionalized with fluorescent dyes. As a result, by rotating the polarization angle of the laser excitation, the alignment of the dye in the MOF is demonstrated. By controlling the functional response to light, this MOF patterning protocol can be used for the microfabrication of optical components for photonic devices.

Gold Nanorod DNA Origami Antennas for 3 Orders of Magnitude Fluorescence Enhancement in NIR.

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.

Ultrasensitive and multiplexed miRNA detection system with DNA-PAINT.

MiRNAs hold great potential as biomarkers for the early detection and monitoring of diseases based on their differential expression profiles. Therefore, the sensitive, specific and accurate detection of miRNAs represents an emerging new tool to improve diagnosis and treatment of several diseases, cancer in particular. DNA origami-based miRNA detection is particularly advantageous as it allows to incorporate multiple attachment sites to capture different target miRNAs at the nanoscale. In this work, we present a DNA origami nanoarray system providing distance-dependent recognition of miRNAs by applying super-resolution microscopy technique; DNA-PAINT (point accumulation for imaging in nanoscale topography). The sensor can detect up to 4 miRNAs either separately or in combination based on the relative distance to the boundary markers on the structure using a single imager strand. The detection is highly sensitive, with a limit of detection down to the low femtomolar range (11 fM - 388 fM) and has a large dynamic range up to 10 nM without need for amplification. Moreover, our detection system can discriminate single base mismatches with low false positive rates. Using our strategy, we demonstrate the detection of endogenous miRNAs from cell extracts of cancer cell lines and plasma from breast cancer patients. Overall, we developed an ultrasensitive and amplification-free, DNA-PAINT imaging-based miRNA detection method using DNA origami nanoarray system for the detection of breast-cancer associated miRNAs which potentially provides a sensitive and accurate alternative to the current multiplexed diagnostic technologies.

Reconfigurable chirality with achiral excitonic materials in the strong-coupling regime.

We introduce and theoretically analyze the concept of manipulating optical chirality via strong coupling of the optical modes of chiral nanostructures with excitonic transitions in molecular layers or semiconductors. With chirality being omnipresent in chemistry and biomedicine, and highly desirable for technological applications related to efficient light manipulation, the design of nanophotonic architectures that sense the handedness of molecules or generate the desired light polarization in an externally controllable manner is of major interdisciplinary importance. Here we propose that such capabilities can be provided by the mode splitting resulting from polaritonic hybridization. Starting with an object with well-known chiroptical response-here, for a proof of concept, a chiral sphere-we show that strong coupling with a nearby excitonic material generates two spectral branches that retain the object's high chirality density, which manifest most clearly through anticrossings in circular-dichroism or differential-scattering dispersion diagrams. These windows can be controlled by the intrinsic properties of the excitonic layer and the strength of the interaction, enabling thus the post-fabrication manipulation of optical chirality. Our findings are further verified via simulations of circular dichroism of a realistic chiral architecture, namely a helical assembly of plasmonic nanospheres embedded in a resonant matrix.

DNA origami book biosensor for multiplex detection of cancer-associated nucleic acids.

DNA nanotechnology provides a promising approach for the development of biomedical point-of-care diagnostic nanoscale devices that are easy to use and cost-effective, highly sensitive and thus constitute an alternative to expensive, complex diagnostic devices. Moreover, DNA nanotechnology-based devices are particularly advantageous for applications in oncology, owing to being ideally suited for the detection of cancer-associated nucleic acids, including circulating tumor-derived DNA fragments (ctDNAs), circulating microRNAs (miRNAs) and other RNA species. Here, we present a dynamic DNA origami book biosensor that is precisely decorated with arrays of fluorophores acting as donors and acceptors and also fluorescence quenchers that produce a strong optical readout upon exposure to external stimuli for the single or dual detection of target oligonucleotides and miRNAs. This biosensor allowed the detection of target molecules either through the decrease of Förster resonance energy transfer (FRET) or an increase in the fluorescence intensity profile owing to a rotation of the constituent top layer of the structure. Single-DNA origami experiments showed that detection of two targets can be achieved simultaneously within 10 min with a limit of detection in the range of 1-10 pM. Overall, our DNA origami book biosensor design showed sensitive and specific detection of synthetic target oligonucleotides and natural miRNAs extracted from cancer cells. Based on these results, we foresee that our DNA origami biosensor may be developed into a cost-effective point-of-care diagnostic strategy for the specific and sensitive detection of a variety of DNAs and RNAs, such as ctDNAs, miRNAs, mRNAs, and viral DNA/RNAs in human samples.

DNA Self-Assembly of Single Molecules with Deterministic Position and Orientation.

An ideal nanofabrication method should allow the organization of nanoparticles and molecules with nanometric positional precision, stoichiometric control, and well-defined orientation. The DNA origami technique has evolved into a highly versatile bottom-up nanofabrication methodology that fulfils almost all of these features. It enables the nanometric positioning of molecules and nanoparticles with stoichiometric control, and even the orientation of asymmetrical nanoparticles along predefined directions. However, orienting individual molecules has been a standing challenge. Here, we show how single molecules, namely, Cy5 and Cy3 fluorophores, can be incorporated in a DNA origami with controlled orientation by doubly linking them to oligonucleotide strands that are hybridized while leaving unpaired bases in the scaffold. Increasing the number of bases unpaired induces a stretching of the fluorophore linkers, reducing its mobility freedom, and leaves more space for the fluorophore to accommodate and find different sites for interaction with the DNA. Particularly, we explore the effects of leaving 0, 2, 4, 6, and 8 bases unpaired and find extreme orientations for 0 and 8 unpaired bases, corresponding to the molecules being perpendicular and parallel to the DNA double-helix, respectively. We foresee that these results will expand the application field of DNA origami toward the fabrication of nanodevices involving a wide range of orientation-dependent molecular interactions, such as energy transfer, intermolecular electron transport, catalysis, exciton delocalization, or the electromagnetic coupling of a molecule to specific resonant nanoantenna modes.

Optical Ultracompact Directional Antennas Based on a Dimer Nanorod Structure.

Controlling directionality of optical emitters is of utmost importance for their application in communication and biosensing devices. Metallic nanoantennas have been proven to affect both excitation and emission properties of nearby emitters, including the directionality of their emission. In this regard, optical directional nanoantennas based on a Yagi-Uda design have been demonstrated in the visible range. Despite this impressive proof of concept, their overall size (~λ2/4) and considerable number of elements represent obstacles for the exploitation of these antennas in nanophotonic applications and for their incorporation onto photonic chips. In order to address these challenges, we investigate an alternative design. In particular, we numerically study the performance of a recently demonstrated "ultracompact" optical antenna based on two parallel gold nanorods arranged as a side-to-side dimer. Our results confirm that the excitation of the antiphase mode of the antenna by a nanoemitter placed in its near-field can lead to directional emission. Furthermore, in order to verify the feasibility of this design and maximize the functionality, we study the effect on the directionality of several parameters, such as the shape of the nanorods, possible defects in the dimer assembly, and different positions and orientations of the nanoemitter. We conclude that this design is robust to structural variations, making it suitable for experimental upscaling.

DNA-Templated Ultracompact Optical Antennas for Unidirectional Single-Molecule Emission.

Optical antennas are nanostructures designed to manipulate light-matter interactions by interfacing propagating light with localized optical fields. In recent years, numerous devices have been realized to efficiently tailor the absorption and/or emission rates of fluorophores. By contrast, modifying the spatial characteristics of their radiation fields remains challenging. Successful phased array nanoantenna designs have required the organization of several elements over a footprint comparable to the operating wavelength. Here, we report unidirectional emission of a single fluorophore using an ultracompact optical antenna. The design consists of two side-by-side gold nanorods self-assembled via DNA origami, which also controls the positioning of the single-fluorophore. Our results show that when a single fluorescent molecule is positioned at the tip of one nanorod and emits at a frequency capable of driving the antenna in the antiphase mode, unidirectional emission with a forward to backward ratio of up to 9.9 dB can be achieved.

An alternative to MINFLUX that enables nanometer resolution in a confocal microscope.

Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity. RASTMIN delivers ~1-2 nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures.

Determining the In-Plane Orientation and Binding Mode of Single Fluorescent Dyes in DNA Origami Structures.

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.

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.

Recent advances in plasmonic nanocavities for single-molecule spectroscopy.

Plasmonic nanocavities are able to engineer and confine electromagnetic fields to subwavelength volumes. In the past decade, they have enabled a large set of applications, in particular for sensing, optical trapping, and the investigation of physical and chemical phenomena at a few or single-molecule levels. This extreme sensitivity is possible thanks to the highly confined local field intensity enhancement, which depends on the geometry of plasmonic nanocavities. Indeed, suitably designed structures providing engineered local optical fields lead to enhanced optical sensing based on different phenomena such as surface enhanced Raman scattering, fluorescence, and Förster resonance energy transfer. In this mini-review, we illustrate the most recent results on plasmonic nanocavities, with specific emphasis on the detection of single molecules.

In Situ Photothermal Response of Single Gold Nanoparticles through Hyperspectral Imaging Anti-Stokes Thermometry.

Several fields of applications require a reliable characterization of the photothermal response and heat dissipation of nanoscopic systems, which remains a challenging task for both modeling and experimental measurements. Here, we present an implementation of anti-Stokes thermometry that enables the in situ photothermal characterization of individual nanoparticles (NPs) from a single hyperspectral photoluminescence confocal image. The method is label-free, potentially applicable to any NP with detectable anti-Stokes emission, and does not require any prior information about the NP itself or the surrounding media. With it, we first studied the photothermal response of spherical gold NPs of different sizes on glass substrates, immersed in water, and found that heat dissipation is mainly dominated by the water for NPs larger than 50 nm. Then, the role of the substrate was studied by comparing the photothermal response of 80 nm gold NPs on glass with sapphire and graphene, two materials with high thermal conductivity. For a given irradiance level, the NPs reach temperatures 18% lower on sapphire and 24% higher on graphene than on bare glass. The fact that the presence of a highly conductive material such as graphene leads to a poorer thermal dissipation demonstrates that interfacial thermal resistances play a very significant role in nanoscopic systems and emphasize the need for in situ experimental thermometry techniques. The developed method will allow addressing several open questions about the role of temperature in plasmon-assisted applications, especially ones where NPs of arbitrary shapes are present in complex matrixes and environments.

DNA Origami as Emerging Technology for the Engineering of Fluorescent and Plasmonic-Based Biosensors.

DNA nanotechnology is a powerful and promising tool for the development of nanoscale devices for numerous and diverse applications. One of the greatest potential fields of application for DNA nanotechnology is in biomedicine, in particular biosensing. Thanks to the control over their size, shape, and fabrication, DNA origami represents a unique opportunity to assemble dynamic and complex devices with precise and predictable structural characteristics. Combined with the addressability and flexibility of the chemistry for DNA functionalization, DNA origami allows the precise design of sensors capable of detecting a large range of different targets, encompassing RNA, DNA, proteins, small molecules, or changes in physico-chemical parameters, that could serve as diagnostic tools. Here, we review some recent, salient developments in DNA origami-based sensors centered on optical detection methods (readout) with a special emphasis on the sensitivity, the selectivity, and response time. We also discuss challenges that still need to be addressed before this approach can be translated into robust diagnostic devices for bio-medical applications.

Directing Single-Molecule Emission with DNA Origami-Assembled Optical Antennas.

We demonstrate the capability of DNA self-assembled optical antennas to direct the emission of an individual fluorophore, which is free to rotate. DNA origami is used to fabricate optical antennas composed of two colloidal gold nanoparticles separated by a predefined gap and to place a single Cy5 fluorophore near the gap center. Although the fluorophore is able to rotate, its excitation and far-field emission is mediated by the antenna, with the emission directionality following a dipolar pattern according to the antenna main resonant mode. This work is intended to set out the basis for manipulating the emission pattern of single molecules with self-assembled optical antennas based on colloidal nanoparticles.