Detection Of Microtubules Research Articles

Prospects for single-molecule electrostatic detection in molecular motor gliding motility assays

Molecular motor gliding motility assays based on myosin/actin or kinesin/microtubules are of interest for nanotechnology applications ranging from cargo-trafficking in lab-on-a-chip devices to novel biocomputation strategies. Prototype systems are typically monitored by expensive and bulky fluorescence microscopy systems. The development of integrated, direct electric detection of single filaments would strongly benefit applications and scale-up. We present estimates for the viability of such a detector by calculating the electrostatic potential change generated at a carbon nanotube transistor by a motile actin filament or microtubule under realistic gliding assay conditions. We combine this with detection limits based on previous state-of-the-art experiments using carbon nanotube transistors to detect catalysis by a bound lysozyme molecule and melting of a bound short-strand DNA molecule. Our results show that detection should be possible for both actin and microtubules using existing low ionic strength buffers given good device design, e.g., by raising the transistor slightly above the guiding channel floor. We perform studies as a function of buffer ionic strength, height of the transistor above the guiding channel floor, presence/absence of the casein surface passivation layer for microtubule assays and the linear charge density of the actin filaments/microtubules. We show that detection of microtubules is a more likely prospect given their smaller height of travel above the surface, higher negative charge density and the casein passivation, and may possibly be achieved with the nanoscale transistor sitting directly on the guiding channel floor.

Detection of microtubules in vivo using antibody-immobilized nanoneedles

We present here an alternative, force-based measurement method for the detection of intracellular cytoskeletal proteins in the live cell. High aspect ratio nanoneedles of 200 nm in diameter were functionalized with anti-tubulin antibodies and inserted, using an atomic force microscope (AFM), into live NIH3T3 cells, without affecting cell viability. Force curves were recorded during insertion and evacuation of nanoneedles from the cells, and used to analyse intracellular interactions of the nanoneedles with the microtubule cytoskeleton during evacuation from the cell. Disruption of microtubules led to a correlated time-dependent decrease in the measured intracellular binding forces, pointing to the high-sensitivity and high-specificity of this detection method. This analytical technique allows for real-time evaluation of the microtubule network in the live cell, without the need to use potentially harmful molecular markers as do conventional detection methods, and may prove beneficial in the diagnosis and investigation of cytoskeleton-associated diseases.