Langmuir 2002, 18, 6935-6939
6935
Investigating the Mechanical Effects of Adsorption of Ca2+ Ions on a Silicon Nitride Microcantilever Surface Suman Cherian, Adosh Mehta, and Thomas Thundat* Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6123 Received April 4, 2002. In Final Form: June 7, 2002 Adsorption characteristics of calcium ions on silicon nitride surfaces have been investigated using microcantilevers. Both resonance frequency and cantilever bending undergo variation due to calcium ion adsorption. The adsorption behavior was found to fit a Langmuir isotherm. From the linearized form of the isotherm, the binding constant of adsorption, K, was found to be 116 M-1 and the stress change at maximum coverage, ∆σmax, was 0.46 N m-1. The Gibbs free energy change was estimated to be -11.8 kJ mol-1. A calcium-binding protein, calmodulin, was used to check for the presence of adsorbed Ca2+ ions on the cantilever surface. Fluorescence measurements with Alexa Fluor 488 conjugated calmodulin gave confirmatory evidence of the presence of Ca2+ ions on the silicon nitride side of the cantilever surface. These results indicate that the presence of Ca2+ ions and probably other cations in the solution can cause cantilever resonance frequency shifts and bending. It is therefore critical to correct for these contributions even when the reaction of interest is confined to the gold side.
Introduction In recent years, there has been a great interest in the development of microelectromechanical devices for various applications. Microcantilevers, typically used in atomic force microscopy, are being developed as a new class of sensitive stress sensors. When a cantilever consists of two different surfaces, processes occurring preferentially on one side cause a differential surface stress resulting in cantilever bending.1-8 Microcantilevers have been used for different applications such as infrared detectors9 and mercury5,10 and humidity11 sensors. In the liquid medium, they have been used as chemical12-14 and biological sensors.15,16 When measuring cantilever bending and frequency shifts due to analyte-cantilever interaction, it is important to understand and correct for the signals originating from interfering species. * Corresponding author. E-mail:
[email protected]. (1) Gimzewski, J. K.; Gerber, C.; Meyer, E.; Schlittler, R. R. Chem. Phys. Lett. 1994, 217, 589. (2) Thundat, T.; Warmack, R. J.; Chen, G. Y.; Allison, D. P. Appl. Phys. Lett. 1994, 64, 2894. (3) Barnes, J. R.; Stephenson, R. J.; Welland, M. E.; Gerber, C.; Gimzewski, J. K. Nature 1994, 372, 79. (4) Butt, H.-J. J. Colloid Interface Sci. 1996, 180, 251. (5) Thundat, T.; Oden, P. I.; Warmack, R. J. Microscale Thermophys. Eng. 1997, 1, 185. (6) Raiteri, R.; Nelles, G.; Butt, H.-J.; Knoll, W.; Skladal, P. Sens. Actuators, B 1999, 61, 213. (7) Raiteri, R.; Bu¨tt, H.-J.; Grattarola, M. Electrochim. Acta 2000, 46, 157. (8) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Gu¨ntherodt, H.-J.; Gerber, C.; Gimzewski, J. K. Science 2000, 288, 316. (9) Datskos, P. G.; Oden, P. I.; Thundat, T.; Wachter, E. A.; Warmack, R. J.; Hunter, S. R. Appl. Phys. Lett. 1996, 69, 2986. (10) Thundat, T.; Wachter, E. A.; Sharp, S. L.; Warmack, R. J. Appl. Phys. Lett. 1995, 66, 1695. (11) Thundat, T.; Chen, G. Y.; Warmack, R. J.; Allison, D. P.; Wachter, E. A. Anal. Chem. 1995, 67, 519. (12) Berger, R.; Delamarche, E.; Lang, H. P.; Gerber, C.; Gimzewski, J. K.; Meyer, E.; Gu¨ntherodt, H.-J. Science 1997, 276, 2021. (13) Lang, H. P.; Baller, M. K.; Berger, R.; Gerber, C.; Gimzewski, J. K.; Battiston, F. M.; Fornaro, P.; Ramseyer, J. P.; Meyer, E.; Gu¨ntherodt, H.-J. Anal. Chim. Acta 1999, 393, 59. (14) Ji, H.-F.; Thundat, T.; Dabestani, R.; Brown, G. M.; Britt, P. F.; Bonnesen, P. V. Anal. Chem. 2001, 73, 1572. (15) Moulin, A. M.; O’Shea, S. J.; Badley, R. A.; Doyle, P.; Welland, M. E. Langmuir 1999, 15, 8776. (16) Hansen, K. M.; Ji, H.-F.; Wu, G.; Datar, R.; Cote, R.; Majumdar, A.; Thundat, T. Anal. Chem. 2001, 73, 1567.
In the present study, we investigated the adsorption characteristics of calcium ion adsorption on a silicon nitride/gold cantilever using variation in resonance frequency and cantilever bending. No chemically selective layer was applied on any of the cantilever surfaces. It was observed that even an unmodified cantilever underwent bending and frequency shifts in the presence of CaCl2 solutions. The Ca2+ ions were found to interact with the silicon nitride side, causing the bending and frequency shifts. A Ca-binding protein, calmodulin, was used to verify the presence of Ca2+ ions on the cantilever surface. To confirm the results of the cantilever bending, fluorescence measurements were done on the cantilever surfaces using fluorescently tagged calmodulin. The results show that Ca2+ ions chemisorb to the silicon nitride surface of the cantilever. This implies that when using cantilever sensors for quantitative estimation of analyte species, it is critical to know the contribution of the different ions in the medium to the overall bending and frequency shifts. Experimental Section All experiments were performed in a fluid cell on silicon nitride cantilevers (Digital Instruments, Santa Barbara, CA). The dimensions and spring constant of the cantilever specified by the manufacturer are 193 µm long, 20 µm wide, 0.6 µm thick, and 0.06 N/m, respectively. The cantilevers were first stripped of the gold and chromium and recoated by electron beam evaporation with 2.5 nm of chromium and 25 nm of gold to obtain a fresh, uniform layer of gold. A Nanoscope III atomic force microscope (AFM) head (Digital Instruments) with a flow cell was used to measure cantilever deflection. A syringe infusion pump (IITC Inc., Woodland Hills, CA) connected to a six-port injection valve (Upchurch Scientific, Inc., WA) was used to introduce the different solutions into the flow cell. CaCl2 was from Sigma. The bovine calmodulin was a gift from Professor Daniel Roberts (University of Tennessee, Knoxville), and Alexa Fluor 488 conjugate was obtained from Molecular Probes Inc., Eugene, OR. Ultrapure deionized water (18.2 MΩ cm) from a Barnstead ultrapure water system was used to prepare all solutions. Cantilever deflections were obtained from the vertical difference signal of the position-sensitive detector (PSD) of the AFM head. The signals from the PSD were fed into a custom-made “controller/signal processor” box. The normalized vertical deflection in volts was acquired using a HP data acquisition/switch
10.1021/la025806m CCC: $22.00 © 2002 American Chemical Society Published on Web 08/03/2002
6936
Langmuir, Vol. 18, No. 18, 2002
Cherian et al.
Figure 1. (a) Surface stress changes in a silicon nitride cantilever coated with gold on one side as a function of CaCl2 concentration. The stress change-concentration curve fits the Langmuir adsorption isotherm, eq 2. (b) Variation in fundamental resonance frequency of the cantilever with CaCl2 concentration. unit (model 34970A). The fundamental resonance frequency of the cantilever was measured by analyzing the ac component of the PSD signal using a fast Fourier transform (FFT) spectrum analyzer (Stanford Research Systems, model 760, Sunnyvale, CA.)
∆σ )
Results and Discussion A triangular silicon nitride cantilever was mounted in the flow cell. The spring constant of the cantilever as specified by the manufacturer was 0.06 N m-1. Deionized water was flushed through the system for about 20 h to obtain a stable baseline. The fundamental resonance frequency of the cantilever in water was measured to be 13.718 kHz. The change in surface stress (∆σ) of the cantilever in the presence of CaCl2 solution was measured by sequential injections of 5, 10, 25, 30, 50, 70, and 100 mM solutions of CaCl2 in deionized (DI) water through the flow cell. DI water was flushed through the cell between injections. The change in surface stress, ∆σ, was calculated from the vertical deflection of the cantilever using Stoney’s equation17 (eq 1) and is shown in Figure 1a,
∆σ1 - ∆σ2 ≈
zEt2 4L2(1 - υ)
(1)
where z is the deflection, E is Young’s modulus, t is thickness, L is the length of the cantilever, and υ is Poisson’s ratio. ∆σ1 and ∆σ2 are the stress changes on either side of the cantilever. (17) Stoney, G. G. Proc. R. Soc. London, Ser. A 1909, 82, 172.
The vertical deflection signals in volts were corrected to take into account the apparent deflection signals due to refractive index changes of the solutions. It was observed that even when the laser beam was focused on the substrate on which the cantilever is attached, a deflection signal was measured when the fluids changed. This is not due to deflection but rather due to a change in the refractive index of the medium. This apparent deflection varied as the depth of the reflective surface in the fluid changed. The path length of the laser beam when it reflects off the substrate is less than when it reflects off the cantilever. To take geometric effects into account, another substrate was attached where the cantilever is located. The ratio of the apparent deflection from the bottom substrate to that from the top substrate was found to be 0.5. Therefore, the corrected vertical deflection was obtained as [measured signal - (signal from top substrate × 0.5)]. The deflection signal was then converted to stress change in N/m using Stoney’s equation. The direction of the deflection signal indicated that the cantilever bending was toward the silicon nitride side. A similar behavior was observed recently with NaCl solutions.18 This implied that the surface stress of the silicon nitride side increased or that of the gold decreased.4 Since the silicon nitride side is polar due to the presence of amine and hydroxyl groups, it is likely that most of the surface interactions are taking place on that side. During the equilibration period in water, the amine groups on the silicon nitride side hydrolyze to hydroxyl groups.19 The exact mechanism of interaction of the Ca2+ ions with the surface that causes the cantilever to bend is unclear at present. However, it is possible that the Ca2+ ions may be interacting with the hydroxylated silicon nitride surface via the electrified double layer. The ions could either be in contact adsorption with the surface or be in the outer diffuse layer. In either case, the interfacial potential must be changing due to the presence of the Ca ions causing the cantilever to bend. The surface stress-concentration curve was found to fit the Langmuir adsorption isotherm,
∆σmaxKC 1 + KC
(2)
where ∆σmax is the maximum stress change, which is proportional to the maximum available number of binding sites, K is the equilibrium binding constant, and C is the solute concentration. The linearized form of eq 3 is represented as 1/∆σ ) 1/∆σmax + 1/(∆σmaxKC). When 1/∆σ was plotted versus 1/C, the value of the maximum stress change, ∆σmax, was found to be 0.46 N m-1, and the binding constant, K, was 116 M-1. From the equilibrium constant of adsorption K, the Gibbs free energy change was calculated using the equation ∆G°(T) ) -RT ln(K) and found to be -11.8 kJ mol-1. This indicates that the binding of Ca2+ ions to the silicon nitride surface is a spontaneous process. This value is comparable to the Gibbs free energy change of -28.3 kJ/mol for the adsorption of Ca2+ ions from CaCl2 solutions onto the surface of CaCO3 particles.20 The resonance frequency of the cantilever was monitored as each solution was introduced into the flow cell and is shown in Figure 1b. The frequency shifted to lower values up to a CaCl2 concentration of ca. 0.06 M and thereafter does not vary significantly. The resonance frequency (18) Cherian, S.; Thundat, T. Appl. Phys. Lett. 2002, 80, 2219. (19) Jaffrezic-Renault, N.; De, A.; Clechet, P.; Maaref, A. Colloids Surf. 1989, 36, 59. (20) Huang, Y. C.; Fowkes, F. M.; Lloyd, T. B.; Sanders, N. D. Langmuir 1991, 7, 1742.
Adsorption of Ca2+ on Silicon Nitride Surfaces
Langmuir, Vol. 18, No. 18, 2002 6937
Figure 2. Noisy signals observed when Na3PO4 solution was injected sequentially after CaCl2 injection. The noisy signals may be due to the formation of Ca3(PO4)2 precipitate on the cantilever surface.
response also indicates that mass loading takes place at lower concentrations and then reaches saturation. The surface excess Γ, as a function of the concentration, C, and a change in surface free energy, γ, is given by the Gibbs equation:21
Γ)-
C dγ RT dC
( )
(3)
The change in surface stress, dσ, is assumed to be equal to the change in surface free energy, dγ. From the linear region of Figure 1a, dγ/dC was found to be 26.5 N m-1 M-1. This value is significantly higher than that reported recently18 for Na+ ions which was 0.43 N m-1 M-1, indicating a higher binding affinity of Ca2+ ions with the silicon nitride surface. Two different experiments were carried out to check for the presence of adsorbed calcium ions on the cantilever surface. In one experiment, 0.1 M CaCl2 was injected into the flow cell containing the cantilever. The cantilever deflected toward the silicon nitride side when exposed to the CaCl2 solution. After the CaCl2 solution flowed through the system, deionized water was allowed to flow for a sufficiently long time so as to flush all unbound Ca2+ ions and obtain a stable baseline again. A 0.1 M solution of Na3PO4 was then injected. The signal observed was extremely noisy as seen in Figure 2. This observation is attributed to the formation of small particulates or precipitate of Ca3(PO4)2 near the surface of the cantilever, where the phosphate ions encounter the adsorbed Ca2+ ions. It is also possible that this noisy signal reflects the heat released from the formation of Ca3(PO4)2 near the interface. The free energy changes accompanying the salt formation at the interface could change the interfacial stress and result in the bending. The resulting signal is noisy probably because the interface structure is changing rapidly, or it could be because of light scattering due to the presence of precipitate or particles around the cantilever. When a Na3PO4 solution was injected into the flow cell containing a fresh cantilever that had not been exposed to CaCl2, a deflection signal (similar to that observed for CaCl2 solution) with no noise was observed. This experiment indicated that all of the bound Ca2+ ions are not desorbed upon flushing with water. In the second experiment, a calcium-binding protein, calmodulin, was used to check for the presence of Ca2+ ions. Calmodulin is a 148 amino acid polypeptide chain (21) Chattoraj, D. K.; Birdi, K. S. Adsorption and the Gibbs Surface Excess; Plenum Press: New York, 1984.
Figure 3. (a) Cantilever bending toward the silicon nitride side (increased stress on that side) due to CaCl2 injection. The CaCl2 solution passes through the flow cell in 15 min, and the deflection comes down in ca. 18 min. (b) Bovine calmodulin (10 µg/mL) is injected sequentially. The bending direction is reversed (decrease in stress on the silicon nitride side) and is also of a shorter duration, ca. 6 min. (c) A second injection of calmodulin results in a bending toward the silicon nitride side (expanding gold side) and occurs for over 2 h. Thereafter, when a CaCl2 solution is injected, two effects of varying durations are observed. First (i) is the deflection toward the silicon nitride side that lasts about 15 min. The second (ii) is the deflection toward the gold side and takes place over hours. The first deflection is due to Ca2+ ions interacting with the silicon nitride side, and the second is the protein conformational change taking place at a slower rate upon complexing with the Ca2+ ions.
containing four Ca2+-binding sites.22 Calmodulin has significantly different conformations in its free (apo) and Ca2+-bound forms.22-24 So the binding of calmodulin to Ca2+ ions on the cantilever surface should induce a change in surface stress, and a deflection should be observed. It is assumed that if Ca2+ ions are present on the surface, calmodulin will bind to them and remove the ions from the surface, due to its significantly higher binding affinity to Ca2+ ions (K ) 5 × 106 to 5 × 107 M-1).23 Therefore, a silicon nitride cantilever was allowed to equilibrate in deionized water and a 1.5 mM CaCl2 solution was injected, Figure 3a. Deionized water was allowed to flush through the system for more than 1 h to remove any physisorbed (22) Babu, Y. S.; Sack, J. S.; Greenhough, T. J.; Bugg, C. E.; Means, A. R.; Cook, W. J. Nature 1985, 315, 37. (23) Chin, D.; Means, A. R. Trends Cell Biol. 2000, 10, 322. (24) Maune, J. F.; Klee, B. C.; Beckingham, K. J. Biol. Chem. 1992, 267, 5286.
6938
Langmuir, Vol. 18, No. 18, 2002
Cherian et al.
Figure 4. Fluorescence images from (a) a gold wafer immersed in calmodulin and rinsed with water; (b) a gold wafer immersed in a CaCl2 solution, rinsed with water several times, and then immersed in calmodulin solution; (c) a silicon nitride wafer immersed in calmodulin solution and rinsed with water; and (d) a silicon nitride wafer immersed in CaCl2 solution, rinsed in water, and then immersed in calmodulin solution and rinsed with water. Fluorescence is observed on the silicon nitride side only after CaCl2 treatment, whereas it is observed on the gold surface even before CaCl2 treatment.
ions. Thereafter, 1 mL of 10 µg/mL bovine calmodulin (BC) was injected. The deflection behavior has two major differences from that observed with a CaCl2 injection (Figure 3b). One is that the cantilever bent toward the gold side, and the second is that it started to return to baseline in 6 min. At a flow rate of 4 mL/h and with a 1 mL injection loop, the solution has a minimum residence time of 15 min in the reservoir. For example, it took 1618 min for the deflection to approach the initial baseline value after the CaCl2 injections, as seen in Figure 3a. The shorter duration of the deflection signal when BC was injected is explained to be the result of Ca2+ ion removal from the silicon nitride surface due to complexation with BC. The direction of deflection indicates a reduction in stress on the silicon nitride side. If the BC injection removes Ca2+ ions from the silicon nitride surface, a second injection of BC is expected to show a different behavior. Therefore, BC was injected again. The cantilever response this time was significantly different from the previous injection, Figure 3c. The deflection was toward the silicon nitride side, indicating an expanding gold side. The deflection continued for over 2 h. This observation is attributed to the fact that interaction of BC with the gold surface mainly contributes to the change in surface stress. BC has several methionines in its sequence that can bind to the gold surface. The cantilever response to the presence of BC was very slow.
A similar observation has been made by Welland et al. for bovine serum albumin (BSA) injection where the deflection continued for several hours.15 Proteins when adsorbed on surfaces can undergo conformational changes.25,26 It is possible that it is this slow process that is manifested as the deflection over a long time. One milliliter of 1.5 mM CaCl2 solution was then introduced into the system at 17.6 × 103 s. Two effects are seen as a result of this injection, indicated by (i) and (ii) in Figure 3c. The first is a bending toward the silicon nitride side (starting at ca. 17.77 × 103 s, (i) in Figure 3c) with a duration of ca. 15 min. This is similar to the bending observed in Figure 3a. This must be due to Ca2+ ions interacting with the silicon nitride side. The second effect is the bending toward the gold side and takes place over a much longer time scale ((ii) in Figure 3c). It is known that when Ca2+ ions bind to the apo calmodulin, its conformation changes.23 On this basis, we interpret the cantilever bending after CaCl2 injection ((ii) in Figure 3c) as due to stress changes resulting from protein conformational changes. The experiments mentioned above point to the fact that the deflection and frequency shifts observed when the silicon nitride/gold cantilever is exposed to CaCl2 solutions (25) Caruso, F.; Vukusic, P. S.; Matsuura, K.; Urquhart, R. S.; Furlong, D. N.; Okahata, Y. Colloids Surf., A 1995, 103, 147. (26) Caruso, F.; Furlong, D. N.; Kingshott, P. J. Colloid Interface Sci. 1997, 186, 129.
Adsorption of Ca2+ on Silicon Nitride Surfaces
are due to interaction of Ca2+ ions on the silicon nitride surface. In addition, the Ca2+-calmodulin experiments also indicate that microcantilevers may be used to observe protein conformational changes taking place on their surfaces. To corroborate the cantilever deflection data, fluorescence measurements were carried out on gold and silicon nitride wafers using bovine brain calmodulin conjugated with AlexaFluor (BC-F) as a fluorescent tag. A clean gold wafer and a silicon nitride wafer were immersed in a 10 µg/mL solution of BC-F. They were withdrawn after 10 min, rinsed with deionized water several times, and fluorescence imaged from both surfaces. Fluorescence was observed on the gold wafer but not on the silicon nitride wafer (Figure 4a,c). Another set of gold and silicon nitride wafers were immersed in a 3 mM CaCl2 solution, withdrawn, and rinsed with deionized water several times. They were then immersed in a 10 µg/mL solution of BC-F, withdrawn after 2 min, rinsed with deionized water, and fluorescence imaged. They were withdrawn after 2 min because the deflection response indicates that the Cacalmodulin complex gets desorbed after about 6 min (Figure 3b). In this case, fluorescence was observed on both the gold and silicon nitride sides (Figure 4b,d). The results of the fluorescence measurements show that calmodulin remains adsorbed to the gold surface even after rinsing with water but not to the silicon nitride surface. However, a pretreatment with CaCl2 solution makes the calmodulin “stick” to the silicon nitride surface. This is due to the affinity of calmodulin to Ca2+ ions. This confirms that some Ca2+ ions are indeed adsorbed to the silicon nitride surface even after rinsing with water. The deflection and frequency shifts observed when the silicon nitride cantilever coated with gold was exposed to CaCl2 solutions
Langmuir, Vol. 18, No. 18, 2002 6939
are thus attributed mainly to the interaction of Ca2+ ions with the silicon nitride surface. Conclusions This study shows that the concentration-dependent stress and frequency changes of a silicon nitride cantilever coated with gold on one side in the presence of CaCl2 solutions are due to the interaction of Ca2+ ions with the silicon nitride side of the cantilever. The adsorption binding constant determined from the Langmuir adsorption isotherm was 116 M-1, and the Gibbs free energy change of adsorption was -11.8 kJ mol-1. The results of the deflection data were corroborated by fluorescence data using Alexa Fluor conjugated calmodulin. These results demonstrate that cations adsorb onto the silicon nitride surface of the cantilever. Therefore, passivating or using an appropriate reference is critical in quantifying analyteinduced stress changes taking place on cantilever surfaces. Acknowledgment. S.C. and A.M. acknowledge support from the ORAU Postdoctoral program. We thank Professor Daniel Roberts (University of Tennessee, Knoxville) for the calmodulins and Dr. Mike Barnes (ORNL) for the use of the inverted microscope for fluorescence measurements. We also thank Leah Downing and Abena Appiah-kubi for their participation in the project. This research was supported by the DOE Office of Biological and Environmental Research (OBER). Oak Ridge National Laboratory is managed by UT-Bettelle, LLC, for the U.S. Department of Energy under Contract DE-AC0500OR22725. LA025806M
