Tiean Zhou

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.

Quartz Crystal Microbalance Study of Endothelial Cell Number Dependent Differences in Initial Adhesion and Steady-State Behavior: Evidence for Cell-Cell Cooperativity in Initial Adhesion and Spreading

The quartz crystal microbalance (QCM) technique has been applied to the real time monitoring of endothelial cell (EC) adhesion and spreading on the QCM gold surface. We previously showed that the measured QCM Δf and ΔR shifts were due to cells adhering to the gold crystal surface, requiring proteolytic enzyme treatment to be removed from the surface, in order for the Δf and ΔR shifts to return to zero. In the present report, we demonstrate the quantitative dependence and saturation of the measured Δf and ΔR shifts on the number of firmly attached ECs as measured by electronic counting of the cells. We demonstrate through a light microscope simulation experiment that the different Δf and ΔR regions of the QCM temporal response curve correspond to the incident ECs contacting the surface, followed by their adhesion and spreading, which reflect cellular mass distribution and cytoskeletal viscoelasticity changes. Also, we demonstrate that the dose response curve of Δf and ΔR values versus attached EC number is more sensitive and possesses less scatter for the hydrophilically treated surface compared to the native gold surface of the QCM. For both surfaces, a Δf and ΔR versus trypsinized, attached EC number plot 1 h post‐seeding exhibits a sigmoid curve shape whereas a similar plot 24 h post‐seeding exhibits a hyperbolic curve shape. This number dependence suggests cell‐cell cooperativity in the initial cell adhesion and spreading processes. These QCM data and our interpretation are corroborated by differences in cell appearance and spreading behavior we observed for ECs in a light microscope fluorescence simulation experiment of the cell density effect. For a stably attached EC monolayer at 24 h post‐addition, steady‐state Δf and ΔR values are higher and exhibit saturation behavior for both the hydrophilically treated gold surface as compared to the untreated surface. The steady‐state 24 h Δf and ΔR values of stably attached ECs are shifted from the 1 h attached ECs. The 24 h values are characteristic of a more energy‐dissipative structure. This is consistent with the time‐dependent elaboration of surface contacts in anchorage‐dependent ECs via the attachment of intregrins to underlying extracellular matrix. It is also in agreement with the known energy dissipation function of the ECs that cover the interior of blood vessels and are exposed to continuous pulsatile blood flow.

Quartz Crystal Microbalance Measurement of Self-Assembled Micellar Tubules of the Amphiphilic Decyl Ester of d-Tyrosine and Their Enzymatic Polymerization

Amphiphilic decyl derivatives of d‐tyrosine self‐assemble into long rodlike or tubular aggregate structures in aqueous buffered solution. In this report we demonstrate the novel use of the quartz crystal microbalance (QCM) to measure the presence in solution, and subequent enzymatic polymerization, of long rodlike monomer aggregates of the decyl ester of d‐tyrosine (DEDT) as a function of their formation and increasing surface binding level as pH values increase from 3 to 7. From these data, using the Sauerbray equation to calculate the effective elastic mass surface binding of deprotonated DEDT aggregates, a pKapp of 8.3 is obtained for the DEDT α‐NH2 group protonation−deprotonation and subsequent aggregation equilibrium. Furthermore, once aggregates are bound to the QCM surface, we initiate and subsequently monitor enzymatic polymerization of the DEDT monomers by horseradish peroxidase through the measurement of significant changes in the quartz crystal frequency and motional resistance. Following the onset of polymerization, the viscoelastic properties of the bound monomer aggregates change. A final polymerized state is achieved in which the altered physical properties of the polymerized rodlike aggregates make the solution immediately above the QCM surface−solution interface behave as a Newtonian fluid, producing a nearly pure viscosity−density energy dissipative effect on the measured crystal frequency and motional resistance values.

Comparative study of electropolymerization versus adsorption of tyrosine and the decyl ester of tyrosine on platinum electrodes

Abstract We compared adsorption versus electropolymerization via cyclic voltammetry (CV) of tyrosine, an amino acid, to that of the decyl ester of d -tyrosine (DEDT), on Pt electrodes. DEDT is an amphiphilic compound, capable of self-assembly into long rod like or tubular aggregate structures. Tyrosine and DEDT differ fundamentally in their interactions with the Pt electrode, affecting their electropolymerization behavior. The DEDT exhibits a significant level of adsorption to the Pt surface, resisting desorption via washing to allow a significant peak current I p to be measured. At concentrations 30 times higher than DEDT, tyrosine exhibits no equivalent behavior. The sweep rate dependence of I p measured during the first CV cycle of film formation for the two species indicates very different behavior as well. Tyrosine has a square root dependence indicating a diffusion limiting mechanism, while DEDT has a linear sweep rate dependence indicating that it is a surface confined species and follows an adsorption limiting mechanism. Electrochemical quartz crystal microbalance (EQCM) studies show that in the absence of an applied potential, tyrosine does not adsorb to the Pt electrode since no Δ f shift is observed. On the other hand, DEDT does adsorb to a significant extent, displaying a sizable Δ f shift. When these same samples are then electropolymerized, tyrosine forms an electropolymerized film stepwise with each potential cycling step, while DEDT, already adsorbed to the Pt, exhibits no net mass gain for any of the potential cycles. Therefore, little electropolymerization of DEDT can take place in the presence of a significant adsorption level of this monomer. We ascribe these differences between tyrosine and DEDT largely to the presence of the decyl chain on DEDT, which can bind hydrophobically to the Pt surface and allow DEDT monomers to self-assemble with one another.

Electropolymerized tyrosine-based thin films: selective cell binding via peptide recognition to novel electropolymerized biomimetic tyrosine RGDY films.

We have created thin films by cyclic voltammetry (CV) electropolymerizations of the following phenolic functional group-based monomers: phenol; tyrosineamide; the tetrapeptide RGDY-containing the integrin membrane adhesion protein recognition tripeptide RGD; RDGY, a nonsense control tetrapeptide; and 1:3 mixtures of tyrosineamide with the two tetrapeptide monomers. The film formation process and description of the film properties were obtained by repetitive CV cycling using the oscillating quartz frequency shift, Deltaf, and motional resistance shift, DeltaR, parameters obtained with the electrochemical quartz crystal microbalance technique. Only the poly(phenol) film exhibited close chain packing-based self-limiting behavior, where all film synthesis ceased after approximately 7 CV cycles. All other films continued to form by electropolymerization with successive CV cycles out to the maximum cycle number (30 cycles) we measured. All of the films exhibited little energy dissipation behavior. Using the quartz crystal microbalance, we next compared the time course of cell attachment with the washed films and demonstrated that cells bound best to films in the following order: RGDY sense peptide:tyrosineamide films>RDGY nonsense peptide:tyrosineamide films=tyrosineamide films>phenol films. Cell enumeration after washing and trypsinization revealed firm protein-based cell attachment to the underlying extracellular matrix for the RGDY-containing films. These sense peptide films bound and retained two- to fivefold as many cells as the other films, with cells exhibiting a normal morphology. These results suggest the operation of specific cell attachment to the electropolymerized films via the RGD binding site for cellular integrin membrane proteins. The electropolymerization method we studied here forms a cassette system for creating electropolymerized films tailored to specific attachment of different cell types by varying the electropolymerized Y(tyrosine)-containing recognition peptide.

Potential Dependent Endothelial Cell Adhesion, Growth and Cytoskeletal Rearrangements

Normal endothelial cells (ECs), lining the blood vessels, are influenced by their interaction with the underlying potentially piezoelectric extracellular matrix (ECM). That this interaction may affect the EC metabolic state and functions in vivo prompted us to study the subsequent response of cultured ECs on indium-tin oxide (ITO) glass electrodes subjected to 1 hr of constant DC surface potential ranging from -0.3 to +0.6 V (vs. Ag/AgCl). We measured, relative to controls, cellular viability, growth rate and changes in actin microfilament organization in ECs over a subsequent 6 days in culture. The growth rate of ECs was stimulated by negative potential and inhibited by positive potential. Differences could be detected as early as three days post-potential. We also observed a potential dependent cellular shape change and actin microfilament rearrangement at positive potentials within four days of treatment. ECs changed in average cell surface area and assumed a polygonal cell shape in response to treatment. Using NBD-phalloidin stain for actin and fluorescence microscopy, microfilaments were observed to re-distribute to the periphery of the cell at positive potential, indicative of cellular stress.