Rate-Engineered Plasmon-Enhanced Fluorescence for Real-Time Microsecond Dynamics of Single Biomolecules.

Single-molecule fluorescence has revealed a wealth of biochemical processes but does not give access to submillisecond dynamics involved in transient interactions and molecular dynamics. Here we overcome this bottleneck and demonstrate record-high photon count rates of >107 photons/s from single plasmon-enhanced fluorophores. This is achieved by combining two conceptual novelties: first, we balance the excitation and decay rate enhancements by the antenna's volume, resulting in maximum fluorescence intensity. Second, we enhance the triplet decay rate using a multicomponent surface chemistry that minimizes microsecond blinking. We demonstrate applications to two exemplary molecular processes: we first reveal transient encounters and hybridization of DNA with a 1 µs temporal resolution. Second, we exploit the field gradient around the nanoparticle as a molecular ruler to reveal microsecond intramolecular dynamics of multivalent complexes. Our results pave the way toward real-time microsecond studies of biochemical processes using an implementation compatible with existing single-molecule fluorescence methods.

Single-Particle Plasmon Sensor to Monitor Proteolytic Activity in Real Time.

We have established a label-free plasmonic platform that monitors proteolytic activity in real time. The sensor consists of a random array of gold nanorods that are functionalized with a design peptide that is specifically cleaved by thrombin, resulting in a blueshift of the longitudinal plasmon. By monitoring the plasmon of many individual nanorods, we determined thrombin's proteolytic activity in real time and inferred relevant kinetic parameters. Furthermore, a comparison to a kinetic model revealed that the plasmon shift is dictated by a competition between peptide cleavage and thrombin binding, which have opposing effects on the measured plasmon shift. The dynamic range of the sensor is greater than two orders of magnitude, and it is capable of detecting physiologically relevant levels of active thrombin down to 3 nM in buffered conditions. We expect these plasmon-mediated label-free sensors to open the window to a range of applications stretching from the diagnostic and characterization of bleeding disorders to fundamental proteolytic and pharmacological studies.

Real-Time Optical Tracking of Protein Corona Formation on Single Nanoparticles in Serum.

The formation of a protein corona, where proteins spontaneously adhere to the surface of nanomaterials in biological environments, leads to changes in their physicochemical properties and subsequently affects their intended biomedical functionalities. Most current methods to study protein corona formation are ensemble-averaging and either require fluorescent labeling, washing steps, or are only applicable to specific types of particles. Here we introduce real-time all-optical nanoparticle analysis by scattering microscopy (RONAS) to track the formation of protein corona in full serum, at the single-particle level, without any labeling. RONAS uses optical scattering microscopy and enables real-time and in situ tracking of protein adsorption on metallic and dielectric nanoparticles with different geometries directly in blood serum. We analyzed the adsorbed protein mass, the affinity, and the kinetics of the protein adsorption at the single particle level. While there is a high degree of heterogeneity from particle to particle, the predominant factor in protein adsorption is surface chemistry rather than the underlying nanoparticle material or size. RONAS offers an in-depth understanding of the mechanisms related to protein coronas and, thus, enables the development of strategies to engineer efficient bionanomaterials.

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.