Single Macromolecule Research Articles

Entanglements of Macromolecules and Their Influence on Rheological and Mechanical Properties of Polymers.

Flexible macromolecules easily become entangled with neighboring macromolecules. The resulting network determines many polymer properties, including rheological and mechanical properties. Therefore, a number of experimental and modeling studies were performed to describe the relationship between the degree of entanglement of macromolecules and polymer properties. The introduction presents general information about the entanglements of macromolecule chains, collected on the basis of studies of equilibrium entangled polymers. It is also shown how the density of entanglements can be reduced. The second chapter presents experiments and models leading to the description of the movement of a single macromolecule. The next part of the text discusses how the rheological properties change after partial disentangling of the polymer. The results on the influence of the degree of chain entanglement on mechanical properties are presented.

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and opensource Brick-MIC platform will be a valuable tool for many laboratories worldwide.

Chemical Production of Cytotoxic Bispecific Antibodies Using the Ugi Multicomponent Reaction.

Bispecific antibodies (bsAbs) have recently emerged as a promising platform for the treatment of several conditions, most importantly cancer. Based on the combination of two different antigen-binding motifs in a single macromolecule; bsAbs can either display the combined characteristics of their parent antibodies, or new therapeutic features, inaccessible by the sole combination of two distinct antibodies. While bsAbs are traditionally produced by molecular biology techniques, the chemical development of bsAbs holds great promises and strategies have just begun to surface. In this context, we took advantage of a chemical strategy based on the use of the Ugi reaction for the site-selective conjugation of whole antibodies and coupled the resulting conjugates in a bioorthogonal manner with Fab fragments, derived from various antibodies. We thus managed to produce five different bsAbs with 2 : 1 valency, with yields ranging from 20 % to 48 %, and showed that the affinity of the parent antibody was preserved in all bsAbs. We further demonstrated the interest of our strategy by producing two other bsAbs behaving as cytotoxic T cell engagers with IC50 values in the picomolar range in vitro.

A validation strategy to assess the role of phase separation as a determinant of macromolecular localization

Liquid-liquid phase separation (LLPS) of putative assembly scaffolds has been proposed to drive the biogenesis of membraneless compartments. LLPS scaffolds are usually identified through invitro LLPS assays with single macromolecules (homotypic), but the predictive value of these assays remains poorly characterized. Here, we apply a strategy to evaluate the robustness of homotypic LLPS assays. When applied to the chromosomal passenger complex (CPC), which undergoes LLPS invitro and localizes to centromeres to promote chromosome biorientation, LLPS propensity invitro emerged as an unreliable predictor of subcellular localization. Invitro CPC LLPS in aqueous buffers was enhanced by commonly used crowding agents. Conversely, diluted cytomimetic media dissolved condensates of the CPC and of several other proteins. We also show that centromeres do not seem to nucleate LLPS, nor do they promote local, spatially restrained LLPS of the CPC. Our strategy can be adapted to purported LLPS scaffolds of other membraneless compartments.

Electrophoretic Deposition Interferometric Scattering Mass Photometry.

Interferometric scattering microscopy (iSCAT) has rapidly developed as a quantitative tool for the label-free detection of single macromolecules and nanoparticles. In practice, this measurement records the interferometric scattering signal of individual nanoparticles in solution as they land and stick on a coverslip, exhibiting an intensity that varies linearly with particle volume and an adsorption rate that reflects the solution-phase transport kinetics of the system. Together, such measurements provide a multidimensional gauge of the particle size and concentration in solution over time. However, the landing kinetics of particles in solution also manifest a measurement frequency limitation imposed by the slow long-range mobility of particle diffusion to the measurement interface. Here we introduce an effective means to overcome the inherent diffusion-controlled sampling limitation of spontaneous mass photometry. We term this methodology electrophoretic deposition interferometric scattering microscopy (EPD-iSCAT). This approach uses a coverslip supporting a conductive thin film of indium tin oxide (ITO). Charging this ITO film to a potential of around +1 V electrophoretically draws charged nanoparticles from solution and binds them in the focal plane of the microscope. Regulating this potential offers a direct means of controlling particle deposition. Thus, we find for a 0.1 nM solution of 50 nm polystyrene nanoparticles that the application of +1 V to an EPD-iSCAT coverslip assembly drives an electrophoretic deposition rate constant of 1.7 s-1 μm-2 nM-1. Removal of the potential causes deposition to cease. This user control of EPD-iSCAT affords a means to apply single-molecule mass photometry to monitor long-term changes in solution, owing to slow kinetic processes. In contrast with conventional coverslips chemically derivatized with charged thin films, EPD-iSCAT maintains a deposition rate that varies linearly with the bulk concentration.

Non‐linear functional polymers containing selective/cleavable bonds: Synthesis and their biomedical applications

AbstractThe growing interest in functional materials has resulted in the emergence of smart polymers, demonstrating practical performance in various applications especially for biomedical purposes. The properties of functional polymers are mainly determined by the presence of functional groups that differ from those in the main chains. While some polymers are naturally active and considered functional, others require modification for enhanced impact and functionality. Many functional polymers typically have a linear backbone, but very recently the attention has been shifted towards those with specific topologies and architecture, for example, bottlebrushes, stars, dendritic polymers, and gels. Over the past few years, there has been a rising emphasis on integrating selective bonds into complex polymer structures to enhance chain scission in single macromolecules. This review captures the most recent and promising approaches to the design of non‐linear functional polymers containing selective/cleavable bonds and discusses the potential of these materials for biomedical applications.

Superfluorinated, Highly Water-Soluble Polyphosphazenes as Potential 19F Magnetic Resonance Imaging (MRI) Contrast Agents.

"Hot spot" 19F magnetic resonance imaging (MRI) has garnered significant attention recently for its ability to image various disease markers quantitatively. Unlike conventional gadolinium-based MRI contrast agents, which rely on proton signal modulation, 19F-MRI's direct detection has a unique advantage in vivo, as the human body exhibits a negligible background 19F-signal. However, existing perfluorocarbon (PFC) or PFC-based contrast materials suffer from several limitations, including low longitudinal relaxation rates and relatively low imaging efficiency. Hence, we designed a macromolecular contrast agent featuring a high number of magnetically equivalent 19F-nuclei in a single macromolecule, adequate fluorine nucleus mobility, and excellent water solubility. This design utilizes superfluorinated polyphosphazene (PPz) polymers as the 19F-source; these are modified with sodium mercaptoethanesulfonate (MESNa) to achieve water solubility exceeding 360 mg/mL, which is a similar solubility to that of sodium chloride. We observed substantial signal enhancement in MRI with these novel macromolecular carriers compared to non-enhanced surroundings and aqueous trifluoroacetic acid (TFA) used as a positive control. In conclusion, these novel water-soluble macromolecular carriers represent a promising platform for future MRI contrast agents.

Methotrexate Inhibits the Binding of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Receptor Binding Domain to the Host-Cell Angiotensin-Converting Enzyme-2 (ACE-2) Receptor.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus mutates, finding effective drugs becomes more challenging. In this study, we use ultrasensitive frequency locked microtoroid optical resonators in combination with in silico screening to search for COVID-19 drugs that can stop the virus from attaching to the human angiotensin-converting enzyme 2 (hACE2) receptor in the lungs. We found 29 promising candidates that could block the binding site and selected four of them that were likely to bind very strongly. We tested three of these candidates using frequency locked optical whispering evanescent resonator (FLOWER), a label-free sensing method based on microtoroid resonators. FLOWER has previously been used for sensing single macromolecules. Here we show, for the first time, that FLOWER can provide accurate binding affinities and sense the inhibition effect of small molecule drug candidates without labels, which can be prohibitive in drug discovery. One of the candidates, methotrexate, showed binding to the spike protein 1.8 million times greater than that to the receptor binding domain (RBD) binding to hACE2, making it difficult for the virus to enter cells. We tested methotrexate against different variants of the SARS-CoV-2 virus and found that it is effective against all four of the tested variants. People taking methotrexate for other conditions have also shown protection against the original SARS-CoV-2 virus. Normally, it is assumed that methotrexate inhibits the replication and release of the virus. However, our findings suggest that it may also block the virus from entering cells. These studies additionally demonstrate the possibility of extracting candidate ligands from large databases, followed by direct receptor-ligand binding experiments on the best candidates using microtoroid resonators, thus creating a workflow that enables the rapid discovery of new drug candidates for a variety of applications.

Particle trapping with optical nanofibers: a review [Invited

Optical trapping has proven to be an efficient method to control particles, including biological cells, single biological macromolecules, colloidal microparticles, and nanoparticles. Multiple types of particles have been successfully trapped, leading to various applications of optical tweezers ranging from biomedical through physics to material sciences. However, precise manipulation of particles with complex composition or of sizes down to nanometer-scales can be difficult with conventional optical tweezers, and an alternative manipulation tool is desirable. Optical nanofibers, that is, fibers with a waist diameter smaller than the propagating wavelength of light, are ideal candidates for optical manipulation due to their large evanescent field that extends beyond the fiber surface. They have the added advantages of being easily connected to a fibered experimental setup, being simple to fabricate, and providing strong electric field confinement and intense magnitude of evanescent fields at the nanofiber's surface. Many different particles have been trapped, rotated, transported, and assembled with such a system. This article reviews particle trapping using optical nanofibers and highlights some challenges and future potentials of this developing topic.

Registration of Functioning of a Single Horseradish Peroxidase Macromolecule with a Solid-State Nanopore

Currently, nanopore-based technology for the determination of the functional activity of single enzyme molecules continues its development. The use of natural nanopores for studying single enzyme molecules is known. At that, the approach utilizing artificial solid-state nanopores is also promising but still understudied. Herein, we demonstrate the use of a nanotechnology-based approach for the investigation of the enzymatic activity of a single molecule of horseradish peroxidase with a solid-state nanopore. The artificial 5 nm solid-state nanopore has been formed in a 40 nm thick silicon nitride structure. A single molecule of HRP has been entrapped into the nanopore. The activity of the horseradish peroxidase (HRP) enzyme molecule inserted in the nanopore has been monitored by recording the time dependence of the ion current through the nanopore in the course of the reaction of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation reaction. We have found that in the process of ABTS oxidation in the presence of 2.5 mM hydrogen peroxide, individual HRP enzyme molecules are able to retain activity for approximately 700 s before a decrease in the ion current through the nanopore, which can be explained by structural changes of the enzyme.

Lysozyme/tripolyphosphate complex coacervates: Properties, curcumin encapsulation and antibacterial activity

Complex coacervates based on the combination of two oppositely charged biopolymers have been proposed as encapsulating and protective platforms for bioactive agents in food science. Lysozyme, an enzymatic protein used as a natural food preservative, was combined with other macromolecules (proteins and polysaccharides) to generate coacervate formulations. In this study, the design of coacervate-like structures based on lysozyme was extended through their combination with multivalent tripolyphosphate anion, a non-macromolecular food additive. It was observed that direct mixing led to the formation of complex coacervates under certain ranges of pH, ionic strength, and component concentrations. Coacervates presented a suitable capacity to encapsulate poorly water-soluble curcumin. More interestingly, coacervates improved the chemical stability (under long-term storage and light irradiation) and antibacterial activity of curcumin. Therefore, these single macromolecule coacervates could be exploited as an active carrier platform for encapsulation in food applications.

Adding Konjac Glucomannan for Enhancing the Whole Spraying Performance on Superhydrophobic and Hydrophilic Surfaces.

Spraying is a common way to coat solutions onto surfaces evenly. Improving spraying effectiveness can avoid wasting solutions and reduce pollution. In this study, a trace amount of natural polysaccharide, konjac glucomannan (KGM), was added into solutions to regulate the spraying performances including the breakup of liquid jets, size of produced droplets, and collision and spreading of droplets on both superhydrophobic and hydrophilic surfaces. The shear viscosity, extensive viscosity, and surface tension of the KGM solutions were tested. The results of spraying experiments showed that adding KGM inhibited the liquid jet from breaking into small droplets, avoided the breakage of droplets on superhydrophobic surfaces, and promoted the spreading of liquid films on hydrophilic surfaces. The numerical simulation showed the stretching of single macromolecules and quantified the energy stored in molecular chains in a shear-dominated flow field during the spreading of droplets on surfaces and an elongational-dominated flow field during the breakage of a liquid bridge. The storage and dissipation of energy during the stretching and relaxing of KMG macromolecules were important origins of the increase in the colloid viscosity and molecular mechanisms of the effect of the KGM additive on spraying performances. This study provided an understanding and a strategy for optimization and application of spraying additives.

Degradable Bottlebrush Polypeptides and the Impact of their Architecture on Cell Uptake, Pharmacokinetics, and Biodistribution In Vivo.

Bottlebrush polymers are highly promising as unimolecular nanomedicines due to their unique control over the critical parameters of size, shape and chemical function. However, since they are prepared from biopersistent carbon backbones, most known bottlebrush polymers are non-degradable and thus unsuitable for systemic therapeutic administration. Herein, wereport the design and synthesis of novel poly(organo)phosphazene-g-poly(α-glutamate) (PPz-g-PGA) bottlebrush polymers with exceptional control over their structure and molecular dimensions (Dh ≈ 15-50 nm). These single macromolecules show outstanding aqueous solubility, ultra-high multivalency and biodegradability, making them ideal as nanomedicines. While well-established in polymer therapeutics, it has hitherto not been possible to prepare defined single macromolecules of PGA in these nanosized dimensions. A direct correlation wasobserved between the macromolecular dimensions of the bottlebrush polymers and their intracellular uptake in CT26 colon cancer cells. Furthermore, the bottlebrush macromolecular structure visibly enhanced the pharmacokinetics by reducing renal clearance and extending plasma half-lives. Real-time analysis of the biodistribution dynamics showed architecture-driven organ distribution and enhanced tumor accumulation. This work, therefore, introduces a robust, controlled synthesis route to bottlebrush polypeptides, overcoming limitations of current polymer-based nanomedicines and, in doing so, offers valuable insights into the influence of architecture on the in vivo performance of nanomedicines.

Construction of macromolecular model and analysis of oxygen absorption characteristics of Hongyang No. 2 coal mine

Understanding the molecular structure characteristics of coal from the molecular level is of great significance for realizing rational utilization and efficient transformation of coal. The molecular structure of coal samples from 1304 working face in No.13 coal seam of Hongyang No.2 Mine (HY) was studied by industrial analysis, elemental analysis, Nuclear magnetic resonance carbon spectroscopy (13C NMR) and Fourier transform infrared spectroscopy (FTIR). The results show that the aromatic compounds in coal samples of HY are mainly naphthalene ring structures. The aliphatic structure mainly consists of methyl, ethyl side chains and cycloalkanes. The ratio of aromatic bridge carbon to weekly carbon in molecular structure is 0.17. Oxygen atoms exist in the form of carbonyl, hydroxyl, and ether bonds, nitrogen atoms exist in the form of pyridine and pyrrole respectively, and sulfur atoms exist in the form of thiophene. Based on this, the planar structure model of coal macromolecule in HY is constructed, and its molecular formula is C129H88O29N2S, and its molecular weight is 2160. The structure optimization and annealing kinetics simulation of a single macromolecule model were carried out, and a stable three-dimensional coal model of HY was obtained. In addition, the oxygen absorption characteristics of coal samples in HY were studied by molecular dynamics and quantum chemistry. The results show that the adsorption capacity of the Wiser model is less than that of HY coal model. This is because, compared with the Wiser model, the content of oxygenated aliphatic hydrocarbons in the molecular structure of HY coal is higher, and the condensation degree of polycyclic aromatic hydrocarbons is lower. Due to the stronger physical adsorption capacity of hydroxyl, ether bond, and carbonyl on O2, HY Mine has stronger physical adsorption capacity on O2.

Tuning dynamic DNA- and peptide-driven self-assembly in DNA-peptide conjugates.

DNA-peptide conjugates offer an opportunity to marry the benefits of both biomolecular classes, combining the high level of programmability found with DNA, with the chemical diversity of peptides. These hybrid systems offer potential in fields such as therapeutics, nanotechnology, and robotics. Using the first DNA-β-turn peptide conjugate, we present three studies investigating the self-assembly of DNA-peptide conjugates over a period of 28 days. Time-course studies, such as these have not been previously conducted for DNA-peptide conjugates, although they are common in pure peptide assembly, for example in amyloid research. By using aging studies to assess the structures produced, we gain insights into the dynamic nature of these systems. The first study explores the influence varying amounts of DNA-peptide conjugates have on the self-assembly of our parent peptide. Study 2 explores how DNA and peptide can work together to change the structures observed during aging. Study 3 investigates the presence of orthogonality within our system by switching the DNA and peptide control on and off independently. These results show that two orthogonal self-assemblies can be combined and operated independently or in tandem within a single macromolecule, with both spatial and temporal effects upon the resultant nanostructures.

Adsorption of Wormlike Chains onto Partially Permeable Membranes.

Reversible adsorption of a single stiff wormlike macromolecule to flat membranes with various permeabilities is considered theoretically. It is shown that the adsorbed layer microstructure is significantly different from either a flexible chain or a stiff chain adsorption at a solid surface. Close to the critical point, the adsorbing wormlike chain forms a strongly anisotropic proximal layer near the membrane in addition to a nearly isotropic distal layer. The proximal layer is characterized by the algebraic monomer concentration profile, c(x)∝x-β, due to the self-similar distribution of aligned polymer loops. For a perfectly penetrable membrane, β=1 which is different from β=4/3 obtained for semiflexible chain adsorption at a solid surface. Moreover, we establish that the critical exponent for a partially permeable membrane depends on its properties (porosity w) and propose an asymptotically exact theory (based on the generalized Edwards equation) predicting this dependence, β=β(w). We also develop a scaling theory elucidating, in particular, an intricate competition of loops and tails in both proximal and distal sublayers.

Effect of Coriolis and buoyancy forces on three-dimensional flow of chemically reactive tangent hyperbolic fluid subject to variable viscosity

Thermophoresis effect has wide range of applications in electro-static precipitators and in biology for calculating single biological macro molecules, such as genomic-length DNA and HIV virus in the micro channels. Current study deal with effects of Coriolis and buoyancy forces on the three-dimensional boundary layer flow of tangent hyperbolic fluid with thermo-migration and haphazard motion of nano-sized particles. Arrhenius kind of chemical reaction is taken along an exponentially stretchable surface. The main focus of current exploration is to execute shear thinning nano-liquid flow past an exponentially rotating stretchable surface under the influence of variable viscosity, mixed convection and activation energy. We are motivated to explore the features of three-dimensional shear thinning model combined under the features of mixed convention, variable viscosity, and activation energy. The mathematical model is designed to generate PDEs and converted them into ODEs by employing fractious transformation. The numerical outcomes are exhibited via graphs by employing Bvp4c numerical technique whereas the values of skin friction coefficient are calculated by monopolizing shooting method. Characteristics of the parameters appearing in modeling like the viscosity parameter, power-law index, local Weissenberg number, mix convection parameter, rotation parameter, Prandtl number and chemical reaction parameter are comprehensively analyzed through graphical behavior. The impact of governing parameters on skin friction, heat and mass transfer rates is illustrated through tables. The detail analysis anticipates that the elevation in Weissenberg number and porosity caused decline in velocity. Further, the temperature behaves doppositely analogous to development Prandtl besides the thermophoresis parameter.

Understanding and Modeling Polymers: The Challenge of Multiple Scales

Polymer materials are multiscale systems by definition. Already the description of a single macromolecule involves a multitude of scales, and cooperative processes in polymer assemblies are governed by their interplay. Polymers have been among the first materials for which systematic multiscale techniques were developed, yet they continue to present extraordinary challenges for modellers. In this Perspective, we review popular models that are used to describe polymers on different scales and discuss scale-bridging strategies such as static and dynamic coarse-graining methods and multiresolution approaches. We close with a list of hard problems which still need to be solved in order to gain a comprehensive quantitative understanding of polymer systems.

Conformation Dynamics of Single Polymer Strands in Solution.

Conformational changes in macromolecules significantly affect their functions and assembly into high-level structures. Despite advances in theoretical and experimental studies, investigations into the intrinsic conformational variations and dynamic motions of single macromolecules remain challenging. Here, liquid-phase transmission electron microscopy enables the real-time tracking of single-chain polymers. Imaging linear polymers, synthetically dendronized with conjugated aromatic groups, in organic solvent confined within graphene liquid cells, directly exhibits chain-resolved conformational dynamics of individual semiflexible polymers. These experimental and theoretical analyses reveal that the dynamic conformational transitions of the single-chain polymer originate from the degree of intrachain interactions. In situ observations also show that such dynamics of the single-chain polymer are significantly affected by environmental factors, including surfaces and interfaces.

Low-dose shift- and rotation-invariant diffraction recognition imaging

A low-dose imaging technique which uses recognition rather than recording of a full high-resolution image is proposed. A structural hypothesis is verified by probing the object with only a few particles (photons, electrons). Each scattered particle is detected in the far field and its position on the detector is analysed by applying Bayesian statistics. Already a few detected particles are sufficient to confirm a structural hypothesis at a probability exceeding 95%. As an example, the method is demonstrated as an application in optical character recognition, where a hand-written number is recognized from a set of different written numbers. In other provided examples, the structural hypothesis of a single macromolecule is recognized from a diffraction pattern acquired at an extremely low radiation dose, less than one X-ray photon or electron per Å2, thus leaving the macromolecule practically without any radiation damage. The proposed principle of low-dose recognition can be utilized in various applications, ranging from optical character recognition and optical security elements to recognizing a certain protein or its conformation.