Supramolecular polymers form tactoids through liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) of biopolymers has recently been shown to play a central role in the formation of membraneless organelles with a multitude of biological functions1-3. The interplay between LLPS and macromolecular condensation is part of continuing studies4,5. Synthetic supramolecular polymers are the non-covalent equivalent of macromolecules but they are not reported to undergo LLPS yet. Here we show that continuously growing fibrils, obtained from supramolecular polymerizations of synthetic components, are responsible for phase separation into highly anisotropic aqueous liquid droplets (tactoids) by means of an entropy-driven pathway. The crowding environment, regulated by dextran concentration, affects not only the kinetics of supramolecular polymerizations but also the properties of LLPS, including phase-separation kinetics, morphology, internal order, fluidity and mechanical properties of the final tactoids. In addition, substrate-liquid and liquid-liquid interfaces proved capable of accelerating LLPS of supramolecular polymers, allowing the generation of a myriad of three-dimensional-ordered structures, including highly ordered arrays of micrometre-long tactoids at surfaces. The generality and many possibilities of supramolecular polymerizations to control emerging morphologies are demonstrated with several supramolecular polymers, opening up a new field of matter ranging from highly structured aqueous solutions by means of stabilized LLPS to nanoscopic soft matter.

Refractive index sensing using quasi-bound states in the continuum in silicon metasurfaces.

This work presents a bulk refractive index sensor based on quasi-bound states in the continuum (BICs) induced by broken symmetries in metasurfaces. The symmetry is broken by detuning the size and position of silicon particles periodically arranged in an array, resulting in multiple quasi-BIC resonances. We investigate the sensing characteristics of each of the resonances by measuring the spectral shift in response to changes in the refractive index of the surrounding medium. In addition, we reveal the sensing range of the different resonances through simulations involving a layer of deviating refractive index of increasing thickness. Interestingly, the resonances show very different responses, which we describe via the analysis of the near-field. This work contributes to the development of highly sensitive and selective BIC-based sensors that can be used for a wide range of applications.

Spectrally PAINTing a Single Chain Polymeric Nanoparticle at Super-Resolution.

Folding a polymer chain into a well-defined single-chain polymeric nanoparticle (SCPN) is a fascinating approach to obtaining structured and functional nanoparticles. Like all polymeric materials, SCPNs are heterogeneous in their nature due to the polydispersity of their synthesis: the stochastic synthesis of polymer backbone length and stochastic functionalization with hydrophobic and hydrophilic pendant groups make structural diversity inevitable. Therefore, in a single batch of SCPNs, nanoparticles with different physicochemical properties are present, posing a great challenge to their characterization at a single-particle level. The development of techniques that can elucidate differences between SCPNs at a single-particle level is imperative to capture their potential applications in different fields such as catalysis and drug delivery. Here, a Nile Red based spectral point accumulation for imaging in nanoscale topography (NR-sPAINT) super-resolution fluorescence technique was implemented for the study of SCPNs at a single-particle level. This innovative method allowed us to (i) map the small-molecule binding rates on individual SCPNs and (ii) map the polarity of individual SCPNs for the first time. The SCPN designs used here have the same polymeric backbone but differ in the number of hydrophobic groups. The experimental results show notable interparticle differences in the binding rates within the same polymer design. Moreover, a marked polarity shift between the different designs is observed. Interestingly, interparticle polarity heterogeneity was unveiled, as well as an intraparticle diversity, information which has thus far remained hidden by ensemble techniques. The results indicate that the addition of hydrophobic pendant groups is vital to determine binding properties and induces single-particle polarity diversity. Overall, NR-sPAINT represents a powerful approach to quantifying the single-particle polarity of SCPNs and paves the way to relate the structural heterogeneity to functionality at the single-particle level. This provides an important step toward the aim of rationally designing SCPNs for the desired application.

Real-Time Interfacial Nanothermometry Using DNA-PAINT Microscopy.

Biofunctionalized nanoparticles are increasingly used in biomedical applications including sensing, targeted delivery, and hyperthermia. However, laser excitation and associated heating of the nanomaterials may alter the structure and interactions of the conjugated biomolecules. Currently no method exists that directly monitors the local temperature near the material's interface where the conjugated biomolecules are. Here, a nanothermometer is reported based on DNA-mediated points accumulation for imaging nanoscale topography (DNA-PAINT) microscopy. The temperature dependent kinetics of repeated and reversible DNA interactions provide a direct readout of the local interfacial temperature. The accuracy and precision of the method is demonstrated by measuring the interfacial temperature of many individual gold nanoparticles in parallel, with a precision of 1 K. In agreement with numerical models, large particle-to-particle differences in the interfacial temperature are found due to underlying differences in optical and thermal properties. In addition, the reversible DNA interactions enable the tracking of interfacial temperature in real-time with intervals of a few minutes. This method does not require prior knowledge of the optical and thermal properties of the sample, and therefore opens the window to understanding and controlling interfacial heating in a wide range of nanomaterials.

Correlative microscopy of single self-assembled nanorod dimers for refractometric sensing.

Single metallic particles and dimers of nanospheres have been used extensively for sensing, but dimers of particles provide attractive advantages because they exhibit multiple modes that can be tuned by the dimer geometry. Here, we employ correlative microscopy of single self-assembled dimers of gold nanorods to study their performance as refractometric sensors. The correlation between atomic force microscopy and single-particle white-light spectroscopy allows us to relate the measured sensitivity to numerical simulations taking into account the exact geometry of the construct. The sensitivity of the antibonding mode is in good agreement with simulations, whereas the bonding mode exhibits a reduced sensitivity related to the accessibility of the gap region between the particles. We find that the figure of merit is a trade-off between the resonance linewidth and its refractive index sensitivity, which depend in opposite ways on the interparticle angle. The presence of two narrow plasmon resonances in the visible to near-infrared wavelength regime makes nanorod dimers exciting candidates for multicolor and multiplexed sensing.

Plasmonic Assemblies for Real-Time Single-Molecule Biosensing.

Their tunable optical properties and versatile surface functionalization have sparked applications of plasmonic assemblies in the fields of biosensing, nonlinear optics, and photonics. Particularly, in the field of biosensing, rapid advances have occurred in the use of plasmonic assemblies for real-time single-molecule sensing. Compared to individual particles, the use of assemblies as sensors provides stronger signals, more control over the optical properties, and access to a broader range of timescales. In the past years, they have been used to directly reveal single-molecule interactions, mechanical properties, and conformational dynamics. This review summarizes the development of real-time single-molecule sensors built around plasmonic assemblies. First, a brief overview of their optical properties is given, and then recent applications are described. The current challenges in the field and suggestions to overcome those challenges are discussed in detail. Their stability, specificity, and sensitivity as sensors provide a complementary approach to other single-molecule techniques like force spectroscopy and single-molecule fluorescence. In future applications, the impact in real-time sensing on ultralong timescales (hours) and ultrashort timescales (sub-millisecond), time windows that are difficult to access using other techniques, is particularly foreseen.

A Robust and General Approach to Quantitatively Conjugate Enzymes to Plasmonic Nanoparticles.

Bioconjugates of plasmonic nanoparticles have received considerable attention due to their potential biomedical applications. Successful bioconjugation requires control over the number and activity of the conjugated proteins and the colloidal stability of the particles. In practice, this requires reoptimization of the conjugation protocol for each combination of protein and nanoparticle. Here, we report a robust and general protocol that allows for the conjugation of a range of proteins to different types of nanoparticles using very short polyethylene-glycol(PEG) linkers, while simultaneously preserving protein activity and colloidal stability. The use of short linkers ensures that the protein is located close to the particle surface, where the refractive index sensitivity and near-field enhancement are maximal. We demonstrate that the use of a Tween20 containing stabilizing buffer is critical in maintaining colloidal stability and protein function throughout the protocol. We obtain quantitative control over the average number of enzymes per particle by either varying the number of functional groups on the particle or the enzyme concentration during incubation. This new route of preparing quantitative protein-nanoparticle bioconjugates paves the way to develop rational and quantitative strategies to functionalize nanoparticles for applications in sensing, medical diagnostics, and drug delivery.

Plasmon Rulers as a Probe for Real-Time Microsecond Conformational Dynamics of Single Molecules.

Biopolymers such as DNA, RNA, and proteins exploit conformational changes to modulate their function. Although state-of-the-art single-molecule approaches enable identification of conformational states, the transition path and metastable intermediates often remain elusive because they occur on microsecond time scales. Here we introduce a method to probe conformational dynamics with microsecond integration times based on a heterodimer of plasmonic particles. By combining Brownian dynamics and electromagnetic simulations, we find that integration times of 1 µs can be routinely achieved, providing the capability to identify short-lived intermediates and transition paths at the single-molecule level in real-time. Importantly, plasmon rulers require no specialized equipment but can be probed on existing fluorescence microscopes equipped with a fast camera. The approach combines the advantages of fluorescent probes (zero-force, parallelization) and mechanical probes such as optical tweezers (continuous microsecond integration times). They offer a unique opportunity to study conformational dynamics and compare measurements to full-atom simulations, where computational demands limit the simulation time.

Heterogeneous Kinetics in the Functionalization of Single Plasmonic Nanoparticles.

The functionalization of gold nanoparticles with DNA has been studied extensively in solution; however, these ensemble measurements do not reveal particle-to-particle differences. Here we study the functionalization of gold nanorods with thiolated single-stranded DNA (ssDNA) at the single-particle level. We exploit the sensitivity of the plasmon resonance to the local refractive index to study the functionalization in real time using single-particle spectroscopy. We find particle-to-particle variations of the plasmon shift that are attributed to the particle size distribution and variations in ssDNA coverage. We find that the ssDNA coverage varies by ∼10% from particle to particle, beyond the expected variation due to Poisson statistics. Surprisingly, we find binding rates that differ from particle to particle by an order of magnitude, even though the buffer conditions are identical. We ascribe this heterogeneity to a distribution of activation energies caused by particle-to-particle variations in effective surface charge. These results yield insight into the kinetics of biofunctionalization at the single particle level and highlight that significant kinetic heterogeneity has to be taken into account in applications of functional particles. The presented methodology is easily extended to any nanoparticle coating and can be used to optimize functionalization protocols.

Tip-Specific Functionalization of Gold Nanorods for Plasmonic Biosensing: Effect of Linker Chain Length.

Gold nanorods are promising platforms for label-free biosensing. We have functionalized gold nanorods with biotin thiol linkers of increasing chain length and evaluated their ability in the molecular detection of streptavidin. We have found an unexpected effect of the increase in linker length, which resulted in a substantial improvement of the plasmon response at surface saturation. The plasmon peak shift increased from 5 to 14 nm, i.e., more than twice the response, between the short and long biotin linkers. This effect is observed only for site-selective tip functionalization, whereas for a full biotin coating there is no improvement observed with the linker length. The improved plasmon response for tip functionalization is attributed to low biotin coverage but is directed to the most sensitive regions, which, combined with a longer chain linker, reduces the steric hindrance for streptavidin binding on the rod's surface. The model sensors were further characterized by measuring their dose-response curves and binding kinetic assays. Simulations of the discrete dipole approximation give theoretical plasmon shifts that compare well with the experimental ones for the long linker but not with those of the short linker, thus suggesting that steric hindrance affects the latter. Our results highlight the importance of specifically functionalizing the plasmonic hot spots in nanoparticle sensors with the adequate density of receptors in order to maximize their response.

Stochastic protein interactions monitored by hundreds of single-molecule plasmonic biosensors.

We present a plasmonic biosensor based on hundreds of individual gold nanorods with single-molecule sensitivity that are simultaneously monitored in real-time within a dark-field microscopy setup. The approach allows for the statistical analysis of single-molecule interactions without requiring any labeling of the analyte. We study an antibody-antigen interaction and find that the waiting-time distribution is concentration-dependent and obeys Poisson statistics. The ability to probe hundreds of nanoparticles simultaneously will provide a sensor with a dynamic range of 7 decades in concentration and will enable the study of heterogeneity in molecular interactions.

Five-dimensional optical recording mediated by surface plasmons in gold nanorods.

Multiplexed optical recording provides an unparalleled approach to increasing the information density beyond 10(12) bits per cm(3) (1 Tbit cm(-3)) by storing multiple, individually addressable patterns within the same recording volume. Although wavelength, polarization and spatial dimensions have all been exploited for multiplexing, these approaches have never been integrated into a single technique that could ultimately increase the information capacity by orders of magnitude. The major hurdle is the lack of a suitable recording medium that is extremely selective in the domains of wavelength and polarization and in the three spatial domains, so as to provide orthogonality in all five dimensions. Here we show true five-dimensional optical recording by exploiting the unique properties of the longitudinal surface plasmon resonance (SPR) of gold nanorods. The longitudinal SPR exhibits an excellent wavelength and polarization sensitivity, whereas the distinct energy threshold required for the photothermal recording mechanism provides the axial selectivity. The recordings were detected using longitudinal SPR-mediated two-photon luminescence, which we demonstrate to possess an enhanced wavelength and angular selectivity compared to conventional linear detection mechanisms. Combined with the high cross-section of two-photon luminescence, this enabled non-destructive, crosstalk-free readout. This technique can be immediately applied to optical patterning, encryption and data storage, where higher data densities are pursued.

Probing silver deposition on single gold nanorods by their acoustic vibrations.

Acoustic vibrations of single gold nanorods coated with silver were investigated. We used single-particle pump-probe spectroscopy to monitor the silver deposition through the particle vibrations. Two vibration modes, the breathing mode and extensional mode, are observed, and the vibrational frequencies are measured as functions of the amount of silver deposited on single gold nanorods. The breathing mode frequency was found to decrease with silver deposition, while the extensional mode frequency was almost constant for silver shells up to 6 nm. The frequency changes agree with a model based on continuum mechanics and on the assumption of a uniform silver coating. The quality factors for the breathing mode and the extensional mode are hardly affected by silver deposition, indicating that the introduced interface between gold and silver contributes negligibly to the damping of the particle vibrations. Finally, we demonstrated that an atomic layer of silver can be detected using the particle acoustic vibrations.

Damping of acoustic vibrations of immobilized single gold nanorods in different environments.

We present measurements of the acoustic vibrations of single gold nanorods deposited on a glass substrate immersed in air and water by ultrafast pump-probe spectroscopy. The nanorods display two vibration modes, the breathing mode and the extensional mode. The damping time of the two modes is influenced by the environment, and a reduction of the quality factor is observed when the particles are immersed in water. The reduced quality factor of the breathing mode is in good agreement with a model that takes into account viscous damping and radiation of sound waves into the medium. The extension mode, however, is heavily damped when the particles are immersed in water, which is attributed to hydrodynamic lubrication forces between the nanoparticle and the glass substrate. Our results identify a new mode of damping in supported nanoparticles and indicate that the immersion medium can have different effects on different modes of vibration.

Continuous Monitoring Biosensing Mediated by Single-Molecule Plasmon-Enhanced Fluorescence in Complex Matrices.

Continuous detection of critical markers directly at the point of interest and in undiluted biological fluids represents the next fundamental step in biosensing. The goal of realizing such a platform is utterly challenging because it requires a reversible biosensor that enables the tracking of pico- to nanomolar molecular concentrations over long time spans in a compact device. Here we describe a sensing method based on plasmon-enhanced fluorescence capable of single-molecule detection of unlabeled analyte by employing biofunctionalized gold nanoparticles. The very strong plasmon-enhanced fluorescence signals allow for single-molecule sensing in unaltered biological media, while the use of low-affinity interactions ensures the continuous tracking of increasing and decreasing analyte concentrations with picomolar sensitivity. We demonstrate the use of a sandwich assay for a DNA cancer marker with a limit of detection of picomolar and a time response of 10 min. The enhanced single-molecule signals will allow for miniaturization into a small and cheap platform with multiplexing capability for application in point-of-care diagnostics, monitoring of industrial processes, and safe keeping of the environment.

Biomolecular interactions on densely coated nanoparticles: a single-molecule perspective.

DNA-modified gold nanoparticles (AuNPs) play a pivotal role in bio-nanotechnology, driving advancements in bio-sensing, bio-imaging, and drug delivery. Synthetic protocols have focused on maximizing the receptor density on particles by fine-tuning chemical conditions, particularly for DNA. Despite their significance, the understanding of hybridization kinetics on functionalized AuNPs is lacking, particularly how this kinetics depends on DNA density and to what extent it varies from particle-to-particle. This study explores the molecular mechanisms of DNA hybridization on densely coated AuNPs by employing a combination of single-molecule microscopy and coarse-grained molecular dynamics simulations providing a quantification of the molecular rate constants for single particles. Our findings demonstrate that DNA receptor density and the presence of spacer strands profoundly impact association kinetics, with short spacers enhancing association rates by up to ∼15-fold. In contrast, dissociation kinetics are largely unaffected by receptor density within the studied range. Single-particle analysis directly reveals variability in hybridization kinetics, which is analyzed in terms of intra- and inter-particle heterogeneity. A coarse-grained DNA model that quantifies hybridization kinetics on densely coated surfaces further corroborates our experimental results, additionally shedding light on how transient base pairing within the DNA coating influences kinetics. This integrated approach underscores the value of single-molecule studies and simulations for understanding DNA dynamics on densely coated nanoparticle surfaces, offering guidance for designing DNA-functionalized nanoparticles in sensor applications.

Damping of acoustic vibrations of single gold nanoparticles optically trapped in water.

We combine ultrafast pump-probe spectroscopy with optical trapping to study homogeneous damping of the acoustic vibrations of single gold nanospheres (80 nm diameter) and nanorods (25 nm diameter by 60 nm length) in water. We find a significant particle-to-particle variation in damping times. Our results indicate that vibrational damping occurs not only by dissipation into the liquid, but also by damping mechanisms intrinsic to the particle. Our experiment opens the study of mechanisms of intrinsic mechanical dissipation in metals at frequencies 1-1000 GHz, a range that has been difficult to access thus far.

Luminescence quantum yield of single gold nanorods.

We study the luminescence quantum yield (QY) of single gold nanorods with different aspect ratios and volumes. Compared to gold nanospheres, we observe an increase of QY by about an order of magnitude for particles with a plasmon resonance >650 nm. The observed trend in QY is further confirmed by controlled reshaping of a single gold nanorod to a spherelike shape. Moreover, we identify two spectral components, one around 500 nm originating from a combination of interband transitions and the transverse plasmon and one coinciding with the longitudinal plasmon band. These components are analyzed by correlating scattering and luminescence spectra of single nanorods and performing polarization sensitive measurements. Our study contributes to the understanding of luminescence from gold nanorods. The enhanced QY we report can benefit applications in biological and soft matter studies.

Single-Molecule Optical Biosensing: Recent Advances and Future Challenges.

In recent years, the sensitivity and specificity of optical sensors has improved tremendously due to improvements in biochemical functionalization protocols and optical detection systems. As a result, single-molecule sensitivity has been reported in a range of biosensing assay formats. In this Perspective, we summarize optical sensors that achieve single-molecule sensitivity in direct label-free assays, sandwich assays, and competitive assays. We describe the advantages and disadvantages of single-molecule assays and summarize future challenges in the field including their optical miniaturization and integration, multimodal sensing capabilities, accessible time scales, and compatibility with real-life matrices such as biological fluids. We conclude by highlighting the possible application areas of optical single-molecule sensors that include not only healthcare but also the monitoring of the environment and industrial processes.

Integrated Signal Amplification on a Fiber Optic SPR Sensor Using Duplexed Aptamers.

Throughout the past decades, fiber optic surface plasmon resonance (FO-SPR)-based biosensors have proven to be powerful tools for both the characterization of biomolecular interactions and target detection. However, as FO-SPR signals are generally related to the mass that binds to the sensor surface, multistep processes and external reagents are often required to obtain significant signals for low molecular weight targets. This increases the time, cost, and complexity of the respective bioassays and hinders continuous measurements. To overcome these requirements, in this work, cis-duplexed aptamers (DAs) were implemented on FO-SPR sensors, which underwent a conformational change upon target binding. This induced a spatial redistribution of gold nanoparticles (AuNPs) upon specific target binding and resulted in an amplified and concentration-dependent signal. Importantly, the AuNPs were covalently conjugated to the sensor, so the principle does not rely on multistep processes or external reagents. To implement this concept, first, the thickness of the gold fiber coating was adapted to match the resonance conditions of the surface plasmons present on the FO-SPR sensors with those on the AuNPs. As a result, the signal obtained due to the spatial redistribution of the AuNPs was amplified by a factor of 3 compared to the most commonly used thickness. Subsequently, the cis-DAs were successfully implemented on the FO-SPR sensors, and it was demonstrated that the DA-based FO-SPR sensors could specifically and quantitatively detect an ssDNA target with a detection limit of 230 nM. Furthermore, the redistribution of the AuNPs was proven to be reversible, which is an important prerequisite for continuous measurements. Altogether, the established DA-based FO-SPR bioassay holds much promise for the detection of low molecular weight targets in the future and opens up possibilities for FO-SPR-based continuous biosensing.