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Microcantilever Sensing and Actuation with End-Grafted Stimulus-Responsive Elastin-Like Polypeptides† Alexei Valiaev,‡,|,⊥ Nehal I. Abu-Lail,‡,|,⊥ Dong Woo Lim,§ Ashutosh Chilkoti,§,| and Stefan Zauscher*,‡,| Department of Mechanical Engineering and Materials Science and Department of Biomedical Engineering, Duke UniVersity, Durham, North Carolina 27708, and Center for Biologically Inspired Materials and Materials Systems, Durham, North Carolina 27708 ReceiVed June 11, 2006. In Final Form: September 22, 2006 Stimulus-responsive elastin-like polypeptides (ELPs) grafted onto surfaces are of significant technical interest because they can be exploited for force generation, in sensing applications, or as molecular switches with tunable properties. Changes in the conformational state of grafted ELPs, induced by a phase transition or changes in osmotic pressure, lead to significant changes in the surface stress in the ELP graft layer and translate into detectable changes in microcantilever deflection. In this study, we investigate the conformational mechanics of ELPs in response to changes in solution pH and ionic strength using atomic force microscopy (AFM) microcantilever deflection and quartz crystal microbalance (QCM) measurements. We show that the use of genetically encoded, surface-grafted ELPs is exciting for cantilever actuation and sensing because commonly available microfabricated cantilever springs offer a simple and nonintrusive way to detect changes in solvent type, temperature, and pH, promising great potential for sensing applications in microfluidic devices.
Introduction In recent years, there has been a proliferation of microcantilever-based sensors (biosensors,1-6 chemical sensors,7-13 and physicochemical sensors14-19) because of their simplicity and † Part of the Stimuli-Responsive Materials: Polymers, Colloids, and Multicomponent Systems special issue. * Corresponding author. E-mail:
[email protected]. Phone: (919) 6605360. Fax: (919) 660-5409. ‡ Department of Mechanical Engineering and Materials Science, 144 Hudson Hall, Duke University. § Department of Biomedical Engineering, Duke University. | Center for Biologically Inspired Materials and Materials Systems. ⊥ These authors contributed equally to this work.
(1) Butt, H. J. J. Colloid Interface Sci. 1996, 180, 251-260. (2) 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, 316-318. (3) Wu, G. H.; Datar, R. H.; Hansen, K. M.; Thundat, T.; Cote, R. J.; Majumdar, A. Nat. Biotechnol. 2001, 19, 856-860. (4) Pei, J. H.; Tian, F.; Thundat, T. Anal. Chem. 2004, 76, 292-297. (5) Calleja, M.; Tamayo, J.; Johansson, A.; Rasmussen, P.; Lechuga, L. M.; Boisen, A. Sensor Lett. 2003, 1, 20-24. (6) Hilt, J. Z.; Gupta, A. K.; Bashir, R.; Peppas, N. A. Biomed. MicrodeVices 2003, 5, 177-184. (7) Rogers, B.; Manning, L.; Jones, M.; Sulchek, T.; Murray, K.; Beneschott, B.; Adams, J. D.; Hu, Z.; Thundat, T.; Cavazos, H.; Minne, S. C. ReV. Sci. Instrum. 2003, 74, 4899-4901. (8) Ji, H. F.; Finot, E.; Dabestani, R.; Thundat, T.; Brown, G. M.; Britt, P. F. Chem. Commun. 2000, 457-458. (9) Ji, H. F.; Thundat, T.; Dabestani, R.; Brown, G. M.; Britt, P. F.; Bonnesen, P. V. Anal. Chem. 2001, 73, 1572-1576. (10) Jung, M. Y.; Kim, D. W.; Choi, S. S.; Kang, C. J.; Kuk, Y. Jpn. J. Appl. Phys. 1999, 38, 6555-6557. (11) Bashir, R.; Hilt, J. Z.; Elibol, O.; Gupta, A.; Peppas, N. A. Appl. Phys. Lett. 2002, 81, 3091-3093. (12) Pinnaduwage, L. A.; Gehl, A.; Hedden, D. L.; Muralidharan, G.; Thundat, T.; Lareau, R. T.; Sulchek, T.; Manning, L.; Rogers, B.; Jones, M.; Adams, J. D. Nature (London) 2003, 425, 474-474. (13) Fritz, J.; Baller, M. K.; Lang, H. P.; Strunz, T.; Meyer, E.; Guntherodt, H. J.; Delamarche, E.; Gerber, C.; Gimzewski, J. K. Langmuir 2000, 16, 96949696. (14) Thundat, T.; Warmack, R. J.; Chen, G. Y.; Allison, D. P. Appl. Phys. Lett. 1994, 64, 2894-2896. (15) Thundat, T.; Chen, G. Y.; Warmack, R. J.; Allison, D. P.; Wachter, E. A. Anal. Chem. 1995, 67, 519-521. (16) Hillier, A. C.; Bard, A. J. ReV. Sci. Instrum. 1997, 68, 2082-2090. (17) Chen, G. Y.; Thundat, T.; Wachter, E. A.; Warmack, R. J. J. Appl. Phys. 1995, 77, 3618-3622.
ability to function in small sample volumes. Microcantilever sensors typically consist of two lamina that differ in their physical or chemical nature13,20 so that physical adsorption, chemical reactions, or biomolecular binding events preferentially occur at one of the two surfaces, causing a differential stress that induces cantilever bending. The deflection of a cantilever is typically recorded as a function of time (dc method).2,13,20 Although the dc method is widely used, it suffers from long thermal equilibration times.2,13,20 To overcome this limitation, an ac detection approach can be used that allows the measurement of frequency shifts of the vibrating microcantilever due to changes in its mass.21 The implementation of such ac measurements in aqueous environments and with picogram mass resolution is, however, technically still challenging,22 making the dc method still attractive. Stimulus-responsive, elastin-like polypeptides (ELPs)23-25 and their hierarchical assemblies on surfaces show great promise for actuation,26,27 sensing,26,28,29 and biotechnological applications.23,25,30 ELPs are genetically engineered and thus have a (18) Lalinsky, T.; Hascik, S.; Mozolova, Z.; Burian, E.; Drzik, M. Sens. Actuators, A 1999, 76, 241-246. (19) Berger, R.; Gerber, C.; Gimzewski, J. K.; Meyer, E.; Guntherodt, H. J. Appl. Phys. Lett. 1996, 69, 40-42. (20) Wu, G. H.; Ji, H. F.; Hansen, K.; Thundat, T.; Datar, R.; Cote, R.; Hagan, M. F.; Chakraborty, A. K.; Majumdar, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1560-1564. (21) Tamayo, J.; Humphris, A. D. L.; Malloy, A. M.; Miles, M. J. Ultramicroscopy 2001, 86, 167-173. (22) Braun, T.; Barwich, V.; Ghatkesar, M. K.; Bredekamp, A. H.; Gerber, C.; Hegner, M.; Lang, H. P. Phys. ReV. E 2005, 72, 031907/1-031907/9. (23) Meyer, D. E.; Chilkoti, A. Nat. Biotechnol. 1999, 17, 1112-1115. (24) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2002, 3, 357-367. (25) Meyer, D. E.; Trabbic-Carlson, K.; Chilkoti, A. Biotechnol. Prog. 2001, 17, 720-728. (26) Hoffmann, J.; Plotner, M.; Kuckling, D.; Fischer, W. J. Sens. Actuators, A 1999, 77, 139-144. (27) Kuckling, D.; Hoffmann, J.; Plotner, M.; Ferse, D.; Kretschmer, K.; Adler, H. J. P.; Arndt, K. F.; Reichelt, R. Polymer 2003, 44, 4455-4462. (28) Trabbic-Carlson, K.; Setton, L. A.; Chilkoti, A. Biomacromolecules 2003, 4, 572-580. (29) Hyun, J.; Lee, W. K.; Nath, N.; Chilkoti, A.; Zauscher, S. J. Am. Chem. Soc. 2004, 126, 7330-7335. (30) Betre, H.; Setton, L. A.; Meyer, D. E.; Chilkoti, A. Biomacromolecules 2002, 3, 910-916.
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Scheme 1. Schematic Representation of the Compositional Structure of the Three ELP Constructs Used in the Experiments
controllable amino acid sequence, can be expressed with precise molecular weight, and can be easily fused with other proteins and peptides.24 The work presented in this article differs significantly from our previous work where we used nanopatterned ELPs as molecular switches to capture ELP fusion proteins from solution.29 Here we demonstrate that the conformational changes associated with the phase transition or changes in the ionization state of ELPs can be employed for the mechanical actuation of microcantilevers. We use two techniques, atomic force microscopy (AFM) and the quartz crystal microbalance with dissipation monitoring (QCM-D), to characterize and assess the potential of ELPs for microcantilever sensing and actuation. Specifically, we investigate the performance of (a) pH-responsive ELPs with (i) two different charge densities but comparable molecular weight and with (ii) constant charge density at three different molecular weights and (b) ionic strength-responsive, charge-neutral ELPs for microcantilever sensing applications. Our results show that ELPs offer a tunable means to transduce and amplify changes in the solvent environment, such as changes in pH or ionic strength, for microcantilever-based sensing. Materials and Methods ELP Synthesis. We used the overexpression of a plasmid-borne synthetic gene in Escherichia coli to synthesize the ELPs used in this study.23 In brief, cells harboring a plasmid that encodes for the ELP were grown in 50 mL of CircleGrow culture media (Bio101, CA) supplemented with 100 µg/mL ampicillin with shaking at 300 rpm at 37 °C for 24 h without induction. After incubation, the cells were harvested from the culture medium by centrifugation (2500g, 4 °C, and 15 min) and resuspended in 5 mL of phosphate-buffered saline solution (PBS) (140 mM NaCl and 2.7 mM KCl). The cells were lysed by sonication and centrifuged at 16 000g for 20 min, and the supernatant containing the ELP was collected for purification. The ELPs were purified by inverse transition cycling, as described elsewhere.23,25 The ELP constructs used in this study (Scheme 1) consist of repeats of the pentapeptide sequence Val-Pro-Gly-XaaGly (VPGXG) (where Xaa can be any amino acid except Pro) and include a leader peptide composed of Ser-Lys-Gly-Pro-Gly and a trailer Trp-Pro dipeptide. ELP1-Z is uncharged and contains Val, Ala, and Gly at the guest residue position of the pentapeptide, in a 5:2:3 ratio, and consists of Z ) 150 and 180 pentapeptide repeats with total molecular weights of 59.4 and 71.2 kDa, respectively. The charged ELP13-X includes Lys, Val, and Phe at the guest residue
position of the pentapeptide in a 1:2:1 ratio and consist of X ) 32, 64, or 128 pentapeptide repeats with total molecular weights of 14.6, 28.3, and 55.7 kDa, respectively. The charged ELP12-Y contains Lys, Val, and Phe at the guest residue positions of the pentapeptide in a 1:7:1 ratio and consists of Y ) 144 pentapeptide repeats, with a total molecular weight of 61.1 kDa. We defined the inverse phase transition temperature, Tt, as the temperature at which the first derivative of optical density with respect to temperature reaches its maximum. The phase transition of ELPs in solution was measured by UV-visible spectrophotometry (Cary300 Bio; Varian; Melbourne, Australia). The transition temperature for ELP1-180 was measured as a function of ionic strength at a fixed ELP concentration of 1.7 mg/mL. Transition temperatures for ELP12-144 and ELP13-128 as a function of pH were measured at an ELP concentration of 0.625 mg/mL in 20 mM phosphate buffer. Sample Preparation for Cantilever Deflection and QCM-D Measurements. For microcantilever deflection measurements, we used commercially available V-shaped Si3N4 microcantilevers (∼196 µm long and ∼600 nm thick, Veeco) that were gold coated on their back side, thus facilitating thiol immobilization. For all measurements, we selected microcantilevers that were co-located in the same region of one wafer to minimize differences in their mechanical responses. We determined the spring constant (i.e., stiffness) of all Si3N4 microcantilevers used in the experiments from the power spectral density of the thermal noise fluctuations in solution.31 The spring constant values were 70 ( 10 pN/nm. For QCM-D experiments, we used gold-coated, polished piezoelectric quartz crystals (Q-Sense, Sweden, part number QSX 301) with a fundamental resonance frequency of 5 MHz. Prior to use, the substrates were exhaustively rinsed with ethanol and water and dried under a flow of nitrogen. Microcantilevers were functionalized with a self-assembled monolayer (SAM) of mercaptohexadecanoic acid (MHA) by immersing the substrate in a 2 mM ethanol solution of the thiol for 2 h while QCM crystals were modified by covering the sensor surface with a drop of the same thiol solution. Before ELP grafting, the acid groups were activated by reaction with 1-ethyl-3-(dimethylamino)propyl carbodiimide EDAC (0.4 M, Aldrich) and N-hydroxysuccinimide (NHS) (0.1 M, Aldrich) for 30 min. Freeze-dried ELPs were dissolved in 20 mM phosphate buffer solution (pH ∼6.8), diluted to the desired concentration (about 300 µM), and were then surface grafted at room temperature (T ) 25 ( 1 °C, i.e., a temperature below the LCST of the ELPs used here) by placing a drop of ELP solution on a substrate surface for a period of 2 to 3 h. Physisorbed ELPs were removed by immersing the substrates in 0.05% sodium (31) Hutter, J. L.; Bechhoefer, J. ReV. Sci. Instrum. 1993, 64, 1868-1873.
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Figure 1. Net microcantilever deflection plotted as a function of time for two different ionic strengths (PBS and PBS + 1.5 M NaCl). Net deflection is determined as the difference between the deflection of a microcantilever with end-grafted ELP1-180 in PBS or in PBS + 1.5 M NaCl and deflections of a bare reference microcantilever under the same solution conditions. ∆d indicates the effective difference in cantilever deflection at steady state. dodecyl sulfate (SDS, Pierce) solution for 20 min, followed by thorough washing with Milli-Q-grade water. Measurement of Cantilever Deflection. For convenience, we performed deflection measurements in the fluid cell of an atomic force microscope (MultiMode AFM with Nanoscope IIIa controller, Digital Instruments, Veeco). For each experiment, the sensitivity of the photodetector was determined from the constant compliance regime after engaging the cantilever on the substrate surface; typical values of photodetector sensitivity were around 90 nm/V. To reduce thermal drift effects during measurements, the setup was thermally equilibrated for about 1 h before measurements. The microcantilever was positioned at least 100 µm above the substrate surface to exclude unfavorable long-range interactions between the lever and surface. Deflection was measured by monitoring the position of a laser beam reflected from a cantilever’s free end onto a photosensitive detector. The deflection voltage was recorded (National Instruments, PCI6052E data acquisition card) every 10 ms until the cantilever deflection reached equilibrium. At that point, the deflection data were averaged every 100 points using a running mean filter. All deflection measurements were conducted at room temperature (23 ( 1 °C). Reference Experiments. Even small differences in the temperature between the cantilever and the surrounding fluid medium lead to thermal drift effects; for example, local cantilever heating occurs from the laser used for the deflection measurements.19,32 In our experimental setup, these thermally induced deflections typically stabilized within approximately 15 min, and a steady-state deflection was reached (Figure 1). To assess this thermally induced deflection and to deconvolute it from that induced by changes in polypeptide conformation, we measured, under the same solution conditions, the deflection of a reference cantilever functionalized with MHA but without ELP. This sequential measurement approach was necessary because, in our measurement setup, a reference cantilever could not be monitored simultaneously during an experiment. However, repeated experiments showed that we were able to align cantilevers consistently and that for cantilevers from the same batch essentially the same thermal deflection response was obtained. Surface Stress. The deflection of microcantilevers can be converted to surface stress if their geometrical and physical characteristics are known. According to Stoney’s formula, the integral surface stress in a polymer layer that is thin compared with the microcantilever thickness is related to the microcantilever deflection by eq 11 (32) Butt, H. J.; Jaschke, M. Nanotechnology 1995, 6, 1-7.
∆dEt2 4L2(1 - ν)
(1)
where d is the deflection signal measured in the experiment, E is the Young modulus of the microcantilever taken as 150 GN/m2,1 ν is the Poisson’s ratio of silicon nitride taken as 0.2,1 ∆σ is the difference in stress values at the two cantilever faces, and L and t are the length (190.7 ( 2.7 µm) and thickness (600 ( 55 nm) of the microcantilever, respectively, estimated as the average value obtained from scanning electron microscopy images of 10 microcantilevers. For the stress estimate, we neglect the effect of the bilayer structure (silicon nitride and gold) of the cantilever. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). To study the hydration and conformational behavior of ELPs, we also used a quartz crystal microbalance with dissipation monitoring (QCM-D, model Q-Sense D-300, Gothenburg, Sweden). This instrument allows the investigation of water-rich polymers on surfaces33 and simultaneously measures changes in the resonance frequency and dissipation at the fundamental frequency and several overtones of a QCM-D quartz crystal. Using QCM-D, we measured the frequency and dissipation response at the fundamental (f ) 5 MHz) and several overtones (f ) 15, 25, and 35 MHz) as a function of time. Data obtained with the QCM-D were used to estimate the change in coupled mass33 (ELP mass plus the mass of the viscously coupled solvent) as a function of solution ionic strength and pH. Reference experiments were performed on the same crystal before grafting ELP molecules to determine the crystal’s frequency and dissipation response to changes in the solvent condition (bulk effect). To assess the conformational and mass changes associated with the ELP response to changes in the solvent environment more accurately, the bare crystal’s response was subtracted from all measurements. All experiments were carried out in a temperature-controlled environment (22.5 ( 0.01 °C), and prior to every measurement, the crystal was thermally equilibrated in the solution of interest. If changes in dissipation are small, then shifts in the resonance frequency (∆f) can be related to changes in the coupled mass (∆m) by the Sauerbrey relation33 ∆m )
( )
CQCM ∆f n
(2)
where CQCM ) 17.7 ng cm-2 Hz-1 is the mass sensitivity constant33 and n is the overtone number. Environmental Stimuli Used in the Measurements. Directly increasing the solution temperature above the ELP transition temperature (Tt) is perhaps the simplest means to trigger the hydrophobic collapse of an ELP; however, increasing temperature in an AFM fluid cell or the QCM-D chamber causes large thermal drifts, thus increasing measurement error. Hydrophobic folding of grafted ELPs was therefore triggered isothermally by increasing the ionic strength of the solvent medium.34 We demonstrated the effectiveness of this approach by measuring thermal profiles of ELP1180 in solution as a function of ionic strength (data not shown). From these measurements, we found that Tt decreases to below room temperature upon addition of 1.5 M NaCl to a PBS buffer. To study the effect of solution pH on polypeptide conformation, we performed experiments in PBS solutions over a range of pH (3 to 12), prepared by adding small amounts of 0.1 M HCl or 0.1 M NaOH to the solution. To demonstrate measurement repeatability and reversibility of the ELP stimulus response, microcantilever deflection experiments were performed with at least two solvent exchange cycles.
Results and Discussion Effect of Thermal Drift on Microcantilever Deflection. An AFM cantilever is typically constructed from an unbalanced (33) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. 2001, 73, 5796-5804. (34) Urry, D. W. Prog. Biophys. Mol. Biol. 1992, 57, 23-57.
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Figure 3. Transition temperature (determined by UV-visible spectrophotometry) plotted as a function of solution pH for two pH-sensitive ELPs (ELP12-144 and ELP13-128) at a solution concentration of 625 µg/mL.
Figure 2. (a) Frequency and dissipation changes associated with the phase transition of ELP13-128 upon increasing the pH from 3.3 to 10.7 and those of ELP1-150 upon the addition of 1.5 M NaCl to PBS. Frequency and dissipation values were measured at 5 MHz (fundamental). Error bars represent an uncertainty due to measurement drift and reflect the average of two independent measurements. (b) Frequency plotted as a function of time for ELP1-150 and for two solution-exchange cycles (i.e., from PBS + 1.5 M NaCl (collapsed state) to PBS).
laminate of a thick layer of silicon nitride and a thin gold film. Thermal stresses, caused by the mismatch between the coefficients of thermal expansion of the two layers, cause the cantilever to bend in much the same way as a bimetallic strip in a thermostat.16 After a while (on the order of minutes), however, the thermally induced deflection of the cantilever stabilizes, and a steady-state deflection is reached (Figure 1). Cantilever vibrations caused by Brownian motion also occur but are much smaller in amplitude and can be neglected in these experiments.35 Importantly, the thermally induced deflections of reference cantilevers were significantly smaller than those induced by changes in the ELP graft conformation. Effect of Ionic Strength on Microcantilever Deflection. Thermodynamically reversible LCST transitions of ELPs in solution are most commonly triggered either by temperature changes or, isothermally, by changes in the type and concentration of salts.36 For experimental convenience, we opted for the latter and added 1.5 M NaCl to trigger the LCST transition of endgrafted ELP1-180. In general, grafting ELPs to microcantilevers resulted in increased equilibrium deflection (i.e., downward or negative deflection) in response to changes in all experimental solution environments (pH or salt concentrations) when compared to that of the unmodified reference cantilevers. This suggests that the grafted ELPs on the top surface of an AFM cantilever (35) Ma, H.; Jimenez, J.; Rajagopalan, R. Langmuir 2000, 16, 2254-2261. (36) Kontturi, K.; Mafe, S.; Manzanares, J.; Svarfvar, B.; Viinikka, P. Macromolecules 1996, 29, 5740-5746.
cause lateral steric interactions (driven by the osmotic swelling pressure of the grafted ELP layer) that lead to increases in the cantilever surface stress and increase the cantilever deflection compared to that of unmodified control cantilevers. Figure 1 shows that the addition of 1.5 M NaCl to PBS decreases the steady-state cantilever deflection significantly (∆d ≈ 70 nm). This is consistent with the conformational collapse of the ELP graft that results from an expulsion of waters of hydration and a concomitant reduction in osmotic pressure within the graft layer. Using eq 1, we can estimate the change in integral surface stress associated with the ELP phase transition, ∆σ ≈ 37 pN/nm. Effect of Ionic Strength on the Frequency and Dissipation Behavior of ELP-Decorated QCM-D Crystals. To corroborate the solvent-induced conformational changes of end-grafted ELPs, we also analyzed the effect of changes in salt concentration on the frequency and dissipation response in QCM-D measurements (Figure 2a). The addition of 1.5 M salt causes an increase in the resonance frequency of about 30 Hz (measured at 5 MHz) and a concomitant decrease in the dissipation response. The increase in resonance frequency indicates a decrease in viscously coupled solvent mass that can be explained by the loss of waters of hydrophobic hydration37 and the associated conformational collapse of the ELP graft layer. The decrease in the dissipation response is consistent with this mechanism and suggests an ELP graft conformation that is more compact and elastic than that of the same ELP in the hydrated state. We used the Sauerbrey equation (eq 2) to estimate the mass change due to an increase in ionic strength of the solvent when ELP1-150 was grafted onto the crystal surface. Equation 2 (with n ) 1) predicts a mass reduction of ∼530 ng/cm2 that is associated with the loss of water of hydration. The use of the Sauerbrey equation is justified here because the estimated ELP1-150 graft height on the surface is on the order of about 12 nm and changes in the measured dissipation are relatively small.38 By simply approximating the water of hydration associated with the ELP as a uniform water layer on top of the sample surface, this mass decrease corresponds to a reduction in thickness of the water layer of about 5 nm. This value is quite reasonable, considering that not only the water of hydration but also viscously coupled water is accounted for by the mass change. Figure 2b shows that the swelling/collapse behavior of ELP1150 grafted to the QCM crystal is reversible (i.e., the frequency changes were approximately the same for two successive exchange cycles between PBS and PBS + 1.5 M NaCl). (37) Li, B.; Alonso, D. O. V.; Bennion, B. J.; Daggett, V. J. Am. Chem. Soc. 2001, 123, 11991-11998. (38) Rodahl, M.; Hook, F.; Fredriksson, C.; Keller, C. A.; Krozer, A.; Brzezinski, P.; Voinova, M.; Kasemo, B. Faraday Discuss. 1997, 229-246.
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Figure 5. Deflection response plotted as a function of time for a microcantilever decorated with ELP13-128 and upon cycling between pH 5.9 and 11.9. The deflection response is reproducible for several pH cycles (∆d1 ≈ ∆d2 ≈ 100 nm).
Figure 6. Steady-state deflection values for microcantilevers decorated with ELP13, plotted as a function of ELP13 molecular weight, and at two pH values (3.3 and 10.7). Error bars reflect the standard deviation in the deflection signal at steady state.
Figure 4. Microcantilever deflection, measured at steady state, plotted as a function of solution pH. Cantilever decorated with (a) pH-sensitive ELP13-128 and (b) ELP12-144. (c) Deflection response of a control cantilever decorated with non-pH-sensitive ELP1-150 and response of a reference cantilever decorated with a SAM of MHA and that of a bare reference cantilever. Error bars reflect the standard deviation in the deflection signal at steady state. The dashed lines in a and b indicate the deflection sensitivity.
Effect of a Change in pH on Microcantilever Deflection. Figure 3 shows that the transition temperatures of ELP12-144 and ELP13-128 in dilute solution depend on the solution pH and suggests that significant hydrophobic collapse of the ELP occurs above the transition temperature. On the basis of the solution data, all of our microcantilever deflection measurements and QCM-D measurements (see below) were conducted at room temperature and over a pH range in which we expect that we stayed below the transition temperatures. The effect of solvent pH on cantilever deflection for ELP12144 (16 Lys, 56 kDa) and ELP13-128 (32 Lys, 61 kDa) is shown in Figures 4a and 4b, respectively. Cantilever deflection decreases (becomes less negative) with increasing solution pH in the range from pH 6 to 11. This is due to ELP collapse with increasing solvent pH and results from the decreasing ionization of the NH3+ of the lysine side groups (pKa ∼10.54).39 The ELP surface (39) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman & Company: New York, 1995.
conformation then depends on the balance between restoring elastic forces in the hydrated graft layer and repulsive electrostatic forces (ionic osmotic pressure). As the concentration of protons decreases when the solution pH increases from ∼6 to ∼11, NH3+ groups become increasingly deprotonated, which results in decreasing osmotic pressure in the ELP graft layer. As a consequence of this pressure decrease, the ELP graft layer collapses until the remaining restoring elastic forces compensate for the new, lower osmotic swelling pressure. A swelling maximum (deflection maximum) is reached at pH ∼4.5 (data not shown), suggesting full protonation, and a further increase in the proton concentration did not affect the cantilever deflection. Figure 4a,b also shows that the minimum deflection around pH 11 is consistent with the pKa (∼10.6) of the NH3+ groups. Finally, Figure 4c shows that there is effectively no pH dependence of the deflection response of a bare cantilever and a cantilever decorated with a charge-neutral ELP graft (ELP1-150) of comparable molecular weight. Sensitivity of ELP-Decorated Microcantilevers for pH Sensing. Surprisingly, the pH sensitivities (deflection response) of 20.7 nm/pH unit for ELP13-128 and 23.8 nm/pH-unit for ELP12-144, estimated from the slope of the linear deflection regime between pH 6 and 10 in Figures 4a,b, are nearly the same, although ELP13-128 contains twice as many Lys residues (32) as ELP12-144 (16) along approximately the same polypeptide chain length. To account for this observation, we argue that the presence and density of positively charged Lys groups affects the grafting behavior and surface conformation of Lys-containing ELPs. Considering that we use a negatively charged MHA SAM on the cantilever surface, it is likely that the positively charged,
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Lys-containing ELPs have more than one surface attachment point due to charge compensation. ELP13-128, containing a larger number of positively charged Lys groups than ELP12-144 could thus adopt a flatter surface conformation and develop less osmotic swelling pressure. To corroborate this argument, we carried out AFM colloidal probe measurements to directly measure the conformational changes of the two surface-grafted, pH-sensitive ELPs (ELP12-144 and ELP13-128) as a function of pH (Supporting Information). As expected, the extent of the steric interaction distance depends strongly on the solvent pH. At low pH, when the charge groups are ionized, the repulsive regime is extended and decreases with increasing pH (i.e., increasing deprotonation). Our measurements show that the conformational changes associated with changes in the pH are less pronounced for ELP13-128 (i.e., the ELP with higher charge density). These observations, at least qualitatively, support our argument that the potential for larger osmotic swelling pressures (i.e., larger surface stresses and consequently larger cantilever deflection) in the ELP of larger charge density is offset by a flatter surface conformation. A more systematic study of the grafting behavior of charged ELPs as a function of surface charge density and the solvent conditions is, however, outside the scope of this article. Reversible Microcantilever Deflection Triggered by pH Cycling. Repeated solvent-exchange cycles using solutions with pH 5.0 and 11.9 (i.e., below and above the pKa of Lys) showed that the time-dependent deflection response of a microcantilever decorated with ELP13-128 is reversible (Figure 5). Using eq 1, we estimated the change in integral surface stress associated with cycling the solution pH between 5.9 and 11.9 to be ∆σ ≈ 55 pN/nm, which corresponds to a deflection change of ∆d ≈ 105 nm. This stress value is much smaller than the one observed for poly(N-isopropylacrylamide)-co-vinylimidazole (pNIPAAMVI) brushes polymerized from the microcantilever surfaces.40 The larger stress values for pNIPAAM-VI are mainly due to much thicker brushes and much higher grafting densities (“grafting from” for pNIPAAM-VI as opposed to “grafting to” for ELPs). Our results show that reversible switching of ELP conformation on a cantilever surface leads to reversible cantilever bending, and the magnitude of cantilever actuation can be modulated by the level of the applied external stimulus. Together, these results suggest that pH-sensitive autonomous actuators and sensors can be designed using microcantilevers decorated with pH-sensitive ELPs. Effect of pH on the Frequency and Dissipation Behavior of ELP-Decorated QCM-D Crystals. The frequency change for grafted ELP13-128 observed in QCM-D measurements upon increasing the pH from 3.3 to 10.7 suggests the collapse of the ELP conformation. Notably, the observed frequency change upon switching the pH is significantly smaller than that associated with switching the ionic strength of the solvent. This is consistent with the notion that the latter reflects a significant change in the state of hydrophobic hydration of the whole grafted ELP whereas the former arises from osmotic pressure changes in the vicinity of the charged groups distributed along the ELP backbone. Furthermore, the smaller frequency response may also point to (40) Abu-Lail, N. I.; Kaholek, M.; LaMattina, B.; Clark, R. L.; Zauscher, S. Sens. Actuators, B 2006, 114, (1), 371-378. (41) Raiteri, R.; Butt, H. J.; Grattarola, M. Electrochim. Acta 2000, 46, (2-3), 157-163.
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an overall flatter conformational state of ELP13-128 on the surface. (See the discussion above.) Effect of Molecular Weight. Figure 6 shows the deflection response for ELP13 plotted as a function of molecular weight, measured at two pH values. As expected, the overall cantilever deflection increases with increasing molecular weight, which we suggest is related to increased graft layer thickness (assuming that the efficiency of grafting is independent of ELP chain length over the range of ELP MWs studied here). Furthermore, the deflection change in response to a pH change of the solvent increases with increasing molecular weight. These results suggest that the magnitude of microcantilever deflection can be tuned by engineering ELPs with precisely controlled molecular weights.23,24
Conclusions We have shown that changes in the conformational state of surface-grafted ELPs can be triggered by changes in salt concentration or pH and lead to significant changes in the surface stress in the ELP graft layer that translates into measurable changes in microcantilever deflection. We found that cantilever deflection was reversible and repeatable upon cycling the solution pH for ELPs that contain ionizable Lys residues periodically distributed along the polypeptide backbone. Furthermore, we showed that the magnitude of steady-state deflection of the AFM cantilever increases with increasing molecular weight and that the net deflection in response to pH increases weakly with molecular weight. QCM-D measurements corroborated our findings on microcantilevers and lent insight to ELP graft conformation and hydration response. We found that the apparent mass change was about 3 times larger upon cycling the ELP through the phase transition by adding NaCl than for experiments where we cycled the pH through the pKa of the side-chain amine groups in the Lys residues of ELP13-128 and ELP12-144. We attribute this observation to a more pronounced effect of ionic strength on the state of hydrophobic hydration of the ELP and to a flatter conformation state of pH-sensitive ELPs. Our results suggest that microcantilever-based sensors, having ELPs as a responsive coating, have an advantage over similar sensors that use other polymers11,41 because the response of ELPs to environmental stimuli can be tuned over wide ranges of pH, temperature, and ionic strength by incorporating different amino acids at the guest residue position through genetic engineering. Furthermore, ELPs offer an exciting advantage over synthetic analogues, such as pNIPAAm, in that they can be easily fused with proteins and ligands, which opens up the intriguing possibility of ELP-based actuators that are triggered by biomolecular binding events. These prospects are currently under investigation. Acknowledgment. We acknowledge funding through NSF EEC-021059 NIRT (A.C. and S.Z.) and NSF DMR-0239769 Career (S.Z.). Supporting Information Available: Sample preparation for colloidal probe measurements. Preparation of colloidal probes. Force plotted as a function of separation at several pH values for ELlP12-14 and ELP13-128. This material is available free of charge via the Internet at http://pubs.acs.org. LA0616698
