Nanomechanical Properties of Globular Proteins: Lactate Oxidase

Lactate oxidase (LOx) has been chosen as a model enzyme since there are few ... AT-cut quartz crystals (5.0 MHz) of 25 mm diameter with Au electrodes ...
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Langmuir 2007, 23, 2747-2754

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Nanomechanical Properties of Globular Proteins: Lactate Oxidase Ana Parra,† Elena Casero,† Encarnacio´n Lorenzo,† Fe´lix Pariente,† and Luis Va´zquez*,‡ Departamento de Quı´mica Analı´tica y Ana´ lisis Instrumental, UniVersidad Auto´ noma de Madrid, 28049 Madrid, Spain, and Instituto de Ciencia de Materiales de Madrid (CSIC), C/Sor Juana Ine´ s de la Cruz No. 3, 28049 Madrid, Spain ReceiVed September 29, 2006. In Final Form: NoVember 15, 2006 We report on the study of the nanomechanical properties of a lactate oxidase (LOx) monolayer immobilized on gold substrates by atomic force microscopy techniques operating under buffer conditions. Topographical contact mode imaging evidenced the protein deformation under the applied tip load. We performed approaching force curves with both stiff and soft cantilevers by imposing maximum loads of 1.6 nN and 400 pN, respectively. We found that the experimental data were well fitted by the Hertz model for a conical indenter. The use of two types of cantilevers allowed us to check further the consistency of the applicability of the Hertz model to the experimental data. After analyzing 180 curves, we obtained an average value of Young’s modulus for the LOx layer in the 0.5-0.8 GPa range. These results agreed with those obtained for LOx submonolayer deposits on mica substrates, which allows discarding any important contribution from the underlying substrate on the measured properties. This range of values is closer to those obtained by other techniques on other globular proteins in comparison with those reported in previous AFM studies on similar systems. We found that for our experimental conditions the force curves can be, in principle, well fitted by the Hertz model for both conical and spherical indenter geometries. However, as the Young’s modulus obtained for both geometries can differ appreciably, it becomes necessary to assess which indenter geometry is more adequate to explain the experimental data. For such purpose a systematic study of the indentation versus applied force curves obtained from both fittings for all the experimental curves was done.

1. Introduction The study of the mechanical properties of single cells, subcellular components, and biomolecules has undergone a rapid progress in recent years. These studies have proved to be important for the fundamental of biology and biochemistry.1 In fact, there is an increasing need to provide systematic data to deepen the understanding of molecular biomechanics. The attainment of such data has become more accessible since the emergence of new techniques for measuring forces and displacements with high resolution as well as the development of bioimaging techniques.2,3 In particular, the investigation of the mechanical properties, deformability, and flexibility of proteins is important since their functions are related to their three-dimensional conformations. Thus, the study of these issues can improve our understanding of enzyme catalysis,4,5 changes in cellular structure,6 and the ways in which drugs exert biological effects.7 Among the high-resolution techniques, atomic force microscopy (AFM) has proved to be powerful enough to obtain reliable data on the mechanical properties of organic samples. Thus, in AFMbased experiments, we can distinguish roughly two methodologies based on the pulling8 or pushing3 of the organic material, respectively. Here, we focus on the second method that provides data on the mechanical properties of organic samples through the measurement of approaching force curves. The mechanical * Corresponding author. E-mail: [email protected]. † Universidad Auto ´ noma de Madrid. ‡ CSIC. (1) Bao, G. J. Mech. Phys. Solids 2002, 50, 2237. (2) Finer, J. T.; Simmons, R. M.; Spudich, J. A. Nature 1994, 368, 113. (3) Vinckier, A.; Semenza, G. FEBS Lett. 1998, 430, 12. (4) Eisenmesser, E. Z.; Bosco, D. A.; Akke, M.; Kern, D. Science 2002, 295, 1520. (5) Shlyk-Kerner, O.; Samish, I.; Kaftan, D.; Holland, N.; Maruthi Sai, P. S.; Kless, H.; Scherz, A. Nature 2006, 442, 827. (6) Bao, G.; Suresh, S. Nat. Mater. 2003, 2, 715. (7) Teague, S. J. Nat. ReV. 2003, 2, 527. (8) Janshoff, A.; Neitzert, M.; Oberdo¨rfer, Y.; Fuchs, H. Angew. Chem., Int. Ed. 2000, 39, 3212.

properties of cells,9 bacteria,10 and chromosomes11 have been evaluated by these procedures. In contrast, the studies on globular protein systems are relatively scarce. Since the pioneering studies on lysozyme,12 only a few reports have appeared on other proteins, such as bovine carbonic anhydrase II.13 Within this framework, our present work aims to study the mechanical properties of a lactate oxidase layer deposited onto flat gold substrates by means of AFM measurements. Lactate oxidase (LOx) has been chosen as a model enzyme since there are few studies concerning its physicochemical behavior when it is immobilized on metallic surfaces. In addition, LOx is one of the enzymes typically used for the development of bioanalytical devices for lactate determination, which is of great importance in the food industry for the control of dairy products and in clinical analysis. To obtain information on the mechanical properties of LOx layers, we have performed approaching AFM force curves, which have been successfully analyzed within the framework of the Hertz model. 2. Experimental Section 2.1. Materials and Methods. Lactate oxidase (EC 232-841-6 from Pediococcus species) lyophilized powder containing 41 units mg-1 solid was obtained from the Sigma Chemical Co. (St. Louis, MO). Stock solution was prepared dissolving 1.3 mg of the LOx lyophilized powder in 250 µL of 0.1 M phosphate buffer (pH 6.5), aliquoted (10 µL) and stored at -30 °C. Under these conditions the enzymatic activities remain stable for several weeks. 2.2. Quartz Crystal Microbalance Measurements. AT-cut quartz crystals (5.0 MHz) of 25 mm diameter with Au electrodes deposited over a Ti adhesion layer (Maxtek Inc., Santa Fe Springs, CA) were used for quartz crystal microbalance (QCM) measure(9) Touhami, A.; Nysten, B.; Dufrene, Y. F. Langmuir 2003, 19, 4539. (10) Schaer-Zammaretti, P.; Ubbink, J. Ultramicroscopy 2003, 97, 199. (11) Jiao, Y.; Scha¨ffer, T. E. Langmuir 2004, 20, 10038. (12) Radmacher, M.; Fritz, M.; Cleveland, J. P.; Walters, D. A.; Hansma, P. K. Langmuir 1994, 10, 3809. (13) Afrin, R.; Alam, M. T.; Atsushi, I. Protein Sci. 2005, 14, 1447.

10.1021/la062864p CCC: $37.00 © 2007 American Chemical Society Published on Web 01/30/2007

2748 Langmuir, Vol. 23, No. 5, 2007 ments. An asymmetric keyhole electrode arrangement was used, in which the circular electrode geometric areas were 1.370 (front) and 0.317 cm2 (back). The electrode surfaces were overtone polished. Prior to use, the quartz crystals were cleaned by immersion in piranha solution, H2SO4/H2O2 (3:1 v/v). Caution: Piranha solution is extremely reactiVe!. They were subsequently rinsed with water and dried in air. The quartz crystal resonator was set in a probe (TPS550, Maxtek) made of Teflon in which the oscillator circuit was included, and the quartz crystal was held vertically. The probe was connected to a cell by a homemade Teflon joint which was immersed in a water-jacketed beaker thermostated at the assay temperature with a thermostatic bath (Digital Temperature Controller Haake F6). The frequency was measured with a plating monitor (PM-740, Maxtek Inc.) and simultaneously recorded by a personal computer. For monitoring the mass changes associated with the lactate oxidase adsorption onto the gold substrates, a QCM probe was immersed in 10 mL of phosphate buffer solution (pH 6.5) and the frequency of the quartz crystal was monitored as a function of time. After the temperature and frequency had stabilized, an aliquot of the enzyme stock solution was added to a final concentration of 0.2 µM. The computation of the amount of protein immobilized on the gold substrate is based on the fact that the decrease of frequency, ∆f, in the QCM is due only to the change in mass arising from the adsorption of the enzyme. Thus, we can calculate the amount and surface coverage, Γ, of the adsorbed LOx monolayer by using the Sauerbrey equation:14 ∆m ) -Cf ∆f, where ∆m is the mass change (ng cm-2) and Cf (17.7 ng Hz-1 cm-2) is a proportionality constant for the 5.0 MHz crystals used in this study. 2.3. AFM Measurements. The AFM measurements were performed with Nanoscope IIIa equipment (Veeco) operating in contact mode in a fluid cell under 0.1 M phosphate buffer at pH 6.5. The protein was immobilized on commercial gold supports consisting of glass substrates (1.1 × 1.1 cm2) covered with a chromium layer (1-4 nm thick) on which a gold layer (200-300 nm thick) was deposited (Arrandee, Germany). Prior to use, gold surfaces were annealed for 2 min in a gas flame to obtain Au(111) terraces. Also, mica surfaces (Veeco) were used as substrates for protein adsorption. In this case, the mica was cleaved just before the immobilization step. The substrate was first imaged in buffer solution to ensure that the surface was flat and clean before the protein immobilization was carried out. Both sample and cantilever were located within a Plexiglas fluid cell where extremely small volumes (∼50 µL) of buffer were added. The enzyme adsorption was carried out by immersing the support into a solution of LOx (0.2 µM in 0.1 M phosphate buffer at pH 6.5). Before being imaged by AFM, the samples were rinsed thoroughly with the buffer solution to remove any loosely bound residue of LOx. Silicon nitride oxide sharpened cantilevers (DNP-S from Veeco) were employed. Two different types of cantilevers were used with nominal spring constants of 0.38 N/m (stiff cantilever) and 0.06 N/m (soft cantilever), respectively. To characterize the tip convolution effects, we have measured gold nanoparticles (from Sigma Aldrich) deposited on the gold substrates with tips used in the LOx measurements. We have chosen the nanoparticle diameter to be close to 5 nm due to its similarity to the LOx size. The force curves, consisting of 2048 data points, were obtained at a frequency close to 4 Hz and imposing a maximum applied force of 1.6 and 0.4 nN for the stiff and soft cantilevers, respectively. The results presented here have been obtained after analyzing 180 and 80 force curves for LOx deposits on gold and mica substrates, respectively. To obtain the indentation values, we calibrated the tip, which was used in the LOx layer force-curve measurements, on hard substrates. For the LOx/mica system this procedure was done simultaneously with the experimental measurements since submonolayer LOx deposits were studied. In the determination of the Young’s modulus, we have considered that the cantilever spring constant is that one provided by the manufacturer. When tapping mode imaging in buffer conditions was performed, we employed stiff cantilevers with free oscillation amplitudes of A0 (14) Sauerbrey, G. Z. Phys. 1959, 155, 206.

Parra et al. ) 10-15 nm at a resonance frequency in the 9-10 kHz range. To image the protein structures at the minimum load, we employed operating set points as close as possible to the free oscillation amplitude (i.e., compatible with stable imaging conditions). 2.4. Data Analysis of Force Curves. It is known that when an approaching force curve is performed on a soft sample, the tip, which moves exactly the piezo displacement z, will indent the sample by a magnitude δ. In this case, the cantilever deflection, d, is related to δ and z through the relationship d ) z - δ. Thus, the deflection of the cantilever obtained on a soft sample is smaller than that obtained on a rigid surface, which explains the smaller slope on the repulsive part of the force curve for soft samples. The force applied on the sample is F ) kd ) k(z - δ), where k is the spring constant of the cantilever. Among the different theories describing the elastic deformation of a soft sample by a rigid indenter, we have analyzed our experimental data within the framework of the Hertz model.15 This approximation has been used previously in similar systems.12,13 The Hertz model studied the elastic deformation of two spherical surfaces touching under load, and Sneddon extended this calculation to other geometries.16 The applied force and the indentation for a soft sample indented by a hard tip are related through the following expression: F ) A[E/(1 - ν2)]δm

(1)

where E is the Young’s modulus of the sample, ν is the Poisson ratio of the sample, and A is a constant that depends on the indenter radius and shape. Also, m is an exponent depending on the indenter geometry, with values of 2, 1.5, and 1 for conical, parabolic or spherical, and flat-ended cylindrical tips, respectively.17 In particular, for a conical tip the specific relationship between applied load and indentation is18 F ) [2E tan R/(π(1 - ν2))]δ2

(2)

where R is the half opening angle of the conical tip. Thus, to obtain the value of the Young’s modulus, we have to fit the experimental data of deflection versus tip-sample relative distance with this model. This implies that z - z0 ) d - d0 + [(kπ(1 - ν2)/(2E tan R))(d - d0)]1/2

(3)

Here, d0 is the zero deflection, which usually corresponds to the deflection value measured on the approaching force curve in the horizontal region at large tip-sample distances, and z0 is the contact point. Similarly, for a spherical indenter the following relationship between the applied force and the indentation holds: F ) [4ER1/2/(3(1 - ν2))]δ3/2

(4)

where R is the tip radius. In this case z - z0 ) d - d0 + [(3k(1 - ν2)/(4ER1/2))(d - d0)]2/3

(5)

The fitting of the experimental curve with the Hertz model (eq 3 or 5, depending on the indenter geometry) provides the contact point value (z0). Once the z0 point in the force curve is determined, we calculate the indentation by computing the lateral (i.e., along the x-axis) deviation of the curve with respect to one obtained on a hard reference surface with the same tip, which consists of a straight curve with slope equal to 1 that has to be plotted from the z0 point.3 This type of analysis becomes necessary in order to unambiguously assess which indenter geometry fits better our experimental data. From such operations we obtain the δ versus d curves and, therefore, (15) Hertz, H. J. Reine Angew. Math. 1882, 92, 156. (16) Sneddon, I. N. Int. J. Eng. Sci. 1965, 3, 47. (17) Weisenhorn, A. L.; Khorsandi, M.; Kasas, S.; Gotzos, V.; Butt, H. J. Nanotechnology 1993, 4, 106. (18) Domke, J.; Radmacher, M. Langmuir 1998, 14, 3320.

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Figure 1. Time dependence of the frequency changes of a bare gold substrate resonator in 0.1 M phosphate buffer solution upon addition of LOx to a final concentration of 0.2 µM. the δ versus F plots for both tip geometries. Finally, the δ versus F plots are fitted by the functions δ ) [π(1 - ν2)/(2E tan R)]1/2F1/2) acF1/2

(6)

for a conical indenter, and δ ) [3(1 - ν2)/(4ER1/2)]2/3F2/3 ) asF2/3

(7)

for a spherical geometry. From the value of ac or as we can determine the value of E.17

3. Results and Discussion 3.1. LOx/Au System: Characterization by QCM and AFM Imaging. The monolayer deposits were obtained as described in the Experimental Section and were characterized by QCM analysis and by atomic force microscopy in order to assess the full coverage of the gold substrate. In Figure 1, we show the frequency change measured by QCM for the deposition of LOx under the conditions described in the Experimental Section. Upon addition of LOx a rapid decrease in frequency is observed during the first 5 min; afterward the frequency decreases more slowly until a steady state is reached. From this change, we can estimate the amount of LOx adsorbed on the gold surface. By using the Sauerbrey equation, and assuming a molecular mass of 80 kDa for LOx, a surface coverage value close to 2.2 × 10-12 mol cm-2 is obtained, which is consistent with coverage of one monolayer. In Figure 2a we show a 250 × 250 nm2 contact mode AFM image taken at a relatively low force, i.e., 0.91 nN, of an LOx monolayer deposited on a bare gold surface. Clearly, a compact layer formed by globular structures that we can associate with the LOx molecules is observed. These structures have lateral sizes in the 5-9 nm range. In addition, isolated LOx structures are observed on top of the layer. When we measured the topography of the LOx monolayer, we verified its softness by imaging the same area at higher loads. This experiment was performed with the same cantilever on the same area just by changing the operating set point (i.e., the deflection of the cantilever and, therefore, the applied force). Thus, Figure 2b shows the same area of Figure 2a, but imaged at a higher force (3 nN). The image becomes blurred at the highest force (Figure 2b) since the globular features are not sharply contrasted. The LOx structures in the monolayer appear distorted but still visible. This distortion is more evident for those structures located on top of the layer. The drift of the equipment can be estimated as being close to 0.01 nm/s. The AFM images of the LOx monolayer taken at low load allow us to estimate the lateral dimensions of the LOx proteins, but because of the relatively high packing density of the LOx

Figure 2. 250 × 250 nm2 contact mode AFM images obtained under buffer environment of a LOx monolayer deposit on a bare gold substrate. The images were taken sequentially with the same stiff cantilever on the same area at forces of (a) 0.91 and (b) 3 nN. The vertical gray scale indicates 7 nm.

structures, we cannot know the height of the monolayer. This value becomes important when assessing the mechanical properties of LOx structures (see below). Therefore, we have imaged by AFM under buffer conditions LOx submonolayer deposits on bare gold substrates. However, for this system we have employed the tapping mode operation mode rather than the contact mode because the latter can lead to the lateral shift or distortion of the isolated LOx structures. Figure 3 shows a typical tapping mode image of such a sample. Individual LOx structures, as well as some small LOx aggregates, are scattered onto the substrate surface. After measuring more than 60 of these structures, we obtain an average height value of the LOx protein of 5.0 ( 1.5 nm. It should be noted that, although we have employed set amplitudes as close to the free one as possible (i.e., the lowest applied loads compatible with stable imaging), the proteins can be already deformed to some extent by the tip action. 3.2. LOx/Au System: Analysis of Force Data for Conical and Spherical Indenter Geometries. We demonstrated (Figure 2) that the LOx monolayer is qualitatively affected by the tip load used in the imaging process due to its softness. To quantify the mechanical properties of the LOx layer, in particular to obtain its Young’s modulus, we have performed approach force curve experiments with stiff cantilevers with a force constant of k1 ) 0.38 N/m. In Figure 4a we show a typical force curve displayed

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Figure 3. 490 × 490 nm2 tapping mode AFM image obtained under buffer environment of a LOx submonolayer deposit on a bare gold substrate. Individual LOx proteins are distinguished. The vertical bar indicates 30 nm.

as the cantilever deflection versus z piezo displacement. According with the Experimental Section, we have proceeded to fit the experimental data with the behavior expected for conical (eq 3) and spherical (eq 5) indenters, respectively. In Figure 4a are shown the experimental data together with the best fittings for conical and spherical indenters. Surprisingly, we have found that both types of fittings seem to be qualitatively similar as they overlap over a wide range with the experimental data. In general, the error of the fitting for the conical tip was smaller than that obtained for the spherical tip. The apparent compatibility of the experimental data with these two models was verified for the most part of the experimental curves. To better understand this fact, we have analyzed for which coefficient settings the theoretical functions z ) z (z0, d) for both conical (eq 3) and spherical (eq 5) indenters overlap. In Figure 4b we have plotted (open circles) eq 3 (conical indenter) for d0 ) 0, z0 ) 0 and the coefficient multiplying d1/2 equal to 1. We have found that for a spherical indenter (eq 5) the corresponding function (solid line in Figure 4b) overlaps with that obtained for the conical indenter when d0 ) 0, z0 ) 0.3 nm, and the coefficient multiplying d2/3 has the value of 0.67. These results explain the behavior found for the experimental data in Figure 4a. Under this scenario, in which the experimental data seem to be explained by two different geometries, we have applied the following strategy to unambiguously assess which indenter geometry is best fitted to explain our data. We have used the procedure proposed by Weisenhorn et al.17 In particular, we have systematically fitted our experimental data with eqs 3 and 5, i.e., with the expected behavior for a conical indenter and a spherical indenter, respectively. Once we had established the z0 value for each experimental curve and both indenter geometries, we obtained the corresponding indentation versus applied force curves. Then, we fitted these curves with the power function δ ) aFb. The exponent b reveals the best approximation for the tip shape (b ) 0.5 or 0.66 for a conical or spherical indenter, respectively). In Figure 4c is shown the δ versus F curve obtained from Figure 4a for a conical tip. Also plotted is the best δ ) aFb fit (i.e., a and b are both fitting parameters), which leads to b ) 0.49. This value is very close to that expected for a conical geometry. Similarly, in Figure 4d is shown the δ versus F curve

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obtained from Figure 4a for a spherical tip together with the best δ ) aFb fitting, which in this case results in b ) 0.59. This value is quite different from that expected for a spherical indenter. These results suggest that the conical geometry better fits the experimental data. This finding is confirmed when all experimental data are analyzed following the same procedure. The histogram of the b values obtained from this analysis is shown in Figure 4e. It is clear that the experimental data are better explained by the conical indenter geometry, as more of the 75% of the obtained values of b are in the 0.49-0.51 range. In contrast, the histogram obtained after analyzing the data for a spherical indenter is rather wide and not centered on the expected value of 0.66. According to these findings, we have assumed a conical geometry for the indenter in our analysis of the mechanical properties of LOx monolayers. 3.3. LOx/Au System: Analysis of Force Data Taken with Stiff and Soft Cantilevers. We have obtained approaching force curves with two types of silicon nitride cantilevers with nominal force constants of k1 ) 0.38 N/m and k2 ) 0.06 N/m, respectively. Representative approaching force curves obtained (open symbols) with cantilevers k1 and k2 are displayed in parts a and b, respectively, of Figure 5. The solid lines correspond to the best fit of the data using the Hertz model for a conical indenter. From the deflection versus z piezo displacement curves, and the determination, through the fitting of the Hertz model, of the corresponding contact point, we obtain the dependence of the indentation, δ, on the deflection, d. It should be noted that we have restricted the fitting to a relatively reduced range of deflection (i.e., forces) values in order to study relatively small deflection and force values. In particular, the range of forces considered for the stiff and soft cantilevers was 1.6 nN and 400 pN, respectively. Typical examples of these results are displayed in Figure 6a for the stiff (top data) and soft (bottom data) cantilevers, respectively. Along with the data points is drawn a solid line indicating the fitting following the Hertz model for a conical indenter. For the soft cantilever the noise level is higher because we are dealing with indentations just in the 1 nm range and forces below 400 pN. It is clear, after eq 2 in Experimental Section, that δ ∝ k1/2d1/2, where the constant of proportionality depends on the soft material properties and the tip geometry. Assuming this constant equal for both cantilevers, the ratio between the preexponential factors of the corresponding fittings following the Hertz model for the stiff and soft cantilever data should be equal to (0.38/0.06)1/2 ≈ 2.5. From the fittings, we obtain an experimental ratio close to 2.2. This value, within the experimental error, is consistent with the expected one. Moreover, this checking is also consistent with the assumption, which was derived from the histogram depicted in Figure 4e, of the system obeying the Hertz model for a conical indenter. From these data, it is straightforward to obtain the plot of δ versus the applied load, F, for both cantilevers (Figure 6b) provided that we know the k value, which we have assumed to be that given by the manufacturer. Clearly, the behavior for both cantilevers is consistent with the Hertz model for a conical tip (see the solid line indicating the best fit). Moreover, the two plots follow quantitatively the same behavior despite the fact that the data were collected with tips of different stiffnesses. It is worth noting that the use of two types of cantilevers with different stiffnesses allows checking the consistency of the assumptions made in the analysis concerning the model employed for the fitting and the tip shape. 3.4. LOx/Au System: Determination of Young’s Modulus. After analyzing 120 and 60 experimental curves obtained with

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Figure 4. (a) Typical approach force curve obtained with a stiff cantilever on a LOx monolayer. Together with the experimental data (open symbols) are displayed the best Hertzian fittings for conical (9) and spherical (solid line) indenter geometries. (b) Theoretical z ) d + d1/2 and z ) 0.3 + d + 0.67d2/3 curves for conical (open symbols) and spherical (solid line) indenters, respectively. (c) δ versus F plot obtained from (a) assuming a conical indenter geometry. The solid line corresponds to the best δ ) aFb fitting (b ) 0.49). (d) δ versus F plot obtained from (a) assuming a spherical indenter geometry. The solid line corresponds to the best δ ) aFb fitting (b ) 0.59). (e) Histogram of the values of exponent b obtained after analyzing the same experimental data with the Hertzian model for conical (shaded blocks) and spherical (nonshaded blocks) indenters. The vertical solid and dashed lines indicate the theoretical expected values of b for both geometries, respectively.

the stiff and soft cantilevers, respectively, we can obtain the average value of Young’s modulus. Accordingly, we fitted the δ versus F curves with the function δ ) acF1/2.17 From the ac value we obtain the value of Young’s modulus, provided that we know ν and tan R [E ) π(1 - ν2)/(2ac2 tan R)]. For the former we have assumed that it is equal to 1/3.12 Regarding the value of tan R, we have to estimate the tip size in order to obtain its reliable value. We have attempted to analyze the tip geometry by scanning electron microscopy (SEM). However, we found charge effects even at low acceleration voltages that precluded reliable tip geometry analysis. Besides, we are concerned in this study with the tip geometry at the very tip apex, in the nanometer range, which is difficult to assess by SEM. To know the tip size, we have taken into account the tip convolution effects induced by the tip geometry on the AFM images. It is well-known how the tip geometry, for either a spherical or a conical geometry, broadens the lateral size of the structures imaged by AFM.19 Accordingly, we have analyzed (19) Odin, C.; Aime´, J. P.; El Kaakour, Z.; Bouhacina, T. Surf. Sci. 1994, 317, 321.

the AFM morphology of those protein structures appearing isolated on top of the LOx monolayer. These structures have heights in the 4-5 nm range and lateral sizes, Lm, in the 13-15 nm range. Assuming a conical geometry for the tip, as the force data suggest, and a spherical geometry of radius 2.5-3 nm for the LOx structure, as the AFM images suggest, we can determine the tip half opening angle, R, by using the formula that relates the lateral size measured, Lm, with the real one, L, through the expression19

Lm ) L(1 + sin R)/cos R

(8)

With the values of Lm (13-15 nm) and L (5-6 nm), we obtain R ≈ 50°. It should be noted that this value is consistent with that obtained directly from the surface profiles of the isolated LOx structures imaged by AFM. In principle, the slope of the tip has to be equal to or higher than the highest slope of the measured surface morphology. Thus, we can obtain a limit for the value of R by analyzing the highest surface slope of these profiles. In addition, we have low-pass filtered the surface profiles to rule out any important artifact in the estimation of this slope coming

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Figure 5. Deflection versus z piezo displacement curves obtained on LOx monolayers with (a) stiff and (b) soft cantilevers. The solid lines in both cases indicate the best fitting following the Hertz model for a conical indenter.

from the high-frequency noise present during the measurements. After analyzing these profiles for the different tips, we obtain typical values of R in the 45°-55° range, which agrees with that one obtained from the analysis of the tip convolution on the imaged LOx structures. We want to note that, as we are dealing with soft LOx structures, we have made the same analysis on isolated hard gold nanoparticles with size (close to 5 nm) similar to the LOx proteins to further assess the tip geometry. The corresponding AFM image (Figure 7a) shows different aggregates of gold nanoparticles together with some isolated nanoparticles. The aggregation of the nanoparticles was also observed in parallel studies by transmission electron microscopy (not shown). We have analyzed the surface profiles of those nanoparticles that are not aggregated. One example is shown in Figure 7b together with that one obtained previously with the same tip on one isolated LOx structure. We have checked that the measured lateral sizes of both the gold nanoparticle and the LOx structure are consistent with a conical tip with a half opening angle of R ≈ 50°. A similar analysis can be done if we assume that the tip is spherical with a radius R.19 In this case, the relationship between the real and measured lateral sizes of an isolated spherical particle of diameter L is Lm ) 2L(2R/L)1/2. In our case, this leads to a value of R ≈ 5 nm. This value will be useful to compare the value of the LOx Young’s modulus for both tip geometries (see below). After these analyses, we obtain for both types of cantilevers E0.38 ) 0.7 ( 0.2 GPa and E0.06 ) 0.5 ( 0.3 GPa. It is clear that the errors are relatively large and that they are larger for the soft cantilever. Taking into account the errors involved, the values obtained with cantilevers of different stiffnesses on the LOx monolayer are consistent. However, the stiff ones prove to be more adequate, under our experimental conditions, to study the LOx nanomechanical properties. At this point, it may be interesting to contrast these values, particularly those obtained with the stiff cantilevers as they have

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Figure 6. (a) Indentation versus deflection curves obtained on LOx monolayers with stiff (top data, open circles) and soft (bottom data, open squares) cantilevers. The solid lines in both cases indicate the best fitting following the Hertz model for a conical indenter (i.e., δ ∝ d0.5). (b) Indentation versus load curves obtained with stiff (open circles) and soft (filled circles) cantilevers. The solid line indicates the best fitting following the Hertz model for a conical indenter (i.e., δ ∝ F0.5) for the data obtained with the stiff cantilever.

smaller errors, with that obtained by assuming a spherical geometry for the tip. This can be done following a procedure similar to that used for the conical geometry. By fitting the corresponding δ versus F curves with a function δ ) asF0.66, from the value of as that of the Young’s modulus can be derived, provided that we know the value of R. As commented above, we can assume that R ≈ 5 nm, which leads to E0.38 ) 0.28 ( 0.08 GPa. Thus, for the same set of experimental data the assumption of a spherical geometry for the indenter leads to an Es value that is 40% of that obtained by assuming a conical geometry. This fact is important since the scarce values of Young’s modulus of globular proteins12,13 obtained by AFM were derived by assuming a spherical indenter shape. In fact, in these works E values in the 0.08-0.5 GPa range are reported. It is worth mentioning that the value of the LOx Young’s modulus obtained on our system assuming a spherical geometry, namely Es ) 0.28 ( 0.08 GPa, is in agreement with this range of reported data. However, these values are smaller than those obtained by other techniques on other globular proteins, which are in the range of 1-2 GPa.20-23 For instance, a Young’s modulus of 2.5 GPa23 has been obtained by surface force apparatus for lysozyme deposits. The E values obtained by AFM considering a conical (20) Rosser, R. W.; Schrag, J. L.; Ferry, J. D.; Greaser, M. Macromolecules 1977, 10, 978. (21) Tamura, Y.; Suzuki, N.; Mihashi, K. Biophys. J. 1993, 65, 1899. (22) Suda, H.; Sugimoto, M.; Chiba, M.; Uemura, C. Biochem. Biophys. Res. Commun. 1995, 211, 219. (23) Gauthier-Manuel, B.; Gallinet, J. P. Biochimie 1998, 80, 391.

Nanomechanical Properties of Lactate Oxidase

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Figure 7. (a) 490 × 490 nm2 tapping mode AFM image of gold nanoparticles of 5 nm diameter deposited on a gold surface. The vertical bar indicates 150 nm. Surface profiles of a gold nanoparticle (b) and an isolated LOx structure (c) obtained with the same tip.

shape are closer to those obtained by other techniques. Thus, the assessment of which tip geometry better fits the experimental data becomes very important when analyzing nanoindentation data in globular proteins, since different geometries can lead to quite different values for the Young’s modulus. 3.5. LOx/Mica System: Analysis of Force Data Taken with Stiff Cantilevers. One question that can arise when studying the nanomechanical properties of nanometer-sized proteins deposited on solid substrates is the possible influence of the substrate on the measured properties. In our case, the measurements of the average LOx height do not seem to indicate a strong deformation of the protein during the adsorption process. Moreover, the obtained LOx height value, close to 5 nm, is slightly larger than that observed for the lysozyme experiments, in which indentations in the 1-1.5 nm range were also employed.12 These facts allow, in principle, discarding important effects of the substrate on the measured data. However, to verify this fact, we have also performed experiments on the nanomechanical properties of LOx deposits on a mica surface, which has a smaller surface energy than gold. Thus, we have studied LOx deposits on mica with a submonolayer surface coverage. This fact allowed us to visualize within the same image by tapping mode AFM under buffer conditions the protein deposit and mica, LOx-free, nanometer areas (Figure 8a). Then, we performed a force volume measurement in contact mode of the same area in order to obtain the typical force curves on the LOx structures and on the mica surface. The latter was used as the reference hard substrate for the study of the LOx nanomechanical properties. The measurements were done with the stiff cantilevers. Figure 8b shows a typical approach force curve obtained on the LOx structures together with a typical curve obtained previously on the LOx/Au system. It is clear that the qualitative behavior is similar in both cases. This is confirmed when the data were fitted by the Hertz model for a conical indenter. Thus, Figure 8c displays the typical indentation versus force plot

Figure 8. (a) 490 × 490 nm2 top view tapping mode AFM image of a LOx submonolayer deposit on mica. The vertical gray scale bar indicates 9 nm. (b) Deflection versus z piezo displacement curves obtained on the LOx/Au (open circles) and LOx/mica (filled circles) systems with the stiff cantilever. (c) Indentation versus load curves obtained with the stiff cantilevers for the LOx/Au (open circles) and LOx/mica (filled circles) systems. The solid line indicates the best fitting following the Hertz model for a conical indenter (i.e., δ ∝ F1/2) for the data obtained with the stiff cantilever for the LOx/Au system.

obtained for the LOx/mica system together with a characteristic one obtained for the LOx/Au system. Clearly, the data are consistent with those obtained previously on LOx monolayers on gold substrates. After analyzing all data measured for the LOx/mica system following the same procedures applied for the LOx/Au system, we obtain an average value for the Young’s modulus of the LOx structures of 0.6 ( 0.2 GPa. These values agree with those obtained with the same type of cantilever on the LOx/Au system (Table 1). These results confirm that the nanomechanical properties of the LOx structures are independent of the substrate and that the Young’s modulus of the LOx proteins is in the 0.5-0.8 GPa range.

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Parra et al.

Table 1. Values of Young’s Modulus Obtained for LOx/Au and LOx/Mica Systems Assuming Conical Tip Geometry LOx/Au LOx/Au LOx/mica

k (N/m)

E (GPa)

0.38 0.06 0.38

0.7 ( 0.2 0.5 ( 0.3 0.6 ( 0.2

The fact that the Young’s modulus values for a LOx monolayer and submonolayer deposits are quite similar suggests that the mechanical behavior of the monolayer is mainly due to that of the LOx proteins. In principle, this fact is not evident as the monolayer packing confers a certain mechanical stability to the individual LOx proteins. However, it has been recently proposed24 that the hardness (internal cohesion) of protein molecules determines predominantly the mechanical behavior of adsorbed protein layers, which is consistent with our experimental observations.

4. Conclusions We have studied in a fluid environment the mechanical properties of LOx monolayers by atomic force microscopy. Contact mode images proved qualitatively the protein deformation under the action of the tip load. To analyze quantitatively the mechanical properties of these layers, we performed force curve (24) Martin, A. H.; Cohen Stuart, M. A.; Bos, M. A.; van Vliet, T. Langmuir 2005, 21, 4083.

measurements on them with both stiff and soft cantilevers. We restricted the maximum applied force in these experiments to 1.6 and 0.4 nN for stiff and soft cantilevers, respectively. The data followed the behavior expected by the Hertz model for a conical indenter. From this analysis, we obtained a Young’s modulus in the 0.5-0.8 GPa range. Moreover, these values agree with those obtained by AFM on LOx submonolayer deposits on mica substrates. This fact allows us to discard any important contribution from the substrate on the nanomechanical properties measured on the LOx deposits. This range of values is higher than others obtained by AFM on other globular proteins and closer to other reported values obtained by different techniques. Finally, we have found that for this nanometer range of indentations there is a certain ambiguity concerning the best indenter geometry that explains the data within the framework of the Hertz model. This ambiguity has to be addressed since the Young’s modulus obtained for both geometries can differ significantly. Thus, a systematic analysis of the indentation versus applied force data was done to determine which indenter geometry is more adequate to interpret the experimental data. Acknowledgment. This work has been partially supported by the Ministerio de Educacio´n y Ciencia of Spain, Project Nos. CTQ2005-02816/BQU, and FIS2006-12253-C06-03, and by Comunidad Auto´noma de Madrid-Universidad Auto´noma de Madrid through Project No. 12/TES/001. LA062864P