- Email: [email protected]
Real time monitoring of the effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cell adhesion process using thickness shear mode (TSM) sensor
Biosensors and Bioelectronics 22 (2007) 2256–2260
Real time monitoring of the effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cell adhesion process using thickness shear mode (TSM) sensor E. Ergezen ∗ , S. Hong, K.A. Barbee, R. Lec School of Biomedical Engineering, Science and Health Systems, Drexel University, United States Received 2 May 2006; received in revised form 3 October 2006; accepted 9 November 2006 Available online 18 December 2006
Abstract The effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cellular interactions of the cell membrane with different substrates to determine the kinetics of cell adhesion was studied using thickness shear mode (TSM) sensor. The TSM sensor was operated at its first, third, fifth and seventh harmonics. Since the penetration depth of the shear wave decreases with increases in frequency, the multi-resonance operation of the TSM sensor was used to monitor the changes in the kinetics of the cell–substrate interaction at different distances from the sensor surface. During the sedimentation and the initial attachment of the cells on the sensor surface, the changes in the sensor resonant frequency and the magnitude response were monitored. First, HSPGs were partially digested with the enzyme Heparinase III to evaluate the effect of HSPG on the cell adhesion process. The results indicated that HSPG did not have any effect on the kinetics of the initial attachment, but it did reduce the strength of steady-state cell adhesion. Next, we investigated the effect of the electrostatic interactions of the cell membrane with the substrate on the cell adhesion. In this case, the sensor surface was coated with positively charged Poly-d-Lysine (PDL). It was observed that electrostatic interaction of the negatively charged cell membrane with the PDL surface promoted the initial cell adhesion but did not support long-term cell adhesion. The multi-resonant TSM technique was shown to be a very promising method for monitoring specific interfacial effects involving in cell adhesion process in real-time. © 2006 Elsevier B.V. All rights reserved. Keywords: Thickness shear mode sensor; Cell adhesion; Multi-resonance; Heparan Sulfate Proteoglycan
1. Introduction The cell adhesion process has been studied extensively due to its vital role in many biological research areas such as tissue engineering and biomaterials. Several techniques have been developed to elucidate the cell adhesion dynamics (CozensRoberts et al., 1990; Garcia et al., 1997). Because these techniques involve detaching the cells from the surface, separate experiments must be performed for each time point of interest making them labor intensive and precluding detailed analysis of the kinetics and mechanics of the complex cell adhesion process. Recently, the thickness shear mode (TSM) sensor method has been introduced to investigate the complex mechanism of the cell adhesion process. It was shown that a shift in the resonant frequency during the cell adhesion process depends on the cell type
∗
Corresponding author. Tel.: +1 2155314296. E-mail address: [email protected] (E. Ergezen).
0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.11.005
that forms a monolayer on the TSM sensor surface (Wegener et al., 1998). It was shown that a change in the resistance in the electrical motional arm of a TSM sensor was strongly related to the evolving surface coverage during attachment and spreading of cells (Haider and Gindre, 2004). Marx et al. (2001) investigated the effect of nocodazole on the steady-state cell adhesion strength on TSM surfaces treated to be hydrophilic. Rodahl et al. (1997) used the multi-resonance operation of TSM to monitor the adsorption of human serum albumin on hydrophobic and hydrophilic surfaces. They showed that the change in dissipation versus the change in frequency curves for 5 MHz and 15 MHz showed different patterns. The focus of this study is to understand the effects of the surface charges and HSPG on the cell adhesion strength by monitoring changes in the magnitude and the resonant frequency during the cell adhesion process. Furthermore, the TSM sensor was operated at not only the fundamental frequency but also its third, fifth, and seventh harmonics to detect the cell adhesion process at different distances from the substrate.
E. Ergezen et al. / Biosensors and Bioelectronics 22 (2007) 2256–2260
2257
2. Materials and methods
3. Results and discussions
2.1. Acoustic measurement system
3.1. Influence of HSPGs on cell adhesion process
A disk-shaped AT-cut quartz crystal with a fundamental frequency of 5 MHz was used in the experiments. The crystal is 14 mm in diameter and has gold electrodes on either side that are 7 mm in diameter. A Teflon cylinder, with an 8 mm inner diameter was attached to the quartz to form the measurement chamber. Only one side of the quartz was exposed to the loading layer of liquid. The sensor was placed in a humidified, 37 ◦ C incubator with 5% CO2 in air. It was connected to a network analyzer (NA) (HP 4395A), and a personal computer was used to control the experiment using a custom program and Labview software. Throughout the experiments, maximum magnitude and frequency corresponding to the maximum magnitude were monitored at 5 MHz, 15 MHz, 25 MHz and 35 MHz. Initially 0.1 ml of serum-free media was placed in the TSM sensor chamber and allowed to equilibrate to 37 ◦ C. Then 0.4 ml of cell suspension (37 ◦ C, 3.75 × 105 cells/ml) was added into the chamber. The final concentration of the cells was 3 × 105 cells/ml.
Fig. 1 shows the changes in the resonant frequency (f) and magnitude (α) during the adhesion of untreated and Heparinase III-treated cells on a gelatin-coated TSM sensor surface. Initially, 100 l DMEM was added to the sensor surface, and f and α were allowed to reach stable baseline value. The cell suspension was added to the chamber at time zero. Two parallel measurement setups were used—one for the control sample, and one for the treatment sample (Heparinase III). Both control and Heparinase III treated cells showed the similar three distinctive zones for the cell adhesion process (Fig. 1a) (Hong et al., 2006); (1) initial drop for 1.30 h, (2) plateau for 1 h, and (3) second slower drop. The Heparinase III treatment did not change the initial kinetics of the cell adhesion but it decreased the steady-state cell adhesion strength. This can be seen in both resonant frequency and the magnitude responses (Fig. 1b and d). The reason for this decreased f and α in the steady state response can be attributed to the important role of HSPGs in the formation of focal adhesion in cell adhesion process. In addition they stabilize and strengthen the cell adhesion to the extra-cellular matrix (ECM) (Bernfield et al., 1999). Due to the reduction in HSPG on the cell surface, the steady state strength of the cell adhesion and focal adhesion formation are decreased, and this effect can be easily seen in sensor responses of each harmonic.
2.2. Preparation of endothelial cell culture The bovine aortic endothelial cells (BAECs) were cultured in complete media [Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Herndon, VA), 100 units/ml penicillin, 100 g/ml streptomycin, 250 ng/ml amphotericin B (Sigma Chemical Co., St. Louis, MO), 2 mM/ml l-glutamine and 10% heat-inactivated calf serum (Invitrogen Co., Carlsbad, CA)] in a humidified, 37 ◦ C incubator with 5% CO2 in air. Passage numbers of the cells were between 6 and 14. The confluent monolayer of BAECs was treated with 0.25% trypsin for 90 s, centrifuged at 200 × g for 5 min, and resuspended in serum-free media [Dulbecco’s modified Eagle’s medium (Mediatech, Inc.), 100 units/ml penicillin, 100 g/ml streptomycin, 250 ng/ml amphotericin B (Sigma Chemical Co.), 2 mM/ml l-glutamine (Invitrogen Co.)]. 2.3. Heparinase III treatment The monolayer of BAECs was incubated in Heparinase III (30 mU/ml) in phenol red free Minimal Essential Media (MEM) [Sigma Chemical Co., 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM/ml l-glutamine, and 1% BSA (Sigma Chemical Co.)] for 3 h (Florian et al., 2003). After the incubation, the monolayer was rinsed with MEM twice, and the cell was resuspended as described above. 2.4. Surface coating of TSM sensor For the gelatin coating, a 0.5% (v/v) gelatin solution (Sigma Chemical Co.) was applied to the TSM sensor for 30 min at room temperature. The surface was then rinsed two times with PBS. For the PDL coating of the surface, a TSM sensor surface was exposed to PDL solution (2 mg/ml in PBS) for 20 min. It was then rinsed with PBS twice and allowed to dry overnight.
3.2. Influence of PDL coated surface on cell adhesion process To understand the effects of the surface charges on the cell adhesion process, one of the sensors was coated with positively charged PDL, while the other sensor was coated with gelatin, which serves as the control. It was expected that this treatment of the sensor surface would promote the initial adhesion of cells on the surface due to the interactions of the positively charged PDL surface with the negatively charged cell surface. Consistent with this hypothesis, cell adhesion on PDL coated surface showed a steeper change in magnitude response in the initial stage (zone 1) of the cell adhesion process (Fig. 2) for all harmonics (only third harmonic data is shown) However, at the end of the experiment the magnitude response for the cells on the PDL-coated surface returned to its initial value (Fig. 3b). This result is also consistent with the fact that PDL does not support integrin binding and focal adhesion formation (Jacobson and Branton, 1977). After 15 h of running the experiment, it was observed by visual inspection that the cells had detached (data not shown). As seen from Fig. 3a, magnitude and frequency change response of control experiment shows the typical cell adhesion process response consisting of three zones. In the resonant frequency changes for cell adhesion on the PDL-coated surface (Fig. 3d), the f of the fifth and seventh harmonics decrease faster than the control experiment (Fig. 3c) but this is not seen in the first and third harmonics. The possible explanation could
2258
E. Ergezen et al. / Biosensors and Bioelectronics 22 (2007) 2256–2260
Fig. 1. The change in the magnitude and the resonant frequency during the cell adhesion process on a gelatin-coated TSM sensor at 5 MHz, 15 MHz, 25 MHz and 35 MHz. (a) Magnitude response of the control experiment. (b) Magnitude response of Heparinase III treated cells. (c) Response of resonant frequency of the control experiment. (d) Response of resonant frequency of Heparinase III treated cells.
be that the fifth and seventh harmonics, due to the fact that their depth of penetration is shorter than the first and third harmonics, are the most sensitive to cell binding process. The resonant frequency responses of cell adhesion on the PDL surface show distinct profiles depending on the harmonic (Fig. 3d). The first harmonic is not significantly different from the control (Fig. 3b). The initial frequency change for the third harmonic was similar to the control but then decayed with time
Fig. 2. Comparison of α responses of the cells on gelatin and PDL-coated surfaces for the first 2 h during the cell adhesion process at 15 MHz (third harmonic).
such that it was approximately 200 Hz different from control at steady state. The higher harmonics (fifth and seventh) showed significantly greater initial frequency changes that persisted through the first plateau period, but then decreased to values similar to the controls. From a multi-resonance TSM sensor modeling point of view, it has been shown that the cell adhesion process does not show rigid mass loading. Hence, the well-known Sauerbrey equation cannot be applied (Janshoff et al., 1996). It was suggested that the cell adhesion process should be modeled as a multi-layer model in which each layer shows different rheological properties (Wegener et al., 2000). In this respect, the use of a multi-resonant TSM sensor to monitor the cell adhesion process brings high quality information useful for cell adhesion modeling. As seen from Fig. 3c, the steady state f’s for the cells on a gelatin-coated surface are 300 Hz, 575 Hz, 825 Hz, and 1000 Hz for first, third, fifth and seventh harmonics, respectively. We have calculated that, for example, in the case of the first harmonic, the measured 300 Hz frequency change would result in the value of the cell effective viscosity of 2.5 mPa s. In turn, this value of the viscosity leads to the amplitude change of −2.5 dB. However, the measured value of the amplitude for the first harmonic was −3.7 dB (Fig. 3a), a significant difference of −1.2 dB. This supports the hypothesis that cell adhesion is a complex process involving a change in the density, viscosity and elasticity of the cell–surface system.
E. Ergezen et al. / Biosensors and Bioelectronics 22 (2007) 2256–2260
2259
Fig. 3. The change in the magnitude and the resonant frequency during the cell adhesion process on a gelatin-coated TSM sensor at 5 MHz, 15 MHz, 25 MHz and 35 MHz. (a) Magnitude response of the control experiment on gelatin-coated surface. (b) Magnitude response of cells on PDL coated surface. (c) Response of resonant frequency of the control experiment on gelatin-coated surface. (d) Response of resonant frequency for cells on a PDL coated surface.
Furthermore, if we apply the same steady-state viscosity value to the other harmonics, we observe that the f’s should be 840 Hz, 1280 Hz, and 1600 Hz for third, fifth and seventh harmonics. In addition, the α’s should be −1.79 dB, −1.21 dB, and −0.8 dB. These results are calculated by using transmission line model (TLM) of TSM sensor (Edwards and Martin, 1994). It can easily be seen that these values differ greatly from the experimental results (−2.2 dB, −1.0 dB and −0.5 dB for third, fifth and seventh harmonics, respectively). The same analysis of the other experiments (Figs. 1a–d and 3a and d) shows a similar discrepancy between the experimental and theoretical data. Therefore, the cell adhesion process needs to be described as a full viscoelastic interfacial phenomenon. 4. Conclusion In this paper, a multi-resonant thickness shear mode sensor was used to monitor the effect of HSPGs and surface charges on the cell adhesion process in real-time. We demonstrated that while HSPGs have an effect on the strength of the steady-state cell adhesion, PDL treated surface promoted the initial attachment of the cells. In addition, the multi-resonance operation of TSM sensor provided a better understanding of processes involving cell–surface interactions.
We have showed that the cell adhesion process cannot be described as a purely viscous or a mass loading phenomenon. It is a complex process involving change in viscosity, elasticity and density at the interface. By using a multi-resonant technique, we demonstrated that acoustic shear propagating waves, operating at different harmonic frequencies exhibited, due to their depth of penetration, unique features related to underlying interfacial processes involving cell and various substrates. Acknowledgements Support is acknowledged from NSF grant BES-9984276 & NSF grant DBI-0242662. We are thankful to Himanshu Mehta, Mark Mattuicci and Robert Weisbein for their productive criticisms. References Bernfield, M., et al., 1999. Annu. Rev. Biochem. 68, 729–777. Cozens-Roberts, C., Quinn, J.A., Lauffenburger, D.A., 1990. Biophys. J. 58 (4), 857–872. Edwards, V., Martin, S.J., 1994. J. Appl. Phys. 75 (3), 1319–1329. Florian, J.A., et al., 2003. Circ. Res. 93 (10), 136–142.
2260
E. Ergezen et al. / Biosensors and Bioelectronics 22 (2007) 2256–2260
Garcia, A.J., Ducheyne, P., Boettiger, D., 1997. Biomaterials 18 (16), 1091–1098. Haider, L., Gindre, M., 2004. IEEE Sens. J. 4 (5). Hong, et al., 2006. Biomaterials 27 (34), 5813–5820, Epub. Jacobson, B.S., Branton, D., 1977. Science 195, 302–304.
Janshoff, A., et al., 1996. Eur. Biophys. J. 25, 93–103. Marx, K.A., et al., 2001. Biosens. Bioelectron. 16, 773–782. Rodahl, et al., 1997. Faraday Discuss. 107, 229–246. Wegener, J., et al., 2000. Biophys. J. 78, 2821–2833. Wegener, J., Janshoff, A., Galla, H.J., 1998. Eur. Biophys. J. 28 (1), 26–37.
Real time monitoring of the effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cell adhesion process using thickness shear mode (TSM) sensor E. Ergezen ∗ , S. Hong, K.A. Barbee, R. Lec School of Biomedical Engineering, Science and Health Systems, Drexel University, United States Received 2 May 2006; received in revised form 3 October 2006; accepted 9 November 2006 Available online 18 December 2006
Abstract The effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cellular interactions of the cell membrane with different substrates to determine the kinetics of cell adhesion was studied using thickness shear mode (TSM) sensor. The TSM sensor was operated at its first, third, fifth and seventh harmonics. Since the penetration depth of the shear wave decreases with increases in frequency, the multi-resonance operation of the TSM sensor was used to monitor the changes in the kinetics of the cell–substrate interaction at different distances from the sensor surface. During the sedimentation and the initial attachment of the cells on the sensor surface, the changes in the sensor resonant frequency and the magnitude response were monitored. First, HSPGs were partially digested with the enzyme Heparinase III to evaluate the effect of HSPG on the cell adhesion process. The results indicated that HSPG did not have any effect on the kinetics of the initial attachment, but it did reduce the strength of steady-state cell adhesion. Next, we investigated the effect of the electrostatic interactions of the cell membrane with the substrate on the cell adhesion. In this case, the sensor surface was coated with positively charged Poly-d-Lysine (PDL). It was observed that electrostatic interaction of the negatively charged cell membrane with the PDL surface promoted the initial cell adhesion but did not support long-term cell adhesion. The multi-resonant TSM technique was shown to be a very promising method for monitoring specific interfacial effects involving in cell adhesion process in real-time. © 2006 Elsevier B.V. All rights reserved. Keywords: Thickness shear mode sensor; Cell adhesion; Multi-resonance; Heparan Sulfate Proteoglycan
1. Introduction The cell adhesion process has been studied extensively due to its vital role in many biological research areas such as tissue engineering and biomaterials. Several techniques have been developed to elucidate the cell adhesion dynamics (CozensRoberts et al., 1990; Garcia et al., 1997). Because these techniques involve detaching the cells from the surface, separate experiments must be performed for each time point of interest making them labor intensive and precluding detailed analysis of the kinetics and mechanics of the complex cell adhesion process. Recently, the thickness shear mode (TSM) sensor method has been introduced to investigate the complex mechanism of the cell adhesion process. It was shown that a shift in the resonant frequency during the cell adhesion process depends on the cell type
∗
Corresponding author. Tel.: +1 2155314296. E-mail address: [email protected] (E. Ergezen).
0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.11.005
that forms a monolayer on the TSM sensor surface (Wegener et al., 1998). It was shown that a change in the resistance in the electrical motional arm of a TSM sensor was strongly related to the evolving surface coverage during attachment and spreading of cells (Haider and Gindre, 2004). Marx et al. (2001) investigated the effect of nocodazole on the steady-state cell adhesion strength on TSM surfaces treated to be hydrophilic. Rodahl et al. (1997) used the multi-resonance operation of TSM to monitor the adsorption of human serum albumin on hydrophobic and hydrophilic surfaces. They showed that the change in dissipation versus the change in frequency curves for 5 MHz and 15 MHz showed different patterns. The focus of this study is to understand the effects of the surface charges and HSPG on the cell adhesion strength by monitoring changes in the magnitude and the resonant frequency during the cell adhesion process. Furthermore, the TSM sensor was operated at not only the fundamental frequency but also its third, fifth, and seventh harmonics to detect the cell adhesion process at different distances from the substrate.
E. Ergezen et al. / Biosensors and Bioelectronics 22 (2007) 2256–2260
2257
2. Materials and methods
3. Results and discussions
2.1. Acoustic measurement system
3.1. Influence of HSPGs on cell adhesion process
A disk-shaped AT-cut quartz crystal with a fundamental frequency of 5 MHz was used in the experiments. The crystal is 14 mm in diameter and has gold electrodes on either side that are 7 mm in diameter. A Teflon cylinder, with an 8 mm inner diameter was attached to the quartz to form the measurement chamber. Only one side of the quartz was exposed to the loading layer of liquid. The sensor was placed in a humidified, 37 ◦ C incubator with 5% CO2 in air. It was connected to a network analyzer (NA) (HP 4395A), and a personal computer was used to control the experiment using a custom program and Labview software. Throughout the experiments, maximum magnitude and frequency corresponding to the maximum magnitude were monitored at 5 MHz, 15 MHz, 25 MHz and 35 MHz. Initially 0.1 ml of serum-free media was placed in the TSM sensor chamber and allowed to equilibrate to 37 ◦ C. Then 0.4 ml of cell suspension (37 ◦ C, 3.75 × 105 cells/ml) was added into the chamber. The final concentration of the cells was 3 × 105 cells/ml.
Fig. 1 shows the changes in the resonant frequency (f) and magnitude (α) during the adhesion of untreated and Heparinase III-treated cells on a gelatin-coated TSM sensor surface. Initially, 100 l DMEM was added to the sensor surface, and f and α were allowed to reach stable baseline value. The cell suspension was added to the chamber at time zero. Two parallel measurement setups were used—one for the control sample, and one for the treatment sample (Heparinase III). Both control and Heparinase III treated cells showed the similar three distinctive zones for the cell adhesion process (Fig. 1a) (Hong et al., 2006); (1) initial drop for 1.30 h, (2) plateau for 1 h, and (3) second slower drop. The Heparinase III treatment did not change the initial kinetics of the cell adhesion but it decreased the steady-state cell adhesion strength. This can be seen in both resonant frequency and the magnitude responses (Fig. 1b and d). The reason for this decreased f and α in the steady state response can be attributed to the important role of HSPGs in the formation of focal adhesion in cell adhesion process. In addition they stabilize and strengthen the cell adhesion to the extra-cellular matrix (ECM) (Bernfield et al., 1999). Due to the reduction in HSPG on the cell surface, the steady state strength of the cell adhesion and focal adhesion formation are decreased, and this effect can be easily seen in sensor responses of each harmonic.
2.2. Preparation of endothelial cell culture The bovine aortic endothelial cells (BAECs) were cultured in complete media [Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Herndon, VA), 100 units/ml penicillin, 100 g/ml streptomycin, 250 ng/ml amphotericin B (Sigma Chemical Co., St. Louis, MO), 2 mM/ml l-glutamine and 10% heat-inactivated calf serum (Invitrogen Co., Carlsbad, CA)] in a humidified, 37 ◦ C incubator with 5% CO2 in air. Passage numbers of the cells were between 6 and 14. The confluent monolayer of BAECs was treated with 0.25% trypsin for 90 s, centrifuged at 200 × g for 5 min, and resuspended in serum-free media [Dulbecco’s modified Eagle’s medium (Mediatech, Inc.), 100 units/ml penicillin, 100 g/ml streptomycin, 250 ng/ml amphotericin B (Sigma Chemical Co.), 2 mM/ml l-glutamine (Invitrogen Co.)]. 2.3. Heparinase III treatment The monolayer of BAECs was incubated in Heparinase III (30 mU/ml) in phenol red free Minimal Essential Media (MEM) [Sigma Chemical Co., 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM/ml l-glutamine, and 1% BSA (Sigma Chemical Co.)] for 3 h (Florian et al., 2003). After the incubation, the monolayer was rinsed with MEM twice, and the cell was resuspended as described above. 2.4. Surface coating of TSM sensor For the gelatin coating, a 0.5% (v/v) gelatin solution (Sigma Chemical Co.) was applied to the TSM sensor for 30 min at room temperature. The surface was then rinsed two times with PBS. For the PDL coating of the surface, a TSM sensor surface was exposed to PDL solution (2 mg/ml in PBS) for 20 min. It was then rinsed with PBS twice and allowed to dry overnight.
3.2. Influence of PDL coated surface on cell adhesion process To understand the effects of the surface charges on the cell adhesion process, one of the sensors was coated with positively charged PDL, while the other sensor was coated with gelatin, which serves as the control. It was expected that this treatment of the sensor surface would promote the initial adhesion of cells on the surface due to the interactions of the positively charged PDL surface with the negatively charged cell surface. Consistent with this hypothesis, cell adhesion on PDL coated surface showed a steeper change in magnitude response in the initial stage (zone 1) of the cell adhesion process (Fig. 2) for all harmonics (only third harmonic data is shown) However, at the end of the experiment the magnitude response for the cells on the PDL-coated surface returned to its initial value (Fig. 3b). This result is also consistent with the fact that PDL does not support integrin binding and focal adhesion formation (Jacobson and Branton, 1977). After 15 h of running the experiment, it was observed by visual inspection that the cells had detached (data not shown). As seen from Fig. 3a, magnitude and frequency change response of control experiment shows the typical cell adhesion process response consisting of three zones. In the resonant frequency changes for cell adhesion on the PDL-coated surface (Fig. 3d), the f of the fifth and seventh harmonics decrease faster than the control experiment (Fig. 3c) but this is not seen in the first and third harmonics. The possible explanation could
2258
E. Ergezen et al. / Biosensors and Bioelectronics 22 (2007) 2256–2260
Fig. 1. The change in the magnitude and the resonant frequency during the cell adhesion process on a gelatin-coated TSM sensor at 5 MHz, 15 MHz, 25 MHz and 35 MHz. (a) Magnitude response of the control experiment. (b) Magnitude response of Heparinase III treated cells. (c) Response of resonant frequency of the control experiment. (d) Response of resonant frequency of Heparinase III treated cells.
be that the fifth and seventh harmonics, due to the fact that their depth of penetration is shorter than the first and third harmonics, are the most sensitive to cell binding process. The resonant frequency responses of cell adhesion on the PDL surface show distinct profiles depending on the harmonic (Fig. 3d). The first harmonic is not significantly different from the control (Fig. 3b). The initial frequency change for the third harmonic was similar to the control but then decayed with time
Fig. 2. Comparison of α responses of the cells on gelatin and PDL-coated surfaces for the first 2 h during the cell adhesion process at 15 MHz (third harmonic).
such that it was approximately 200 Hz different from control at steady state. The higher harmonics (fifth and seventh) showed significantly greater initial frequency changes that persisted through the first plateau period, but then decreased to values similar to the controls. From a multi-resonance TSM sensor modeling point of view, it has been shown that the cell adhesion process does not show rigid mass loading. Hence, the well-known Sauerbrey equation cannot be applied (Janshoff et al., 1996). It was suggested that the cell adhesion process should be modeled as a multi-layer model in which each layer shows different rheological properties (Wegener et al., 2000). In this respect, the use of a multi-resonant TSM sensor to monitor the cell adhesion process brings high quality information useful for cell adhesion modeling. As seen from Fig. 3c, the steady state f’s for the cells on a gelatin-coated surface are 300 Hz, 575 Hz, 825 Hz, and 1000 Hz for first, third, fifth and seventh harmonics, respectively. We have calculated that, for example, in the case of the first harmonic, the measured 300 Hz frequency change would result in the value of the cell effective viscosity of 2.5 mPa s. In turn, this value of the viscosity leads to the amplitude change of −2.5 dB. However, the measured value of the amplitude for the first harmonic was −3.7 dB (Fig. 3a), a significant difference of −1.2 dB. This supports the hypothesis that cell adhesion is a complex process involving a change in the density, viscosity and elasticity of the cell–surface system.
E. Ergezen et al. / Biosensors and Bioelectronics 22 (2007) 2256–2260
2259
Fig. 3. The change in the magnitude and the resonant frequency during the cell adhesion process on a gelatin-coated TSM sensor at 5 MHz, 15 MHz, 25 MHz and 35 MHz. (a) Magnitude response of the control experiment on gelatin-coated surface. (b) Magnitude response of cells on PDL coated surface. (c) Response of resonant frequency of the control experiment on gelatin-coated surface. (d) Response of resonant frequency for cells on a PDL coated surface.
Furthermore, if we apply the same steady-state viscosity value to the other harmonics, we observe that the f’s should be 840 Hz, 1280 Hz, and 1600 Hz for third, fifth and seventh harmonics. In addition, the α’s should be −1.79 dB, −1.21 dB, and −0.8 dB. These results are calculated by using transmission line model (TLM) of TSM sensor (Edwards and Martin, 1994). It can easily be seen that these values differ greatly from the experimental results (−2.2 dB, −1.0 dB and −0.5 dB for third, fifth and seventh harmonics, respectively). The same analysis of the other experiments (Figs. 1a–d and 3a and d) shows a similar discrepancy between the experimental and theoretical data. Therefore, the cell adhesion process needs to be described as a full viscoelastic interfacial phenomenon. 4. Conclusion In this paper, a multi-resonant thickness shear mode sensor was used to monitor the effect of HSPGs and surface charges on the cell adhesion process in real-time. We demonstrated that while HSPGs have an effect on the strength of the steady-state cell adhesion, PDL treated surface promoted the initial attachment of the cells. In addition, the multi-resonance operation of TSM sensor provided a better understanding of processes involving cell–surface interactions.
We have showed that the cell adhesion process cannot be described as a purely viscous or a mass loading phenomenon. It is a complex process involving change in viscosity, elasticity and density at the interface. By using a multi-resonant technique, we demonstrated that acoustic shear propagating waves, operating at different harmonic frequencies exhibited, due to their depth of penetration, unique features related to underlying interfacial processes involving cell and various substrates. Acknowledgements Support is acknowledged from NSF grant BES-9984276 & NSF grant DBI-0242662. We are thankful to Himanshu Mehta, Mark Mattuicci and Robert Weisbein for their productive criticisms. References Bernfield, M., et al., 1999. Annu. Rev. Biochem. 68, 729–777. Cozens-Roberts, C., Quinn, J.A., Lauffenburger, D.A., 1990. Biophys. J. 58 (4), 857–872. Edwards, V., Martin, S.J., 1994. J. Appl. Phys. 75 (3), 1319–1329. Florian, J.A., et al., 2003. Circ. Res. 93 (10), 136–142.
2260
E. Ergezen et al. / Biosensors and Bioelectronics 22 (2007) 2256–2260
Garcia, A.J., Ducheyne, P., Boettiger, D., 1997. Biomaterials 18 (16), 1091–1098. Haider, L., Gindre, M., 2004. IEEE Sens. J. 4 (5). Hong, et al., 2006. Biomaterials 27 (34), 5813–5820, Epub. Jacobson, B.S., Branton, D., 1977. Science 195, 302–304.
Janshoff, A., et al., 1996. Eur. Biophys. J. 25, 93–103. Marx, K.A., et al., 2001. Biosens. Bioelectron. 16, 773–782. Rodahl, et al., 1997. Faraday Discuss. 107, 229–246. Wegener, J., et al., 2000. Biophys. J. 78, 2821–2833. Wegener, J., Janshoff, A., Galla, H.J., 1998. Eur. Biophys. J. 28 (1), 26–37.