From Bulk to Single Molecules: Surface-Enhanced Raman Scattering of Cytochrome C Using Plasmonic DNA Origami Nanoantennas.

Cytochrome C, an evolutionarily conserved protein, plays pivotal roles in cellular respiration and apoptosis. Understanding its molecular intricacies is essential for both academic inquiry and potential biomedical applications. This study introduces an advanced single-molecule surface-enhanced Raman scattering (SM-SERS) system based on DNA origami nanoantennas (DONAs), optimized to provide unparalleled insights into protein structure and interactions. Our system effectively detects shifts in the Amide III band, thereby elucidating protein dynamics and conformational changes. Additionally, the system permits concurrent observations of oxidation processes and Amide bands, offering an integrated view of protein structural and chemical modifications. Notably, our approach diverges from traditional SM-SERS techniques by de-emphasizing resonance conditions for SERS excitation, aiming to mitigate challenges like peak oversaturation. Our findings underscore the capability of our DONAs to illuminate single-molecule behaviors, even within aggregate systems, providing clarity on molecular interactions and behaviors.

Antiviral action of a functionalized plastic surface against human coronaviruses.

Viruses may persist on solid surfaces for long periods, which may contribute to indirect transmission. Thus, it is imperative to develop functionalized surfaces that will lower the infectious viral load in everyday life. Here, we have tested a plastic surface functionalized with tall oil rosin against the seasonal human coronavirus OC43 as well as severe acute respiratory syndrome coronavirus 2. All tested non-functionalized plastic surfaces showed virus persistence up to 48 h. In contrast, the functionalized plastic showed good antiviral action already within 15 min of contact and excellent efficacy after 30 min over 90% humidity. Excellent antiviral effects were also observed at lower humidities of 20% and 40%. Despite the hydrophilic nature of the functionalized plastic, viruses did not adhere strongly to it. According to helium ion microscopy, viruses appeared flatter on the rosin-functionalized surface, but after flushing away from the rosin-functionalized surface, they showed no apparent structural changes when imaged by transmission electron microscopy of cryogenic or negatively stained specimens or by atomic force microscopy. Flushed viruses were able to bind to their host cell surface and enter endosomes, suggesting that the fusion with the endosomal membrane was halted. The eluted rosin from the functionalized surface demonstrated its ability to inactivate viruses, indicating that the antiviral efficacy relied on the active leaching of the antiviral substances, which acted on the viruses coming into contact. The rosin-functionalized plastic thus serves as a promising candidate as an antiviral surface for enveloped viruses.IMPORTANCEDuring seasonal and viral outbreaks, the implementation of antiviral plastics can serve as a proactive strategy to limit the spread of viruses from contaminated surfaces, complementing existing hygiene practices. In this study, we show the efficacy of a rosin-functionalized plastic surface that kills the viral infectivity of human coronaviruses within 15 min of contact time, irrespective of the humidity levels. In contrast, non-functionalized plastic surfaces retain viral infectivity for an extended period of up to 48 h. The transient attachment on the surface or the leached active components do not cause major structural changes in the virus or prevent receptor binding; instead, they effectively block viral infection at the endosomal stage.

Nanoscale Hotspot-Induced Emitters in DNA Origami-Assisted Nanoantennas.

We report the observation of hotspot-induced emitters and photoluminescence enhancement of up to 42-fold from DNA origami-assisted plasmonic dimer nanoantennas upon excess polarized laser illumination. The presence of DNA and laser polarization alignment along the dimer axis are critical for the generation of bright emitters responsible for the observed PL increase. The emission spectrum reveals characteristic Raman peaks of amorphous carbon, suggesting the formation of carbon-based emitters in the nanoantenna due to the plasmonic hotspots at the longitudinal antenna resonance.

The Effect of Nanoparticle Composition on the Surface-Enhanced Raman Scattering Performance of Plasmonic DNA Origami Nanoantennas.

A versatile generation of plasmonic nanoparticle dimers for surface-enhanced Raman scattering (SERS) is presented by combining a DNA origami nanofork and spherical and nonspherical Au or Ag nanoparticles. Combining different nanoparticle species with a DNA origami nanofork to form DNA origami nanoantennas (DONAs), the plasmonic nanoparticle dimers can be optimized for a specific excitation wavelength in SERS. The preparation of such nanoparticle dimers is robust enough to enable the characterization of SERS intensities and SERS enhancement factors of dye-modified DONAs on a single dimer level by measuring in total several thousands of dimers from five different dimer designs, each functionalized with three different Raman reporter molecules and measured at four different excitation wavelengths. Based on these data, SERS enhancement factor (EF) distributions have been determined for each dimer design and excitation wavelengths. The structures and measurement conditions with the highest EFs are suitable for single-molecule SERS (SM-SERS), which is realized by placing single dye molecules into hot spots. We demonstrate that the probability of placing single molecules in a strongly enhancing hot spot for SM-SERS can be increased by using anisotropic nanoparticles with several sharp edges, such as nanoflowers. Combining a Ag nanoparticle with a Au particle in one dimer structure allows for a broadband excitation covering almost the whole visible range. The most versatile plasmonic dimer structure for SERS combines a spherical Ag nanoparticle with a Au nanoflower. Employing the discontinuous Galerkin time domain method, we numerically investigate the bare, symmetric dimers with respect to spectral and near-field properties, showing that, indeed, the nanoflowers induce multiple hot spots located at the edges which surpass the intensity of the spherical dimers, indicating the possibility for SM-SERS. The presented DONA structures and SERS data provide a robust basis for applying such designs as versatile SERS tags and as substrates for SM-SERS measurements.

Creation of ordered 3D tubes out of DNA origami lattices.

Hierarchical self-assembly of nanostructures with addressable complexity has been a promising route for realizing novel functional materials. Traditionally, the fabrication of such structures on a large scale has been achievable using top-down methods but with the cost of complexity of the fabrication equipment versus resolution and limitation mainly to 2D structures. More recently bottom-up methods using molecules like DNA have gained attention due to the advantages of low fabrication costs, high resolution and simplicity in an extension of the methods to the third dimension. One of the more promising bottom-up techniques is DNA origami due to the robust self-assembly of arbitrarily shaped nanostructures with feature sizes down to a few nanometers. Here, we show that under specific ionic conditions of the buffer, the employed plus-shaped, blunt-ended Seeman tile (ST) origami forms elongated, ordered 2D lattices, which are further rolled into 3D tubes in solution. Imaging structures on a surface by atomic force microscopy reveals ribbon-like structures, with single or double layers of the origami lattice. Further studies of the double-layered structures in a liquid state by confocal microscopy and cryo-TEM revealed elongated tube structures with a relatively uniform width but with a varying length. Through meticulous study, we concluded that the assembly process of these 3D DNA origami tubes is heavily dependent on the concentration of both mono- and divalent cations. In particular, nickel seems to act as a trigger for the formation of the tubular assemblies in liquid.

Elongation and plasmonic activity of embedded metal nanoparticles following heavy ion irradiation.

Shape modification of embedded nanoparticles by swift heavy ion (SHI) irradiation is an effective way to produce nanostructures with controlled size, shape, and orientation. In this study, randomly oriented gold nanorods embedded in SiO2 are shown to re-orient along the ion beam direction. The degree of orientation depends on the irradiation conditions and the nanorod's initial size. SHI irradiation was also applied to modify spherical metallic nanoparticles embedded in Al2O3. The results showed that they elongate due to the irradiation comparably to those embedded in SiO2. Metallic nanostructures embedded in dielectric matrices can exhibit localized surface plasmon (LSP) modes. The elongated nanoparticles investigated by means of dark-field spectroscopy showed two discrete peaks which correspond to longitudinal and transverse modes.

Molecular states and spin crossover of hemin studied by DNA origami enabled single-molecule surface-enhanced Raman scattering.

The study of biologically relevant molecules and their interaction with external stimuli on a single molecular scale is of high importance due to the availability of distributed rather than averaged information. Surface enhanced Raman scattering (SERS) provides direct chemical information, but is rather challenging on the single molecule (SM) level, where it is often assumed to require a direct contact of analyte molecules with the metal surface. Here, we detect and investigate the molecular states of single hemin by SM-SERS. A DNA aptamer based G-quadruplex mediated recognition of hemin directs its placement in the SERS hot-spot of a DNA Origami Nanofork Antenna (DONA). The configuration of the DONA structure allows the molecule to be trapped at the plasmonic hot-spot preferentially in no-contact configuration with the metal surface. Owing to high field enhancement at the plasmonic hot spot, the detection of a single folded G-quadruplex becomes possible. For the first time, we present a systematic study by SM-SERS where most hemin molecule adopt a high spin and oxidation state (III) that showed state crossover to low spin upon strong-field-ligand binding. The present study therefore, provides a platform for studying biologically relevant molecules and their properties at SM sensitivity along with demonstrating a conceptual advancement towards successful monitoring of single molecular chemical interaction using DNA aptamers.

Plasmonic reactivity of halogen thiophenols on gold nanoparticles studied by SERS and XPS.

Localized surface plasmon resonances on noble metal nanoparticles (NPs) can efficiently drive reactions of adsorbed ligand molecules and provide versatile opportunities in chemical synthesis. The driving forces of these reactions are typically elevated temperatures, hot charge carriers, or enhanced electric fields. In the present work, dehalogenation of halogenated thiophenols on the surface of AuNPs has been studied by surface enhanced Raman scattering (SERS) as a function of the photon energy to track the kinetics and identify reaction products. Reaction rates are found to be surprisingly similar for different halothiophenols studied here, although the bond dissociation energies of the C-X bonds differ significantly. Complementary information about the electronic properties at the AuNP surface, namely, work-function and valence band states, has been determined by x-ray photoelectron spectroscopy of isolated AuNPs in the gas-phase. In this way, it is revealed how the electronic properties are altered by the adsorption of the ligand molecules, and we conclude that the reaction rates are mainly determined by the plasmonic properties of the AuNPs. SERS spectra reveal differences in the reaction product formation for different halogen species, and, on this basis, the possible reaction mechanisms are discussed to approach an understanding of opportunities and limitations in the design of catalytical systems with plasmonic NPs.

Synthesis of nanostructured protein-mineral-microcapsules by sonication.

We propose a simple and eco-friendly method for the formation of composite protein-mineral-microcapsules induced by ultrasound treatment. Protein- and nanoparticle-stabilized oil-in-water (O/W) emulsions loaded with different oils are prepared using high-intensity ultrasound. The formation of thin composite mineral proteinaceous shells is realized with various types of nanoparticles, which are pre-modified with Bovine Serum Albumin (BSA) and subsequently characterized by EDX, TGA, zeta potential measurements and Raman spectroscopy. Cryo-SEM and EDX mapping visualizations show the homogeneous distribution of the densely packed nanoparticles in the capsule shell. In contrast to the results reported in our previous paper,1 the shell of those nanostructured composite microcapsules is not cross-linked by the intermolecular disulfide bonds between BSA molecules. Instead, a Pickering-Emulsion formation takes place because of the amphiphilicity-driven spontaneous attachment of the BSA-modified nanoparticles at the oil/water interface. Using colloidal particles for the formation of the shell of the microcapsules, in our case silica, hydroxyapatite and calcium carbonate nanoparticles, is promising for the creation of new functional materials. The nanoparticulate building blocks of the composite shell with different chemical, physical or morphological properties can contribute to additional, sometimes even multiple, features of the resulting capsules. Microcapsules with shells of densely packed nanoparticles could find interesting applications in pharmaceutical science, cosmetics or in food technology.

Raman Enhancement of Nanoparticle Dimers Self-Assembled Using DNA Origami Nanotriangles.

Surface-enhanced Raman scattering is a powerful approach to detect molecules at very low concentrations, even up to the single-molecule level. One important aspect of the materials used in such a technique is how much the signal is intensified, quantified by the enhancement factor (EF). Herein we obtained the EFs for gold nanoparticle dimers of 60 and 80 nm diameter, respectively, self-assembled using DNA origami nanotriangles. Cy5 and TAMRA were used as surface-enhanced Raman scattering (SERS) probes, which enable the observation of individual nanoparticles and dimers. EF distributions are determined at four distinct wavelengths based on the measurements of around 1000 individual dimer structures. The obtained results show that the EFs for the dimeric assemblies follow a log-normal distribution and are in the range of 106 at 633 nm and that the contribution of the molecular resonance effect to the EF is around 2, also showing that the plasmonic resonance is the main source of the observed signal. To support our studies, FDTD simulations of the nanoparticle's electromagnetic field enhancement has been carried out, as well as calculations of the resonance Raman spectra of the dyes using DFT. We observe a very close agreement between the experimental EF distribution and the simulated values.

A Versatile DNA Origami-Based Plasmonic Nanoantenna for Label-Free Single-Molecule Surface-Enhanced Raman Spectroscopy.

DNA origami technology allows for the precise nanoscale assembly of chemical entities that give rise to sophisticated functional materials. We have created a versatile DNA origami nanofork antenna (DONA) by assembling Au or Ag nanoparticle dimers with different gap sizes down to 1.17 nm, enabling signal enhancements in surface-enhanced Raman scattering (SERS) of up to 1011. This allows for single-molecule SERS measurements, which can even be performed with larger gap sizes to accommodate differently sized molecules, at various excitation wavelengths. A general scheme is presented to place single analyte molecules into the SERS hot spots using the DNA origami structure exploiting covalent and noncovalent coupling schemes. By using Au and Ag dimers, single-molecule SERS measurements of three dyes and cytochrome c and horseradish peroxidase proteins are demonstrated even under nonresonant excitation conditions, thus providing long photostability during time-series measurement and enabling optical monitoring of single molecules.

About the mechanism of ultrasonically induced protein capsule formation.

In this paper, we propose a consistent mechanism of protein microcapsule formation upon ultrasound treatment. Aqueous suspensions of bovine serum albumin (BSA) microcapsules filled with toluene are prepared by use of high-intensity ultrasound following a reported method. Stabilization of the oil-in-water emulsion by the adsorption of the protein molecules at the interface of the emulsion droplets is accompanied by the creation of the cross-linked capsule shell due to formation of intermolecular disulfide bonds caused by highly reactive species like superoxide radicals generated sonochemically. The evidence for this mechanism, which until now remained elusive and was not proven properly, is presented based on experimental data from SDS-PAGE, Raman spectroscopy and dynamic light scattering.

Surface Characteristics Control the Attachment and Functionality of (Chimeric) Avidin.

The physical adsorption (physisorption) of proteins to surfaces is an important but incompletely understood factor in many biological processes and is of increasing significance in bionanotechnology as well. Avidin is an important protein because of strong avidin-biotin binding, which has been exploited in numerous applications. We have undertaken thorough experimentation on the physisorption of avidin, to chemically different flat surfaces of Si and graphite and also to the curved version of the latter, on multiwalled carbon nanotubes (MWNTs) of different diameters. The difference in the behavior of avidin on Si versus graphite is drastic; on Si, avidin deposits as single globular tetrameric units and maintains functionality, whereas on graphite, it forms irregular networks of two-layer thick filaments, where the first layer has lost its biological activity. On MWNTs, avidin also deposits as one-dimensional formations, or stripes, but these appear to order in a perpendicular arrangement to the MWNT axis. A better understanding of protein-surface interactions is essential for the development of robust and reliable methods for biofunctionalization of materials. This work also provides insights into the importance of the nanoscale surface architecture.

A DNA-nanoparticle actuator enabling optical monitoring of nanoscale movements induced by an electric field.

Merging biological and non-biological matter to fabricate nanoscale assemblies with controllable motion and function is of great interest due to its potential application, for example, in diagnostics and biosensing. Here, we have constructed a DNA-based bionanoactuator that interfaces with biological and non-biological matter via an electric field in a reversibly controllable fashion. The read-out of the actuator is based on motion-induced changes in the plasmon resonance of a gold nanoparticle immobilized to a gold surface by single stranded DNA. The motion of the gold nanoparticle and thus the conformational changes of the DNA under varying electric field were analyzed by dark field spectroscopy. After this basic characterization, another actuator was built utilizing hairpin-DNA coated gold nanoparticles, where the hairpin-DNA induced discrete transitions between two specific open-loop and folded-loop states. These two states and the transition dynamics between them were clearly visible in the actuator behavior. The demonstrated nanoactuator concept could be readily extended to inspection of conformational changes of other biomolecules as well. Besides, this concept enables other possibilities in applications like surface-enhanced Raman spectroscopy and fluorescence enhancement, since the specific wavelength of the plasmon resonance of the actuator can be tuned by the external voltage.

Plasmonic nanostructures through DNA-assisted lithography.

Programmable self-assembly of nucleic acids enables the fabrication of custom, precise objects with nanoscale dimensions. These structures can be further harnessed as templates to build novel materials such as metallic nanostructures, which are widely used and explored because of their unique optical properties and their potency to serve as components of novel metamaterials. However, approaches to transfer the spatial information of DNA constructions to metal nanostructures remain a challenge. We report a DNA-assisted lithography (DALI) method that combines the structural versatility of DNA origami with conventional lithography techniques to create discrete, well-defined, and entirely metallic nanostructures with designed plasmonic properties. DALI is a parallel, high-throughput fabrication method compatible with transparent substrates, thus providing an additional advantage for optical measurements, and yields structures with a feature size of ~10 nm. We demonstrate its feasibility by producing metal nanostructures with a chiral plasmonic response and bowtie-shaped nanoantennas for surface-enhanced Raman spectroscopy. We envisage that DALI can be generalized to large substrates, which would subsequently enable scale-up production of diverse metallic nanostructures with tailored plasmonic features.

Toward Single Electron Nanoelectronics Using Self-Assembled DNA Structure.

DNA based structures offer an adaptable and robust way to develop customized nanostructures for various purposes in bionanotechnology. One main aim in this field is to develop a DNA nanobreadboard for a controllable attachment of nanoparticles or biomolecules to form specific nanoelectronic devices. Here we conjugate three gold nanoparticles on a defined size TX-tile assembly into a linear pattern to form nanometer scale isolated islands that could be utilized in a room temperature single electron transistor. To demonstrate this, conjugated structures were trapped using dielectrophoresis for current-voltage characterization. After trapping only high resistance behavior was observed. However, after extending the islands by chemical growth of gold, several structures exhibited Coulomb blockade behavior from 4.2 K up to room temperature, which gives a good indication that self-assembled DNA structures could be used for nanoelectronic patterning and single electron devices.

Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology.

A straightforward solution-based method to modify the biofunctionality of stainless steel (SS) using heterobifunctional silane-polyethylene glycol (silane-PEG) overlayers is reported. Reduced nonspecific biofouling of both proteins and bacteria onto SS and further selective biofunctionalization of the modified surface were achieved. According to photoelectron spectroscopy analyses, the silane-PEGs formed less than 10 Å thick overlayers with close to 90% surface coverage and reproducible chemical compositions. Consequently, the surfaces also became more hydrophilic, and the observed non-specific biofouling of proteins was reduced by approximately 70%. In addition, the attachment of E. coli was reduced by more than 65%. Moreover, the potential of the overlayer to be further modified was demonstrated by successfully coupling biotinylated alkaline phosphatase (bAP) to a silane-PEG-biotin overlayer via avidin-biotin bridges. The activity of the immobilized enzyme was shown to be well preserved without compromising the achieved antifouling properties. Overall, the simple solution-based approach enables the tailoring of SS to enhance its activity for biomedical and biotechnological applications.

DNA-Based Enzyme Reactors and Systems.

During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme) cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

Metallic Nanostructures Based on DNA Nanoshapes.

Metallic nanostructures have inspired extensive research over several decades, particularly within the field of nanoelectronics and increasingly in plasmonics. Due to the limitations of conventional lithography methods, the development of bottom-up fabricated metallic nanostructures has become more and more in demand. The remarkable development of DNA-based nanostructures has provided many successful methods and realizations for these needs, such as chemical DNA metallization via seeding or ionization, as well as DNA-guided lithography and casting of metallic nanoparticles by DNA molds. These methods offer high resolution, versatility and throughput and could enable the fabrication of arbitrarily-shaped structures with a 10-nm feature size, thus bringing novel applications into view. In this review, we cover the evolution of DNA-based metallic nanostructures, starting from the metallized double-stranded DNA for electronics and progress to sophisticated plasmonic structures based on DNA origami objects.

Influence of Nitrogen Doping on Device Operation for TiO2-Based Solid-State Dye-Sensitized Solar Cells: Photo-Physics from Materials to Devices.

Solid-state dye-sensitized solar cells (ssDSSC) constitute a major approach to photovoltaic energy conversion with efficiencies over 8% reported thanks to the rational design of efficient porous metal oxide electrodes, organic chromophores, and hole transporters. Among the various strategies used to push the performance ahead, doping of the nanocrystalline titanium dioxide (TiO2) electrode is regularly proposed to extend the photo-activity of the materials into the visible range. However, although various beneficial effects for device performance have been observed in the literature, they remain strongly dependent on the method used for the production of the metal oxide, and the influence of nitrogen atoms on charge kinetics remains unclear. To shed light on this open question, we synthesized a set of N-doped TiO2 nanopowders with various nitrogen contents, and exploited them for the fabrication of ssDSSC. Particularly, we carefully analyzed the localization of the dopants using X-ray photo-electron spectroscopy (XPS) and monitored their influence on the photo-induced charge kinetics probed both at the material and device levels. We demonstrate a strong correlation between the kinetics of photo-induced charge carriers probed both at the level of the nanopowders and at the level of working solar cells, illustrating a direct transposition of the photo-physic properties from materials to devices.