Nature Communications, 11(4513), September 2020 (article)
Chiral plasmonic nanoparticles can exhibit strong chiroptical signals compared to the corresponding molecular response. Observations are, however, generally restricted to measurements on stationary single particles with a ﬁxed orientation, which complicates the spectral analysis. Here, we report the spectroscopic observation of a freely diffusing single chiral nanoparticle in solution. By acquiring time-resolved circular differential scattering signals we show that the spectral interpretation is signiﬁcantly simpliﬁed. We experimentally demonstrate the equivalence between time-averaged chiral spectra observed for an individual nanostructure and the corresponding ensemble spectra, and thereby demonstrate the ergodic principle for chiroptical spectroscopy. We also show how it is possible for an achiral particle to yield an instantaneous chiroptical response, whereas the time-averaged signals are an unequivocal measure of chirality. Time-resolved chiroptical spectroscopy on a freely moving chiral nanoparticle advances the ﬁeld of single-particle spectroscopy, and is a means to obtain the true signature of the nanoparticle’s chirality.
ACS Nano, 13, pages: 11453-11459, September 2019 (article)
Plasmonic molecules are building blocks of metallic nanostructures that give rise to intriguing optical phenomena with similarities to those seen in molecular systems. The ability to design plasmonic hybrid structures and molecules with nanometric resolution would enable applications in optical metamaterials and sensing that presently cannot be demonstrated, because of a lack of suitable fabrication methods allowing the structural control of the plasmonic atoms on a large scale. Here we demonstrate a wafer-scale “lithography-free” parallel fabrication scheme to realize nanogap plasmonic meta-molecules with precise control over their size, shape, material, and orientation. We demonstrate how we can tune the corresponding coupled resonances through the entire visible spectrum. Our fabrication method, based on glancing angle physical vapor deposition with gradient shadowing, permits critical parameters to be varied across the wafer and thus is ideally suited to screen potential structures. We obtain billions of aligned dimer structures with controlled variation of the spectral properties across the wafer. We spectroscopically map the plasmonic resonances of gold dimer structures and show that they not only are in good agreement with numerically modeled spectra, but also remain functional, at least for a year, in ambient conditions.
Enzyme-based biocatalysis exhibits multiple advantages over inorganic catalysts, including the biocompatibility and the unchallenged specificity of enzymes towards their substrate. The recovery and repeated use of enzymes is essential for any realistic application in biotechnology, but is not easily achieved with current strategies. For this purpose, enzymes are often immobilized on inorganic scaffolds, which could entail a reduction of the enzymes’ activity. Here, we show that immobilization to a nano-scaled biological scaffold, a nanonetwork of end-to-end cross-linked M13 bacteriophages, ensures high enzymatic activity and at the same time allows for the simple recovery of the enzymes. The bacteriophages have been genetically engineered to express AviTags at their ends, which permit biotinylation and their specific end-to-end self-assembly while allowing space on the major coat protein for enzyme coupling. We demonstrate that the phages form nanonetwork structures and that these so-called nanonets remain highly active even after re-using the nanonets multiple times in a flow-through reactor.
ACS Nano, 13, pages: 5810–5815, March 2019 (article)
Catalytically active colloids are model systems for chemical motors and active matter. It is desirable to replace the inorganic catalysts and the toxic fuels that are often used, with biocompatible enzymatic reactions. However, compared to inorganic catalysts, enzyme-coated colloids tend to exhibit less activity. Here, we show that the self-assembly of genetically engineered M13 bacteriophages that bind enzymes to magnetic beads ensures high and localized enzymatic activity. These phage-decorated colloids provide a proteinaceous environment for directed enzyme immobilization. The magnetic properties of the colloidal carrier particle permit repeated enzyme recovery from a reaction solution, while the enzymatic activity is retained. Moreover, localizing the phage-based construct with a magnetic field in a microcontainer allows the enzyme-phage-colloids to function as an enzymatic micropump, where the enzymatic reaction generates a fluid flow. This system shows the fastest fluid flow reported to date by a biocompatible enzymatic micropump. In addition, it is functional in complex media including blood where the enzyme driven micropump can be powered at the physiological blood-urea concentration.
The diffusion of enzymes is of fundamental importance for many biochemical processes. Enhanced or directed enzyme diffusion can alter the accessibility of substrates and the organization of enzymes within cells. Several studies based on fluorescence correlation spectroscopy (FCS) report enhanced diffusion of enzymes upon interaction with their substrate or inhibitor. In this context, major importance is given to the enzyme fructose-bisphosphate aldolase, for which enhanced diffusion has been reported even though the catalysed reaction is endothermic. Additionally, enhanced diffusion of tracer particles surrounding the active aldolase enzymes has been reported. These studies suggest that active enzymes can act as chemical motors that self-propel and give rise to enhanced diffusion. However, fluorescence studies of enzymes can, despite several advantages, suffer from artefacts. Here we show that the absolute diffusion coefficients of active enzyme solutions can be determined with Pulsed Field Gradient Nuclear Magnetic Resonance (PFG-NMR). The advantage of PFG-NMR is that the motion of the molecule of interest is directly observed in its native state without the need for any labelling. Further, PFG-NMR is model-free and thus yields absolute diffusion constants. Our PFG-NMR experiments of solutions containing active fructose-bisphosphate aldolase from rabbit muscle do not show any diffusion enhancement for the active enzymes nor the surrounding molecules. Additionally, we do not observe any diffusion enhancement of aldolase in the presence of its inhibitor pyrophosphate.
Accounts of Chemical Research, 51(9):1911-1920, August 2018 (article)
Self-propelled chemical motors are chemically powered micro- or nanosized swimmers. The energy required for these motors’ active motion derives from catalytic chemical reactions and the transformation of a fuel dissolved in the solution. While self-propulsion is now well established for larger particles, it is still unclear if enzymes, nature’s nanometer-sized catalysts, are potentially also self-powered nanomotors. Because of its small size, any increase in an enzyme’s diffusion due to active self-propulsion must be observed on top of the enzyme’s passive Brownian motion, which dominates at this scale. Fluorescence correlation spectroscopy (FCS) is a sensitive method to quantify the diffusion properties of single fluorescently labeled molecules in solution. FCS experiments have shown a general increase in the diffusion constant of a number of enzymes when the enzyme is catalytically active. Diffusion enhancements after addition of the enzyme’s substrate (and sometimes its inhibitor) of up to 80\% have been reported, which is at least 1 order of magnitude higher than what theory would predict. However, many factors contribute to the FCS signal and in particular the shape of the autocorrelation function, which underlies diffusion measurements by fluorescence correlation spectroscopy. These effects need to be considered to establish if and by how much the catalytic activity changes an enzyme’s diffusion.We carefully review phenomena that can play a role in FCS experiments and the determination of enzyme diffusion, including the dissociation of enzyme oligomers upon interaction with the substrate, surface binding of the enzyme to glass during the experiment, conformational changes upon binding, and quenching of the fluorophore. We show that these effects can cause changes in the FCS signal that behave similar to an increase in diffusion. However, in the case of the enzymes F1-ATPase and alkaline phosphatase, we demonstrate that there is no measurable increase in enzyme diffusion. Rather, dissociation and conformational changes account for the changes in the FCS signal in the former and fluorophore quenching in the latter. Within the experimental accuracy of our FCS measurements, we do not observe any change in diffusion due to activity for the enzymes we have investigated.We suggest useful control experiments and additional tests for future FCS experiments that should help establish if the observed diffusion enhancement is real or if it is due to an experimental or data analysis artifact. We show that fluorescence lifetime and mean intensity measurements are essential in order to identify the nature of the observed changes in the autocorrelation function. While it is clear from theory that chemically active enzymes should also act as self-propelled nanomotors, our FCS measurements show that the associated increase in diffusion is much smaller than previously reported. Further experiments are needed to quantify the contribution of the enzymes’ catalytic activity to their self-propulsion. We hope that our findings help to establish a useful protocol for future FCS studies in this field and help establish by how much the diffusion of an enzyme is enhanced through catalytic activity.
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