Label-free affinity assays by rapid detection of immune complexes in submicrometer pores.
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Antibodies, Monoclonal↗
Biomedical subjects
Alan J Hunt
Publications and source records attributed to Alan J Hunt.
Submicrometer pore-based characterization and quantification of antibody-virus interactions.
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Animals↗
Microtubule movements on the arms of mitotic chromosomes: polar ejection forces quantified in vitro.
During mitosis, "polar ejection forces" (PEFs) are hypothesized to direct prometaphase chromosome movements by pushing chromosome arms toward the spindle equator. PEFs are postulated to be caused by (i) plus-end-directed microtubule (MT)-based motor proteins on the chromosome arms, namely chromokinesins, and (ii) the polymerization of spindle MTs into the chromosome. However, the exact role of PEFs is unclear, because little is known about their magnitude or their forms (e.g., impulsive vs. sustained, etc.). In this study, we employ optical tweezers to bring about the lateral interaction between chromosome arms and MTs in vitro to directly measure the speed and force of the PEFs developed on chromosome arms. We find that forces are unidirectional and frequently exceed 1 pN, with maximum forces of 2-3 pN and peak velocities of 63 +/- 41 nm/s; the movements are ATP-dependent and exhibit a characteristic noncontinuous motion that includes displacements of >50 nm, stalls, and backwards slippage of the MT even under low loads. We perform experiments using antibodies to the chromokinesins Kid and KIF4 that identify Kid as the principal force-producing agent for PEFs. At first glance, this motor activity appears surprisingly weak and erratic, but it explains how PEFs can guide chromosome movements without severely deforming or damaging the local chromosome structure.
Adenosine Triphosphate↗
Rapidly prototyped three-dimensional nanofluidic channel networks in glass substrates.
Microfluidic and nanofluidic technologies have long sought a fast, reliable method to overcome the creative limitations of planar fabrication methods, the resolution limits of lithography, and the materials limitations for fast prototyping. In the present work, we demonstrate direct 3D machining of submicrometer diameter, subsurface fluidic channels in glass, via optical breakdown near critical intensity, using a femtosecond pulsed laser. No postexposure etching or bonding is required; the channel network (or almost any arbitrary-shaped cavity below the surface) is produced directly from "art-to-part". The key to this approach is to use very low energy, highly focused, pulses in the presence of liquid. Microbubbles that result from laser energy deposition gently expand and extrude machining debris from the channels. These bubbles are in a highly damped, low Reynolds number regime, implying that surface spalling due to bubble collapse is unimportant. We demonstrate rapid prototyping of three-dimensional "jumpers", mixers, and other key components of complex 3D microscale analysis systems in glass substrates.
Glass↗
Microtubules: mechanical meets chemical.
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Binding Sites↗
A theoretical model of a molecular-motor-powered pump.
The motion of a cylindrical bead in a fluid contained within a two-dimensional channel is investigated using the boundary element method as a model of a biomolecular-motor-powered microfluidics pump. The novelty of the pump lies in the use of motor proteins (kinesin) to power the bead motion and the few moving parts comprising the pump. The performance and feasibility of this pump design is investigated using two model geometries: a straight channel, and a curved channel with two concentric circular walls. In the straight channel geometry, it is shown that increasing the bead radius relative to the channel width, increases the flow rate at the expense of increasing the force the kinesins must generate in order to move the bead. Pump efficiency is generally higher for larger bead radii, and larger beads can support higher imposed loads. In the circular channel geometry, it is shown that bead rotation modifies the force required to move the bead and that shifting the bead inward slightly reduces the required force. Bead rotation has a minimal effect on flow rate. Recirculation regions, which can develop between the bead and the channel walls, influence the stresses and force on the bead. These results suggest this pump design is feasible, and the kinesin molecules provide sufficient force to deliver pico- to atto- l/s flows.
Computer Simulation↗
Micropatterned structures for studying the mechanics of biological polymers.
Studying the mechanics of nanometer-scale biomolecules presents many challenges; these include maintaining light microscopy image quality and avoiding interference with the laser used for mechanical manipulation, that is, optical tweezers. Studying the pushing forces of a polymerizing filament requires barriers that meet these requirements and that can impede and restrain nanoscale structures subject to rapid thermal movements. We present a flexible technique that meets these criteria, allowing complex barrier geometries with undercut sidewall profiles to be produced on #1 cover glass for the purpose of obstructing and constraining polymerizing filaments, particularly microtubules. Using a two-layer lithographic process we are able to separate the construction of the primary features from the construction of a depth and shape-controlled undercut. The process can also be extended to create a large uniform gap between an SU-8 photoresist layer and the glass substrate. This technique can be easily scaled to produce large quantities of shelf-stable, reusable microstructures that are generally applicable to microscale studies of the interaction of cellular structures with defined microscale features.
Biomechanical Phenomena↗
Optics at critical intensity: applications to nanomorphing.
Laser-induced optical breakdown by femtosecond pulses is extraordinarily precise when the energy is near threshold. Despite numerous applications, the basis for this deterministic nature has not been determined. We present experiments that shed light on the basic mechanisms of light-matter interactions in this regime, which we term "optics at critical intensity." We find that the remarkably sharp threshold for laser-induced material damage enables the structure or properties of materials to be modified with nanometer precision. Through detailed study of the minimum ablation size and the effects of polarization, we propose a fundamental framework for describing light-matter interactions in this regime. In surprising contrast to accepted damage theory, multiphoton ionization does not play a significant role. Our results also reject the use of the Keldysh parameter in predicting the role of multiphoton effects. We find that the dominant mechanism is Zener ionization followed by a combination of Zener and Zener-seeded avalanche ionization. We predict that the minimum feature size ultimately depends on the valence electron density, which is sufficiently high and uniform, to confer deterministic behavior on the damage threshold even at the nanoscale. This behavior enables nanomachining with high precision, which we demonstrate by machining highly reproducible nanometer-sized holes and grooves in dielectrics.
Journal Article↗
Does axonemal dynein push, pull, or oscillate?
Dynein is the molecular motor that provides motive force in cilia and flagella. Dynein is anchored to the A-subtubule of the outer doublets by a club-shaped extension called the stem, which supports the large globular head of the molecule. Dynein forms an attachment or cross-bridge to the B-subtubule of the adjacent outer doublet through a slender appendage extending from the head that is called the stalk or alternately the B-link. It is generally thought that the B-link mediates the interdoublet transfer of force that bends the flagellum. This requires that energy released at the site of ATP hydrolysis, located in the globular head, be transferred as mechanical work to the microtubule binding site at the tip of the B-link. It has been proposed that this is accomplished by a sideways or rotational translocation of the B-link caused by a rotation of the globular head. An estimate of the stiffness of the B-link and stem derived from the recently published data of Burgess et al. [2003: Nature 421:715-718] yields a maximum stiffness of 0.47 pN/nm for the B-link and 0.1 pN/nm for the stem. The B-link stiffness would allow transfer of 3.8 pN of force in response to an 8-nm displacement of the B-link tip. However, if as proposed the globular head of the dynein heavy chain is supported by the stem, the B-link and stem elasticity are in series. Thus, the flexibility of the stem would limit the force that can be transferred laterally by the entire dynein heavy chain to 0.6 pN at 8 nm displacement. This force is insufficient to support flagellar motility. So, if the stem were the only support for the globular head, then force would have to be transmitted linearly along the axis defined by the stem and B-link. Because this configuration is never observed, the hypothesis that dynein generates force by lateral displacement of the B-link is more attractive, but requires that the globular head of the dynein is stabilized by an additional means of support during the power stroke. We propose that the microtubule affinity of the tip of the B-link is independent of the ATP-dependent powerstroke, and that detachment from the B-subtubule is regulated by tension. A dynein cross-bridge cycle that incorporates an anchored head, together with a ratchet-like mechanism for microtubule translocation by the B-link, would have distinct advantages. This mechanism may reconcile dynein oscillation and interdoublet sliding within one cross-bridge mechanism.
Adenosine Triphosphate↗
Advanced optical tweezers for the study of cellular and molecular biomechanics.
Optical tweezers are an important tool for studying cellular and molecular biomechanics. We present a robust optical tweezers device with advanced features including: multiple optical traps, acousto-optic trap steering, and back focal plane interferometry position detection. We integrate these features into an upright microscope, with no compromise to its capabilities (differential interference contrast microscopy, fluorescence microscopy, etc.). Acousto-optic deflectors (AODs) steer each beam and can create multiple time-shared traps. Position detection, force calibrations and AOD performance are presented. The system can detect subnanometer displacements and forces below 0.1 pN.
Biomechanical Phenomena↗
A simple, mechanistic model for directional instability during mitotic chromosome movements.
During mitosis, chromosomes become attached to microtubules that emanate from the two spindle poles. Thereafter, a chromosome moves along these microtubule "tracks" as it executes a series of movements that bring it to the spindle equator. After the onset of anaphase, the sister chromatids separate and move to opposite spindle poles. These movements are often characterized by "directional instability" (a series of runs with approximately constant speed, punctuated by sudden reversals in the direction of movement). To understand mitosis, it is critical to describe the physical mechanisms that underlie the coordination of the forces that drive directional instability. We propose a simple mechanistic model that describes the origin of the forces that move chromosomes and the coordination of these forces to produce directional instability. The model demonstrates that forces, speeds, and direction of motion associated with prometaphase through anaphase chromosome movements can be predicted from the molecular kinetics of interactions between dynamic microtubules and arrays of microtubule binding sites that are linked to the chromosome by compliant elements.
Anaphase↗