A Quartz Crystal Microbalance Cell Biosensor: Detecting Nocodazole Dependent Microtubule Disruption Dynamics In Living C
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A Quartz Crystal Microbalance Cell Biosensor: Detecting Nocodazole Dependent Microtubule Disruption Dynamics In Living Cells Kenneth A. Marx*, Tiean Zhou, Anne Montrone# and Susan J. Braunhut# Center for Intelligent Biomaterials, Departments of Chemistry and Biological Sciences# University of Massachusetts, Lowell, MA 01854 * corresponding author: [email protected] ; phone (978) 934-3658 ABSTRACT A Quartz Crystal Microbalance (QCM) was used to create a biosensor utilizing living adherent endothelial cells (ECs) as the biological sensing element. This EC QCM biosensor detected the effect of varying concentrations of nocodazole, a microtubule binding and disrupting drug, on the adherent cells as they altered the underlying QCM device state frequency, ∆f, and motional Resistance, ∆R, shift values. Over the dose range 0.11-15 µM nocodazole, the ∆f shift values decreased significantly in magnitude in a dose dependent fashion over a 5-6 hr incubation period following drug addition to a limiting value, with a 900 nM midpoint. This effect is consistent with nocodazole's known dose dependent effect on the disruption of microtubules. At all drug concentrations, the relative ∆f decrease with time was found to be very similar and well fit by a single exponential decay equation. For all nocodazole doses, t 0.5 was invariant, averaging t 0.5 = 0.83 ± 0.14 hr. These data demonstrate that a single dynamic sensing system within the cell, the microtubules, responds to the addition of nocodazole and its response can be quantified by the biosensor. These results indicate that the EC QCM biosensor can be used to detect EC cytoskeletal alterations and dynamics and may be a valuable screening method for all classes of biologically active drugs or biological macromolecules that affect cytoskeleton perturbations or cellular attachment. INTRODUCTION The development of new measurement techniques represents one of the major driving forces in biotechnology. Much recent activity has focused on whole cell based assay systems using optical techniques, especially fluorescence and chemileuminescence. We have developed a whole cell assay utilizing an alternative signal transduction mechanism, the piezoelectric mechanism of the Quartz Crystal Microbalance (QCM). Via automated recording of the oscillating crystal frequency, f, and motional resistance, R, the QCM sensitively and continuously detects the adhering surface mass of living cells and detects alterations of cellular viscoelastic behavior on the QCM surface. We call this new tool the cellular QCM biosensor. Aside from the QCM's initial use measuring chemicals in the gas phase[1] and as a more recent tool in analytical electrochemistry [2,3], in the past dozen years the QCM has been used to create biosensors [4-6]. Infrequently, whole cells have been studied at the QCM surface [7-10]. While these studies establish that adherent cells produce a reversible QCM crystal frequency shift, the considerable variability in reported ∆f shift values has not been understood. However, these studies clearl
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