**1.3. Probing mechanical response of cells with quartz crystal microbalance with dissipation monitoring**

In contrast to AFM, the quartz crystal microbalance with dissipation monitoring (QCM-D) has not been widely used in characterization of cell mechanics. The QCM-D is an ultrasensitive piezoelectric device (Hook F. et al., 1998; Rodahl Michael et al., 1996; Rodahl M. & Kasemo, 1996) that is able to detect mass coupled (adsorbed or adhered) to the surface of the sensing element. The sensing element is a single piezoelectric quartz crystal in the form of a thin disc with a metal electrode deposited on its underside. This sensor crystal is set into free vibration in shear mode by means of a pulse of current. In air, the sensor crystal has a characteristic resonant frequency; this frequency is changed when any material, liquid or solid, is coupled or attached to it by adsorption or adhesion.

The mass of an ultrathin and elastic adsorbed layer, which exhibits negligible dissipation, is given by the Sauerbrey equation:

$$
\Delta m = \frac{\text{C}}{n \times \Delta f} \tag{2}
$$

Dynamic Mechanical Response of Epithelial Cells to Epidermal Growth Factor 175

al., 1997; Lee H. et al., 2010), to detect nucleic acid hybridization (Furtado & Thompson,

**Figure 2.** Diagram of a layer of cells attached to the surface of a sensor crystal in the QCM-D technique. The vibrational wave originating from the piezoelectric sensor crystal penetrates the cells from the bottom and diminishes with distance above the sensor surface. This figure is adapted with permission

In the field of cell biology, the QCM technique has become particularly attractive for its capability to study cells in a label-free manner (Heitmann et al., 2007; Janshoff et al., 1996; Matsuda et al., 1992; Redepenning et al., 1993; Wegener et al., 1998). Importantly, the technique is non-invasive to mammalian cells when the amplitude of shear oscillation is kept under 1 nm (Heitmann & Wegener, 2007). It has been used for determining the kinetics of cell attachment and spreading (Fredriksson et al., 1998; Nimeri et al., 1998) and for monitoring the long term growth of cells (Otto et al., 1999; Reipa et al., 2006). More recently, the QCM has been applied to characterization of cell viscoelasticity (Alessandrini et al., 2006; Galli Marxer et al., 2003; Li et al., 2008; Marx et al., 2005; Pax et al., 2005; Voinova et al., 2004). When the instrument used has the capability for monitoring the change in dissipation factor as well as frequency, the technique is termed QCM-D (QCM with dissipation monitoring). Because the acoustic signal diminishes exponentially with distance above the surface of the quartz crystal oscillator on which the cells are deposited, the QCM probes primarily the basal area of the cell monolayer (Heitmann et al., 2007; Le Guillou-Buffello et al., 2011). The test configuration is shown in Figure 2. Thus, ∆*f* and ∆*D*, for adherent cells measured by the QCM-D technique can be expected to be related to mechanical changes in basal area of the cells and may be related to strength and quality of adhesion between the

cell and the surface it contacts (Fredriksson et al., 1998; Rodahl M. et al., 1997).

**2.1. Mechanical behavior of an A431 cell monolayer probed with AFM** 

**monolayer in response to EGF** 

**2. Investigation of the dynamic mechanical response of an A431 cell** 

An example of the use of the AFM to assess change in the mechanical response of cells upon exposure to a biologically active molecule is illustrated in this section. Figure 3 shows the Young's moduli, obtained at a probe speed of 5.8 µm/s and indentation depth of ~500 nm, for two hundred randomly selected A431 cells before and after the treatment with a 40-nM

1998), and to study immunoresponse (Aizawa et al., 2001).

from (Chen et al., 2011). Copyright 2011, American Chemical Society.

where ∆*m* is mass, *C* is the instrument sensitivity constant in ng/cm2 of the crystal surface area, *n* is the frequency overtone number (*n* = 1, 3, 5, …), and ∆*f* is the simplified representation of ∆*fn*, the change in resonant frequency at the overtone number *n*, caused by the attached mass (Sauerbrey, 1959). The acoustic shear wave from the vibrating sensor penetrates the attached layer without disrupting it and the instrument monitors ∆*f* of the layer at multiple overtones (Hook Fredrik et al., 2001) as a function of time. The instrument simultaneously monitors the change in dissipation factor, ∆*D*, which is defined as the ratio of the dissipated energy to the stored (elastic) energy per vibrational cycle. An ultrathin, elastic layer exhibits a negligible value of ∆*D*, but a layer that is viscoelastic exhibits a nonnegligible and measureable value of ∆*D*. Both ∆*f* and ∆*D* provide information about the mechanical response of a layer attached to the surface of the sensor crystal, and any type of change in the layer produces changes in these quantities.

The QCM has been used to assess the changes in mass and mechanical properties of a layer of biomolecules immobilized on the surface of the quartz crystal (Dixon, 2008). Specific examples include the use of the QCM to show protein adsorption (Hook F. et al., 1998), to indicate changes in an immobilized layer caused by ligand–receptor interactions (Janshoff et al., 1997; Lee H. et al., 2010), to detect nucleic acid hybridization (Furtado & Thompson, 1998), and to study immunoresponse (Aizawa et al., 2001).

174 Viscoelasticity – From Theory to Biological Applications

**dissipation monitoring** 

given by the Sauerbrey equation:

retraction curves (Alcaraz et al., 2003), as shown in Figure 1.

or solid, is coupled or attached to it by adsorption or adhesion.

change in the layer produces changes in these quantities.

This dissipative behavior is manifested as a loss (as heat to the surroundings) of some of the energy stored during loading, and can be seen in the indentation process as hysteresis in a cycle of force displacement (Figure 1). In a cell, energy dissipation is believed to be accomplished by internal friction and/or viscous damping mechanisms (Alcaraz et al., 2003; Smith et al., 2005). In AFM, the mechanical energy dissipated per cycle of indentation is given quantitatively by the area of the hysteresis loop enclosed by the approach and

**1.3. Probing mechanical response of cells with quartz crystal microbalance with** 

In contrast to AFM, the quartz crystal microbalance with dissipation monitoring (QCM-D) has not been widely used in characterization of cell mechanics. The QCM-D is an ultrasensitive piezoelectric device (Hook F. et al., 1998; Rodahl Michael et al., 1996; Rodahl M. & Kasemo, 1996) that is able to detect mass coupled (adsorbed or adhered) to the surface of the sensing element. The sensing element is a single piezoelectric quartz crystal in the form of a thin disc with a metal electrode deposited on its underside. This sensor crystal is set into free vibration in shear mode by means of a pulse of current. In air, the sensor crystal has a characteristic resonant frequency; this frequency is changed when any material, liquid

The mass of an ultrathin and elastic adsorbed layer, which exhibits negligible dissipation, is

 *C*

where ∆*m* is mass, *C* is the instrument sensitivity constant in ng/cm2 of the crystal surface area, *n* is the frequency overtone number (*n* = 1, 3, 5, …), and ∆*f* is the simplified representation of ∆*fn*, the change in resonant frequency at the overtone number *n*, caused by the attached mass (Sauerbrey, 1959). The acoustic shear wave from the vibrating sensor penetrates the attached layer without disrupting it and the instrument monitors ∆*f* of the layer at multiple overtones (Hook Fredrik et al., 2001) as a function of time. The instrument simultaneously monitors the change in dissipation factor, ∆*D*, which is defined as the ratio of the dissipated energy to the stored (elastic) energy per vibrational cycle. An ultrathin, elastic layer exhibits a negligible value of ∆*D*, but a layer that is viscoelastic exhibits a nonnegligible and measureable value of ∆*D*. Both ∆*f* and ∆*D* provide information about the mechanical response of a layer attached to the surface of the sensor crystal, and any type of

The QCM has been used to assess the changes in mass and mechanical properties of a layer of biomolecules immobilized on the surface of the quartz crystal (Dixon, 2008). Specific examples include the use of the QCM to show protein adsorption (Hook F. et al., 1998), to indicate changes in an immobilized layer caused by ligand–receptor interactions (Janshoff et

*n f*

(2)

*m*

**Figure 2.** Diagram of a layer of cells attached to the surface of a sensor crystal in the QCM-D technique. The vibrational wave originating from the piezoelectric sensor crystal penetrates the cells from the bottom and diminishes with distance above the sensor surface. This figure is adapted with permission from (Chen et al., 2011). Copyright 2011, American Chemical Society.

In the field of cell biology, the QCM technique has become particularly attractive for its capability to study cells in a label-free manner (Heitmann et al., 2007; Janshoff et al., 1996; Matsuda et al., 1992; Redepenning et al., 1993; Wegener et al., 1998). Importantly, the technique is non-invasive to mammalian cells when the amplitude of shear oscillation is kept under 1 nm (Heitmann & Wegener, 2007). It has been used for determining the kinetics of cell attachment and spreading (Fredriksson et al., 1998; Nimeri et al., 1998) and for monitoring the long term growth of cells (Otto et al., 1999; Reipa et al., 2006). More recently, the QCM has been applied to characterization of cell viscoelasticity (Alessandrini et al., 2006; Galli Marxer et al., 2003; Li et al., 2008; Marx et al., 2005; Pax et al., 2005; Voinova et al., 2004). When the instrument used has the capability for monitoring the change in dissipation factor as well as frequency, the technique is termed QCM-D (QCM with dissipation monitoring). Because the acoustic signal diminishes exponentially with distance above the surface of the quartz crystal oscillator on which the cells are deposited, the QCM probes primarily the basal area of the cell monolayer (Heitmann et al., 2007; Le Guillou-Buffello et al., 2011). The test configuration is shown in Figure 2. Thus, ∆*f* and ∆*D*, for adherent cells measured by the QCM-D technique can be expected to be related to mechanical changes in basal area of the cells and may be related to strength and quality of adhesion between the cell and the surface it contacts (Fredriksson et al., 1998; Rodahl M. et al., 1997).
