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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.

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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 blue-shift 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, 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 open the window to a range of applications stretching from diagnostic and characterization of bleeding disorders to fundamental proteolytic and pharmacological studies.

Significant advances in synthesis and functionalization have provided state-of-the-art technology in controlling the physico-chemical properties of nanomaterials. These are finding numerous applications including in the biomedical field whereby nanoparticles are injected in vivo for medical imaging, theranostics and biosensing. However, interactions with proteins contained in biological fluids lead to the formation of a shell on the surface of the nanoparticles called "protein corona" (PC). PC plays a detrimental role for the intended applications as it may modify the interface of the nanoparticle and thereby block functional groups needed for recognition. It is therefore essential to understand the mechanisms of formation of these PCs in order to control the surface chemistry of the nanoparticles in complex biological fluids. Current characterization techniques can identify and quantify the composition of PCs using mass spectroscopy and electrophoresis. However, most of them do not enable real-time measurement in complex media because they require washing steps to remove excess protein. Finally, most techniques provide ensemble averages and are unable to access inter-particle heterogeneity. Here, we demonstrate the use of single-particle scattering microscopy combined with a microfluidic system to study PC formation in real-time at the single-nanoparticle level. The method is label-free and operates in undiluted blood serum. We probe PC formation on both, metallic and dielectric nanoparticles with different surface chemistries. Analysis of protein adsorption revealed unexpectedly strong heterogeneity whereby the amount of accumulated protein varies by up to a factor of 10 between the particles. Furthermore, it is found that the surface roughness of the nanoparticles affects the kinetics of the PC formation. The results of this in-situ characterization are a powerful tool to optimize the surface chemistry in order to minimize the formation of PCs and thus increase the efficiency of nanoparticles for applications such as targeted drug delivery.