Gotowa bibliografia na temat „Microrobotics, Magnetic Tweezers, Atomic Force Microscopy”

In this study, we investigated the DNA condensation induced by polyethylene glycol (PEG) with different molecular weights (PEG 600 and PEG 6000) in the presence of NaCl or MgCl₂ by using magnetic tweezers (MT) and atomic force microscopy (AFM).

Single-molecule technologies have expanded our ability to detect biological events individually, in contrast to ensemble biophysical technologies, where the result provides averaged information. Recent developments in atomic force microscopy have not only enabled us to distinguish the heterogeneous phenomena of individual molecules, but also allowed us to view up to the resolution of a single covalent bond. Similarly, optical tweezers, due to their versatility and precision, have emerged as a potent technique to dissect a diverse range of complex biological processes, from the nanomechanics of ClpXP protease–dependent degradation to force-dependent processivity of motor proteins. Despite the advantages of optical tweezers, the time scales used in this technology were inconsistent with physiological scenarios, which led to the development of magnetic tweezers, where proteins are covalently linked with the glass surface, which in turn increases the observation window of a single biomolecule from minutes to weeks. Unlike optical tweezers, magnetic tweezers use magnetic fields to impose torque, which makes them convenient for studying DNA topology and topoisomerase functioning. Using modified magnetic tweezers, researchers were able to discover the mechanical role of chaperones, which support their substrate proteinsby pulling them during translocation and assist their native folding as a mechanical foldase. In this article, we provide a focused review of many of these new roles of single-molecule technologies, ranging from single bond breaking to complex chaperone machinery, along with the potential to design mechanomedicine, which would be a breakthrough in pharmacological interventions against many diseases.

G-quadruplexes (G4s) are stable secondary nucleic acid structures that play crucial roles in many fundamental biological processes. The folding/unfolding dynamics of G4 structures are associated with the replication and transcription regulation functions of G4s. However, many DNA G4 sequences can adopt a variety of topologies and have complex folding/unfolding dynamics. Determining the dynamics of G4s and their regulation by proteins remains challenging due to the coexistence of multiple structures in a heterogeneous sample. Here, in this mini-review, we introduce the application of single-molecule force-spectroscopy methods, such as magnetic tweezers, optical tweezers, and atomic force microscopy, to characterize the polymorphism and folding/unfolding dynamics of G4s. We also briefly introduce recent studies using single-molecule force spectroscopy to study the molecular mechanisms of G4-interacting proteins.

The mechanical properties of proteins can be studied with single molecule force spectroscopy (SMFS) using optical tweezers, atomic force microscopy and magnetic tweezers. It is common to utilize a flexible linker between the protein and trapped probe to exclude short-range interactions in SMFS experiments. One of the most prevalent linkers is DNA due to its well-defined properties, although attachment strategies between the DNA linker and protein or probe may vary. We will therefore provide a general overview of the currently existing non-covalent and covalent bioconjugation strategies to site-specifically conjugate DNA-linkers to the protein of interest. In the search for a standardized conjugation strategy, considerations include their mechanical properties in the context of SMFS, feasibility of site-directed labeling, labeling efficiency, and costs.

There are relatively few technologies for measurement at the single-molecule scale. Fluorescent imaging, for example, can be used to directly visualize molecules and their interactions, but diffraction limitations and labeling requirements may push the system from its native state. Although recent advances in super-resolution imaging have been able to break this resolution barrier, important challenges remain. Atomic force microscopy (AFM) is capable of imaging molecules at high resolution and at high speed. However, AFM imaging is a surface technique, requiring sample preparation and some immobilization. Other technologies such as optical tweezers and magnetic tweezers are capable of molecular manipulation and spectroscopy to great effect but require a significant apparatus and have limited inherent analytical capabilities.

In higher organisms, the professional phagocytes of the immune system (dendritic cells, neutrophils, monocytes, and macrophages) are responsible for pathogen clearance, the development of immune responses via cytokine secretion and presentation of antigens derived from internalized material, and the normal turnover and remodelling of tissues and disposal of dead cells. These functions rely on the ability of phagocytes to migrate and adhere to sites of infection, dynamically probe their environments to make contact with phagocytic targets, and perform phagocytosis, a mechanism of internalization of large particles, microorganisms, and cellular debris for intracellular degradation. The cell-generated forces that are necessary for the professional phagocytes to act in their roles as ‘first responders’ of the immune system have been the subject of mechanical studies in recent years. Methods of force measurement such as atomic force microscopy, traction force microscopy, micropipette aspiration, magnetic and optical tweezers, and exciting new variants of these have accompanied classical biological methods to perform mechanical investigations of these highly dynamic immune cells.

Abstract A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

In this work, the preparation procedure and properties of anionic magnetic microgels loaded with antitumor drug doxorubicin are described. The functional microgels were produced via the in situ formation of iron nanoparticles in an aqueous dispersion of polymer microgels based on poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAM-PAA). The composition and morphology of the resulting composite microgels were studied by means of X-ray diffraction, Mössbauer spectroscopy, IR spectroscopy, scanning electron microscopy, atomic-force microscopy, laser microelectrophoresis, and static and dynamic light scattering. The forming nanoparticles were found to be β-FeO(OH). In physiological pH and ionic strength, the obtained composite microgels were shown to possess high colloid stability. The average size of the composites was 200 nm, while the zeta-potential was −27.5 mV. An optical tweezers study has demonstrated the possibility of manipulation with microgel using external magnetic fields. Loading of the composite microgel with doxorubicin did not lead to any change in particle size and colloidal stability. Magnetic-driven interaction of the drug-loaded microgel with model cell membranes was demonstrated by fluorescence microscopy. The described magnetic microgels demonstrate the potential for the controlled delivery of biologically active substances.

Micro-robotic systems are used in various fields of science and technology for the manipulation of objects of size less than a millimeter. Magnetic tweezers can be considered as micro-robotic systems due to their ability to manipulate samples of size in the range of few micrometers. In magnetic tweezers, a magnetic microparticle is manipulated by applying magnetic fields near the particle. Magnetic tweezers are popular for manipulating biological samples due to their high specificity, bio-compatibility and having an untethered end effector that enables them to manipulate inside the samples. Despite these benefits, magnetic tweezers suffer from limitations such as non-linearity in actuation, poor actuation bandwidth, the measurement strategy demanding the particle to be clearly visible and finally, the necessity of sophisticated control strategies for controlling the position of magnetic particles. This thesis investigates the design and development of a micro-robotic system with force sensing capability that addresses the actuation, measurement and control limitations of magnetic tweezers system. In order to address the actuation and measurement limitations of the magnetic tweezers, a current carrying micro-actuator is proposed to apply magnetic forces to the magnetic particles while an integrated force sensor measures the applied force. A simple analytical model for the force of interaction between the micro-actuator and magnetic particle is proposed and employed to show that force is proportional to the actuation current and position of the magnetic particle in three-dimensions (3-D). Further, simple models for mechanical stiffness and rise in temperature due to ohmic heating of the micro-actuator with force sensing capability are proposed. Subsequently, systematic guidelines are proposed for the design of the micro-actuator with force sensing capability. The designed micro-actuator with force sensing capability is fabricated and evaluated. The micro-actuator has an electrical bandwidth of about 1 MHz. The ability of the micro-actuator to apply 3-D forces to a magnetic particle is demonstrated by actuating permanent-magnet microparticles attached to micro-cantilever beams. The force sensing capability of micro-actuator is demonstrated by measuring the deflection of the micro-actuator while it is actuating a permanent-magnet microparticle. The applicability of the micro-actuator with force sensing capability is shown by employing it for the development of a magnetometer to estimate the magnetic moment of micrometer-scale magnetic particles in 3-D. The developed magnetometer is evaluated by measuring magnetic moments of both hard and soft ferromagnetic particles and untethered magnetic particles. The measured magnetic moments agree well with their theoretical counterpart with an average error of 18%. Finally, an open loop control strategy is proposed for controlling the position of magnetic particles in 2-D and 3-D respectively by applying appropriate actuation currents to the micro-actuator. Also, the force sensing capability is utilized to estimate the position of the magnetic particle along the out-of-plane axis of the micro-actuator. The estimated position of the magnetic particle is used to develop a novel scanning probe microscope (SPM) with an untethered probe. The motion of the magnetic particle in 3-D due to actuation current to the micro-actuator is estimated numerically and analyzed by using Mathieu’s equation with Dehmelt’s approximation. The control of the position of magnetic particle is demonstrated by moving the magnetic particles in pre-defined trajectories along 2-D and 3-D respectively. Further, a new strategy is developed to push samples of dimensions much smaller than the size of the magnetic particle. Finally, the imaging capability of the developed SPM is shown by imaging artificially generated topographies using a tethered probe.

Single Molecule Science (SMS) has emerged from developing, using and combining technologies such as super-resolution microscopy, atomic force microscopy, and optical and magnetic tweezers, alongside sophisticated computational and modelling techniques. This comprehensive, edited volume brings together authoritative overviews of these methods from a biological perspective, and highlights how they can be used to observe and track individual molecules and monitor molecular interactions in living cells. Pioneers in this fast-moving field cover topics such as single molecule optical maps, nanomachines, and protein folding and dynamics. A particular emphasis is also given to mapping DNA molecules for diagnostic purposes, and the study of gene expression. With numerous illustrations, this book reveals how SMS has presented us with a new way of understanding life processes. A must-have for researchers and graduate students, as well as those working in industry, primarily in the areas of biophysics, biological imaging, genomics and structural biology.

Traditional structural biology ensemble techniques such as x-ray crystallography, electron cryomicroscopy with angular reconstruction, electron microscopy, nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) provide detailed information on the structure of biological macromolecules. In cases where the crystal form of the macromolecule is available, the structure is known with the ultimate atomic resolution. The knowledge of the static structure can provide some insight to the macromolecule function, especially if it is coupled with other biochemical measurements, but in general the structure-function relationship is to a large extent unknown. With the aid of recently developed techniques such as patch clamp, atomic force microscopy (AFM) and optical tweezers, ionic current fluctuations in individual ion channels and forces and/or displacements generated during single molecular motor reaction were measured. Such measurements furnish information about function, but do not provide local, dynamical structural information.