Marlene Bachand

In vitro Capture, Transport, and Detection of Protein Analytes Using Kinesin‐Based Nanoharvesters

Miniaturization of lab-on-a-chip devices to nanoscale dimensions necessitates a level of systems integration currently found primarily in biological systems. Such devices will require new modes of transportingmacromolecularmaterials at nanometer length scales. In cells, efficient cytoplasmic transport is achieved by energy-consuming, active transport systems in which motor proteins transport cargo along cytoskeletal filaments. For example, the motor protein kinesin-1 carries cell organelles and macromolecules over considerable distances along microtubule filaments. Microtubules are hollow protein polymeric filaments with a diameter of 25 nm and tens of micrometers in length that form a 3D transportation network within the cell. Small groups of kinesin transport cargo at rates up to 12mms , with a catalytic efficiency (i.e., conversion of chemical energy into work) of 50%. Together, this transport system provides a highefficiency means of transporting macromolecular cargo through the highly viscous medium of cytoplasm. The intriguing and powerful properties of kinesin-based transport have spurred its application in hybrid nanoscale systems. Early work focused on applying microfabrication technologies and surface functionalization to guide the kinesinbased transport of molecular shuttles (i.e., stabilized microtubule filaments) and achieve directed transport ofmaterials at the nanoscale. In this mode of application, commonly referred to as the inverted or glidingmotility geometry, kinesin motor proteins are bound on a solid surface such that their catalytic and microtubule-binding domains extend into the solution. In the presence ofATP,microtubule filaments bind to

Biomolecular motors in nanoscale materials, devices, and systems

Biomolecular motors are a unique class of intracellular proteins that are fundamental to a considerable number of physiological functions such as DNA replication, organelle trafficking, and cell division. The efficient transformation of chemical energy into useful work by these proteins provides strong motivation for their utilization as nanoscale actuators in ex vivo, meso- and macro-scale hybrid systems. Biomolecular motors involved in cytoskeletal transport are quite attractive models within this context due to their ability to direct the transport of nano-/micro-scale objects at rates significantly greater than diffusion, and in the absence of bulk fluid flow. As in living organisms, biomolecular motors involved in cytoskeletal transport (i.e., kinesin, dynein, and myosin) function outside of their native environment to dissipatively self-assemble biological, biomimetic, and hybrid nanostructures that exhibit nonequilibrium behaviors such as self-healing. These systems also provide nanofluidic transport function in hybrid nanodevices where target analytes are actively captured, sorted, and transported for autonomous sensing and analytical applications. Moving forward, the implementation of biomolecular motors will continue to enable a wide range of unique functionalities that are presently limited to living systems, and support the development of nanoscale systems for addressing critical engineering challenges.

Directed Attachment of Antibodies to Kinesin-Powered Molecular Shuttles

Biomolecular motors, such as kinesin, have been used to shuttle a range of biological and synthetic cargo in microfluidic architectures. A critical gap in this technology is the ability to controllably link macromolecular cargo on microtubule (MT) shuttles without forming extraneous byproducts that may potentially limit their application. Here we present a generalized approach for functionalizing MTs with antibodies in which covalent bonds are formed between the carbohydrate in Fc region of polyclonal antibodies and the positively charged amino acids on the MT surface using the crosslinker succinimidyl 4‐hydrazidoterephthalate hydrochloride (SHTH). Antibody‐functionalized MTs (Ab‐MTs) produced through this approach maintained motility characteristics and antigenic selectivity, and did not produce undesirable byproducts common to other approaches. We also demonstrate and characterize the application of these Ab‐MTs for capturing and transporting bacterial and viral antigens. While this approach cannot be applied to monoclonal antibodies, which lack a carbohydrate moiety, it may be used for selectively functionalizing MT shuttles with a variety of carbohydrate‐containing cargoes. Biotechnol. Bioeng. 2009; 104: 1182–1188.

A continuous network of lipid nanotubes fabricated from the gliding motility of kinesin powered microtubule filaments.

Synthetic interconnected lipid nanotube networks were fabricated on the millimeter scale based on the simple, cooperative interaction between phospholipid vesicles and kinesin-microtubule (MT) transport systems. More specifically, taxol-stabilized MTs, in constant 2D motion via surface absorbed kinesin, extracted and extended lipid nanotube networks from large Lα phase multilamellar liposomes (5-25 μm). Based on the properties of the inverted motility geometry, the total size of these nanofluidic networks was limited by MT surface density, molecular motor energy source (ATP), and total amount and physical properties of lipid source material. Interactions between MTs and extended lipid nanotubes resulted in bifurcation of the nanotubes and ultimately the generation of highly branched networks of fluidically connected nanotubes. The network bifurcation was easily tuned by changing the density of microtubules on the surface to increase or decrease the frequency of branching. The ability of these networks to capture nanomaterials at the membrane surface with high fidelity was subsequently demonstrated using quantum dots as a model system. The diffusive transport of quantum dots was also characterized with respect to using these nanotube networks for mass transport applications.

Directed self-assembly of 1D microtubule nano-arrays

Microtubules (MTs) are biological polymer filaments that display unique polymerization dynamics, and serve as inspiration for developing synthetic nanomaterials that exhibit similar assembly-derived behaviours. Here we explore an assembly process in which extended 1D nano-arrays (NAs) are formed through the directed, head-to-tail self-assembly of MT filaments. In particular, we demonstrate that the elongation of NAs over time is due to directed self-assembly of MTs by a process that is limited by diffusion and follows second-order rate kinetics. We further described a mechanism, both experimental and through molecular dynamics simulations, where stable junctions among MT building blocks are formed by alignment and adhesion of opposing filament ends, which is followed by formation of a stable junction through the incorporation of free tubulin and the removal of lattice vacancies. The fundamental principles described in this directed self-assembly process provide a promising basis for new approaches to manufacturing complex, heterostructured nanocomposites.