Response to Comment on "Following Molecular Mobility during Chemical Reactions: No Evidence for Active Propulsion" and "Molecular Diffusivity of Click Reaction Components: The Diffusion Enhancement Question".

In their Comment (DOI: 10.1021/jacs.2c02965) on two related publications by our groups (J. Am. Chem. Soc. 2021, 143, 20884-20890; DOI: 10.1021/jacs.1c09455) and another (J. Am. Chem. Soc. 2022, 144, 1380-1388; DOI: 10.1021/jacs.1c11754), Huang and Granick discuss the diffusion NMR measurements of molecules during a copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction. Here we respond to these comments and maintain that no diffusion enhancement was observed for any species during the reaction. We show that the relaxation agent does not interfere with the CuAAC reaction kinetics nor the diffusion of the molecules involved. Similarly, the gradient pulse length and diffusion time do not affect the diffusion coefficients. Peak overlap was completely removed in our study with the use of hydrazine as the reducing agent. The steady-state assumption does not hold for these diffusion measurements that take several minutes, which is the reason monotonic gradient orders are not suitable. Finally, we discuss the other reactions where similar changes in diffusion have been claimed. Our conclusions are fully supported by the results represented in our original JACS Article and the corresponding Supporting Information.

Diffusion mechanisms of DNA in agarose gels: NMR studies and Monte Carlo simulations.

We report on the diffusion mechanism of short, single-stranded DNA molecules with up to 100 nucleobases in agarose gels with concentrations of up to 2.0% with the aim to characterize the DNA-agarose interaction. The diffusion coefficients were measured directly, i.e., without any model assumptions, by pulsed field gradient nuclear magnetic resonance (PFG-NMR). We find that the diffusion coefficient decreases, as expected, with an increase in both DNA strand length and gel concentration. In addition, we performed Monte Carlo simulations of particle diffusion in a model network of polymer chains, considering our experimental conditions. Together, the Monte Carlo simulations and the PFG-NMR results show that the decrease in diffusion coefficients in the presence of the agarose gel is due to a temporary adhesion of the DNA molecules to the surface of gel fibers. The average adhesion time to a given gel fiber increases with the length of the DNA strands but is independent of the number of gel fibers. The corresponding magnitude of the binding enthalpies of DNA strands to gel fibers indicates that a mixture of van der Waals interactions and hydrogen bonding contributes to the decreased diffusion of DNA in agarose gels.

Following Molecular Mobility during Chemical Reactions: No Evidence for Active Propulsion.

The reported changes in self-diffusion of small molecules during reactions have been attributed to "boosted mobility". We demonstrate the critical role of changing concentrations of paramagnetic ions on nuclear magnetic resonance (NMR) signal intensities, which led to erroneous measurements of diffusion coefficients. We present simple methods to overcome this problem. The use of shuffled gradient amplitudes allows accurate diffusion NMR measurements, even with time-dependent relaxation rates caused by changing concentrations of paramagnetic ions. The addition of a paramagnetic relaxation agent allows accurate determination of both diffusion coefficients and reaction kinetics during a single experiment. We analyze a copper-catalyzed azide-alkyne cycloaddition "click" reaction, for which boosted mobility has been claimed. With our methods, we accurately measure the diffusive behavior of the solvent, starting materials, and product and find no global increase in diffusion coefficients during the reaction. We overcome NMR signal overlap using an alternative reducing agent to improve the accuracy of the diffusion measurements. The alkyne reactant diffuses slower as the reaction proceeds due to binding to the copper catalyst during the catalytic cycle. The formation of this intermediate was confirmed by complementary NMR techniques and density functional theory calculations. Our work calls into question recent claims that molecules actively propel or swim during reactions and establishes that time-resolved diffusion NMR measurements can provide valuable insight into reaction mechanisms.

Comment on "Using NMR to Test Molecular Mobility during a Chemical Reaction".

A study reported in The Journal of Physical Chemistry Letters (Wang et al., 2021, 12, 2370) of "boosted mobility" measured by diffusion NMR experiments contains significant errors in data analysis and interpretation. We carefully reanalyzed the same data and find no evidence of boosted mobility, and we identify several sources of error.

Light- and magnetically actuated FePt microswimmers.

Externally controlled microswimmers offer prospects for transport in biological research and medical applications. This requires biocompatibility of the swimmers and the possibility to tailor their propulsion mechanisms to the respective low Reynolds number environment. Here, we incorporate low amounts of the biocompatible alloy of iron and platinum (FePt) in its [Formula: see text] phase in microstructures by a versatile one-step physical vapor deposition process. We show that the hard magnetic properties of [Formula: see text] FePt are beneficial for the propulsion of helical micropropellers with rotating magnetic fields. Finally, we find that the FePt coatings are catalytically active and also make for Janus microswimmers that can be light-actuated and magnetically guided.

Comment on "Boosted molecular mobility during common chemical reactions".

The apparent "boosted mobility" observed by Wang et al (Reports, 31 July 2020, p. 537) is the result of a known artifact. When signal intensities are changing during a nuclear magnetic resonance (NMR) diffusion measurement for reasons other than diffusion, the use of monotonically increasing gradient amplitudes produces erroneous diffusion coefficients. We show that no boosted molecular mobility is observed when shuffled gradient amplitudes are applied.

Chiroptical spectroscopy of a freely diffusing single nanoparticle.

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.

Arrays of Plasmonic Nanoparticle Dimers with Defined Nanogap Spacers.

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.

Absolute diffusion measurements of active enzyme solutions by NMR.

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 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. Furthermore, 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.

Self-Assembled Phage-Based Colloids for High Localized Enzymatic Activity.

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 concentrations.

Diffusion Measurements of Swimming Enzymes with Fluorescence Correlation Spectroscopy.

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.