A quartz crystal microbalance cell biosensor: detection of microtubule alterations in living cells at nM nocodazole concentrations.
The quartz crystal microbalance (QCM) was used to create a piezoelectric biosensor utilizing living endothelial cells (ECs) as the biological signal transduction element. ECs adhere to the hydrophilically treated gold QCM surface under growth media containing serum. At 24 h following cell addition, calibration curves were constructed relating the steady state Deltaf and DeltaR shift values observed to the numbers of electronically counted cells requiring trypsinization to be removed from the surface. We then utilized this EC QCM biosensor for the detection of the effect of [nocodazole] on the steady state Deltaf and DeltaR shift values. Nocodazole, a known microtubule binding drug, alters the cytoskeletal properties of living cells. At the doses used in these studies (0.11-15 microM), nocodazole, in a dose dependent fashion, causes the depolymerization of microtubules in living cells. This leads a monolayer of well spread ECs 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. We observed the negative Deltaf shift values and the positive DeltaR shift values to increase significantly in magnitude over a 4-h incubation period following nocodazole addition, in a dose dependent fashion, with a transition midpoint of 900 nM. 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. These results indicate that the EC QCM biosensor can be used for the study of EC attachment and to detect EC cytoskeletal alterations. We suggest the potential of this cellular biosensor for the real time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment, regardless of their molecular mechanism of action.
Marx
K A
KA
Department of Chemistry, Center for Intelligent Biomaterials, University of Massachusetts, Lowell, MA 01854, USA. kenneth_marx@uml.edu
Zhou
T
T
Montrone
A
A
Schulze
H
H
Braunhut
S J
SJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation*; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects*; D015739:Nocodazole / Q000494:pharmacology*; D011791:Quartz; D015398:Signal Transduction
D000199:Actins; D011791:Quartz; D015739:Nocodazole