Surface Stress Changes Induced by the Conformational Change of

has potential for use in label-free, protein-analyte screening applications. During the past decade, increasing interest has been ... stress on the ca...
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Langmuir 2006, 22, 11241-11244

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Surface Stress Changes Induced by the Conformational Change of Proteins Xiaodong Yan,† Kalisha Hill,‡ Hongyan Gao,‡ and Hai-Feng Ji*,† Chemistry Institute for Micromanufacturing, Louisiana Tech UniVersity, Ruston, Louisiana 71272, and Department of Chemistry, Yili Normal UniVersity, Xin Jiang, China 835000 ReceiVed February 24, 2006. In Final Form: September 24, 2006 A potential binding assay based on conformational-change-induced micromechanical motion is described. Calmodulin was used to modify a microcantilever (MCL) by a self-assembled layer-by-layer approach. The results showed that the modified MCL bent when the proteins changed their conformation upon binding with Ca2+. The cantilever deflection amplitudes were different under different ionic strengths, indicating different degrees of conformational change of the proteins in these conditions. On the contrary, cantilevers modified by proteins, such as hemoglobin and myoglobin, that do not change conformations upon binding with analytes do not cause the cantilever deflection. These results suggest that the conformational changes of proteins may be used to develop cantilever biosensors, and the MCL system has potential for use in label-free, protein-analyte screening applications.

During the past decade, increasing interest has been given to surface stress and processes that are affected by surface stress. It has been recognized that surface stress can provide a qualitative and sometimes quantitative understanding of the microscopic and mesoscopic surface processes. Surface stress changes have been used to develop novel micro/nanomechanical sensors. One such example is microcantilevers (MCLs), which have been proven to be an outstanding platform for chemical and biological sensors.1-7 MCLs are the simplest microelectromechanical systems (MEMS) device that can be micromachined and massproduced.8 MCLs can also be incorporated into multichannel MCL chips that offer improved dynamic response, greatly reduced size, high precision, increased reliability, and integration of micromechanical components with on-chip electronic circuitry.9 MCLs can undergo bending due to molecular adsorption or absorption by confining the adsorption and absorption to one side of the cantilever.10-12 Surface stress studies have led to the development of extremely sensitive MCL sensors for species in both air and aqueous solutions. The surface stress change can be calculated according to equation11

∆z )

3(1 - ν)L2 δs Et2

(1)

where ∆z is the observed deflection at the end of the cantilever, * Corresponding author. E-mail: [email protected]. † Louisiana Tech University. ‡ Yili Normal University. (1) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Guntherodt, H.-J.; Gerber, C.; Gimzewski, J. K. Science 2000, 288, 316. (2) Butt, H. J. J. Colloid Interface Sci. 1996, 180, 251. (3) Thundat, T.; Warmack, R. J.; Chen, G. Y.; Allison, D. P. Appl. Phys. Lett. 1994, 64, 2894. (4) Gimzewski, J. K.; Gerber, C.; Meyer, E.; Schlittler, R. R. Chem. Phys. Lett. 1994, 217, 589. (5) Chen, G. Y.; Warmack, R. J.; Thundat, T.; Allison, D. P.; Huang, A. ReV. Sci. Instrum. 1994, 65, 2532. (6) Lang, H. P.; Baller, M. K.; Berger, R.; Gerber, C.; Gimzewski, J. K.; Battiston, F. M.; Fornaro, P.; Ramseyer, J. P.; Meyer, E.; Guntherodt, H. J. Anal. Chim. Acta 1999, 393, 59. (7) Raiteri, R.; Grattar, M.; Butt, H.-J.; Skladal, P. Sens. Actuators, B 2001, 79, 115. (8) Tang, Y.; Fang, J.; Yan, X.; Ji, H.-F. Sens. Actuators, B 2004, 97, 109. (9) Lange, D.; Hagleitner, C.; Hierlemann, A.; Brand, O.; Baltes, H. Anal. Chem. 2002, 74, 3084.

ν and E are Poisson’s ratio (0.2152) and Young’s modulus (155.8 GPa) for the silicon substrate, respectively, t and L are the thickness (1 µm) and length (180 µm) of the cantilever, respectively, and δs is the differential stress on the cantilever. From a molecular point of view, the binding results in electrostatic repulsion,1 attraction,11 steric effects, intermolecular interactions,13 or a combination of these, which alters the surface stresses on the cantilever. As a sensor platform, one advantage of the cantilever platform is that no label compounds are needed since the MCL’s deflection is derived from molecular-bindinginduced surface stress changes. Recently, several protein assays based on antibody-protein interaction on MCLs demonstrated that biomarker proteins can be detected via measurement of surface stress generated by antigen-antibody recognition.13,14 Although not specifically addressed, the conformation change of the biomarker proteins and the antibodies might contribute to the surface stress change on the cantilever surface. Moulin et al. demonstrated15 that adsorbed proteins on the cantilever surface changed the surface stress on the cantilever surface because of the conformation change of the proteins on the surface from solutions. It is well received that many proteins undergo conformational changes upon complexation with analytes, such as small metal ions or molecules. In this paper, we report that the conformational changes of proteins upon molecular recognition could contribute to the surface stress changes, and this concept may be used for label-free, cantileverbased bioassay or screening. In our experiments, we used commercially available silicon MCLs (Veeco Instruments, Santa Barbara, CA). The dimensions of the V-shaped silicon MCLs were 180 µm in length, and 2 µm in thickness. One side of these cantilevers was covered with a thin film of chromium (3 nm), followed by a 20-nm layer of (10) Tipple, C. A.; Lavrik, N. V.; Culha, M.; Headrick, J.; Datskos, P.; Sepaniak, M. J. Anal. Chem. 2002, 74, 3118. (11) Yang, Y.; Ji, H.-F.; Thundat, T. J. Am. Chem. Soc. 2003, 115, 460. (12) Alvarez, M.; Carrascosa, L. G.; Moreno, M.; Calle, A.; Zaballos, A.; Lechuga, L. M.; Martinez-A, C.; Tamayo, J. Langmuir 2004, 20, 9663. (13) Wu, G.; Datar, R. H.; Hansen, K. M.; Thundat, T.; Cote, R. J.; Majumdar, A. Nat. Biotechnol. 2001, 19, 856. (14) Arntz, Y.; Seelig, J. D.; Lang, H. P.; Zhang, J.; Hunziker, P.; Ramseyer, J. P.; Meyer, E.; Hegner, M.; Gerber, C. Nanotechnology 2003, 14, 86. (15) Moulin, A. M.; O’Shea, S. J.; Badley, R. A.; Doyle, P.; Welland, M. W. Langmuir 1999, 15, 8776.

10.1021/la0605337 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/10/2006

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gold, both deposited by e-beam evaporation. Since gold film does not stick well to SiO2, the thin chromium layer was used to improve adhesion. The deflection experiments were performed in a flow-through glass cell (Veeco, CA) similar to those used in atomic force microscopy (AFM). For continuous flow-through experiments, initially, the electrolyte solution was circulated through the cell using a syringe pump. A constant flow rate was maintained during each experiment. The cantilever was immersed in the electrolyte solution until a baseline was obtained, and the voltage of the position-sensitive detector was set as background corresponding to 0 nm. Experimental solutions containing different concentrations of CaCl2 were injected directly into the flowing fluid stream via a low-pressure injection port sample loop arrangement with a loop volume of 2.0 mL. This arrangement allows for continuous exposure of the cantilever to the desired solution without disturbing the flow cell or changing the flow rate. Since the volume of the glass cell, including the tubing, was only 0.3 mL, a relatively fast replacement of the liquid in contact with the cantilever was achieved. MCL deflection measurements were determined using the optical beam deflection method. The bending of the cantilever was measured by monitoring the position of a laser beam reflected from the gold-coated side of the cantilever onto a four-quadrant AFM photodiode. We define bending toward the gold side as ‘bending up”; “bending down” refers to bending toward the silicon side. In the case where the adsorption occurs on the gold surface, in general, the downward bending is caused by the repulsion or expansion of molecules on the gold surface, which is so-called compressive stress; vice versa, the upward bending is caused by the attraction or contraction of molecules on the gold surface, which is called tensile surface stress. Three cantilevers were prepared for each of the individual experiments to allow statistical comparison of repeatability and efficiency between devices. To eliminate thermomechanical motion of the silicon cantilever caused by temperature fluctuations, we mounted the fluid cell on thermoelectric coolers so that the temperature of the fluid cell could be controlled to 20 ( 0.2 °C. We used a nanoassembly layer-by-layer (LBL) technique to immobilize the proteins on the cantilever surface. The LBL technique allows the formation of ultrathin organized films on almost any surface through the alternate adsorption of oppositely charged components, such as linear polyions and enzymes.16-19 Polyethyleneimine (PEI, a polycation), poly(sulfonate styrene) (PSS, a polyanion), and proteins (Calmodulin (CaM), hemoglobin, and myoglobin studied in this work) were used for modification of the cantilever surfaces by using the LBL technique. Since MCL bending is generated from adsorption-induced surface stress from one side of the MCL, the key surface modification technology is to control the formation of multilayers on only one surface of the MCL by choosing appropriate surface materials. In a typical multilayer formation procedure, the aimed target was alternately dipped into a polycation and polyanion solution, and the process was repeated several times for multilayer formation. When this procedure was applied, multilayer nanoassembly film formation was found on both sides of the cantilever. Recently, we reported a modified multilayer growing process20 taking advantage of the hydrophobic/lipophobic properties of the perfluorocarbon materials. In this method, (tridecafluoro(16) Lvov, Y.; Decher, G.; Mo¨hwald, H. Langmuir 1993, 9, 481. (17) Decher, G. Science 1997, 227, 1232. (18) Yoo, D.; Shiratori, S.; Rubner, M. Macromolecules 1998, 31, 4309. (19) Dubas, S.; Schlenoff, J. Macromolecules 1999, 32, 8153. (20) Yan, X.; Lvov, Y.; Thundat, T.; Ji, H.-F. Org. Biomol. Chem. 2003, 1, 460.

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1,1,2,2-tetrahydrooctyl)triethoxysilane was used to develop a thin perfluorocarbon monolayer on a silicon surface using a typical silicon surface modification procedure,21 and the polymeric multilayers were found only on the gold surface of the cantilever. The modified LBL procedure specific for MCL surface modification used in this experiment is as follows: (A) A monolayer of 2-mercaptoethane sulfonic acid (MES) was self-assembled on the gold surface of an MCL by immersing the MCL in a 5mM MES solution for 12 h, and then rinsing with EtOH three times followed by deionized water three times. (B) The MCL was immersed in a PEI solution for 10 min and then rinsed with flowing water at a speed faster than 1 m/min over the cantilever surface for 1 min. The MCL was then immersed in the opposite polyelectrolyte for 10 min, followed by another rinsing of flowing water. (C) This cycle was repeated several times until the desired number of multilayers was reached. CaM was used in our experiments to verify the conformationalchange-induced surface stress. CaM, a small (17 kD), heat stable, acidic protein with approximately 148 amino acid residues, is an abundant and ubiquitous calcium binding protein that serves as an activator of numerous cellular enzymes. In the absence of Ca2+, CaM consists of a short dumbbell structure with two globular domains connected by a helical linker. Each of the globular domains contains two EF hands (helix/12-residue-loop/ helix motif), which bind to a Ca2+ ion at the loop region with intermediate affinity (KD from 1 to 10 µM).22 Upon binding with four Ca2+ per molecule of CaM, CaM undergoes large conformational changes and becomes more elongated. The conformational change of CaM upon Ca2+ complexation is due to a combination of electrostatic interactions, steric effects, and other intramolecular interactions. This altered conformation of the Ca2+-CaM complex enables it to interact with other proteins, and this action is critical to various aspects of cell metabolism. It is expected that these enzyme conformational changes can result in a surface stress change that can be detected from the consequent bending response of the cantilever. Many methods based on CaM have been used to detect Ca2+ in cellular concentrations. These methods include calcium fluorescent probes and calcium electrode sensors.23,24 Many of these sensors are excellent for the purpose of Ca2+ analysis. However, the weight of most of these systems is not sufficiently low to be placed in certain applications, such as in a space shuttle where light weight is a requirement. An MCL sensor device may provide a lightweight, portable, and sensitive tool for these applications. In our experiments, the multilayers on the MCLs were (PEI/ PSS)3, followed by three bilayers of CaM/PEI on the gold surface. The first three bilayers of PEI/PSS provided a solid base for subsequent enzyme immobilization. The formation of multilayers was confirmed by contact angle and Fourier transform infrared experiments. The MCL was initially exposed to a constant flow (4 mL/h) of a 0.01 M solution of NaCl in a fluid cell. Figure 1 shows a cantilever deflection profile when the modified cantilever is exposed to 10-5 M CaCl2 in the 0.01 M NaCl solution. A 2.0 mL aliquot of Ca2+ solution was switched into the fluid cell at the marked time. It took approximately 30 min for the injected Ca2+ concentration to flow through the fluid cell, and the NaCl electrolyte solution was circulated back through the fluid cell. (21) Wasserman, S. R.; Tao, Y.-T.; Whitesides, G. M. Langmuir 1989, 5, 1074. (22) Seaton, B. A.; Head, J. F.; Engelman, D. M.; Richards, F. M. Biochemistry 1985, 24, 6740. (23) Chin, D.; Means, A. R. Trends Cell Biol. 2000, 10, 322. (24) Oliver, A. E.; Baker, G. A.; Fugate, R. D.; Tablin, F.; Crowe, J. H. Biophys. J. 2000, 78, 2116.

Protein-Conformation-Induced MCL Modification

Figure 1. Bending responses of (PEI/CaM)3 multilayer-modified MCLs to a 10-5 M concentration of Ca2+ and other metal ions in a 0.01 M NaCl solution.

Figure 2. Maximum bending amplitude for a (PEI/CaM)3-modified MCL as a function of the change in concentration of Ca2+ in a 0.01 M NaCl solution.

When the Ca2+ solution was replaced by the original NaCl electrolyte solution, the cantilever bent gradually backward to its original position, suggesting that the proteins returned to their original conformation. Figure 2 shows the bending amplitudes of a CaM-multilayer-modified cantilever to various concentrations of Ca2+. The cantilever deflection increased as the concentrations of Ca2+ increased. The detection limit for Ca2+ measurement was approximately 10-7 M. The binding constant using a Langmuir-model-based nonlinear cure fitting method could not be obtained with the data in Figure 2 since each CaM protein could bind four Ca2+ molecules. Control experiments showed that a CaM-multilayer-modified cantilever did not deflect upon exposure to other metal ions, such as K+ and Mg2+, which either do not complex or weakly complex with CaM (Figure 1). Other control experiments were performed by exposing a (PSS/PEI)6-modified MCL to a 10-5

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Figure 3. Bending responses of a (PEI/CaM)3 multilayer-modified MCL to a 10-5 M concentration of Ca2+ in 0.01, 0.001, and 1 M NaCl solutions.

M solution of Ca2+. No deflection of the cantilever was observed (figure not shown), which ruled out possible interaction of Ca2+ with PDDA or PSS polymers. Figure 3 compares the cantilever deflection profiles when the modified cantilever is exposed to a 10-5 M Ca2+ solution under different ionic strength. In these experiments, the ionic strengths before and after injection were about the same. For instance, a 10-5 M Ca2+ in 0.001 M NaCl solution was injected to replace the 0.001 M NaCl solution; a 10-5 M Ca2+ in 0.01 M NaCl solution was injected to replace the 0.01 M NaCl solution. It was observed that the cantilever bending amplitude in lower ionic strength solution was higher in solutions with higher ionic strength. The bending response of the cantilever to 10-5 M Ca2+ in a 1 M NaCl solution was too weak to be detected (also shown in Figure 3). The larger bending amplitude suggested a larger conformational change of CaM in lower ionic strength solutions, and vice versa. Similar observations have been reported from other investigators,22 and one possible explanation has been that the ions may partially neutralize CaM’s negative charges, reducing the electrostatic interaction between the CaM and Ca2+ and minimizing the conformational change that occurs upon Ca2+ binding in solutions with higher ionic strength. The Langmuir isotherm model does not fit the concentration versus deflection data very well. This may primarily be due to multiple binding sites in CaM since each CaM can bind four Ca2+ and the binding constants are different for the different binding sites. For the 12 nm deflection, the surface stress change is calculated to be 0.098 N/m, i.e., 1.76 × 10-5 N on the cantilever according to eq 1. Calculations based on the curvature of the cantilever showed that the length of the gold side surface was approximately 1.3 nm longer than that of the silicon surface. This result showed that the multilayer side of the cantilever extended 0.001% due to the conformational change of the CaM. It was known that, in the absence of calcium, the reported dimensions of the CaM were 2.06 nm for the radius of gyration and 5.8 nm for the maximum vector length; in the presence of calcium, this radius of gyration increased to 2.15 nm, and the maximum vector length increased to 6.2 nm. The density of CaM in one PEI/CaM bilayer

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film was 3.20 ( 0.13 µg/cm2, measured by quartz crystal microbalance, suggesting the CaM closely packed up on the cantilever surface. At this density, the maximum average extension of the surface upon complexation with Ca2+ is approximately 5% when all the CaM is converted to CaM-Ca2+ complex. The comparisons between the calculated and the measured results indicated that the conformational change of CaM in a polymer film might be complicated and significantly different from that in solution. Further characterization and determination of the molecular and structural change of CaM in a multilayer film will be essential for future biosensor improvement. In the cases where some proteins do not change their conformations upon complexation with specific analytes, such as complexation of hemoglobin or myoglobin with oxygen at the heme group sites, no cantilever bending is observed upon binding with oxygen with hemoglobin- or myoglobin-multilayer-modified cantilevers (figure not shown). In these cases, the binding will not result in any other intermolecular interactions such as electrostatic repulsion or attraction, steric effects, etc., thus, the binding will not generate any surface stress on the cantilever surface and subsequent cantilever deflections. In conclusion, this paper has demonstrated that the conformational change of proteins may be used to develop surface stress change-based biosensors. The surface stress change phenomenon due to the conformational change of proteins offers unique opportunities in the design of small and sensitive analytical methods. The scope of this analytical method for other analytes includes the detection of chemicals or environmental change

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that could alter the conformational change of the proteins, including metal ions, biomarker molecules, drug candidates, and so forth. A state-of-the-art screening microchip capable of developing and running biochemical assays such as enzyme inhibition as well as binding assays and cell-based assays could be established. The general concept and method will find applications in the environment, clinical diagnostics, homeland security, and drug discovery. Furthermore, a small, inexpensive instrument, such as an MCL chip, that can detect conformational changes would be valuable in studying receptor mechanisms and enable the biosensor to distinguish between compounds producing different conformational changes in the protein. This instrument provides an alternative, label-free bioassay to study the protein-ligand interaction under varying conditions of ionic strength or electrolyte identity. Understanding how the proteins change their conformation under varying conditions should give new insight into improving these biosensors. The simplicity with which such a label-free method is currently being developed also significantly increases the likelihood of the application of this technology and reduces the costs. Acknowledgment. This work was supported by the NSF Sensor and Sensor Network ECS-0428263. K. Hill was supported by a NASA LURA-2004 project. H. Gao was supported by the Chinese Scholarship Council. LA0605337