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Schematic of flow cell (a) top view and (b) cross-section for monitoring biomolecular motor (myosin or HMM) activity (RhPh F-actin motion) by fluorescence microscopy. Solutions are exchanged in the flow cell by pipetting [yellow tip at right of (a)]. Molecular components in (b) are not drawn to scale.
Source publication
Biomolecular motors such as the muscle protein myosin with its partner protein actin hold great promise for actuation in hybrid nanoscale biomicroelectromechanical systems devices (bio-MEMS), particularly for future biomedical applications that involve highly localized delivery of biomolecules over short distances (e.g., micrometers) to specific ti...
Contexts in source publication
Context 1
... Cell Preparation and Fluorescence Microscopy: To prepare flow cells for motility assays, #1 glass coverslips were coated with 0.1% nitrocellulose (NC) in amyl acetate and allowed to dry. Treated coverslips were then mounted on microscope slides with #1 glass spacers and silicone grease to hold the spacers in place (Fig. 1). Solutions were applied to the flow cell at room temperature as described [15], [20] in the following order. Rabbit HMM (0.25-0.5 mg/mL) or fish myosin (1-5 mg/mL) was incubated in the flow cell for 1 or 3 min, respectively. Preliminary experiments in which incuba- tion of fish myosin within a flow cell was increased from one to three ...
Context 2
... also attempted to develop techniques to stabilize proteins within flow cells. High concentrations of sucrose or glycerol stabilize protein structure and function [26] during storage at 20 C or lower. High-viscosity solutions containing glycerol (50%) were impractical to use in the restricted spaces of our flow cells [ Fig. 1(a)]. Sucrose (1.2 M) preserved function for up to one month, the longest duration tested, when flow cells were stored at 20 C, but did not preserve function for much shorter times at room temperature. Despite this initial success with storage in sucrose, we continued to examine additional conditions because we are seeking conditions that ...
Citations
... To overcome these problems it has been proposed that biological molecular motors, with their inherent extensive miniaturization, biodegradability and self-propelling features, may be used to transport analytes e.g. from recognition to detection chambers, achieving separation, concentration as well as certain forms of detection [6-8]. Several important steps towards a functional molecular motor driven diagnostic device have also been realized (reviewed in [8-13]) such as: (i) attachment of antibodies to cytoskeletal microtubule [14] and actin filament [15] shuttles, followed by molecular motor-driven transportation of analytes (viruses, protein antigens etc.) bound to the antibodies, (ii) nano/microfabrication of devices for guided transportation of the motor propelled shuttles to concentrate analytes at a detector site [6,7,16-18] and (iii) long-term storage of ready-to-use devices without loss of activity [19-21]. ...
Background
Introduction of effective point-of-care devices for use in medical diagnostics is part of strategies to combat accelerating health-care costs. Molecular motor driven nanodevices have unique potentials in this regard due to unprecedented level of miniaturization and independence of external pumps. However motor function has been found to be inhibited by body fluids.
Results
We report here that a unique procedure, combining separation steps that rely on antibody-antigen interactions, magnetic forces applied to magnetic nanoparticles (MPs) and the specificity of the actomyosin bond, can circumvent the deleterious effects of body fluids (e.g. blood serum). The procedure encompasses the following steps: (i) capture of analyte molecules from serum by MP-antibody conjugates, (ii) pelleting of MP-antibody-analyte complexes, using a magnetic field, followed by exchange of serum for optimized biological buffer, (iii) mixing of MP-antibody-analyte complexes with actin filaments conjugated with same polyclonal antibodies as the magnetic nanoparticles. This causes complex formation: MP-antibody-analyte-antibody-actin, and magnetic separation is used to enrich the complexes. Finally (iv) the complexes are introduced into a nanodevice for specific binding via actin filaments to surface adsorbed molecular motors (heavy meromyosin). The number of actin filaments bound to the motors in the latter step was significantly increased above the control value if protein analyte (50–60 nM) was present in serum (in step i) suggesting appreciable formation and enrichment of the MP-antibody-analyte-antibody-actin complexes. Furthermore, addition of ATP demonstrated maintained heavy meromyosin driven propulsion of actin filaments showing that the serum induced inhibition was alleviated. Detailed analysis of the procedure i-iv, using fluorescence microscopy and spectroscopy identified main targets for future optimization.
Conclusion
The results demonstrate a promising approach for capturing analytes from serum for subsequent motor driven separation/detection. Indeed, the observed increase in actin filament number, in itself, signals the presence of analyte at clinically relevant nM concentration without the need for further motor driven concentration. Our analysis suggests that exchange of polyclonal for monoclonal antibodies would be a critical improvement, opening for a first clinically useful molecular motor driven lab-on-a-chip device.
... Ca 2+ activates thin filaments by binding to Tn, leading to structural changes that ultimately expose myosin-binding sites on actin subunits [11]. Our previous work on the thermoelectric controller utilized only myosin and unregulated F-actin, that is, Ca 2+ -independent motility achieved using actin filaments without Tn or Tm [5] [6]. This study was initiated because Ca 2+ -regulated thin filaments could have several advantages over unregulated F-actin in a thermoelectric nanoactuator. ...
Microfabricated thermoelectric controllers can be employed to investigate mechanisms underlying myosin-driven sliding of Ca(2+)-regulated actin and disease-associated mutations in myofilament proteins. Specifically, we examined actin filament sliding-with or without human cardiac troponin (Tn) and α-tropomyosin (Tm)-propelled by rabbit skeletal heavy meromyosin, when temperature was varied continuously over a wide range (~20-63°C). At the upper end of this temperature range, reversible dysregulation of thin filaments occurred at pCa 9 and 5; actomyosin function was unaffected. Tn-Tm enhanced sliding speed at pCa 5 and increased a transition temperature (T(t)) between a high activation energy (E(a)) but low temperature regime and a low E(a) but high temperature regime. This was modulated by factors that alter cross-bridge number and kinetics. Three familial hypertrophic cardiomyopathy (FHC) mutations, cTnI R145G, cTnI K206Q, and cTnT R278C, cause dysregulation at temperatures ~5-8°C lower; the latter two increased speed at pCa 5 at all temperatures.
... The conception of new medicines, the synthesis of alloys, the prototyping of bio-nanorobots [1] as well as the transmission of information between two nanomachines [2], [3] , and the development of bio-microelectromechanical system devices (bio- MEMS) [4], [5] are made possible thanks to molecular simulators . Since ease of manipulation is one of the key points of such tools, haptic devices are widely used. ...
This paper presents a novel tool for the analysis of new molecular structures which enables a wide variety of manipulations. It is composed of a molecular simulator and a haptic device. The simulation software deals with systems of hundreds or thousands of degrees of freedom and computes the reconfiguration of the molecules in a few tenths of a second. For the ease of manipulation and to help the operator understand nanoscale phenomena, a haptic device is connected to the simulator. To handle a wide variety of applications, both position and force control are implemented. To our knowledge, this is the first time the applications of force control are detailed for molecular simulation. These two control modes are compared in terms of adequacy with molecular dynamics, transparency and stability sensitivity with respect to environmental conditions. Based on their specificity the operations they can realize are detailed. Experiments highlight the usability of our tool for the different steps of the analysis of molecular structures. It includes the global reconfiguration of a molecular system, the measurement of molecular properties and the comprehension of nanoscale interactions. Compared to most existing systems, the one developed in this paper offers a wide range of possible experiments. The detailed analysis of the properties of the control modes can be easily used to implement haptic feedback on other molecular simulators.
... The molecules can be used as a channel to transmit an information between two nanomachines ([6] and [7]) stephane.redon@inria.fr study of molecules in order to develop biomicroelectromechanical system devices (bio-MEMS) ([8] and [9]). Simulation Haptic device Desired position Manipulated moleculeFig. ...
In this paper, a new tool dedicated to the analysis and the conception of molecules is presented. It is composed of an adaptive simulation software and a haptic device used to interact with molecules while feeling either the forces applied by the environment or the internal forces. The adaptive articulated body algorithm allows fast simulations of complex flexible molecules. To handle the coupling with the force feedback device, two different control schemes designed for nanoscale applications and providing high transparency rendering are proposed and compared.
Mass transport limitations of particulates within conventional microanalytical systems are often cited as the root cause for low sensitivity but can be overcome by directed analyte transport, such as via biomolecular motors or gradient surfaces. An ongoing challenge is the development of materials that are passive in nature (i.e., no external power source required), but can reconfigure to perform work, such as transporting particle‐based analytes. Mimicking biology's concepts of autonomous and reconfigurable materials systems, like the Drosera capensis leaf, reconfigurable fiber networks that effectively concentrate particulates within a localized spot that can act as a detection patch are developed. These networks, prepared by electrohydrodynamic co‐jetting, draw their reconfigurability from a bicompartmental fiber architecture. Upon exposure to neutral pH, a differential swelling of both fiber compartments gives rise to interfacial tension and ultimately results in shape reconfiguration of the fiber network. Compared to free particles, the reconfigurable fiber networks display a 57‐fold increase in analyte detectability, average transport efficiencies of 91.9 ± 2.4%, and separation selectivity between different surface properties of 95 ± 3%. The integration of biomimetic materials into microanalytical systems, exemplified in this study, offers ample opportunities to design novel and effective detection schemes that circumvent mass transport limitations.
A new frontier in the development of prosthetic devices is the design of nanoscale systems which replace, augment, or support individual cells. Similar to cells, such devices will require the ability to generate mechanical movement, either for transport or actuation. Here, the development of nanoscale transport systems, which integrate biomolecular motors, is reviewed. To date, close to 100 publications have explored the design of such "molecular shuttles" based on the integration of synthetic molecules, nano- and micro- particles, and micropatterned structures with kinesin and myosin motors and their associated cytoskeletal filaments, microtubules, and actin filaments. Tremendous progress has been made in addressing the key challenges of guiding, loading, and controlling the shuttles, providing a foundation for the exploration of applications in medicine and engineering
We propose that a thermo-electrical control system for rapid and reversible actuation of biomolecular motors and their partner filaments can also be used to study molecular mechanisms of cardiovascular diseases. We have previously used this device to evaluate the temperature-dependence of unregulated (absence of cardiac Ca2+-regulatory proteins tropomyosin, α-Tm, and troponin, Tn) actin filament sliding powered by myosin motors, which hydrolyze ATP. These assays using the thermo-electric controller can also be applied to regulated thin filaments (F actin plus α-Tm and Tn) to obtain energetic parameters and functional correlates of structural stability at the level of single filaments. This allows us not only to examine Ca2+-dependent sliding of thin filaments, but also to test for altered function of clinically relevant mutations of cardiac myofilament proteins such as those identified in familial hypertrophic cardiomyopathy (FHC).
In recent years, the innovative use of microelectromechanical systems ( MEMSs ) and nanoelectromechanical systems ( NEMSs ) in biomedical applications has opened wide opportunities for precise and accurate human diagnostics and therapeutics. The introduction of nanotechnology in biomedical applications has facilitated the exact control and regulation of biological environments. This ability is derived from the small size of the devices and their multifunctional capabilities to operate at specific sites for selected durations of time. Researchers have developed wide varieties of unique and multifunctional MEMS / NEMS devices with micro and nano features for biomedical applications ( BioMEMS / NEMS ) using the state of the art microfabrication techniques and biocompatible materials. However, the integration of devices with the biological milieu is still a fundamental issue to be addressed. Devices often fail to operate due to loss of functionality, or generate adverse toxic effects inside the body. The in vitro and in vivo performance of implantable BioMEMS such as biosensors, smart stents, drug delivery systems, and actuation systems are researched extensively to understand the interaction of the BioMEMS devices with physiological environments. BioMEMS developed for drug delivery applications include microneedles, microreservoirs, and micropumps to achieve targeted drug delivery. The biocompatibility of BioMEMS is further enhanced through the application of tissue and smart surface engineering. This involves the application of nanotechnology, which includes the modification of surfaces with polymers or the self‐assembly of monolayers of molecules. Thereby, the adverse effects of biofouling can be reduced and the performance of devices can be improved in in vivo and in vitro conditions. WIREs Nanomed Nanobiotechnol 2013, 5:544–568. doi: 10.1002/wnan.1227
This article is categorized under: Diagnostic Tools > Biosensing
Diagnostic Tools > Diagnostic Nanodevices
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
The outcome of a self-assembly process is not only determined by the specified connections between building blocks, but also by the means of bringing building blocks into contact and of testing for the formation of an intended connection. Endowing each building block with the ability to actively move overcomes some limitations of diffusion-driven molecular and nanoscale self-assembly by accelerating transport, reducing unwanted connections, and introducing self-organization phenomena with desirable consequences. Proof-of-principle experiments utilizing biomolecular motors, e.g. motor proteins, to propel nanostructures and the underlying concepts are reviewed, and the potential impact for nanomanufacturing is discussed.
We report the development of "nano-storage wires" (NSWs) which can store chemical species and release them at a desired moment via external electrical stimuli. Here, using the electrodeposition process through an anodized aluminum oxide template, we fabricated multi-segmented nanowires composed of a polypyrrole segment containing adenosine triphosphate (ATP) molecules, a ferromagnetic nickel segment, and a conductive gold segment. Upon the application of a negative bias voltage, the NSWs released ATP molecules for the control of motor protein activities. Furthermore, NSWs can be printed onto various substrates including flexible or three dimensional structured substrates by direct writing or magnetic manipulation strategies to build versatile chemical storage devices. Since our strategy provides a means to store and release chemical species in a controlled manner, it should open up various applications such as drug delivery systems and biochips for the controlled release of chemicals.
