Cooperative dynamics of DNA-grafted magnetic nanoparticles optimize magnetic biosensing and coupling to DNA origami.

Magnetic nanoparticles (MNPs) provide new opportunities for enzyme-free biosensing of nucleic acid biomarkers and magnetic actuation by patterning on DNA origami, yet how the DNA grafting density affects their dynamics and accessibility remains poorly understood. Here, we performed surface functionalization of MNPs with single-stranded DNA (ssDNA) via click chemistry with a tunable grafting density, which enables the encapsulation of single MNPs inside a functional polymeric layer. We used several complementary methods to show that particle translational and rotational dynamics exhibit a sigmoidal dependence on the ssDNA grafting density. At low densities, ssDNA strands adopt a coiled conformation that results in minor alterations to particle dynamics, while at high densities, they organize into polymer brushes that collectively influence particle dynamics. Intermediate ssDNA densities, where the dynamics are most sensitive to changes, show the highest magnetic biosensing sensitivity for the detection of target nucleic acids. Finally, we demonstrate that MNPs with high ssDNA grafting densities are required to efficiently couple to DNA origami. Our results establish ssDNA grafting density as a critical parameter for the functionalization of MNPs for magnetic biosensing and functionalization of DNA nanostructures.

Graphene-on-Glass Preparation and Cleaning Methods Characterized by Single-Molecule DNA Origami Fluorescent Probes and Raman Spectroscopy.

Graphene exhibits outstanding fluorescence quenching properties that can become useful for biophysics and biosensing applications, but it remains challenging to harness these advantages due to the complex transfer procedure of chemical vapor deposition-grown graphene to glass coverslips and the low yield of usable samples. Here, we screen 10 graphene-on-glass preparation methods and present an optimized protocol. To obtain the required quality for single-molecule and super-resolution imaging on graphene, we introduce a graphene screening method that avoids consuming the investigated sample. We apply DNA origami nanostructures to place fluorescent probes at a defined distance on top of graphene-on-glass coverslips. Subsequent fluorescence lifetime imaging directly reports on the graphene quality, as deviations from the expected fluorescence lifetime indicate imperfections. We compare the DNA origami probes with conventional techniques for graphene characterization, including light microscopy, atomic force microscopy, and Raman spectroscopy. For the latter, we observe a discrepancy between the graphene quality implied by Raman spectra in comparison to the quality probed by fluorescence lifetime quenching measured at the same position. We attribute this discrepancy to the difference in the effective area that is probed by Raman spectroscopy and fluorescence quenching. Moreover, we demonstrate the applicability of already screened and positively evaluated graphene for studying single-molecule conformational dynamics on a second DNA origami structure. Our results constitute the basis for graphene-based biophysics and super-resolution microscopy.

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.

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.

DNA-Mediated Self-Assembly of Plasmonic Antennas with a Single Quantum Dot in the Hot Spot.

DNA self-assembly is a powerful tool to arrange optically active components with high accuracy in a large parallel manner. A facile approach to assemble plasmonic antennas consisting of two metallic nanoparticles (40 nm) with a single colloidal quantum dot positioned at the hot spot is presented here. The design approach is based on DNA complementarity, stoichiometry, and steric hindrance principles. Since no intermediate molecules other than short DNA strands are required, the structures possess a very small gap (≈ 5 nm) which is desired to achieve high Purcell factors and plasmonic enhancement. As a proof-of-concept, the fluorescence emission from antennas assembled with both conventional and ultrasmooth spherical gold particles is measured. An increase in fluorescence is obtained, up to ≈30-fold, compared to quantum dots without antenna.

Ideal Dimers of Gold Nanospheres for Precision Plasmonics: Synthesis and Characterization at the Single-Particle Level for Identification of Higher Order Modes.

Ideal dimers comprising gold nanoparticles with a smooth surface and high sphericity are synthesized by a substrate-based assembly strategy with efficient cetyltrimethylammonium bromide removal. An unprecedented structural and plasmonic uniformity at the single-particle level is observed since inhomogeneities resulting from variations in gap morphology are eliminated. Single ideal dimers are analyzed by polarization-resolved dark-field scattering spectroscopy. Contributions from transverse as well as quadrupolar and octupolar longitudinal plasmon coupling modes can be discriminated because of their orthogonal polarization behavior. The assignment of these higher order coupling modes is supported by computer simulations.

Polyalkylcyanoacrylate nanocapsules: variation of membrane permeability by chemical cross-linking.

The permeability of the polymer walls of polyalkylcyanoacrylate nanocapsules varies by different degrees of chemical cross-linking. For this reason, different amounts of bivalent alkylcyanoacrylates are added to the monovalent alkylcyanoacrylate prior to an interfacial polymerization step in order to generate capsules with various cross-linking densities. The obtained nanocapsules are characterized by observing the water molecules via pulsed field-gradient nuclear magnetic resonance using a stimulated echo sequence. The resulting echo decay plots reveal the exchange rate of the water molecules between the free and encapsulated states. The observed dwell times of water molecules in the encapsulated state are characteristic parameters for the permeability of the given capsule membranes. They show a clear dependence on the degree of cross-linking, proving the potential of this approach for a controlled variation of the capsule permeability. Also, the cross-linked nanocapsules exhibit a significantly decreased solubility in tetrahydrofuran which may lead to new applications for polyalkylcyanoacrylate nanocapsules in organic solvents.