Langmuir 1992,8, 1236-1238
1236
Letters Plasma Protein and Antisera Interactions with L-Cysteine and 3-Mercaptoproprionic Acid Monolayers on Gold Surfaces P. Tengvall,' M. Lestelius, B. Liedberg, and I. Lundstrom Laboratory of Applied Physics, University of Linkoping, 24-581 83 Linkoping, Sweden Received December 31, 1991. In Final Form: February 25, 1992 Thiols with varying functionalities are well suited for immobilization onto gold. In the present study surfaces modified with L-cysteine (zwitterionic, neutral) and 3-mercaptoproprionicacid (MPA, negatively charged)were incubated in human plasma, and the antiserabinding patterns of four proteins were determined by ellipsometry. Significant differences among the surfaces were observed. Plasma-treated L-cysteine surfaces bound low amounts of both anti-fibrinogen (a-FG) and anti high molecular weight kininogen (a-HMWK), while MPA surfaces bound increased amounts of a-HMWK but no a-FG. The results demonstrate that the surface biology in complex, but biologically relevant, systems may be conveniently studied through a combination of systematicsurfacemodifications, antisera techniques,and ellipsometry.
Introduction Gold surfaces modified by thiol-containing molecules appear to be very interesting for the development of biosensorsl and for the study of protein-surface interactions.2 Prime and Whitesides concluded that self-assembled monolayers are today the best defined model systems available for the latter purpose.2 In a.ccordance with previous studies they found that the surface free energy is an important parameter in determining the amount of adsorbed protein molecules, with increased amounts adsorbed on to hydrophobic s~rfaces.~aFurthermore, alkanethiole with flexible ethylene glycol chains showed a minimal protein adsorption due to so-called steric stabilization.4 The understanding of the adsorption and cooperative mechanisms of proteins on surfaces is of importance in research of biomaterialss-7 and cell adhesion.8 The dynamic phenomena occurring when blood contacts foreign material are of particular interest. Vroman and co-workers showed by antisera techniques that human plasma proteins apparently displace each other at hydrophilic s u r f a c e ~ . ~ J ~ In later studies ellipsometry in combination with antisera techniques have been used to demonstrate the displacement reactions on silicon3 and metal surfaces." In this communication we present results on plasma protein interactions on gold and gold surfaces modified with Lcysteine (~-Cys) or 3-mercaptopropionic acid (MPA). Both (1) M f b , S.;Johnason, B. J. Chem. SOC.,Chem. Commun. 1990,1526. (2) Prime, K. L.; Whitesides, G. M. Science 1991,252, 1164. (3) Elwing, H.; Welin, S.; Aakendal, A.; Nilsson, U.; Lundstrdm, I. J. , Colloid Interface Sci. 1987, 119, 1. (4) Jeon, I.; Lee, J. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (5) Williams, D. F.; Bagnall, R. G. In Fundamental Aspects of Biocompatibility; Williams, D. F., Ed.; CRC Press: Boca Raton, FL, 1981; Vol. 11, p 113. (6) Hoffman, A. Ann. N.Y. Acac. Sci. 1987, 516, 96. (7) Andrede, J. D.; Hlady, V. C. Ann. N.Y. Acad. Sci. 1987,516, 158. (8) Schankenraad, J. M.; Busscher, H. J. Colloids. Surf. 1989,42,331. (9) Vroman, L.; Adams, A. L. In Proteins at Interfaces; Brash, J., Horbett, T. A., Me.; ACS Symposium Series, American Chemical Society: Washington, DC, 1987; Vol. 343, p 154. (10) Vroman, L.; Adams, A. L.; Fischer, G. C.; Munoz, P. C. Blood 1980,55, 166. (11) Elwing, H.; Tengvall, P.; Askendal, A.; LundstrBm, I. J.Biomater. Sci., Polym. Ed. 1991,3, 7.
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are known to bind strongly to gold via the thiol group.12 The suggested structures of chemisorbed L - C ~and S MPA a t neutral pH are schematically shown in Figure 1. The two molecules thus present either a negatively charged hydrophilic or a zwitterionic noncharged hydrophilic surface to the plasma proteins. It should be pointed out that we are not studying single protein adsorption but rather the interaction of whole plasma with surfaces incorporating both protein-surface and protein-protein induced phenomena. Subsequently it is not necessarily possible to correlate the observed effects with the structure and composition of individual proteins. Instead, we probe the dynamics of a biological system on the surfaces involving displacement phenomena, protein complex formation, and steric blocking as revealed by the antisera probes. The molecules in Figure 1were thus chosen to investigate the influence of surface polarity on the phenomena important for surface-activated blood coagulation.
Materials and Methods Sputtered gold films, 200 nm thick, on glass substrates were used. They were very flat as revealed by scanning force microscopy, which suggested a surface roughness of 16 nm.The surfaces of the gold film were cleaned in five parte distilled water, one part ammonium hydroxide (25%), and one part of hydrogen peroxide (30%) for 10 min at 80 "C. After the cleaning the samples were rinsed and stored in distilled water. The static contact angle with water indicated that the cleaned gold surface was hydrophilic in character (see Table I). Some of the gold films were incubated in aqueous solutions of 1 mM L - C ~ (pH S = 5.3 f 0.3) purchased from Sigma or 1mM MPA (pH = 3.3 0.3) from Fluka for 15 min at 50 "C. The molecular structure of the formed L-CYSand MPA layers was checked by Fourier transform IR reflection-absorption spectroscopy(IRAS)and was compared with earlier measurementa.12 The peak positions and the general appearance of the spectra for the monolayers used in this investigation were similar to those in ref 12. L - Cmono~ layers prepared at pH 5.3 and analyzed in UHV (ultrahigh vacuum) with X-ray photoelectron spectroscopy (XPS)lSshow that the amino and carboxyl groups exist as NHs+and CO1- i.e.,
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(12) Ihs, A.; Liedberg, B. J. Colloid Interface Sci. 1991, 144, 282. (13) Uvdal, K.; Bodd, P.; Liedberg, B. J. Colloid Interface Sci. 1992, 149, 162.
0 1992 American Chemical Society
Letters
Langmuir, VoZ.8, No. 5, 1992 1237
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The L-Cys-modified surface, on the other hand, bound increased amounts of a-IgG and a-LP but relatively low amounts of a-FG and a-HMWK after the plasma incubation.
n
Figure 1. Schematic models of the molecular structure of
L-
cysteine and MPA monolayers on gold surfaces a t neutral pH. in the zwitterionic form. The molecular structures in a buffer at pH = 7.4 are therefore likely to be those shown in Figure 1. The charged species may, of course, be compensated by countercharges from the buffer. The plasma was prepared from the blood of one apparently healthy donor and stored at -80 "C until use. Swine anti-immunoglobulin G (a-IgG) was from Orion Diagnostica, Finland; rabbit anti-fibrinogen (a-FG), goat anti high molecular weight kininogen (a-HMWK)and goat anti-lipoprotein (a-LP)were from Nordic Immunochemical Laboratories, The Netherlands. The surface concentrations of HMWK and L P are generally low and the binding of their antibodies hard to distinguish from the background. Their antisera were therefore amplified by using rabbit anti-goat IgG (RbaGt IgG) from Dakopatts, Denmark. Control tests showed only a small cross reactivity between the antisera and RbaGt IgG displayed low affinity to surfaces incubated in plasma only. Prior to plasma incubations one of six surfaces was rinsed in distilled water and blown dry in flowing nitrogen and its optical properties (substrate complex refractive index) were calculated after measurements a t ten different points (see Table I). The surfaceswere incubated for 10min in 107% citrated plasma in PBS buffer at room conditions. After being rinsed in buffer the pieces were incubated in antisera (a-FG and a-IgG, 1/20; a-HMWK and a-LP, 1/50) for 15 min; the a-HMWK and a-LP immersed samples were then transferred to the RbaGt IgG (1/50 dilution; 15 min). After rinsing and drying in flowing nitrogen the amount of organic material was determined by ellipsometry14J5at five points on each sample. Five samples of each kind of surfacewere analyzed for each antisera. The equivalent organic layer thickness was calculated from the ellipsometric data using an isotropic three-phase model14 and a refractive index of n = 1.465 for the dried organic layer.3
Results The maximum error of the calculated organic layer thicknesses due to variations of the optical properties of the gold substrates was low, h0.3 nm. Furthermore it was possible to estimate the thickness of the L-CYSand MPA layers from the change in the optical properties of the gold surface after modification with the molecules. A summary of the experimental parameters for the three types of surfaces used is given in Table I. Figure 2 summarizesthe amount (equivalent thickness) of deposited organic material on the surfaces after plasma and antisera incubations. The most striking observation is the low total plasma protein adsorption onto the MPA surfacesand the apparent lack of antisera binding, except for large depositions of a-HMWK and its amplification antisera. Pure gold surfaces incubated in plasma deposit both anti-fibrinogenand a-HMWK in significantamounts. (14)Azzam, R.M.A.; Bashara,N. In Ellipsometry and Polarized light; North Holland: Amsterdam, 1987;paperbook edition. (15)Elwing, H.; Welin, S.; Askendal,A.; Nilsson, U.; Lundstrcm, I. J.
Colloid Interface Sci. 1987,119, 203.
Discussion Since an amplification antisera was used for a-HMWK and a-LP detections, the results in Figure 2 should be compared only for each protein and does not allow quantitative comparisons between the proteins on a specific surface. Figure 2 indicates thus relatively small plasma protein adsorption on MPA, large a-HMWK (+amplification) binding and small a-IgG, a-FG, and aLP (+amplification) binding on MPA, relative large binding of a-IgG and a-LP (+amplification) on L-CYS,and so on. In a recent study using diluted human plasma and silicon surfaces having wettability gradients, it was found that a-HMWK deposited mainly at the negatively charged hydrophilic (Si-0-) end of the gradient and a-FG at the methylated hydrophobic end.16 The Vroman sequencegJO apparently did not go to completion on the hydrophobic part of the surface. The low overall a-FG binding on to both hydrophilic surfaces used in this study and the large a-HMWK binding onto the negatively charged MPA surface are therefore not surprising in this context. The simultaneous low a-FG and a-HMWK depositions onto the zwitterionic, hydrophilic, but neutrally charged L-CYS surface are, however, of principal interest. The antisera binding pattern for L-CYSresembles the moderately methylated region of the wettability gradient on silicon.l6 This region had a static water contact angle (e), 50" < 8 < 70°, showed mixed hydrophobic/hydrophilic characteristics, and bound low amounts of all the antisera tested for. In the present study the depositionof a-HMWK followed the expected binding pattern of HMWK, i.e. the histidinerich positively charged portion of HMWK17 adsorbed on to the MPA surface,probably physisorbed via electrostatic interactions. The increased a-IgG deposition onto the LCys surface (see Figure 2) was also noted. At the present time we have no explanation of this finding, although in the Vroman displacement sequence a-IgG binds on to a plasma-incubatedsurface before both a-FG and a-HMWK. The present results on L-CYSshow that a high surface wettability (or high free surface energy) may not be sufficient alone for contact activation but that other characteristics also associated with the surface functionality play an important role. Surface-blood plasma interactions are thus governed by parameters such as net charge, surface dipole density, specific steric accommodations, acid-base strength of the surface functional groups, volume exclusion, and water ordering and binding capacity of the surface. In addition, protein-protein interactions are important in surface-related biological phenomena. The plasma protein antisera binding pattern is most probably related to the in vivo behavior of a material on its initial contact with blood. The results in Figure 2 suggest that the MPA-modified gold surface could be involved in surface-activated coagulation through the intrinsic pathway since it shows increased a-HMWK binding. Since both a-FG and a-HMWK depositions are small on L-Cys-modified surfaces after incubation in (16)Tengvall, P.;Askendal, A.; Lundstrcm, I.; Elwing, H. Biomate-
rials, in press.
(17)Miiller-Esterl,W.;Iwanaga,S.; Nakanishi, S . TIBS 1986, August, 336.
1238 Langmuir, Vol. 8,No. 5, 1992
Letters Table I. Surface Characterization' gold gold + MPA
