Langmuir 2002, 18, 3517-3523
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Adsorption Behavior of Creatine Phosphokinase onto Solid Substrates S. M. Pancera,† E. B. Alvarez,† M. J. Politi,† H. Gliemann,‡ Th. Schimmel,‡,§ and D. F. S. Petri*,† Instituto de Quı´mica, Universidade de Sa˜ o Paulo, P.O. Box 26077, Sa˜ o Paulo, SP, 05513-970, Brazil, Institut fu¨ r Nanotechnologie, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021 Karlsruhe, Germany, and Institut fu¨ r Angewandte Physik, Universita¨ t Karlsruhe (TH), 76128 Karlsruhe, Germany Received July 12, 2001. In Final Form: January 31, 2002 The understanding of biomolecular interactions on solid surfaces is of importance for the design of new biomaterials and medical devices. In this work, the adsorption behavior of creatine phosphokinase (CPK) onto hydrophilic (silicon wafers and amino-terminated surfaces), hydrophobic (polystyrene), and charged (sulfonated polystyrene films) substrates was investigated by means of in situ ellipsometry, contact angle measurements, and atomic force microscopy. CPK is an interesting biomolecule due to its large application in the diagnosis for myocardial infarction and muscle disorders. In the dilute regime (c ∼ 0.005 g/L) the ellipsometric measurements revealed that the kinetics adsorption process of CPK onto silicon wafers and amino-terminated surfaces can be divided into four stages: (i) a diffusive one, (ii) adsorption and rearrangement, (iii) formation of a monolayer, and (iv) continuous and irreversible adsorption caused by relaxation process and cooperative binding. This seems to be the first time that such a behavior has been experimentally observed. For more concentrated solutions, the CPK formed aggregates in solution and, therefore, the adsorption increased continuously with time. CPK adsorbed irreversibly either on hydrophilic or on hydrophobic substrates. The adsorption isotherms showed a preferential adhesion of CPK onto the hydrophilic substrates. Since hydrophilic segments predominate the CPK structure, hydrogen bonding seems to play a major role in the adsorption process.
Introduction There is a great interest in immobilizing enzymes selectively on solid substrates in order to develop biosensors,1 kits for diagnosis,2 and chromatographic columns.3 Questions such as the adsorption rate, the driving forces responsible for the immobilization, and the change of structural conformation have been extensively explored in the literature.4-24 Most proposed mechanisms for * To whom correspondence may be addressed. Telephone: 0055 11 3091 3831. Fax: 0055 11 3815 5579. E-mail: dfsp@ quim.iq.usp.br. † Universidade de Sa ˜ o Paulo. ‡ Forschungszentrum Karlsruhe. § Universita ¨ t Karlsruhe. (1) Eggins, B. Biosensors an Introduction; Wiley & Sons: New York, 1997. (2) Montgomery, R.; Conway, T.; Spector, A. Biochemistry: A caseoriented approach, 5th ed.; The C. V. Mosby Company: St. Louis, MO, 1990. (3) Chibata, I.; Tosa, T.; Sato, T.; Mori, T. Immobilized Enzymes; Wiley & Sons: New York, 1978. (4) Schaaf, P.; Talbot, J. J. Chem. Phys. 1989, 91, 3301. (5) Lundstro¨m, I. Prog Colloid Polym. Sci. 1985, 70, 76. (6) Lyklema, J.; Norde, W. Prog. Colloid Polym. Sci. 1996, 101, 9. (7) Lu, C. F.; Nadarajah, A.; Chittur, K. J. Colloid Interface Sci. 1994, 168, 152. (8) Norde, W.; Lyklema, J. Colloid Surf. 1989, 38, 1. (9) Balladur, V.; Theretz, A.; Mandrand, B. J. Colloid Interface Sci. 1997, 194, 408. (10) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundstro¨m, I. J. Colloid Interface Sci. 1998, 207, 228. (11) Tengvall, P.; Lundstro¨m, I.; Liedberg, B. Biomaterials 1998, 19, 407. (12) Norde, W.; Giacomelli, C. E. Macromol. Symp. 1999, 145, 125. (13) Elgersma, A. V.; Zsom, R. L. J.; Lyklema, J.; Norde, W. Colloid Surf. 1992, 65, 17. (14) Ramsden, J. J. Colloids Surf., A 1998, 141, 287. (15) Tilton, R. D.; Robertson, C. R.; Gast, A. P. J. Colloid Interface Sci. 1990, 137, 192. (16) Wojciechowski, P. W.; Brash, J. L. J. Colloid Interface Sci. 1990, 140, 239.
protein adsorption assume multiple conformational states. The random sequential adsorption4 (RSA) model assumes that the particles are hard spheres which cannot overlap on the surface and that once a particle is adsorbed, it cannot desorb or diffuse on the surface. However, these equations describe the process accurately when the surface coverage becomes less than 0.50. Lundstro¨m5 applied modified Langmuir models to describe reversible and irreversible adsorption of proteins on solid surfaces, based on two different conformational states of the adsorbed entities. However, considering the complex nature of biomacromolecules, describing a general mechanism for the overall adsorption process is not a trivial task and the models do not always fit the experimental results. The orientation of biomacromolecules on the surface may change with time and thus affect the overall adsorption behavior. Regarding protein conformational changes due to adsorption processes, Lyklema and Norde6 considered two types of proteins: the hard and the soft ones. Upon adsorption the hard ones keep their native structure, while the soft ones spread completely on the substrate. In the case of soft proteins, the driving force for the adsorption is an entropic gain (∆adsS) caused by the breakdown of (17) Ladam, G.; Schaaf, P.; Cuisinier, F. J. G.; Decher, G.; Voegel, J. C. Langmuir 2001, 17, 878. (18) Haynes, C. A.; Norde, W. J. Colloid Interface Sci. 1995, 169, 313. (19) Claesson, P. M.; Blomberg, E.; Fro¨berg, J.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57, 161. (20) Malmstem, M. Colloid Surf., B 1995, 3, 297. (21) Malmstem, M. J. Colloid Interface Sci. 1994, 166, 333. (22) Swalen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Israelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (23) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706. (24) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Zhang, X.; Angermaier, L.; Knoll, W.; Spinke, J. J. Biomater. Sci., Polym. Ed. 1994, 6, 481.
10.1021/la0110693 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/27/2002
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tertiary and/or secondary structures. Not only the entropic contribution reduces the adsorption Gibbs energy (∆adsG) but also the enthalpic contribution (∆adsH):
∆adsG ) ∆adsH - T∆adsS
angles, ∆ and Ψ, using the fundamental ellipsometric equation and iterative calculations with Jones matrixes30
ei∆ tan Ψ ) Rp/Rs ) f(nk, dk, λ, φ)
(2)
(1)
In this work the adsorption behavior of creatine phospokinase (CPK) onto solid substrates was investigated by means of null ellipsometry, contact angle measurements, and atomic force microscopy. CPK was chosen due to its large application in the determination of creatinine25 in the diagnosis for myocardial infarction and muscle disorders.25,26 To study preferential adsorption of CPK, hydrophobic, hydrophilic, and charged surfaces were used as substrates. Silicon wafers and amino-terminated surfaces were chosen as hydrophilic substrates, while polystyrene and sulfonated polystyrene films were chosen as hydrophobic and charged substrates, respectively. The influence of ionic strength on the adsorption behavior and on the solution properties of CPK was investigated. Materials and Methods CPK was purchased from Sigma (C3755), St. Louis, MO. Solutions of CPK were prepared in the concentration range of 0.002-1.0 g/L in 0.001 mol/L NaCl. To investigate the effect of ionic strength on the solution properties, experiments were also performed in 0.01 and 0.1 mol/L NaCl. The CPK solutions were also prepared in Tris-HCl buffer, but they presented the disadvantage of the intensive air bubble formation, making ellipsometric measurements impossible. Silicon (100) wafers purchased from Crystec (Berlin, Germany) with a native oxide layer approximately 2 nm thick were used as substrates. The Si wafers (cut in a typical dimension of 1 cm2) were rinsed in a standard manner.27 After this, the surfaces were functionalized with aminopropyltrimethoxysilane, APS (Fluka, Switzerland), following a method described elsewhere.27 This method yields a flat and homogeneous amino-terminated monolayer covalently bound on silicon wafers. Polystyrene (PS, Mw ∼ 200 000 g/mol, kindly supplied by BASF Aktiengesellschaft (Ludwigshafen, Germany)) was spin-coated (3000 rpm, 30 s) on silicon wafers from solutions prepared in toluene at a concentration of 10 g/L. Toluene, hydrogen peroxide, ammonium hydroxide, and sodium chloride were purchased from Nuclear (Sa˜o Paulo, Brazil) and utilized without previous purification. Half of the spincoated PS films suffered surface sulfonation, described elsewhere.28 They were completely covered by a liquid film of sulfuric acid, 96 ( 1% analytical grade (Merck, Brazil), for 30 s. Afterward the samples were washed with distilled water and dried under a stream of N2. The surface reaction yielded SO3groups on the surface, turning the new substrates more hydrophilic than pure PS. If the surface sulfonation is not complete, these surfaces present charged PS-SO3- domains and unmodified PS domains. Ellipsometry. Ellipsometric measurements were performed in a vertical computer-controlled DRE-EL02 ellipsometer (Ratzeburg, Germany). The angle of incidence φ was set to 70.0°, and the wavelength λ of the laser was 632.8 nm. This equipment works as a null ellipsometer, as described elsewhere.29 For the data interpretation, a multilayer model composed of the substrate, the unknown layer, and the surrounding medium should be used. Then the thickness (dx) and refractive index (nx) of the unknown layer can be calculated from the ellipsometric (25) Garret, R. H.; Grisham, C. M. Biochemistry; Saunders College Publishing: Philadelphia, PA, 1995. (26) Wyss, M.; Kaddurah-Daouk, R. Physiol. Rev. 2000, 80, 1107. (27) Petri, D. F. S.; Wenz, G.; Schunk, P.; Schimmel, T. Langmuir 1999, 15, 4520. (28) Siqueira Petri, D. F.; Wenz, G.; Schunk, P.; Schimmel, Th.; Bruns, M.; Dichtl, M. Colloids Polym. Sci. 1999, 277, 673. (29) Fujimoto, J.; Petri, D. F. S. Langmuir 2001, 17, 56.
where Rp and Rs are the overall reflection coefficients for the parallel and perpendicular waves. They are a function of the angle of incidence, φ, the wavelength, λ, of the radiation and of the indices of refraction, and the thickness of each layer of the model, nk, dk. Ellipsometry is a technique which enables the independent determination of the index of refraction and the thickness, only if the optical contrast in the system is big enough or if the layer thickness is thick enough. However, the product nxdx is a constant value and does not depend on the adopted model or concentration profile near the wall.31 First of all, the thickness of the SiO2 layers was determined in air, considering the index of refraction for Si as n˜ ) 3.88 i0.01832 and its thickness as an infinite one; for the surrounding medium (air) the index of refraction was considered as 1.00. Because the native SiO2 layer is very thin, its index of refraction was set as 1.46232 and just the thickness was calculated. The mean SiO2 thickness measured for 50 samples was 1.9 ( 0.1 nm. After the characterization, the Si wafers were functionalized by the silanization reaction with APS.27 The thickness of the aminoterminated monolayer was determined in air, considering the nominal index of refraction of the silane as 1.424. The mean thickness value calculated for the amino-terminated layer was 0.9 ( 0.1 nm. The mean thickness and index of refraction of the PS and PSS films were determined as 65 ( 2 nm and 1.580 ( 0.005 and 48 ( 3 nm and 1.585 ( 0.008, respectively. The adsorption from solution was monitored in situ with the help of a poly(tetrafluoroethylene) cell. This cell has two quartz windows, one for the incident beam and the other for the reflected beam, with the inclination angle of 70.0°, as described elsewhere.29 The measurements were done in a conditioned room, where the temperature ranged from 23 to 25 °C. The adsorbed amount, Γ, is determined from
Γ)
dCPK(nCPK - n0) ) dCPKcCPK dn/dc
(3)
where nCPK and dCPK are the index of refraction and thickness of the adsorbed enzyme, n0 is the index of refraction of the solution measured with an Abbe refractometer, dn/dc is the increment of refractive index determined with a differential refractometer, and cCPK is the average enzyme concentration within the layer.29-31 For our system, n0 was measured for each concentration and dn/dc amounted to 0.16 mL/g at a temperature of 23 °C. From the ellipsometric angles ∆ and Ψ and a multilayer model composed of silicon, silicon dioxide, amino-terminated monolayer or polymeric film, enzyme layer, and bulk solution, it is possible to determine the thickness of the adsorbed enzyme, dCPK, and the enzyme index of refraction, dCPK, if the bulk concentration was higher than 0.5 g/L. Under such conditions, the adsorbed amount was high (Γ > 30 mg/m2) enough to enable the independent determination of nCPK and dCPK. The index of refraction of CPK, nCPK, determined by iterative calculation amounted to 1.495 ( 0.005. However, if the bulk concentration was less than 0.5 g/L, the adsorbed amount was low and the small differences in the indices of refraction of the substrate, enzyme, and solution made an independent determination of nCPK and dCPK impossible. Therefore, in the concentration range of cCPK < 0.5 g/L, nCPK was kept constant at 1.50 and dCPK was calculated. Nevertheless, it is important to remember here that if the index of refraction assumed for the adsorbing layer lies in a reasonable range (between 1.40 and 1.60), the product nCPKdCPK should be a constant value.29-31 Contact angle measurements were performed in a closed quartz cell saturated with water vapor, at 22.5 ( 0.5 °C in a (30) Azzam, R. M.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland Publication: Amsterdam, 1979. (31) Motschmann, H.; Stamm, M.; Toprakcioglu, Ch. Macromolecules 1991, 24, 3229. (32) Edward, D. P., Ed. Handbook of Optical Constants of Solids; Academic Press: London, 1985.
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home-built apparatus equipped with a Casio QV-10 digital camera, which is connected to a computer. Sessile water drops of 4 µL were used for the advancing contact angle (θA), and then the volume was reduced to 2 µL to measure the receding contact angle (θR). At least five samples of the same composition were analyzed at different spots before and after the 16 h of adsorption. To verify aging effects on the surface wettability, advancing contact angles were measured for the same drop in time intervals of 10 min during 1 h. These control measurements indicated the maximum variation in the contact angle of 5°. Atomic force microscopy (AFM) measurements were carried out with an Autoprobe CP Park Scientific Instruments system in the noncontact mode in air at room temperature. V-shaped silicon nitride cantilevers with sharpened pyramidal tips and force constants between 0.03 and 0.1 N/m were applied. All AFM images represent unfiltered original data and are displayed in a linear gray scale. After 16 h of adsorption experiment in the ellipsometric cell, the samples were removed from the solution, washed three times with pure solvent, and dried under a N2 stream. At least three samples of the same composition were analyzed at different areas of the surface. Dynamic light scattering (DLS) measurements were performed at 90° and 25.0 ( 0.5 °C on a Brookhaven Instruments Zeta PAL analyzer equipped with a digital correlator and Nd: YAG laser (λ ) 532 nm). The CPK solutions were filtered in a 0.2 µm filter (Gellman, Acrodisc) prior to measurements.
Results The Influence of Ionic Strength on the CPK Solution Properties. The influence of ionic strength on the CPK solution properties was investigated by DLS. The CPK concentration was set to 1.0 g/L, the medium pH was kept constant at 6.5, while the ionic strength was varied from 0.001 to 0.1 mol/L NaCl. The solutions were filtered prior to the DLS measurements. On increase of the ionic strength from 0.001 to 0.1 mol/L NaCl, a decrease in the mutual diffusion coefficients was observed. The mutual diffusion coefficients, D, were converted into the average CPK hydrodynamic radii, Rh, by the StokesEinstein relation
Rh ) kT/6πηD
Figure 1. Adsorption kinetics measured for CPK (c ) 0.005 g/L) onto silicon wafers at 22.5 ( 0.5° C. The AFM image corresponds to the dried CPK-covered substrate after 4 h of adsorption.
(4)
where k is the Boltzmann’s constant and η is the viscosity of the solvent, assumed as 1 mPa‚s. The values of Rh found at ionic strengths of 0.001, 0.01, and 0.1 mol/L NaCl amounted to 46 ( 9, 76 ( 10, and 140 ( 12 nm, respectively, indicating an aggregation process favored by the presence of salt. This is a usual effect phenomenon,2 which can be explained by the compression of the electrical double layer. In the CPK concentration range of 0.25-1.0 g/L, no dependence of the Rh values could be determined, suggesting that the dominant effect is particle aggregation. It should be added that the scattered intensity for DLS determinations for CPK concentrations below 0.25 g/L were very weak. Measurements performed with CPK solutions prepared in TrisHCl buffer and in the concentration range of 0.25-1.0 g/L yielded the mean value of Rh ) 19 ( 4 nm, indicating aggregation of CPK molecules. Therefore, all adsorption measurements were performed with CPK solutions prepared in 0.001 mol/L NaCl. In this low ionic strength the electrostatic interactions between substrates and CPK molecules are very important, since the screening effect of small ions is negligible. For comparison, the radius of gyration of CPK was estimated as 2.2 nm and its hydrodynamic radius as 2.8 nm from calculations using Protein Data Bank data33 and the Crysol program. Adsorption Kinetics. The adsorption kinetics measured for CPK onto silicon wafers and onto amino-
Figure 2. Adsorption kinetics measured for CPK (c ) 0.005 g/L) onto amino-terminated surfaces at 22.5 ( 0.5 °C.
terminated surfaces are shown in Figure 1 and Figure 2, respectively. These curves present similar features, which can be depicted into four stages: (i) a period with a linear increase of the adsorbed amount Γ as a function of the square root of time, (ii) a period with a nonlinear increase of the adsorbed amount Γ as a function of the square root of time, (iii) an adsorption plateau, and (iv) a stage where the adsorbed amount Γ increased continuously with t0.5. At the beginning the substrate is bare and the adsorption kinetics is governed by the diffusion of the biomolecules from the bulk solution to the surface. All biomolecules that arrive at the substrate are assumed to be immediately adsorbed. This stage was observed in the first 15 min of adsorption for silicon wafers and for amino-terminated substrates. After this, the adsorbed amount increased further in a nonlinear behavior until the adsorption plateau had been achieved. In the case of silicon wafers,
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this second step lasted more than 2 h, while in the case of the amino-terminated substrates it took about 30 min. This second region might be explained considering that the substrate is a new surface composed by free sites and sites occupied by the already adsorbed biomolecules. The arriving biomolecules might stick on the latter, due to cooperative binding. Afterward, they might diffuse to the free sites on the substrate, followed by conformational changes.6 The structural rearrangement of the adsorbed CPK molecules seems to be slower onto silicon wafers than onto amino-terminated surfaces. After this period of time the formation of a monolayer was evidenced by the adsorption plateau. This equilibrium situation remained for a period of four additional hours in the case of Si wafers, and 30 min in the case of amino-terminated substrates. In the last stage, the adsorbed amount Γ increased continuously as a function of time in both cases. Silicon wafers (θA ) 5°) and freshly prepared amino-terminated substrates (θA ) 22°)27 are hydrophilic substrates. The global adsorption process of CPK onto amino-terminated surfaces was faster than adsorption onto Si wafers. This might have been caused by the slower rate of accommodation of CPK molecules onto Si wafers than onto amino-terminated surfaces, leading to a thermodynamically higher adsorption barrier in the former. A similar behavior has been observed by Ortega-Vinuesa.10 In a mixture of serum albumin, immunoglobulin G, and fibrinogen, fibrinogen presented the slowest adsorption rate onto hydrophobic substrates. The analysis of the four regions in Figures 1 and 2 might be done in the following way. The first, second, and third stages have been described by theoretical4,5,7,16 and experimental works.8-13,34 At the initial stages the mass transport might be considered as a Fickian diffusion, and the diffusion coefficient D can be calculated applying the relation29,31
Γ(t) )
2 cbulk(Dt)1/2 π1/2
(5)
From the slopes (Γ/t0.5), in Figures 1b and 2b, the diffusion coefficient was found to be D ∼ (8 ( 1) × 10-7 cm2/s. The diffusion coefficient D and the hydrodynamic radius Rh of CPK might be interrelated by the StokesEinstein relation, eq 4. Considering the viscosity of the solvent as 1 mPa‚s and applying the D value determined by the adsorption kinetics to eq 4, the Rh found amounts to approximately 3 nm. This value is reasonable for the diffusion of a single CPK molecule,26 indicating that the initial stage is governed by a purely diffusive process. For comparison, the radius of gyration of CPK was estimated as 2.2 nm and its hydrodynamic radius as 2.8 nm from calculations using Protein Data Bank data33 and the Crysol program. The AFM image of dried CPK-covered silicon wafer (Figure 1) after 4 h of adsorption revealed the presence of spherical entities with radius ranging from 5 to 15 nm, which are closely packed on the surface forming a film. These findings corroborate with the suggestion that in the very dilute regime isolated CPK molecules diffuse to the substrate and, after a given period of time, they form a monolayer. The formation of a monolayer was evidenced by adsorption plateaus, which amounted to 2.5 and 4.0 mg/m2 in the case of silicon wafers and aminoterminated substrates, respectively. The substrates pre(33) www.rcsb.org/pdb (pdbID ) 2CRK). (34) Siqueira, D. F.; Reiter, J.; Breiner, U.; Stadler, R.; Stamm, M. Langmuir 1996, 12, 972.
sented an area of 1.0 cm2, leading to adsorbed masses of 2.5 × 10-7 and 4.0 × 10-7 g, respectively. To estimate if these quantities correspond to monolayers, the CPK molecules were assumed to adsorb in a spherical shape and the radius of gyration of an adsorbed CPK molecule was assumed to be 2.2 nm.33 The area of an adsorbed CPK molecule was calculated as 61 × 10-18 m2. Therefore, the area of 1.0 cm2 should be occupied by 1.6 × 1012 CPK molecules. The volume of one CPK molecule was calculated as 45 × 10-27 m2. Then the volume occupied by 1.6 × 1012 CPK molecules should be 71 × 10-15 m2 or 7.1 × 10-8 cm2. Considering the CPK density35 as 1.37 g/cm2, the corresponding adsorbed mass should amount to approximately 1.0 × 10-7 g, which is the same order of magnitude of the experimental values. The adsorption plateaus existed for a longer period of time in the case of silicon wafers than in amino-terminated surfaces. This might have been caused by a slower relaxation and accommodation process of the adsorbed CPK molecules onto the former substrate, as discussed above. The relaxed conformation allowed the additional adsorption as a function of time observed in the third region. Ramsden14 explained the irreversible adsorption based on conformational changes. Upon being absorbed, the enzymes or proteins arrive at the substrate in their compact and native conformations, but interactions with the surface lower the energy to a less compact, unfolded conformation, to which the adsorbed protein may slowly relax. This process requires space, but if this space is occupied by already adsorbed molecules, then the relaxation is blocked, resulting in an irreversible adsorption process which increases with increasing concentration. Ramsden proposed a function to describe the probability of an adsorbed molecule to relax, which considers relaxed and native conformations. After the formation of a CPK film, the substrate is no longer the silicon wafer or the amino-terminated surface for the arriving CPK molecules. The continuous adsorption of CPK onto the new substrates might be due to cooperative binding, which is usual among proteins. The first, second, and third stages in the adsorption kinetics are commonly observed for polymers,29,31,34 but the forth one seems to be a special feature of proteins adsorption. To our knowledge this is the first time that such adsorption behavior is experimentally observed. Desorption experiments were performed by ellipsometry exchanging the CPK solution after 16 h of adsorption by pure solvent. Whatever the substrate, the result was the same: after 48 h in contact with pure solvent, a mean decrease of 5% in Γ was observed. This can be attributed to losely attached CPK molecules to the surface, which are washed up from the surface in the presence of pure solvent. These findings showed that the adsorption of CPK on the investigated substrates is irreversible in nature. Tilton15 et al. proposed a mechanism for irreversible adsorption of globular proteins based on many weak segmental attachments to the surface, in their compact and native conformations. It is proposed that interactions with the surface lower the energy of a less compact, unfolded conformation, to which the adsorbed protein may slowly relax. One possible explanation for the irreversible adsorption observed for CPK on the investigated substrates is that the actual desorption rate is so low that even after 48 h no significative reduction in the adsorbed amount could be detected, as suggested by Wojciechowski and Brash.16 (35) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundstro¨m, I. Thin Solid Films 1998, 324, 257.
Adsorption of Creatine Phosphokinase
Figure 3. Adsorption kinetics measured for CPK (c ) 0.5 g/L) onto amino-terminated surfaces at 22.5 ( 0.5 °C.
Figure 4. Adsorption isotherms measured for CPK onto silicon wafers at 22.5 ( 0.5 °C. The adsorbed amounts correspond to 1 h (open symbols) and to 16 h (solid symbols) of adsorption.
Figure 3 shows a typical adsorption kinetics for more concentrated CPK solutions (cCPK ) 0.5 g/L) onto aminoterminated surfaces. The first part of the curve presents a linear dependence of the adsorbed amount Γ with t0.5, from which the diffusion coefficient D could be estimated as 7 × 10-10 cm2/s. Substituting this D value in eq 4, one obtains the Rh value of 3 µm, indicating the diffusion of big aggregates to the substrate. The adsorbed amount Γ increases continuously along the time. Adsorption Isotherms. The adsorption isotherms measured for CPK onto Si wafers and amino-terminated substrates showed a continuously increase of Γ as a function of the bulk concentration, as shown in Figures 4 and 5, respectively. The Γ values in Figure 4 correspond to 1 and 16 h of adsorption, while the Γ values in Figure 5 correspond to 16 h of adsorption. In both cases, no adsorption equilibrium has been achieved over the investigated concentration range because of the formation of CPK aggregates in the solution, as discussed above. The aggregates were also evidenced by AFM. AFM images of CPK-covered amino-terminated substrates obtained from regions I, II, and III, respectively, 0.25, 0.50, and 1.0 g/L in the adsorption isotherms, showed the presence of aggregated CPK adsorbed molecules (Figure 5). The adsorption behavior of CPK was investigated not only onto hydrophilic substrates but also for hydrophobic substrates. PS forms very hydrophobic surfaces (θA ) 88 ( 2°). However, by means of a simple sulfonation reaction28 they can turn moderately hydrophilic (θA ) 53 ( 2°) due to the appearance of SO3- groups on the surface. The adsorption isotherms of CPK on pure PS and on PS-SO3surfaces indicated a higher affinity of CPK for the former after 16 h of adsorption, as shown in parts a and b of
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Figure 6, respectively. Electrostatic repulsion between the charges of SO3- on the surface and the phosphate groups in the enzyme might be the reason for the weaker affinity. However, after 1 h of adsorption the adsorption isotherms of CPK on pure PS and on PS-SO3- showed similar behavior. Enzymes are very complex adsorbates because they possess hydrophobic, hydrophilic, and charged domains. CPK presents 2191 hydrophilic segments and 1188 hydrophobic segments.33 The adsorption isotherms showed a preferential adhesion of CPK aggregates onto the hydrophilic substrates. Since hydrophilic segments predominate the CPK structure, favorable interactions as hydrogen bonding between Si wafers or amino-terminated substrates and the polar hydrophilic CPK segments might be expected to reduce even more the ∆adsG. Hydrophobic bonding between PS and the hydrophobic CPK segments lead to small ∆adsH, and in this case, the entropic gain is the main driving force for the adsorption. In the case of sulfonated PS surfaces, the electrostatic repulsion between the charged PS-SO3- domains and the phosphate groups in the CPK causes the relatively lower Γ values (higher ∆adsG values). This result is in agreement with Ladam and co-workers,17 who have recently reported that when the charges of surface and the protein are similar, one usually observes the formation of protein monolayers, while when the surface and protein are oppositely charged, the formation of thick protein layers is observed. Contact Angle Measurements. Figures 7 and 8 show the advancing and receding angles measured for water drops on the CPK-covered Si wafers and amino-terminated substrates, respectively, as a function of CPK bulk concentration. It is interesting to notice that silicon wafers and amino-terminated substrates turned very hydrophobic after the CPK adsorption, even for very low values of bulk concentration, where no aggregates were detected. The preferential orientation of the hydrophobic sites of CPK to the air should probably lead to a minimization of the system free energy. As proposed by Lyklema and Norde,6 small protein molecules tend to be more hydrophobic than the large ones because they have a large area/volume ratio, making it difficult for them to place all the hydrophobic segments in the interior of the molecules. The hysteresis in the contact angle (∆θ) for pure silicon wafers and pure amino-terminated substrates amounted to 1° and 3°, respectively. After the adsorption, ∆θ amounted to 30 ( 3° over the whole concentration range, in both cases. The high hysteresis values can be explained with the basis on the surface chemical heterogeneity and surface roughness. AFM images of CPK-covered aminoterminated substrates obtained from regions I, II, and III, respectively, 0.25, 0.50, and 1.0 g/L in the adsorption isotherms, showed that the adsorbed CPK molecules form aggregates when dried and exposed to air (Figure 5). These aggregates appeared more frequently and higher as the bulk concentration increased. The presence of CPK aggregates in the solution was detected by DLS measurements, as discussed above. However, their dimensions were less than those observed in the AFM images because the solutions were filtered with a 0.2 µm pore membrane, where big aggregates were probably retained. PS forms very hydrophobic surfaces (θA ) 88 ( 2°). However, by means of a simple sulfonation reaction they can turn hydrophilic (θA ) 53 ( 2°) due to the appearance of SO3- groups on the surface.28 No effect on the contact angle measurements was measured in the case of pure PS. The hydrophobicity remained very high. Contrarily, a significant increase in θA of approximately 35° was
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Figure 5. Adsorption isotherms measured for CPK onto amino-terminated surfaces at 22.5 ( 0.5 °C. The adsorbed amounts correspond to 16 h of adsorption. AFM images correspond to dried samples after adsorption from the bulk concentrations of (I) 0.25 mg/mL, (II) 0.50 mg/mL, and (III) 1.0 mg/mL.
Figure 6. Adsorption isotherms measured at 22.5 ( 0.5 °C for CPK onto (a) PS and (b) PS-SO3- films. The adsorbed amounts correspond to 1 h (open symbols) and to 16 h (solid symbols) of adsorption.
Figure 7. Advancing (solid symbols) and receding (open symbols) angles measured for water drops on dried CPK-covered Si wafers at 22.5 ( 0.5 °C as a function of the bulk concentrations where the samples were obtained (16 h of adsorption).
Figure 8. Advancing (solid symbols) and receding (open symbols) angles measured for water drops on dried CPK-covered amino-terminated substrates at 22.5 ( 0.5 °C as a function of the bulk concentrations where the samples were obtained (16 h of adsorption).
measured for the CPK-covered PS-SO3- surfaces (Figure 9) as a function of CPK bulk concentration. Similarly to Si wafers and amino-terminated surfaces, after the adsorption of CPK, the PS-SO3- surfaces turned hydrophobic.
Conclusions The ellipsometric study on the adsorption kinetics of CPK onto silicon wafers and amino-terminated substrates from very dilute solutions (cCPK > 0.005 g/L) showed four distinct regions: (i) a diffusion controlled one, (ii) adsorp-
Adsorption of Creatine Phosphokinase
Figure 9. Advancing (solid symbols) and receding (open symbols) angles measured for water drops on dried CPK-covered PS-SO3- films at 22.5 ( 0.5 °C as a function of the bulk concentrations where the samples were obtained (16 h of adsorption).
tion and rearrangement, (iii) monolayer formation, and (iv) irreversible adsorption. The appearance of the forth
Langmuir, Vol. 18, No. 9, 2002 3523
region was explained based on relaxed CPK conformation and cooperative binding. For more concentrated solutions, the CPK molecules formed aggregates, as revealed by DLS measurements, contact angle measurements, and AFM. The adsorption isotherms showed a continuous increase of the adsorbed amount with the bulk concentration, either on hydrophobic or on hydrophilic substrates. However, CPK presented preferential adsorption onto the hydrophilic (silicon wafers and amino-terminated surfaces) substrates. It is probably due to the high content of hydrophilic segments in its structure. For the development of biosensors, the results indicated the amino-terminated surface as the most adequate substrate for the immobilization of CPK due to the high affinity and fast adsorption. Acknowledgment. S.M.P. and D.F.S.P. acknowledge FAPESP and CNPq for financial support. LA0110693
