| 1. |
- Kumar, Saroj, et al.
(författare)
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Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 7:10
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Tidskriftsartikel (refereegranskat)abstract
- Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 mu m(-1)). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.
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| 2. |
- Lard, Mercy, et al.
(författare)
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Molecular Motor Transport through Hollow Nanowires
- 2014
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Ingår i: Nano letters (Print). - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 14:6, s. 3041-3046
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Tidskriftsartikel (refereegranskat)abstract
- Biomolecular motors offer self-propelled, directed transport in designed microscale networks and can potentially replace pump-driven nanofluidics. However, in existing systems, transportation is limited to the two-dimensional plane. Here we demonstrate fully one-dimensional (1D) myosin-driven motion of fluorescent probes (actin filaments) through 80 nm wide, Al2O3 hollow nanowires of micrometer length. The motor-driven transport is orders of magnitude faster than would be possible by passive diffusion. The system represents a necessary element for advanced devices based on gliding assays, for example, in lab-on-a-chip systems with channel crossings and in pumpless nanosyringes. It may also serve as a scaffold for bottom-up assembly of muscle proteins into actin ordered contractile units, mimicking the muscle sarcomere.
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| 3. |
- Lard, Mercy, et al.
(författare)
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Nano-structuring for molecular motor control
- 2015
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Ingår i: Nano-Structures for Optics and Photonics. - Dordrecht : Springer. ; , s. 459-459, s. 459-459
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Konferensbidrag (refereegranskat)abstract
- The interaction of self-propelled biological molecular-motors and cytoskeletal filaments holds relevance for a variety of applications such as biosensing, drug screening, diagnostics and biocomputation. The use of these systems for lab-on-a-chip biotechnology applications shows potential for replacement of microfluidic flow by active, molecular-motor driven transport of filaments. The ability to control, confine and detect motile objects in such a system is possible by development of nanostructured surfaces for on-chip applications and fundamental studies of molecular-motors. Here we describe the localized detection (Lard et al., Sci Rep 3:1092, 2013) and fast transport of actin filaments by myosin molecular-motors (Lard et al., Biosens Biolectron 48(0):145–152, 2013), inserted within nanostructures, as a method for biocomputation and molecular concentration. These results include extensive myosin driven concentration of actin filaments on a miniaturized detector, of relevance for use of molecular-motors in a diagnostics platform. Also, we discuss the local enhancement of the fluorescence signal of filaments, relevant for use in a biocomputation device where tracking of potentially thousands of motile objects is of primary significance.
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| 4. |
- Lard, Mercy, et al.
(författare)
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Nanowire-Imposed Geometrical Control in Studies of Actomyosin Motor Function
- 2015
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Ingår i: IEEE Transactions on Nanobioscience. - : IEEE. - 1536-1241 .- 1558-2639. ; 14:3, s. 289-297
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Tidskriftsartikel (refereegranskat)abstract
- Recently, molecular motor gliding assays with actin and myosin from muscle have been realized on semiconductor nanowires coated with Al2O3. This opens for unique nanotechnological applications and novel fundamental studies of actomyosin motor function. Here, we provide a comparison of myosin-driven actin filament motility on Al2O3 to both nitrocellulose and trimethylchlorosilane derivatized surfaces. We also show that actomyosin motility on the less than 200 nm wide tips of arrays of Al2O3-coated nanowires can be used to control the number, and density, of myosin-actin attachment points. Results obtained using nanowire arrays with different inter-wire spacing are consistent with the idea that the actin filament sliding velocity is determined both by the total number and the average density of attached myosin heads along the actin filament. Further, the results are consistent with buckling of long myosin-free segments of the filaments as a factor underlying reduced velocity. On the other hand, the findings do not support a mechanistic role in decreasing velocity, of increased nearest neighbor distance between available myosin heads. Our results open up for more advanced studies that may use nanowire-based structures for fundamental investigations of molecular motors, including the possibility to create a nanowire-templated bottom-up assembly of 3D, muscle-like structures.
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| 5. |
- Lard, Mercy, et al.
(författare)
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Tracking Actomyosin at Fluorescence Check Points
- 2013
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Emerging concepts for on-chip biotechnologies aim to replace microfluidic flow by active, molecular-motor driven transport of cytoskeletal filaments, including applications in bio-simulation, biocomputation, diagnostics, and drug screening. Many of these applications require reliable detection, with minimal data acquisition, of filaments at many, local checkpoints in a device consisting of a potentially complex network of channels that guide filament motion. Here we develop such a detection system using actomyosin motility. Detection points consist of pairs of gold lines running perpendicular to nanochannels that guide motion of fluorescent actin filaments. Fluorescence interference contrast (FLIC) is used to locally enhance the signal at the gold lines. A cross-correlation method is used to suppress errors, allowing reliable detection of single or multiple filaments. Optimal device design parameters are discussed. The results open for automatic read-out of filament count and velocity in high-throughput motility assays, helping establish the viability of active, motor-driven on-chip applications.
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| 6. |
- Lard, Mercy, et al.
(författare)
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Ultrafast molecular motor driven nanoseparation and biosensing
- 2013
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Ingår i: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 48, s. 145-152
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Tidskriftsartikel (refereegranskat)abstract
- Portable biosensor systems would benefit from reduced dependency on external power supplies as well as from further miniaturization and increased detection rate. Systems built around self-propelled biological molecular motors and cytoskeletal filaments hold significant promise in these regards as they are built from nanoscale components that enable nanoseparation independent of fluidic pumping. Previously reported microtubule-kinesin based devices are slow, however, compared to several existing biosensor systems. Here we demonstrate that this speed limitation can be overcome by using the faster actomyosin motor system. Moreover, due to lower flexural rigidity of the actin filaments, smaller features can be achieved compared to microtubule-based systems, enabling further miniaturization. Using a device designed through optimization by Monte Carlo simulations, we demonstrate extensive myosin driven enrichment of actin filaments on a detector area of less than 10 μm2, with a concentration half-time of approximately 40 s. We also show accumulation of model analyte (streptavidin at nanomolar concentration in nanoliter effective volume) detecting increased fluorescence intensity within seconds after initiation of motor-driven transportation from capture regions. We discuss further optimizations of the system and incorporation into a complete biosensing workflow.
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| 7. |
- Lindberg, Frida W., et al.
(författare)
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Design and development of nanoimprint-enabled structures for molecular motor devices
- 2019
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Ingår i: Materials Research Express. - : IOP Publishing. - 2053-1591. ; 6:2
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Tidskriftsartikel (refereegranskat)abstract
- Devices based on molecular motor-driven cytoskeletal filaments, e.g., actin filaments, have been developed both for biosensing and biocomputational applications. Commonly, these devices require nanoscaled tracks for guidance of the actin filaments which has limited the patterning technique to electron beam lithography. Thus, large scale systems become intractable to fabricate at a high throughput within a reasonable time-frame. We have studied the possibility to fabricate molecular motor-based devices using the high throughput, high resolution technique of nanoimprint lithography. Molecular motor-based devices require wide open regions (loading zones) to allow filaments to land for later propulsion into the nanoscale tracks. Such open zones are challenging to fabricate using nanoimprint lithography due to the large amount of material displaced in the process. We found that this challenge can be overcome by introducing nanoscaled pillars inside the loading zones, into which material can be displaced during imprint. By optimising the resist thickness, we were able to decrease the amount of material displaced without suffering from insufficient filling of the stamp. Furthermore, simulations suggest that the shape and positioning of the pillars can be used to tailor the overall cytoskeletal filament transportation direction and behaviour. This is a potentially promising design feature for future applications that however, requires further optimisations before experimental realisation.
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| 8. |
- Meinecke, Christoph R., et al.
(författare)
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Nanolithographic Fabrication Technologies for Network-Based Biocomputation Devices
- 2023
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Ingår i: Materials. - : MDPI. - 1996-1944. ; 16:3
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Tidskriftsartikel (refereegranskat)abstract
- Network-based biocomputation (NBC) relies on accurate guiding of biological agents through nanofabricated channels produced by lithographic patterning techniques. Here, we report on the large-scale, wafer-level fabrication of optimized microfluidic channel networks (NBC networks) using electron-beam lithography as the central method. To confirm the functionality of these NBC networks, we solve an instance of a classical non-deterministic-polynomial-time complete ("NP-complete") problem, the subset-sum problem. The propagation of cytoskeletal filaments, e.g., molecular motor-propelled microtubules or actin filaments, relies on a combination of physical and chemical guiding along the channels of an NBC network. Therefore, the nanofabricated channels have to fulfill specific requirements with respect to the biochemical treatment as well as the geometrical confienement, with walls surrounding the floors where functional molecular motors attach. We show how the material stack used for the NBC network can be optimized so that the motor-proteins attach themselves in functional form only to the floor of the channels. Further optimizations in the nanolithographic fabrication processes greatly improve the smoothness of the channel walls and floors, while optimizations in motor-protein expression and purification improve the activity of the motor proteins, and therefore, the motility of the filaments. Together, these optimizations provide us with the opportunity to increase the reliability of our NBC devices. In the future, we expect that these nanolithographic fabrication technologies will enable production of large-scale NBC networks intended to solve substantially larger combinatorial problems that are currently outside the capabilities of conventional software-based solvers.
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| 9. |
- Månsson, Alf, et al.
(författare)
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Self-organization of motor-propelled cytoskeletal filaments at topographically defined borders.
- 2012
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Ingår i: Journal of Biomedicine and Biotechnology. - : Hindawi Limited. - 1110-7243 .- 1110-7251. ; 2012, s. Article ID: 647265-
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Tidskriftsartikel (refereegranskat)abstract
- Self-organization phenomena are of critical importance in living organisms and of great interest to exploit in nanotechnology. Here we describe in vitro self-organization of molecular motor-propelled actin filaments, manifested as a tendency of the filaments to accumulate in high density close to topographically defined edges on nano- and microstructured surfaces. We hypothesized that this "edge-tracing" effect either (1) results from increased motor density along the guiding edges or (2) is a direct consequence of the asymmetric constraints on stochastic changes in filament sliding direction imposed by the edges. The latter hypothesis is well captured by a model explicitly defining the constraints of motility on structured surfaces in combination with Monte-Carlo simulations [cf. Nitta et al. (2006)] of filament sliding. In support of hypothesis 2 we found that the model reproduced the edge tracing effect without the need to assume increased motor density at the edges. We then used model simulations to elucidate mechanistic details. The results are discussed in relation to nanotechnological applications and future experiments to test model predictions.
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| 10. |
- Nicolau, Dan V., Jr., et al.
(författare)
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Parallel computation with molecular-motor-propelled agents in nanofabricated networks
- 2016
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:10, s. 2591-2596
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Tidskriftsartikel (refereegranskat)abstract
- The combinatorial nature of many important mathematical problems, including nondeterministic-polynomial-time (NP)-complete problems, places a severe limitation on the problem size that can be solved with conventional, sequentially operating electronic computers. There have been significant efforts in conceiving parallel-computation approaches in the past, for example: DNA computation, quantum computation, and microfluidics-based computation. However, these approaches have not proven, so far, to be scalable and practical from a fabrication and operational perspective. Here, we report the foundations of an alternative parallel-computation system in which a given combinatorial problem is encoded into a graphical, modular network that is embedded in a nanofabricated planar device. Exploring the network in a parallel fashion using a large number of independent, molecular-motor-propelled agents then solves the mathematical problem. This approach uses orders of magnitude less energy than conventional computers, thus addressing issues related to power consumption and heat dissipation. We provide a proof-of-concept demonstration of such a device by solving, in a parallel fashion, the small instance {2, 5, 9} of the subset sum problem, which is a benchmark NP-complete problem. Finally, we discuss the technical advances necessary to make our system scalable with presently available technology.
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