Biophysical Applications Research Articles

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual‐Functional Nucleotide Probes

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5‐heterocycle‐modified environment‐sensitive 2′‐deoxyuridine‐5′‐triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual‐app probes, combining a fluorophore with X‐ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real‐time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next‐generation nucleotide probe development.

Recycling Materials for Sustainable DNA Origami Manufacturing.

DNA origami nanotechnology has great potential in multiple fields including biomedical, biophysical, and nanofabrication applications. However, current production pipelines lead to single-use devices incorporating a small fraction of initial reactants, resulting in a wasteful manufacturing process. Here, we introduce two complementary approaches to overcome these limitations by recycling the strand components of DNA origami nanostructures (DONs). We demonstrate reprogramming entire DONs into new devices, reusing scaffold strands. We validate this approach by reprogramming DONs with complex geometries into each other, using their distinct geometries to verify successful scaffold recycling. We reprogram one DON into a dynamic structure and show both pristine and recycled structures display similar properties. Second, we demonstrate the recovery of excess staple strands postassembly and fold DONs with these recycled strands, showing these structures exhibit the expected geometry and dynamic properties. Finally, we demonstrate the combination of both approaches, successfully fabricating DONs solely from recycled DNA components.

Capillary-Assisted Assembly of Polymer Gel-Supported Lipid Bilayers.

The polymer-supported lipid bilayer represents an attractive supramolecular assembly in numerous biophysical and bioanalytical applications. The assembly of polymer-supported membranes with a polymer layer thickness of just a few nanometers is now well-established, but bilayer properties in such a membrane architecture are still influenced by the nearby solid substrate. Polymer-supported lipid bilayer systems with a several micrometers thick polymer layer will overcome this shortcoming. However, formation of a fluid lipid bilayer on a fully hydrated, micrometer thick polymer film using traditional methods (e.g., vesicle fusion and lipid monolayer deposition techniques) remains a challenging task due to the rather unfavorable interfacial conditions for bilayer formation in such a system. Here, we report for the first time on the facile capillary-assisted formation of a lipid bilayer on the surface of a fully hydrated, several micrometers thick polyacrylamide (PAA) gel, in which forced molecular crowding of lipids at the air-water interface of the capillary results in monolayer instability and collapse, thereby forming a lipid bilayer on the top of the polymer gel inside the capillary. Stable bilayer attachment on the surface of the polymer gel can be achieved via physisorption or specific chemical linkages (tethering) on both cross-linked and non-cross-linked PAA films. Unlike the traditional solid-supported lipid bilayer (SLB), the lipid lateral diffusion in the polymer gel-supported lipid bilayer is not anymore perturbed by a solid substrate. Instead, more like a plasma membrane, it is mainly influenced by the properties of the underlying polymer and the nature/distribution of polymer-bilayer attachments. Polymer gel-supported lipid bilayers built using the capillary-assisted assembly approach show attractive self-healing properties, resulting in superior long-term stability relative to the SLB. We hypothesize that the described capillary-assisted assembly method can be applied to a wide range of polymeric materials and lipid compositions, opening exciting opportunities as an advanced model membrane system.

DIB‐BOT: An Open‐Source Hardware Approach for Plate‐Integrated Droplet Interface Bilayer Deposition

AbstractDroplet interface bilayers (DIBs) offer a controlled lipid environment for studying membrane‐bound processes, with applications in artificial cells, biosensing, and biophysics. Current DIB fabrication faces challenges due to time‐consuming processes and specialized equipment, limiting scale‐up and hindering statistical significance in single‐molecule assays. This research introduces “DIB‐BOT,” an open‐source solution combining a nanoinjector and a 3D printer. DIB‐BOT enables rapid, reproducible DIB fabrication, overcoming manual limitations. Using off‐the‐shelf components, DIB‐BOT ensures high spatial reproducibility, minimal user input, and scalable experiments. The system's utility is demonstrated through pairwise droplet assembly and a fluorescence plate‐reader assay. Compared to manual fabrication, DIB‐BOT shows a 10‐fold reduction in droplet volume error, a threefold reduction in positional error, and 100% droplet yield. This method lowers entry barriers to DIB research, expanding its applications and enhancing data quality.

AI-based Prediction of Protein Corona Composition on DNA Nanostructures.

DNA nanotechnology has emerged as a powerful approach to engineering biophysical tools, therapeutics, and diagnostics because it enables the construction of designer nanoscale structures with high programmability. Based on DNA base pairing rules, nanostructure size, shape, surface functionality, and structural reconfiguration can be programmed with a degree of spatial, temporal, and energetic precision that is difficult to achieve with other methods. However, the properties and structure of DNA constructs are greatly altered in vivo due to spontaneous protein adsorption from biofluids. These adsorbed proteins, referred to as the protein corona, remain challenging to control or predict, and subsequently, their functionality and fate in vivo are difficult to engineer. To address these challenges, we prepared a library of diverse DNA nanostructures and investigated the relationship between their design features and the composition of their protein corona. We identified protein characteristics important for their adsorption to DNA nanostructures and developed a machine-learning model that predicts which proteins will be enriched on a DNA nanostructure based on the DNA structures' design features and protein properties. Our work will help to understand and program the function of DNA nanostructures in vivo for biophysical and biomedical applications.

Switchable optical trapping and manipulation enabled by polarization-modulated multifunctional phase-change metasurfaces

Optical trapping, a cutting-edge methodology, is pivotal for contactlessly controlling and exploring microscopic objects. However, it encounters formidable challenges such as multiparticle trapping, flexible control, and seamless integration. Here, we employ a polarization-modulated multi-foci technique for versatile nanoparticle trapping using multifunctional metasurfaces relying on geometric phase. Numerical simulations demonstrate the generation of two focused spots with orthogonal polarization distributions through our metasurfaces when illuminated with linearly polarized light, with their polarization distributions be interchanged by orthogonally switching the incident polarizations. We extend this design to an array of multi-foci metasurface tweezers modulated by polarization, highlighting the versatility and robustness of our approach. Furthermore, we demonstrate the simultaneous generation of two distinct focusing cylindrical vector beams using a monolayer metasurface, showcasing the two vector beams possess the interchange ability of their polarization distributions. By leveraging the Maxwell stress tensor, we assess the distinct contributions of the focused beams to longitudinal and transverse optical forces on SiO2 spheres, validating diverse trapping and manipulation behaviors for nanoparticles with the proposed metasurface designs. By manipulating the phase states of Sb2S3 nanopillars, binary-switchable optical trapping and manipulation are facilitated for all proposed metasurface tweezers. Our work underscores the efficacy of polarization-modulation multifunctional metasurface tweezers in consolidating multiple trapping tasks into a single device, paving the way for innovative lab-on-a-chip optical trapping applications in biophysics, nanotechnology, and photonics.

Fast, approximation-free molecular simulation of the SPC/Fw water model using non-reversible Markov chains

In a world made of atoms, computer simulations of molecular systems such as proteins in water play an enormous role in science. Software packages for molecular simulation have been developed for decades. They all discretize Hamilton’s equations of motion and treat long-range potentials through cutoffs or discretization of reciprocal space. This introduces severe approximations and artifacts that must be controlled algorithmically. Here, we bring to fruition a paradigm for molecular simulation that relies on modern concepts in statistics to explore the thermodynamic equilibrium with an exact and efficient non-reversible Markov process. It is free of all discretizations, approximations, and cutoffs. We explicitly demonstrate that this approach reaches a break-even point with traditional molecular simulation performed at high precision, but without any of its approximations. We stress the potential of our paradigm for crucial applications in biophysics and other fields, and as a practical approach to molecular simulation. We set out a strategy to reach our goal of rigorous molecular simulation.

Statistical Mechanics Approaches for Studying Temperature and Rate Effects in Multistable Systems

Systems with a multistable energy landscape are widespread in physics, biophysics, technology, and materials science. They are strongly influenced by thermal fluctuations and external mechanical actions that can be applied at different rates, moving the system from equilibrium to non-equilibrium regimes. In this paper, we focus on a simple system involving a single breaking phenomenon to describe the various theoretical approaches used to study these problems. To begin with, we propose the exact solution at thermodynamic equilibrium based on the calculation of the partition function without approximations. We then introduce the technique of spin variables, which is able to simplify the treatment even for systems with a large number of coordinates. We then analyze the energy balance of the system to better understand its underlying physics. Finally, we introduce a technique based on transition state theory useful for studying the non-equilibrium dynamical regimes of these systems. This method is appropriate for the evaluation of rate effects and hysteresis loops. These approaches are developed for both the Helmholtz ensemble (prescribed extension) and the Gibbs ensemble (applied force) of statistical mechanics. The symmetry and duality of these two ensembles is discussed in depth. While these techniques are used here for a simple system with theoretical purposes, they can be applied to complex systems of interest for several physical, biophysical, and technological applications.

Cellular and Biophysical Applications of Genetic Code Expansion.

Despite their diverse functions, proteins are inherently constructed from a limited set of building blocks. These compositional constraints pose significant challenges to protein research and its practical applications. Strategically manipulating the cellular protein synthesis system to incorporate novel building blocks has emerged as a critical approach for overcoming these constraints in protein research and application. In the past two decades, the field of genetic code expansion (GCE) has achieved significant advancements, enabling the integration of numerous novel functionalities into proteins across a variety of organisms. This technological evolution has paved the way for the extensive application of genetic code expansion across multiple domains, including protein imaging, the introduction of probes for protein research, analysis of protein-protein interactions, spatiotemporal control of protein function, exploration of proteome changes induced by external stimuli, and the synthesis of proteins endowed with novel functions. In this comprehensive Review, we aim to provide an overview of cellular and biophysical applications that have employed GCE technology over the past two decades.

Capillary-based, multifunctional manipulation of particles and fluids via focused surface acoustic waves

Surface acoustic wave (SAW)-enabled acoustofluidic technologies have recently attracted increasing attention for applications in biology, chemistry, biophysics, and medicine. Most SAW acoustofluidic devices generate acoustic energy which is then transmitted into custom microfabricated polymer-based channels. There are limited studies on delivering this acoustic energy into convenient commercially-available glass tubes for manipulating particles and fluids. Herein, we have constructed a capillary-based SAW acoustofluidic device for multifunctional fluidic and particle manipulation. This device integrates a converging interdigitated transducer to generate focused SAWs on a piezoelectric chip, as well as a glass capillary that transports particles and fluids. To understand the actuation mechanisms underlying this device, we performed finite element simulations by considering piezoelectric, solid mechanic, and pressure acoustic physics. This experimental study shows that the capillary-based SAW acoustofluidic device can perform multiple functions including enriching particles, patterning particles, transporting particles and fluids, as well as generating droplets with controlled sizes. Given the usefulness of these functions, we expect that this acoustofluidic device can be useful in applications such as pharmaceutical manufacturing, biofabrication, and bioanalysis.

Applications of ArcticDEM for measuring volcanic dynamics, landslides, retrogressive thaw slumps, snowdrifts, and vegetation heights

Topographical changes are of fundamental interest to a wide range of Arctic science disciplines faced with the need to anticipate, monitor, and respond to the effects of climate change, including geohazard management, glaciology, hydrology, permafrost, and ecology. This study demonstrates several geomorphological, cryospheric, and biophysical applications of ArcticDEM – a large collection of publicly available, time-dependent digital elevation models (DEMs) of the Arctic. Our study illustrates ArcticDEM's applicability across different disciplines and five orders of magnitude of elevation derivatives, including measuring volcanic lava flows, ice cauldrons, post-failure landslides, retrogressive thaw slumps, snowdrifts, and tundra vegetation heights. We quantified surface elevation changes in different geological settings and conditions using the time series of ArcticDEM. Following the 2014–2015 Bárðarbunga eruption in Iceland, ArcticDEM analysis mapped the lava flow field, and revealed the post-eruptive ice flows and ice cauldron dynamics. The total dense-rock equivalent (DRE) volume of lava flows is estimated to be (1431 ± 2) million m3. Then, we present the aftermath of a landslide in Kinnikinnick, Alaska, yielding a total landslide volume of (400 ± 8) × 103 m3 and a total area of 0.025 km2. ArcticDEM is further proven useful for studying retrogressive thaw slumps (RTS). The ArcticDEM-mapped RTS profile is validated by ICESat-2 and drone photogrammetry resulting in a standard deviation of 0.5 m. Volume estimates for lake-side and hillslope RTSs range between 40,000 ± 9000 m3 and 1,160,000 ± 85,000 m3, highlighting applicability across a range of RTS magnitudes. A case study for mapping tundra snow demonstrates ArcticDEM's potential for identifying high-accumulation, late-lying snow areas. The approach proves effective in quantifying relative snow accumulation rather than absolute values (standard deviation of 0.25 m, bias of −0.41 m, and a correlation coefficient of 0.69 with snow depth estimated by unmanned aerial systems photogrammetry). Furthermore, ArcticDEM data show its feasibility for estimating tundra vegetation heights with a standard deviation of 0.3 m (no bias) and a correlation up to 0.8 compared to the light detection and ranging (LiDAR). The demonstrated capabilities of ArcticDEM will pave the way for the broad and pan-Arctic use of this new data source for many disciplines, especially when combined with other imagery products. The wide range of signals embedded in ArcticDEM underscores the potential challenges in deciphering signals in regions affected by various geological processes and environmental influences.

Assembling large numbers of cell-mimetic giant vesicles in physiological saline for biophysical applications

Assembling large numbers of cell-mimetic giant vesicles in physiological saline for biophysical applications

Biophysical insights into nanomaterial-induced DNA damage: mechanisms, challenges, and future directions

<p>Nanomaterials have garnered significant attention due to their unique properties and wide-ranging applications in medicine and biophysics. However, their interactions with biological systems, particularly DNA, raise critical concerns about genotoxicity and potential long-term health risks. This review delves into the biophysical mechanisms underlying nanomaterial-induced DNA damage, highlighting recent insights, current challenges, and future research directions. We explore how the physicochemical properties of nanomaterials influence their interaction with DNA, the pathways through which they induce damage, and the biophysical methods employed to study these processes.</p>

Electro-thermal analysis of hybrid fluid through wavy divergent channel

In the bio-medical domains, the transportation of physiological fluids within the human body like chyme movement in the gastrointestinal tract, bolus through the esophagus, blood circulation in the small vessels and heart chambers etc., where the channels are nonuniform in nature. Due to diverse applications of nonuniform channel, in the current mathematical analysis the peristaltic flow of hybrid fluid (containing particular nanoparticles) under an applied axial electric field through the nonuniform channel is presented. To observe hybrid fluid performance, a set of modified hybrid fluid that comprise nanoparticles of titanium dioxide, aluminum trioxide and copper with water as a base fluid is considered in the analysis. Taking into consideration the long wavelength and low Reynolds number assumptions. Poisson-Boltzmann equations are linearized by the Debye-Hückel approximation. Solutions for the axial velocity, average flow rate, pressure gradient, pumping phenomena, heat transfer phenomena and volumetric fraction are obtained analytically. The physical impacts of numerous embedded parameters on the above-mentioned features are argued through graphs which are plotted by Mathematica 11.0 Software. Additionally, the influence of porous medium and nonuniform nature of flow geometry are also discussed in detail. The study could be useful in medical as well as the industrial domains since the electro-osmotic phenomenon is observed throughout the human body where through the osmosis, the body absorbs the nutrients from the food and toxins are removed from the blood stream. Also the nanofluids are widely used in medicines for drug delivery. Also in industries, electro-osmosis has many applications in microchips, biophysics, microfluidics, gas and oil manufacture and separations through the membranes etc.

Generalized Michaelis-Menten rate law with time-varying molecular concentrations.

The Michaelis-Menten (MM) rate law has been the dominant paradigm of modeling biochemical rate processes for over a century with applications in biochemistry, biophysics, cell biology, systems biology, and chemical engineering. The MM rate law and its remedied form stand on the assumption that the concentration of the complex of interacting molecules, at each moment, approaches an equilibrium (quasi-steady state) much faster than the molecular concentrations change. Yet, this assumption is not always justified. Here, we relax this quasi-steady state requirement and propose the generalized MM rate law for the interactions of molecules with active concentration changes over time. Our approach for time-varying molecular concentrations, termed the effective time-delay scheme (ETS), is based on rigorously estimated time-delay effects in molecular complex formation. With particularly marked improvements in protein-protein and protein-DNA interaction modeling, the ETS provides an analytical framework to interpret and predict rich transient or rhythmic dynamics (such as autogenously-regulated cellular adaptation and circadian protein turnover), which goes beyond the quasi-steady state assumption.

Cancerous cell viability affected by synergism between electric pulses and a low dose of silver nanoparticle: An adaptive neuro-fuzzy inference system

In the current study, applying simultaneously electroporation and silver nanoparticles (SNPs) are considered. Moreover, one restriction normally assigned to such nanoparticles is their side effects on the vital organs of the body. To mitigate such deleterious effects, it is better to use lower dosages of them. However, this can result in a decline in the technique's effectiveness. To compensate for the lower dose of SNPs, one can use secondary method like electroporation to deliver SNPs directly into the cells and reinforces the effect of electric pulses due to the high electrical conductivity of SNPs while having a minimal cytotoxicity effect on normal cells that are not treated with electroporation. In the present study, synergism effects of both procedures (SNPs and electroporation) experimentally and theoretically are considered to investigate the property of each technique in increasing the performance with respect to both procedures' limitations. To investigate more, adaptive neuro-fuzzy inference system (ANFIS) is used to predict the percent cell viability of cancerous cells affected by both procedures by considering amplitude and duration as inputs affecting on change of cell viability as an output. The results obtained from both experimental and simulation procedures showed that the maximum synergism between nanoparticles and electric pulses was recorded at 700 ​V/cm strength and 100 μs duration. Also, Results indicated high correlation between observed and predicted data (r2 ​= ​0.88). Moreover, the calculated root mean square error for the results of the ANFIS model was equal to 1.1. This implies that the model has practical value and can estimate the percent cell viability of cancerous cells influenced by both procedures with varying electric field amplitude and duration. This method can be proposed for other biophysical or drug delivery applications to save time and resources by utilizing the previous experimental data rather than performing more experiments.

Finite element analysis of a 3D triboelectric nanogenerator for biophysical health monitoring applications

The Triboelectric nanogenerator (TENG) has numerous applications in biophysical health monitoring due to its small size, lightweight, and economical use. The triboelectric effect occurs when two triboelectric materials with opposite polarities come into contact or rub against each other. This paper presents the simulated output performance of a three-dimensional TENG with two opposite dielectric circular layers separated by a distance of 0.05 μm. The bottom negative layer of polytetrafluoroethylene (PTFE) with a 0.5 μm diameter and 0.1 μm thickness has a negative aluminium electrode with the same dimensions. The top of the negative layer has cylindrical surface morphology. A layer of copper serves as both a positive layer and electrode with a diameter of 1 μm and a thickness of 0.1 μm. Furthermore, for analysis, PTFE is replaced by polyvinyl chloride (PVC) and polyethylene and the results are compared. The Finite Element Analysis (FEA) was used to analyze the geometry, employing a combination of Solid mechanics and Electrostatic module of COMSOL Multiphysics 5.6 software. The result shows that a maximum output voltage of 35.9 V was observed for an applied pulse pressure of 3 kPa as input. The results indicate that the TENGs have the potential to be used for various devices and wearable healthcare monitors.

Single-shot measurement of the Jones matrix for anisotropic media using four-channel digital polarization holography.

Dynamic measurement of the Jones matrix is crucial in investigating polarization light fields, which have wide applications in biophysics, chemistry, and mineralogy. However, acquiring the four elements of the Jones matrix instantly is difficult, hindering the characterization of random media and transient processes. In this study, we propose a single-shot measurement method of the Jones matrix for anisotropic media called "four-channel digital polarization holography" (FC-DPH). The FC-DPH system is created by a slightly off-axis superposition of reference light waves, which are modulated by a spatial light modulator (SLM), and signal light waves that pass through a Ronchi grating. The SLM enables flexible adjustment of the spatial carrier frequency, which can be adapted to different anisotropic media. The four elements of the Jones matrix can be obtained from the interferogram through the inverse Fourier transform. Optical experiments on anisotropic objects validate the feasibility and accuracy of the proposed method.

Emerging Mueller matrix microscopy applications in biophysics and biomedicine

AbstractPolarized and wide-field light microscopy has been studied for many years to develop accurate and information-rich images within a focused framework on biophysics and biomedicine. Technological advances and conceptual understanding have recently led to significant results in terms of applications. Simultaneously, developments in label-free methods are opening a new window on molecular imaging at a low dose of illumination. The ability to encode and decode polarized light pixel by pixel, coupled with the computational strength provided by artificial intelligence, is the running perspective of label-free optical microscopy. More specifically, the information-rich content Mueller matrix microscopy through its 16 elements offers multimodal imaging, an original data set to be integrated with other advanced optical methods. This dilates the spectrum of possible and potential applications. Here, we explore the recent advances in basic and applied research towards technological applications tailored for specific questions in biophysics and biomedicine.