Single-molecule Behavior Research Articles

Understanding the Spin of Metal Complexes from a Single-Molecule Perspective.

Compared with aggregate spin behavior, single-molecule spin behavior can be accurately understood, controlled, and applied at the level of basic building blocks. The potential of single-molecule electronic and nuclear spins for monitoring and control represents a beacon of promise for the advancement of molecular spin devices, which are fabricated by connecting a single molecule between two electrodes. Metal complexes, celebrated for their superior magnetic attributes, are widely used in the devices to explore spin effects. Moreover, single-molecule electrical techniques with high signal-to-noise ratio, temporal resolution, and reliability help to understand the spin characteristics. In this review, the focus is on the devices with metal complexes, especially single-molecule magnets, and systematically present experimental and theoretical state of the art of this field at the single-molecule level, including the fundamental concepts of the electronic and nuclear spin and their basic spin effects. Then, several experimental methods developed to regulate the spin characteristics of metal complexes at single-molecule level are introduced, as well as the corresponding intrinsic mechanisms. A brief discussion is provided on the comprehensive applications and the considerable challenges of single-molecule spin devices in detail, along with a prospect on the potential future directions of this field.

Organelle Proximity Analysis for Enhanced Quantification of Mitochondria-Endoplasmic Reticulum Interactions in Single Cells via Super-Resolution Microscopy.

Mitochondria (MT) and the endoplasmic reticulum (ER) maintain lipid and calcium homeostasis through membrane contacts, particularly MT-ER contacts (MERCs), spanning distances from 10 to 50 nm. However, the variation of different distance ranges and the metabolic factors influencing this variation remain poorly understood. This study employed microfluidic chip-based super-resolution microscopy in conjunction with a Moore-Neighbor tracing-incorporated organelle proximity analysis algorithm. This approach enabled precise three-dimensional localization of single-fluorescence protein molecules within narrow and irregular membrane proximities. It achieved lateral localization precision of less than 20 nm, resulting in a minimum MERC distance of approximately 8 nm in spatial and mean distances across multiple threshold ranges. Additionally, we demonstrated that the MERC distance variation was correlated with MT size rather than ER width. The proportion of each distance range varied significantly after the stimuli. Free cholesterol showed a negative correlation with various distances, while distances of 10-30 nm were associated with glucose, glutamine, and pyruvic acid. Furthermore, the 30-40 nm range was influenced by citric acid. These results underscore the role of advanced subcellular organelle analysis in elucidating the single-molecule behavior and organelle morphology in single-cell studies.

From Bulk to Single Molecules: Surface-Enhanced Raman Scattering of Cytochrome C Using Plasmonic DNA Origami Nanoantennas.

Cytochrome C, an evolutionarily conserved protein, plays pivotal roles in cellular respiration and apoptosis. Understanding its molecular intricacies is essential for both academic inquiry and potential biomedical applications. This study introduces an advanced single-molecule surface-enhanced Raman scattering (SM-SERS) system based on DNA origami nanoantennas (DONAs), optimized to provide unparalleled insights into protein structure and interactions. Our system effectively detects shifts in the Amide III band, thereby elucidating protein dynamics and conformational changes. Additionally, the system permits concurrent observations of oxidation processes and Amide bands, offering an integrated view of protein structural and chemical modifications. Notably, our approach diverges from traditional SM-SERS techniques by de-emphasizing resonance conditions for SERS excitation, aiming to mitigate challenges like peak oversaturation. Our findings underscore the capability of our DONAs to illuminate single-molecule behaviors, even within aggregate systems, providing clarity on molecular interactions and behaviors.

Left-right Myosin-Is, Myosin1C, and Myosin1D exhibit distinct single molecule behaviors on the plasma membrane of Drosophila macrophages.

Left-right (LR) asymmetry is crucial for animal development, particularly in Drosophila where LR-asymmetric morphogenesis of organs hinges on cellular-level chirality, termed cell chirality. In this species, two class I myosins, Myosin1D (Myo1D), and Myosin1C (Myo1C), respectively determine dextral (wild type) and sinistral (mirror image) cell chirality. Previous studies demonstrated Myo1D's ability to propel F-actin in leftward circles during in vitro gliding assays, suggesting its mechanochemical role in defining dextral chirality. Conversely, Myo1C propels F-actin without exhibiting LR-directional preference in this assay, suggesting at other properties governing sinistral chirality. Given the interaction of Myo1D and Myo1C with the membrane, we hypothesized that differences in their membrane behaviors might be critical in dictating their dextral or sinistral activities. In this study, employing single-molecule imaging analyses, we investigated the dynamic behaviors of Myo1D and Myo1C on the plasma membrane. Our findings revealed that Myo1C exhibits a significantly greater proportion of slow-diffusing population compared to Myo1D. Importantly, this characteristic was contingent upon both head and tail domains of Myo1C. The distinct diffusion patterns of Myo1D and Myo1C did not exert mutual influence on each other. This divergence in membrane diffusion between Myo1D and Myo1C may be crucial for dictating cell and organ chirality.

Atomic single-molecule imaging by the confinement methods in advanced microscopy

Atomic single-molecule imaging has drawn extensive attention for exploring different behaviors and properties of small molecules. However, current techniques still face challenges, due to the thermal activity and beam sensitivity of small molecules. Recently, ångström-level spatial resolution for single molecules was successfully demonstrated using different imaging methods, including scanning tunneling microscopy, atomic force microscopy, cryogenic and transmission electron microscopy. In this perspective, we focus on different confinement strategies in these single-molecule imaging techniques, and summarize the recent studies of the structures and behaviors of single molecules. Especially, a new concept of spatial confinement at room temperature has been achieved by using microporous materials, such as zeolites, which has made it possible to fix and visualize the configurations of single molecule inside channels. In outlook, we describe what progress we can make along such a confinement strategy, and what new perspectives and discoveries we may find beyond our imagination.

Ultraviolet Organic Laser from Rhombus Microcrystal: Benefits of Single-Molecule Emission from Twisted Structure.

Light-emitting molecular crystals with efficient emission behavior are crucial for fabricating low-threshold ultraviolet organic lasers. Herein, we demonstrated a rhombus microcrystal from a fluorene-based conjugated molecule (CL-1) with robust emission behavior for an ultraviolet organic laser. Due to the synergistic effect of twisted intramolecular conformation and weak π-interaction, the CL-1 single crystal showed an extremely high photoluminescence quantum yield (PLQY) of ∼82%, due to their single-molecule excitonic behavior. Considering the diverse noncovalent interactions, CL-1 molecules easily self-assembled into the rhombus microcrystals. Finally, a low-threshold ultraviolet organic laser was fabricated with a sharp emission at 379 nm, attributed to the 0-1 vibration band of a single CL-1 molecule, also further confirming the single twisted-molecule emission in crystal states. Precisely controlling the intramolecular twisted structure and intermolecular interaction of organic conjugated molecules is a precondition to obtain robust ultraviolet emission for optoelectronic applications.

Theoretical exploration of single-molecule magnetic and single-molecule toroic behaviors in peroxide-bridged double-triangular {MII3LnIII3} (M = Ni, Cu and Zn; Ln = Gd, Tb and Dy) complexes.

Detailed state-of-the-art ab initio and density functional theory (DFT) calculations have been undertaken to understand both Single-Molecule Magnetic (SMM) and Single-Molecule Toroic (SMT) behaviors of fascinating 3d-4f {M3Ln3} triangular complexes having the molecular formula [MII3LnIII3(O2)L3(PyCO2)3](OH)2(ClO4)2·8H2O (with M = Zn; Ln = Dy (1), Tb (2) & Gd (3) and M = Cu; Ln = Dy (4), Tb (5) & Gd (6)) and [Ni3Ln3(H2O)3(mpko)9(O2)(NO3)3](ClO4)·3CH3OH·3CH3CN (Ln = Dy (7), Tb (8), and Gd (9)) [mpkoH = 1-(pyrazin-2-yl)ethanone oxime]. All these complexes possess a peroxide ligand that bridges the {LnIII3} triangle in a μ3-η3:η3 fashion and the oxygen atoms/oxime of co-ligands that connect each MII ion to the {LnIII3} triangle. Through our computational studies, we tried to find the key role of the peroxide bridge and how it affects the SMM and SMT behavior of these complexes. Primarily, ab initio Complete Active Space Self-Consistent Field (CASSCF) SINGLE_ANISO + RASSI-SO + POLY_ANISO calculations were performed on 1, 2, 4, 5, 7, and 8 to study the anisotropic behavior of each Ln(III) ion, to derive the magnetic relaxation mechanism and to calculate the LnIII-LnIII and CuII/NiII-LnIII magnetic coupling constants. DFT calculations were also performed to validate these exchange interactions (J) by computing the GdIII-GdIII and CuII/NiII-GdIII interactions in 3, 6, and 9. Our calculations explained the experimental magnetic relaxation processes and the magnetic exchange interactions for all the complexes, which also strongly imply that the peroxide bridge plays a role in the SMM behavior observed in these systems. On the other hand, this peroxide bridge does not support the SMT behavior. To investigate the effect of bridging ions in {M3Ln3} systems, we modeled a {ZnII3DyIII3} complex (1a) with a hydroxide ion replacing the bridged peroxide ion in complex 1 and considered a hydroxide-bridged {CoIII3DyIII3} complex (10) having the formula [Co3Dy3(OH)4(OOCCMe3)6(teaH)3(H2O)3](NO3)2·H2O. We discovered that as compared to the LoProp charges of the peroxide ion, the greater negative charges on the bridging hydroxide ion reduce quantum tunneling of magnetization (QTM) effects, enabling more desirable SMM characteristics and also leading to good SMT behavior.

Single-molecule electrochemical imaging of 'split waves' in the electrocatalytic (EC') mechanism.

We describe a single-molecule electrochemical imaging strategy to study the electrocatalytic (EC') mechanism. Using the fluorescent molecule ATTO647N at extremely low concentrations as the substrate, we confirmed its catalytic reduction to a nonfluorescent form in the presence of the mediator phenazine methosulfate (PMS) by imaging and counting fluorescent molecules. Conventional electrochemical current in cyclic voltammetry would not have allowed us to infer the existence of an EC' process or the PMS-mediated ATTO647N reduction. Additionally, we observed shifts in the catalytic reduction potential of ATTO647N at various mediator concentrations, which agree with the theoretical predictions by Savéant. Our work offers a new perspective on connecting single-molecule EC' behaviors with the conventional ensemble EC' mechanism, both practically and theoretically.

Visualization, Quantification, and Statistical Evaluation of Dynamics for DNA-Binding Proteins in Bacteria by Single-Molecule Tracking.

All DNA-binding proteins in vivo exist as a population of freely diffusing molecules and of DNA-bound molecules. The molecules bound to DNA can be split into specifically/tightly and nonspecifically bound proteins. Single-molecule tracking (SMT) is a method allowing to visualize protein dynamics in living cells, revealing their behavior in terms of mode of motion, diffusion coefficient/speed, change of dwell times, and unveiling preferred subcellular sites of dwelling. Bleaching-type SMT or fluorescent protein-tagged SMT involves rapid laser-induced bleaching of most fluorophore-labeled molecules. The remaining single fluorescent proteins are then continuously tracked. The trajectories of several fluorescent molecules per cell for a population of cells are analyzed and combined to permit a robust analysis of average behavior of single molecules in live cells, including analyses of protein dynamics in mutant cells or cells exposed to changes in environmental conditions.In this chapter, we describe the preparation of Bacillus subtilis cells, the recording of movies of those cells expressing a monomeric variant of a yellow fluorescent protein (mNeonGreen) fused to a protein of choice, and the subsequent curation of the movie data including the statistical analysis of the protein dynamics. We present a short overview of the analysis program SMTracker 2.0, highlighting its ability to analyze SMT data by non-expert scientists.

Substituent Positioning Effects on the Magnetic Properties of Sandwich-Type Erbium(III) Complexes with Bis(trimethylsilyl)-Substituted Cyclooctatetraenyl Ligands.

Lanthanide complexes with judiciously designed ligands have been extensively studied for their potential applications as single-molecule magnets. With the influence of ligands on their magnetic properties generally established, recent research has unearthed certain effects inherent to site differentiation due to the different types and varying numbers of substituents on the same ligand platform. Using two new sandwich-type Er(III) complexes with cyclooctatetraenyl (COT) ligands featuring two differently positioned trimethylsilyl (TMS) substituents, namely, [Li(DME)Er(COT1,5-TMS2)2]n (Er1) and [Na(DME)3][Er(COT1,3-TMS2)2] (Er2) [COT1,3-TMS2 and COT1,5-TMS2 donate 1,3- and 1,5-bis(trimethylsilyl)-substituted cyclooctatetraenyl ligands, respectively; DME = 1,2-dimethoxyethane], and with reference to previously reported [Li(DME)3][Er(COT1,4-TMS2)2] (A) and [K(DME)2][Er(COT1,4-TMS2)2] (B), any possible substituent position effects have been explored for the first time. The rearrangement of the TMS substituents from the starting COT1,4-TMS2 to COT1,3-TMS2 and COT1,5-TMS2, by way of formal migration of the TMS group, was thermally induced in the case of Er1, while for the formation of Er2, the use of Na+ in the placement of its Li+ and K+ congeners is essential. Both Er1 and Er2 display single-molecule magnetic behaviors with energy barriers of 170(3) and 172(6) K, respectively. Magnetic hysteresis loops, butterfly-shaped for Er1 and wide open for Er2, were observed up to 12 K for Er1 and 13 K for Er2. Studies of magnetic dynamics reveal the different pathways for relaxation of magnetization below 10 K, mainly by the Raman process for Er1 and by quantum tunneling of magnetization for Er2, leading to the order of magnitude difference in magnetic relaxation times and sharply different magnetic hysteresis loops.

Plasmonic Probing of Deoxyribonucleic Acid Hybridization at the Single Base Pair Resolution.

Partial DNA duplex formation greatly impacts the quality of DNA hybridization and has been extensively studied due to its significance in many biological processes. However, traditional DNA sensing methods suffer from time-consuming amplification steps and hinder the acquisition of information about single-molecule behavior. In this work, we developed a plasmonic method to probe the hybridization process at a single base pair resolution and study the relationship between the complementarity of DNA analytes and DNA hybridization behaviors. We measured single-molecule hybridization events with Au NP-modified ssDNA probes in real time and found two hybridization adsorption events: stable and transient adsorption. The ratio of these two hybridization adsorption events was correlated with the length of the complementary sequences, distinguishing DNA analytes from different complementary sequences. By using dual incident angle excitation, we recognized different single-base complementary sequences. These results demonstrated that the plasmonic method can be applied to study partial DNA hybridization behavior and has the potential to be incorporated into the identification of similar DNA sequences, providing a sensitive and quantitative tool for DNA analysis.

Probing conformational kinetics of catalase with and without magnetic field by single-entity collision electrochemistry

The conformational motions of enzymes are crucial for their catalytic activities, but these fluctuations are usually spontaneous and unsynchronized and thus difficult to obtain from ensemble-averaged measurements. Here, we employ label-free single-entity electrochemical measurements to monitor in real time the fluctuating enzymatic behavior of single catalase molecules toward the degradation of hydrogen peroxide. By probing the electrochemical signals of single catalase molecules at a carbon nanoelectrode, we were able to observe three distinct current traces that could be attributed to conformational changes on the sub-millisecond timescale. Whereas, nearly uniform single long peaks were observed for single catalase molecules under a moderate magnetic field due to the restricted conformational changes of catalase. By combining high-resolution current signals with a multiphysics simulation model, we studied the catalytic kinetics of catalase with and without a magnetic field, and further estimated the maximum catalytic rate and conformational transition rate. This work introduces a new complementary approach to existing single-molecule enzymology, giving further insight into the enzymatic reaction mechanism.

"One-Pot" In Situ Tandem Reaction─Dy(III) Coordination-Catalyzed Multicomponent Condensation of Salicylaldehyde Derivatives to Obtain Ketals.

It is difficult to subject simple reaction starting materials to a "one-pot" in situ tandem reaction without post-treatment under mild reaction conditions to obtain multimers with complex structural linkages. In organic synthesis, acetal reactions are often used to protect derivatives containing carbonyl functional groups. Therefore, acetal products tend to have very low stability, and performing multi-step condensation to obtain complex multimeric products is difficult. Herein, we achieved the first efficient multiple condensation of o-vanillin derivatives using Dy(OAc)3·6H2O undergoing a "one-pot" in situ tandem reaction under mild solvothermal conditions to obtain a series of dimers (I and II, clusters 1 and 2) and trimers (I and II, clusters 3 and 4). When methanol or ethanol is used as the solvent, the alcoholic solvent participates in acetal and dehydration reactions to obtain dimers (I and II). Surprisingly, when using acetonitrile as the reaction solvent, the o-vanillin derivatives undergo acetal and dehydration reactions to obtain trimers (I and II). In addition, clusters 1-4 all showed distinct single-molecule magnetic behaviors under zero-field conditions. To the best of our knowledge, this is the first time that multiple acetal reactions catalyzed by coordination-directed catalysis under "one-pot" conditions have been realized, opening a new horizon for the development of fast, facile, green, and efficient synthetic methods for complex compounds.

Transient Adsorption Behavior of Single Fluorophores on an Electrode-Supported Nanobubble.

Here we report the use of a Langmuir isotherm model to analyze and better understand the dynamic adsorption and desorption behavior of single fluorophore molecules at the surface of a hydrogen nanobubble supported on an indium tin oxide (ITO) electrode. Three rhodamine dyes, rhodamine 110 (R110, positively charged), rhodamine 6G (R6G, positively charged), and sulforhodamine G (SRG, negatively charged) were chosen for this study. The use of the Langmuir isotherm model allows us to determine the equilibrium constant and the rate constants for the adsorption and desorption processes. Of the three fluorophores used in this study, SRG was found to have the greatest equilibrium constant. No significant potential dependence was observed on the adsorption characteristics, which suggests the nanobubble size, geometry, and surface properties are relatively constant within the range of potentials used in this study. Our results suggest that the use of the Langmuir isotherm model is a valid and useful means for probing and better understanding the unique adsorption behavior of fluorophores at surface-supported nanobubbles.

Flexible Glass-Based Hybrid Nanofluidic Device to Enable the Active Regulation of Single-Molecule Flows.

Single-molecule studies offer deep insights into the essence of chemistry, biology, and materials science. Despite significant advances in single-molecule experiments, the precise regulation of the flow of single small molecules remains a formidable challenge. Herein, we present a flexible glass-based hybrid nanofluidic device that can precisely block, open, and direct the flow of single small molecules in nanochannels. Additionally, this approach allows for real-time tracking of regulated single small molecules in nanofluidic conditions. Therefore, the dynamic behaviors of single small molecules confined in different nanofluidic conditions with varied spatial restrictions are clarified. Our device and approach provide a nanofluidic platform and mechanism that enable single-molecule studies and applications in actively regulated fluidic conditions, thus opening avenues for understanding the original behavior of individual molecules in their natural forms and the development of single-molecule regulated chemical and biological processes in the future.

DNA-Programed Plasmon Rulers Decrypt Single-Receptor Dimerization on Cell Membrane.

Decrypting the dynamics of receptor dimerization on cell membranes bears great importance in identifying the mechanisms regulating diverse cellular activities. In this regard, long-term monitoring of single-molecule behavior during receptor dimerization allows deepening insight into the dimerization process and tracking of the behavior of individual receptors, yet this remains to be realized. Herein, real-time observation of the receptor tyrosine kinases family (RTKs) at single-molecule level based on plasmon rulers was achieved for the first time, which enabled precise regulation and dynamic monitoring of the dimerization process by DNA programming with excellent photostability. Additionally, those nanoprobes demonstrated substantial application in the regulation of RTKs protein dimerization/phosphorylation and activation of downstream signaling pathways. The proposed nanoprobes hold considerable potential for elucidating the molecular mechanisms of single-receptor dimerization as well as the conformational transitions upon dimerization, providing a new paradigm for the precise manipulation and monitoring of specific single-receptor crosslink events in biological systems.

Optical Trapping of a Single Molecule of Length Sub-1 nm in Solution

Optical Trapping of a Single Molecule of Length Sub-1 nm in Solution

Large Heterogeneity Observed in Single Molecule Measurements of Intramolecular Electron Transfer Rates through DNA

Abstract Current understanding of electron transfer (ET) kinetics is largely based on ensemble measurements that obscure the underlying single-molecule behavior. We previously reported systematic distance-dependent single molecule ET measurements that show a large heterogeneity. Here we study and discuss this heterogeneity in more detail and suggest that it is a rather fundamental phenomenon that is difficult to explain solely by the distribution of DNA conformations. This heterogeneity may arise from the uniqueness of each molecule, or it may reflect the uncertainty of ET kinetics.

First-principles study of chalcogen-bonded self-assembly structures on silicene: Some insight into the fabrication of molecular architectures on surfaces through chalcogen bonding

In this work, the self-assembly structures of a set of chalcogenazolo and chalcogendiazolo derivatives on silicene surface were studied in detail using first-principles DFT calculations, with the aim of providing fundamental insight into the fabrication of supramolecular architectures on surfaces through chalcogen bonding (ChB). The adsorption behaviors of single molecules on the facet were firstly investigated and, subsequently, the dimeric and trimeric complexes consisting of different self-assembled patterns, such as wire-, ribbon- and herringbone-like structures, were examined either in gas phase or on the surface. The assemblies of these molecules are driven by not only intermolecular Ch∙∙∙N bonds but also vdW interactions between the molecules and the substrate. Intermolecular Te∙∙∙N contacts appear to be the primary interactions in the self-assembly systems, although the Te-congeners show an enhanced affinity on the surface. In contrast, intermolecular S∙∙∙N contacts are somewhat weaker with comparable strength to interfacial interactions with the substrate.

Ferroelectric Single-Molecule Magnet with Toroidal Magnetic Moments.

Materials that coexist magnetic and electric properties on the molecular scale in single‐molecule magnets (SMMs) with peculiar quantum behaviors have promise in molecular electronics and spintronics. Nevertheless, such molecular materials are limited in potentials because their magnetic signal cannot be transformed into an electrical signal through magnetoresistance or Hall effects for their high insulativity. The discovery of an entirely new material, ferroelectric SMMs (FE SMMs) is reported. This FE SMM also shows single‐molecule magnetic behaviors, toroidal magnetic moments, and room‐temperature ferroelectricity. The toroidal moment is formed by a vortex distribution of magnetic dipoles in triangular Dy3 clusters. The analysis of ac magnetic susceptibility reveals the coexistence of three distinct magnetic relaxation processes at low temperatures. The ferroelectricity is introduced by incorporating polar alcohol molecules in the structure, which is confirmed by the X‐ray diffraction and optical second harmonic generation (SHG) measurements. Moreover, the dielectric measurements reveal a ferroelectric‐to‐ferroelectric phase transition around 150 K due to the symmetry change from Pc to Pna21. The coexistence of toroidal moment and ferroelectricity along with quantum magnetism in the rare‐earth single‐molecule magnets yields a unique class of multiferroics.