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Optoplasmonic methods capable of single protein detection so far rely on analyte immobilization in order to facilitate detection [1-6]. These detection schemes, even if they facilitate transient single-molecule detection [7,8] via consequent formation and cleavage of chemical bonds, typically exhibit time resolutions on the order of milliseconds. The need for analyte immobilisation is a direct consequence of the minuscule dimensions of plasmonic near fields typically providing sub-attolitre-sized detection volumes which in turn demand sub-microsecond temporal resolution for the direct detection of proteins in motion. Here we show that such temporal resolution can indeed be achieved. We demonstrate the observation of single proteins as small as Hemoglobin (molecular weight: 64 kDa) as they traverse plasmonic near fields of gold nanorods and interact with their surface, all while maintaining signal-to-noise ratios larger than 5 and an unprecedented temporal resolution well below microseconds. This method enables the label-free observation of single-molecule dynamics on previously unaccessible timescales., Comment: 15 pages, 7 figures

Optical detection of individual nanometer-sized analytes, virus particles, and protein molecules holds great promise for understanding and control of biological samples and healthcare applications. As fluorescent labels impose restrictions on detection bandwidth and require lengthy and invasive processes, label-free optical techniques are highly desirable. Powerful label-free optical methods have recently emerged, such as interferometric scattering microscopy, plasmonic nanoparticle-based assays and microcavity-based assays. Although highly sensitive, these methods are so far restricted to integration times in excess of microseconds. This often imposes a requirement to impede analyte motion during these periods via specific molecular tethers, unspecific adsorption or confining arrangements. Here we introduce an optical technique capable of transforming gold nanorods commonly used as photostable labels into highly localized high-speed probes. Our method provides a time resolution well below microseconds. This mitigates the requirement for molecular tethers and allows us to detect single untethered nanoparticles in Brownian motion traversing sub atto-liter sensing volumes. Our method opens a novel gateway for the investigation of highly localized and highly dynamic nanoscale systems and constitutes a first step towards the label-free recognition of single untethered proteins., Comment: Manuscript and suppl. information are on 30 pages and possess 16 figures

Monitoring the kinetics and conformational dynamics of single enzymes is crucial in order to better understand their biological functions as these motions and structural dynamics are usually unsynchronized among the molecules. Detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single molecule basis, however, remain as a challenge with established optical techniques due to the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually non-trivial and the labels themselves might skew the physical properties of the enzyme. Here we demonstrate an optical, label-free method capable of observing enzymatic interactions and the associated conformational changes on the single molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we employ two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches we find that low and high polymerase activities can be clearly discerned via their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities via plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.