Influence of bulky substituents on single-molecule SERS sensitivity.

The surface-enhanced Raman spectroscopy (SERS) detection limit strongly depends on the molecular structure, which we demonstrate for a family of tert-butyl-substituted porphycenes. Even though the investigated species present very similar photophysical properties, the ratio between the SERS signal and fluorescence background depends on the number of bulky tert-butyl groups. Moreover, the probability of single molecule detection systematically drops with the number of the moieties attached to the pyrrole ring. As steric hindrance is the only significantly changing feature among the studied chromophores, we attribute the observed phenomena to the spatial structure. We also show that the sensitivity of the SERS technique can be improved by lowering the temperature. We managed to observe single-molecule spectra for derivatives for which this was unattainable at room temperature.

Synthesis and characterization of Gd2O3: Er3+, Yb3+doped with Mg2+, Li+ions-effect on the photoluminescence and biological applications.

Gd2O3:1% Er3+, 18% Yb3+,x% Mg2+(x = 0; 2.5; 4; 5; 6; 8;10; 20; 25; 50) and Gd2O3:1% Er3+, 18% Yb3+, 2,5% Mg2+,y% Li+(y = 0.5-2.5) nanoparticles were synthesized by homogenous precipitation method and calcined at 900 °C for 3 h in air atmosphere. Powder x-ray diffraction, scanning electron microscopy, cathodoluminescence, transmission electron microscopy, energy dispersive x-ray spectroscopy and photoluminescence techniques were employed to characterize the obtained nanoparticles. We observed a 8-fold increase in red luminescence for samples suspended in DMSO solution for 2.5% of Mg2+doping. The x-ray analysis shows that for the concentration of 2.5% Mg, the size of the crystallites in the NPs is the largest, which is mainly responsible for the increase in the intensity of the upconversion luminescence. But the addition of Li+ions did not improve the luminescence of the upconversion due to decreasing of crystallites size of the NPs. Synthesized nanomaterials with very effective upconverting luminescence, can act as luminescent markers inin vivoimaging. The cytotoxicity of the nanoparticles was evaluated on the 4T1 cell line for the first time.

Structural, optical and magnetic properties of Y3-0.02-xEr0.02Yb x Al5O12 (0 < x < 0.20) nanocrystals: effect of Yb content.

The paramagnetic Y3-0.02-x Er0.02Yb x Al5O12 (x = 0.02, 0.06, 0.10, 0.12, 0.18, 0.20) nanocrystals (NCs) were synthesized by the microwave-induced solution combustion method. The XRD, TEM and SEM techniques were applied to determine the NCs' structures and sizes. The XRD patterns confirmed that the NCs have for the most part a regular structure of the Y3Al5O12 (YAG) phase. The changes of the distance between donor Yb3+ (sensitizer) and acceptor Er3+ (activator) were realized by changing the donor's concentration with a constant amount of acceptor. Under 980 nm excitation, at room temperature, the NCs exhibited strong red emission near 660 and 675 nm, and green upconversion emission at 550 nm, corresponding to the intra 4f transitions of Er3+ (4F9/2, 2H11/2, 4S3/2) â†’ Er3+ (4I15/2). The strongest emission was observed in a sample containing 18% Yb3+ ions. The red and green emission intensities are respectively about 5 and 12 times higher as compared to NCs doped with 2% of Yb3+. In order to prove that the main factor responsible for the increase of the upconversion luminescence efficiency is reduction of the distance between Yb3+ and Er3+, we examined, for the first time the influence of hydrostatic pressure on luminescence and luminescence decay time of the radiative transitions inside donor ion. The decrease of both luminescence intensity and luminescence decay times, with increasing hydrostatic pressure was observed. After applying hydrostatic pressure to samples with e.g. 2% and 6% Yb3+, the distance between the donor and acceptor decreases. However, for higher concentrations of the donor, this distance is smaller, and this leads to the effective energy transfer to Er3+ ions. With increasing pressure, the maximum intensity of near infrared emission is observed at 1029, 1038 and 1047 nm, what corresponds to 2F5/2 â†’ 2F7/2 transition of Yb3+.

Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

Single-step synthesis of Er3+ and Yb3+ ions doped molybdate/Gd2O3 core-shell nanoparticles for biomedical imaging.

Nanostructures as color-tunable luminescent markers have become major, promising tools for bioimaging and biosensing. In this paper separated molybdate/Gd2O3 doped rare earth ions (erbium, Er3+ and ytterbium, Yb3+) core-shell nanoparticles (NPs), were fabricated by a one-step homogeneous precipitation process. Emission properties were studied by cathodo- and photoluminescence. Scanning electron and transmission electron microscopes were used to visualize and determine the size and shape of the NPs. Spherical NPs were obtained. Their core-shell structures were confirmed by x-ray diffraction and energy-dispersive x-ray spectroscopy measurements. We postulated that the molybdate rich core is formed due to high segregation coefficient of the Mo ion during the precipitation. The calcination process resulted in crystallization of δ/ξ (core/shell) NP doped Er and Yb ions, where δ-gadolinium molybdates and ξ-molybdates or gadolinium oxide. We confirmed two different upconversion mechanisms. In the presence of molybdenum ions, in the core of the NPs, Yb3+-[Formula: see text] (∣2F7/2, 3T2〉) dimers were formed. As a result of a two 980 nm photon absorption by the dimer, we observed enhanced green luminescence in the upconversion process. However, for the shell formed by the Gd2O3:Er, Yb NPs (without the Mo ions), the typical energy transfer upconversion takes place, which results in red luminescence. We demonstrated that the NPs were transported into cytosol of the HeLa and astrocytes cells by endocytosis. The core-shell NPs are sensitive sensors for the environment prevailing inside (shorter luminescence decay) and outside (longer luminescence decay) of the tested cells. The toxicity of the NPs was examined using MTT assay.

Expanding the range of graphene energy transfer with multilayer graphene.

The interaction between single emitters and graphene in the context of energy transfer has attracted significant attention due to its potential applications in fields such as biophysics and super-resolution microscopy. In this study, we investigate the influence of the number of graphene layers on graphene energy transfer (GET) by placing single dye molecules at defined distances from monolayer, bilayer, and trilayer graphene substrates. We employ DNA origami nanostructures as chemical adapters to position the dye molecules precisely. Fluorescence lifetime measurements and analysis reveal an additive effect of graphene layers on the energy transfer rate extending the working range of GET up to distances of approximately 50-60 nm. Moreover, we show that switching a DNA pointer strand between two positions on a DNA origami nanostructure at a height of >28 nm above graphene is substantially better visualized with multilayer graphene substrates suggesting enhanced capabilities for applications such as biosensing and super-resolution microscopy for larger systems and distances. This study provides insights into the influence of graphene layers on energy transfer dynamics and offers new possibilities for exploiting graphene's unique properties in various nanotechnological applications.

Thiol-yne click reactions on alkynyl-dopamine-modified reduced graphene oxide.

The large-scale preparation of graphene is of great importance due to its potential applications in various fields. We report herein a simple method for the simultaneous exfoliation and reduction of graphene oxide (GO) to reduced GO (rGO) by using alkynyl-terminated dopamine as the reducing agent. The reaction was performed under mild conditions to yield rGO functionalized with the dopamine derivative. The chemical reactivity of the alkynyl function was demonstrated by post-functionalization with two thiolated precursors, namely 6-(ferrocenyl)hexanethiol and 1H,1H,2H,2H-perfluorodecanethiol. X-ray photoelectron spectroscopy, UV/Vis spectrophotometry, Raman spectroscopy, conductivity measurements, and cyclic voltammetry were used to characterize the resulting surfaces.

Transport of NaYF4:Er3+, Yb3+ up-converting nanoparticles into HeLa cells.

An effective, simple and practically useful method to incorporate fluorescent nanoparticles inside live biological cells was developed. The internalization time and concentration dependence of a frequently used liposomal transfection factor (Lipofectamine 2000) was studied. A user friendly, one-step technique to obtain water and organic solvent soluble Er(3+) and Yb(3+) doped NaYF4 nanoparticles coated with polyvinylpyrrolidone was obtained. Structural analysis of the nanoparticles confirmed the formation of nanocrystals of the desired sizes and spectral properties. The internalization of NaYF4 nanoparticles in HeLa cervical cancer cells was determined at different nanoparticle concentrations and for incubation periods from 3 to 24 h. The images revealed a redistribution of nanoparticles inside the cell, which increases with incubation time and concentration levels, and depends on the presence of the transfection factor. The study identifies, for the first time, factors responsible for an effective endocytosis of the up-converting nanoparticles to HeLa cells. Thus, the method could be applied to investigate a wide range of future 'smart' theranostic agents. Nanoparticles incorporated into the liposomes appear to be very promising fluorescent probes for imaging real-time cellular dynamics.

Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy.

3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques with fluorescence imaging. Here, we achieve 3D super-resolution by combining the 2D localization of pMINFLUX with the axial information of graphene energy transfer (GET) and the single-molecule switching by DNA-PAINT. We demonstrate <2 nm localization precision in all 3 dimension with axial precision reaching below 0.3 nm. In 3D DNA-PAINT measurements, structural features, i.e., individual docking strands at distances of 3 nm, are directly resolved on DNA origami structures. pMINFLUX and GET represent a particular synergetic combination for super-resolution imaging near the surface such as for cell adhesion and membrane complexes as the information of each photon is used for both 2D and axial localization information. Furthermore, we introduce local PAINT (L-PAINT), in which DNA-PAINT imager strands are equipped with an additional binding sequence for local upconcentration improving signal-to-background ratio and imaging speed of local clusters. L-PAINT is demonstrated by imaging a triangular structure with 6 nm side lengths within seconds.

Exploring the Synergies of Single-Molecule Fluorescence and 2D Materials Coupled by DNA.

The world of 2D materials is steadily growing, with numerous researchers attempting to discover, elucidate, and exploit their properties. Approaches relying on the detection of single fluorescent molecules offer a set of advantages, for instance, high sensitivity and specificity, that allow the drawing of conclusions with unprecedented precision. Herein, it is argued how the study of 2D materials benefits from fluorescence-based single-molecule modalities, and vice versa. A special focus is placed on DNA, serving as a versatile adaptor when anchoring single dye molecules to 2D materials. The existing literature on the fruitful combination of the two fields is reviewed, and an outlook on the additional synergies that can be created between them provided.

Nano-bio interactions of upconversion nanoparticles at subcellular level: biodistribution and cytotoxicity.

Background: Modern medicine requires intensive research to find new diagnostic and therapeutic solutions. Recently, upconverting nanoparticles (UCNPs) doped with lanthanide ions have attracted significant attention. Methods: The efficient internalization of UCNPs by cells was confirmed, and their precise cellular localization was determined by electron microscopy and confocal studies. Results: UCNPs colocalized only with specific organelles, such as early endosomes, late endosomes and lysosomes. Furthermore, experiments with chemical inhibitors confirmed the involvement of endocytosis in UCNPs internalization and helped select several mechanisms involved in internalization. Exposure to selected UCNPs concentrations did not show significant cytotoxicity, induction of oxidative stress or ultrastructural changes in cells. Conclusion: This study suggests that UCNPs offer new diagnostic options for biomedical infrared imaging.

Three modes of proton transfer in one chromophore: photoinduced tautomerization in 2-(1H-pyrazol-5-yl)pyridines, their dimers and alcohol complexes.

Studies of 2-(1H-pyrazol-5-yl)pyridine (PPP) and its derivatives 2-(4-methyl-1H-pyrazol-5-yl)pyridine (MPP) and 2-(3-bromo-1H-pyrazol-5-yl)pyridine (BPP) by stationary and time-resolved UV/Vis spectroscopic methods, and quantum chemical computations show that this class of compounds provides a rare example of molecules that exhibit three types of photoreactions: 1) excited-state intramolecular proton transfer (ESIPT) in the syn form of MPP, 2) excited-state intermolecular double-proton transfer (ESDPT) in the dimers of PPP in nonpolar media, as well as 3) solvent-assisted double-proton transfer in hydrogen-bonded 1:1 complexes of PPP and MPP with alcoholic partners. The excited-state processes are manifested by the appearance of a dual luminescence and a bimodal irreversible kinetic coupling of the two fluorescence bands. Ground-state syn-anti equilibria are detected and discussed. The fraction of the higher-energy anti form varies for different derivatives and is strongly dependent on the solvent polarity and hydrogen-bond donor or acceptor abilities.

Substituent screening effect on single-molecule photostability: comparison of three differently substituted porphycenes.

Photobleaching of single molecules has been studied using confocal fluorescence microscopy for porphycene, a porphyrin isomer, and its two derivatives. Fourfold substitution of porphycene with bulkytert-butyl groups leads to the enhancement of photostability, even though the spectral, photophysical, and redox parameters remain similar. We attribute this effect to the increase of the efficiency of physical quenching of the chromophore triplet state by oxygen, compared with the yield of chemical reaction that leads to photobleaching. Analysis of the observed photon fluxes from single emitters embedded in a polymer film shows that the experiment based on fluorescence is biased towards detection of molecules which have oxygen-the triplet quencher-in their vicinity. The distribution of the measured photodegradation quantum yields is very heterogeneous, suggesting that physical and chemical quenching rates exhibit different distance and orientation dependences.

Graphene Energy Transfer for Single-Molecule Biophysics, Biosensing, and Super-Resolution Microscopy.

Graphene is considered a game-changing material, especially for its mechanical and electrical properties. This work exploits that graphene is almost transparent but quenches fluorescence in a range up to ≈40 nm. Graphene as a broadband and unbleachable energy-transfer acceptor without labeling, is used to precisely determine the height of molecules with respect to graphene, to visualize the dynamics of DNA nanostructures, and to determine the orientation of Förster-type resonance energy transfer (FRET) pairs. Using DNA origami nanopositioners, biosensing, single-molecule tracking, and DNA PAINT super-resolution with <3 nm z-resolution are demonstrated. The range of examples shows the potential of graphene-on-glass coverslips as a versatile platform for single-molecule biophysics, biosensing, and super-resolution microscopy.

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.

Yttrium-Doped Iron Oxide Nanoparticles for Magnetic Hyperthermia Applications.

Magnetic nanoparticles of Fe3O4 doped by different amounts of Y3+ (0, 0.1, 1, and 10%) ions were designed to obtain maximum heating efficiency in magnetic hyperthermia for cancer treatment. Single-phase formation was evident by X-ray diffraction measurements. An improved magnetization value was obtained for the Fe3O4 sample with 1% Y3+ doping. The specific absorption rate (SAR) and intrinsic loss of power (ILP) values for prepared colloids were obtained in water. The best results were estimated for Fe3O4 with 0.1% Y3+ ions (SAR = 194 W/g and ILP = 1.85 nHm2/kg for a magnetic field of 16 kA/m with the frequency of 413 kHz). The excellent biocompatibility with low cell cytotoxicity of Fe3O4:Y nanoparticles was observed. Immediately after magnetic hyperthermia treatment with Fe3O4:0.1%Y, a decrease in 4T1 cells' viability was observed (77% for 35 µg/mL and 68% for 100 µg/mL). These results suggest that nanoparticles of Fe3O4 doped by Y3+ ions are suitable for biomedical applications, especially for hyperthermia treatment.

Strong Plasmonic Enhancement of a Single Peridinin-Chlorophyll a-Protein Complex on DNA Origami-Based Optical Antennas.

In this contribution, we fabricate hybrid constructs based on a natural light-harvesting complex, peridinin-chlorophyll a-protein, coupled to dimer optical antennas self-assembled with the help of the DNA origami technique. This approach enables controlled positioning of individual complexes at the hotspot of the optical antennas based on large, colloidal gold and silver nanoparticles. Our approach allows us to selectively excite the different pigments present in the harvesting complex, reaching a fluorescence enhancement of 500-fold. This work expands the range of self-assembled functional hybrid constructs for harvesting sunlight and can be further developed for other pigment-proteins and proteins.

Broadband Fluorescence Enhancement with Self-Assembled Silver Nanoparticle Optical Antennas.

Plasmonic structures are known to affect the fluorescence properties of dyes placed in close proximity. This effect has been exploited in combination with single-molecule techniques for several applications in the field of biosensing. Among these plasmonic structures, top-down zero-mode waveguides stand out due to their broadband capabilities. In contrast, optical antennas based on gold nanostructures exhibit fluorescence enhancement on a narrow fraction of the visible spectrum typically restricted to the red to near-infrared region. In this contribution, we exploit the DNA origami technique to self-assemble optical antennas based on large (80 nm) silver nanoparticles. We have studied the performance of these antennas with far- and near-field simulations and characterized them experimentally with single-molecule fluorescence measurements. We demonstrate that silver-based optical antennas can yield a fluorescence enhancement of more than 2 orders of magnitude throughout the visible spectral range for high intrinsic quantum yield dyes. Additionally, a comparison between the performance of gold and silver-based antennas is included. The results indicate that silver-based antennas strongly outperform their gold counterparts in the blue and green ranges and exhibit marginal differences in the red range. These characteristics render silver-based optical antennas ready for applications involving several fluorescently labeled species across the visible spectrum.

Single molecule Raman spectra of porphycene isotopologues.

Single molecule surface-enhanced resonance Raman scattering (SERRS) spectra have been obtained for the parent porphycene (Pc-d0) and its deuterated isotopologue (Pc-d12), located on gold and silver nanoparticles. Equal populations of "hot spots" by the two isotopologues are observed for 1 : 1 mixtures in a higher concentration range of the single molecule regime (5 × 10(-9) M). For decreasing concentrations, hot spots are preferentially populated by undeuterated molecules. This is interpreted as an indication of a lower surface diffusion coefficient of Pc-d12. The photostability of single Pc molecules placed on nanoparticles is strongly increased in comparison with polymer environments. Trans tautomeric species dominate the spectra, but the analysis of time traces reveals transient intermediates, possibly due to rare cis tautomeric forms.