SUNY at Albany
June 19-23, 2001
A Quarts Crystal Microbalance Cell Biosensor: Detection of Microtubule Altarations in Living Cells at nM Nocodazole Conceatrations
Endothelial cells (ECs), in vivo, stably attached to their underlying extracellular matrix (ECM), represent an important biological state sensitive to flow properties and are regulated by small and large biological molecule effectors. The Quartz Crystal Microbalance (QCM) is a sensitive bound mass and interfacial viscoelastic property sensor based on the piezoelectric effect . ECs form monolayers on the (QCM) gold electrode surface under culture medium that exhibit cell number dependent QCM frequency (Df) and motional resistance (DR) shifts characteristic of energy dissipative behavior [2,3]. This system represents a new piezoelectric cell biosensor. The complex energy transduction mechanisms of this system are represented in a combination of the ECs' internal cytoskeleton linked via membrane bound integrins to the extracellular matrix, which is adhering to the gold QCM electrode. Changes in either the QCM effective surface bound mass of this complex system, or in the energy dissipation behavior of these coupled signal transduction elements bring about corresponding changes in sensor Df and DR output. Small drug or large biomolecules can be sensed by this cellular biosensor, if they effect changes in the mass distribution or viscoelastic properties of ECs following a 24 hr EC attachment period to establish steady state cell properties. We utilized this EC QCM biosensor for the detection of the effect of nocodazole, a known microtubule binding and depolymerizing drug, that alters the cytoskeletal properties of living cells. In a dose dependent fashion, in the range 0.11-15 mM, nocodazole caused negative Df shifts and positive DR shifts over a 5-6 hr incubation period following nocodazole addition. The Df and DR magnitudes changed (up to a 50% final Df change) in a sigmoid shaped dose dependent fashion, with a transition midpoint of 900 nM nocodazole . The nocodazole effect on Df and DR appears fully reversible by 25-30 hr following drug addition. Fluorescence microscopy of the ECs, fixed on the gold QCM surface and stained for actin, demonstrated that the shape and cytoskeleton of ECs were affected by as little as 330 nM nocodazole. Increasing nocodazole concentrations caused an EC monolayer to gradually occupy a smaller area, lose cell to cell contact, exhibit actin stress fibers at the cell periphery and acquire a rounded cell shape, before beginning to detach above 15 mM nocodazole. These results indicate that the EC QCM biosensor can be used for the study of EC attachment and to detect EC cytoskeletal alterations. In addition to basic research, this novel cell biosensor may be useful for the screening of either small molecule drugs or macromolecular therapeutics' effects on adherent cells. In particular, this biosensor would be of great utility for agents targeted to its signal transduction elements: the cytoskeleton, the membrane bound integrins, or the ECM-regardless of their molecular mechanism of action.
Kenneth A. Marx*, Tiean Zhou, Heather Schulze#, Anne Montrone# and Susan Braunhut#
Center for Intelligent Biomaterials, Departments of Chemistry and Biological Sciences#,
University of Massachusetts, Lowell, MA 01854