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- Bunk, Richard
(författare)
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Creation of a Nanometer-Scale Toolbox for Molecular Motor Transport-Circuits
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis presents studies of molecular motors in interaction with nm-scale structures, as well as the development of a set of tools that can be used for the construction of custom-designed nano-transportation systems. In our studies, we have used the latest nanoscale technology and combined this with advanced results from chemistry and biomedical sciences. We have succeeded in transferring biomolecules from their natural habitat to an artificial environment created on a silicon-chip. The molecules - motor proteins myosin and actin - were maintained in their fully functional state by controlling the surface morphology and chemistry of the chip environment with nm-scale precision. These proteins are nanomachines, capable of transforming chemical energy into mechanical work. Our work has been concentrated on the introduction and development of a toolbox concept. A set of nm-scale tools, or components, have been defined and created, each with their unique basic transport function. The custom-designed components have been constructed as independent building blocks that can be combined into any circuit design of for example motor-driven micro-laboratories. The designing can be performed without detailed knowledge of the underlying mechanisms, e.g. lithography or motor protein biochemistry. To some extent, the concept resembles that of micro-electronics. The key components in the toolbox have been constructed of molecular monolayers and lithographic resist. We have found that monolayers of trimethylchlorosilane can be used to make conventional semiconductor materials, such as silicon, biocompatible. Furthermore, we have created a three-dimensional resist structure on the surface of a silicon-chip, that have been used to guide the mechanical motion developed by the motor proteins. With this novel design we have reduced the degrees of freedom for the proteins so that the effective guidance precision has increased successively from millimeter-, to micrometer- and eventually nanometer scale. Principally, electron-beam lithography has been used for the fabrication of the samples, although nano-imprint lithography has also been demonstrated as a powerful tool for parallel massive production on a commercial scale. In a series of experiments we have fine-tuned and characterized the properties of each toolbox component. Tools have been developed to capture and stream the molecular motors, reroute them and to analyze them. We have also demonstrated how cargo can be attached to the filaments, and performed successful experiments with chemically-linked quantum dots.
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| 3. |
- Bunk, Richard, et al.
(författare)
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Guiding molecular motors with nano-imprinted structures
- 2005
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Ingår i: Japanese Journal of Applied Physics. - 0021-4922. ; 44:5A, s. 3337-3340
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Tidskriftsartikel (refereegranskat)abstract
- This work, for the first time, demonstrates that nano-imprinted samples, with 100 nm wide polymer lines, can act as guides for molecular motors consisting of motor proteins actin and myosin. The motor protein function was characterized using fluorescence microscopy and compared to actomyosin motility on non-structured nitrocellulose surfaces. Our results open for further use of the nano-imprint technique in the production of disposable chips for bio-nanotechnological applications and miniaturized biological test systems. We discuss how the nano-imprinted motor protein assay system may be optimized and also how it compares to previously tested assay systems involving low-resolution UV-lithography and low throughput but high-resolution electron beam lithography.
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| 5. |
- Bunk, Richard, et al.
(författare)
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Guiding motor-propelled molecules with nanoscale precision through silanized bi-channel structures
- 2005
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Ingår i: Nanotechnology. - : IOP Publishing. - 0957-4484 .- 1361-6528. ; 16:6, s. 710-717
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Tidskriftsartikel (refereegranskat)abstract
- We report on the design and fabrication of a channel structure for high precision guidance and achieving excellent confinement properties for motor-propelled molecular shuttles. The techniques used to manufacture the channel structure are mainly e-beam lithography and selective monolayer functionalization. The structure consists of two lateral layers of concentric channels on a SiO2 surface made biocompatible with the molecular motors. The quality and advantages of the design are demonstrated by experiments using the motor proteins actin and myosin. The special channel geometry leads to stable biochemical conditions with full motor protein functionality. ATP is sufficiently supplied to all parts of the structure by dedicated service channels, as is the venting of ADP and P-i (inorganic phosphorus). Channels of different widths (100-700 nm) and shapes are fabricated and measurements made on them.
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| 6. |
- Mansson, A, et al.
(författare)
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Actin-based molecular motors for cargo transportation in nanotechnology - Potentials and challenges
- 2005
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Ingår i: IEEE Transactions on Advanced Packaging. - 1521-3323. ; 28:4, s. 547-555
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Tidskriftsartikel (refereegranskat)abstract
- Here, we review the use of actin-based motors, (myosins; e.g., the molecular motor of muscle) in. nanotechnology. The review starts from the viewpoints of the molecular motors as being important devices responsible of cargo transportation in the cell and end in discussions about their employment in nanotechnological applications. First, we describe basic biophysics of the myosin motors with focus on their involvement in cargo transportation in the living cell, leading us over into a discussion about in vitro motility assays. These are biological test systems where the myosin-induced translocation of actin filaments is studied on an artificial surface outside the cell. Then follows a review about modified motility assays for production of ordered motion. Here, we discuss ours and others' work with regards to making micro- and nanostructured surfaces and channels where the position and direction of movement produced by molecular motors is controlled. In this section, we consider the role of the channel size in promoting unidirectional myosin-induced motion of actin filaments. Furthermore, we consider the usefulness of surface modifications, e.g., various silanization procedures in order to promote and hinder molecular motility, respectively. Particularly, we describe our latest test system being both morphologically and chemically nanostructured giving us unsurpassed possibilities to perform functional studies as well as extremely good spatio-temporal control. Then follows a section about nanotechnological cargo transportation systems based on the actomyosin motor system. For instance, we present results of attaching fluorescent quantum dots as cargoes to the actin filaments. In this section, we also discuss the possibilities of having cargo attachment and detachment being performed on demand. Finally, we consider the usefulness of molecular motors for lab-on-a-chip applications and the requirements for incorporating these motors in commercially viable devices. In this context, the significant potential of the actomyosin motor system to overcome traditional limitations of micro- and nanofluidics is stressed.
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| 9. |
- Sundberg, Mark, et al.
(författare)
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Actin filament guidance on a chip: Toward high-throughput assays and lab-on-a-chip applications
- 2006
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 22:17, s. 7286-7295
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Tidskriftsartikel (refereegranskat)abstract
- Biological molecular motors that are constrained so that function is effectively limited to predefined nanosized tracks may be used as molecular shuttles in nanotechnological applications. For these applications and in high-throughput functional assays (e. g., drug screening), it is important that the motors propel their cytoskeletal filaments unidirectionally along the tracks with a minimal number of escape events. We here analyze the requirements for achieving this for actin filaments that are propelled by myosin II motor fragments (heavy meromyosin; HMM). First, we tested the guidance of HMM-propelled actin filaments along chemically defined borders. Here, trimethylchlorosilane (TMCS)-derivatized areas with high-quality HMM function were surrounded by SiO2 domains where HMM did not bind actin. Guidance along the TMCS-SiO2 border was almost 100% for filament approach angles between 0 and 20 degrees but only about 10% at approach angles near 90 degrees. A model (Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Bohringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Langmuir 2003, 19, 10967-10974) accounted for essential aspects of the data and also correctly predicted a more efficient guidance of actin filaments than previously shown for kinesin- propelled microtubules. Despite the efficient guidance at low approach angles, nanosized (< 700 nm wide) TMCS tracks surrounded by SiO2 were not effective in guiding actin filaments. Neither was there complete guidance along nanosized tracks that were surrounded by topographical barriers (walls and roof partially covering the track) unless there was also chemically based selectivity between the tracks and surroundings. In the latter case, with dually defined tracks, there was close to 100% guidance. A combined experimental and theoretical analysis, using tracks of the latter type, suggested that a track width of less than about 200-300 nm is sufficient at a high HMM surface density to achieve unidirectional sliding of actin filaments. In accord with these results, we demonstrate the long- term trapping of actin filaments on a closed-loop track (width < 250 nm). The results are discussed in relation to lab-on-a-chip applications and nanotechnology-assisted assays of actomyosin function.
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