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Langmuir 2004, 20, 9202-9209
Nonlinear Optical Probe of Biopolymer Adsorption on Colloidal Particle Surface: Poly-L-lysine on Polystyrene Sulfate Microspheres Heather M. Eckenrode and Hai-Lung Dai* Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323 Received May 7, 2004. In Final Form: August 1, 2004 A nonlinear optical techniquessecond harmonic generation (SHG)shas been applied to characterize the adsorption of poly-L-lysine on micrometer size polystyrene particles, whose surface is covered with negatively charged sulfonate groups, in aqueous solutions. Adsorption behavior of the biopolymer with two chain lengths (14 and 75 amino acid units; PL14 and PL75) has been examined. Centrifugation experiments were also performed to support the adsorption measurements made using SHG. The adsorption free energies of the two polymers PL75 and PL14 are determined as -16.57 and -14.40 kcal/mol, respectively. The small difference in the adsorption free energies of the two chain lengths, however, leads to dramatic difference in the concentration needed for saturated surface coverage: nearly 50 times higher concentration is needed for the smaller polymer. Under acidic colloidal conditions, polylysine is found to adsorb in a relatively flat conformation on the surface. The surface area that each polylysine molecule occupies is nearly 1 order of magnitude larger than the size of the molecule in its extended form. The low adsorption density is likely a result from Coulombic repulsion between the positive charges on the amino acid units of PL. The measurements demonstrate the utility of SHG as an efficient and sensitive experimental approach for measuring adsorption characteristics of bio/macromolecules on colloidal particles and define surface and colloidal conditions for achieving maximum surface coverage of a widely used biopolymer.
I. Introduction Many biological processes begin with the adsorption of a molecule to the surface of a biological object. As mechanistic understandings of biological processes become ever more important, biomolecular adsorption at interfaces has recently begun to receive a lot of attention as a research topic. For example, adsorption of charged molecules was found to play an important role in blood clotting and immunoadsorption.1 Water-soluble polymers, such as polyL-lysine (PL), have been used in procedures aimed at blocking recognition at biological surfaces including red blood cells and fibroblasts.2 PL adsorption to viruses has been explored for inhibiting replication of HIV3 and for hindering virus attachment to host cells.4 The characteristics of adsorption include such tangible observables as adsorption rate and structure and adsorbate density. Such characteristics are expected to depend on the properties of the adsorbate molecules, the nature of the surface, and the composition and properties of the solvent. For example, adsorption may be influenced by surface charge density, pH, and salt concentration in the solution and the size, shape, and functional groups of the adsorbate molecules. Characterization of the effect these factors have on adsorption structure and kinetics is essential to understand molecular adsorption in biological processes. To achieve this objective, the ability to make quantitative measurements of adsorption characteristics, * To whom correspondence may be addressed. E-mail: dai@ sas.upenn.edu. Tel: (215) 898-5077. Fax: (215) 898-2037. (1) Hesselink, F. T. In Adsorption from Solution at the Solid/Liquid Interface; Parfitt, G. D., Rochester, C. H., Ed.; Academic Press Inc.: New York, 1983. (2) Elbert, D. L.; Hubbell, J. A. Chem. Biol. 1998, 5, 177-183. (3) Hosoya, M.; Neyts, J.; Yamamoto, N.; Schols, D.; Smoeck, R.; Pauwels, R.; De Clercq, E. Antiviral Chem. Chemother. 1991, 2, 243248. (4) Yang, Y. W.; Yang, J. C. Antiviral Res. 1996, 33, 33-39.
including free energy of adsorption and adsorbate density that can be deduced from adsorption isotherms, is essential. The conventional methods for measuring adsorption isotherms, though effective, have been tedious and timeconsuming. In such measurements of adsorption isotherms, for each point on the isotherm, a separate sample must be prepared for different molecular concentrations or solvent/surface conditions. A separation process often involving a centrifuge is needed to allow determination of the molecular concentration in the solution before and after exposure to the surface. In addition to the lengthiness of the procedure, the techniques are not directly sensitive to the molecules adsorbed on the surface and the accuracy of measurements often relies on the completeness of the separation process. To be able to characterize biomolecular adsorption more effectively and efficiently, techniques that are faster, surface sensitive, and in situ need to be developed. We report here the application of a nonlinear optical technique to the characterization of adsorption of a prototypical biopolymer in a colloidal environment. This technique, based on the nonlinear optical phenomenon second harmonic generation (SHG), is capable of quantitative, in situ measurements of molecular adsorption in colloids5 and can be used to investigate adsorption of biological molecules at the solid-liquid interface. The process of SHG, in which the frequency of part of the light in interaction with a medium is converted to twice the original, is sensitive to the symmetry of the medium. For molecules that meet the symmetry requirement for facilitating SHG individually, i.e., they have nonzero second-order polarizability, the polarization of these molecules in solution will be averaged out due to their random orientation, producing no SH signal. When these (5) Wang, H. F.; Yan, E. C.; Borguet, E.; Eisenthal, K. B. Chem. Phys. Lett. 1996, 259, 15-20.
10.1021/la048863j CCC: $27.50 © 2004 American Chemical Society Published on Web 09/14/2004
Poly-L-lysine Adsorption on Colloids
molecules are adsorbed at a surface, the molecules are forced to have an ordered orientation and the ensemble average of the polarization is no longer averaged out. In cases where the coherence length of the SHG process is shorter than the colloidal particle size, the polarizations of molecules adsorbed to opposite sides of the microparticles do not cancel each other, resulting in SHG. This method can thus be used to probe adsorption of molecules with strong second-order polarizability. Eisenthal and coworkers5 first reported the use of SHG in detecting the adsorption of dye molecules in colloidal systems. Monitoring the SHG during the incremental addition of dye to a solution of microspheres resulted in the recording of an adsorption isotherm, which allowed the adsorption free energy and adsorbate density to be determined. Adsorption of molecules that do not have a strong second-order polarizability, which is the case for most of the molecules of interest, may be probed by this technique through monitoring the competition of adsorption with the adsorbed dye molecules. First, the colloidal particle surface can be saturated with a monolayer of dye molecules. This will produce a constant second harmonic (SH) signal level. As the molecule of interest is added to the solution, it displaces the surface dye molecules and results in a decrease of the SH signal, allowing the adsorption isotherm to be obtained for the molecule of interest. Previously, we have reported the use of this technique to make adsorption measurements of surfactants on polystyrene particles in colloids.6 Because of the extensive use of PL adsorption and polystyrene microspheres in the literature, we have chosen to investigate the adsorption of poly-L-lysine to polystyrene sulfate microspheres (PSSM) as a model system for demonstrating the effectiveness of this technique in making in situ adsorption measurements for biomolecules. In addition to the new applications of PL in biological studies mentioned above, there has been a great deal of interest on the adsorption properties of PL in general. PL has been widely used to mediate adherence of cells to glass surfaces.7,8 PL has been used extensively in the production of DNA microarrays.9 PL adsorption has also found use in the production of biocompatible films,10 and its adsorption has been studied as a model polyelectrolyte and biopolymer.11,12 A variety of surfaces have been used in the study of PL adsorption, including glass,13,14 clay minerals and marine sediments,15 silver iodide,16 and polystyrene.12,16,17 Polystyrene is one of the most frequently used particles in colloids, a common environment for biological studies. Many adsorption studies of proteins or other molecules of biological interest have been done using this polymer (6) Wang, H.; Troxler, T.; Yeh, A. G.; Dai, H. L. Langmuir 2000, 16, 2475-2481. (7) Mazia, D.; Schatten, G.; Sale, W. J. Cell Biol. 1975, 66, 198-200. (8) Linssen, K. F.; Jacobs, J. A.; Nieman, F.; Cornelissen, L. E.; Drent, M. Anal. Quant. Cytol. Histol. 2003, 25, 281-284. (9) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 70, 467-470. (10) Picart, C.; Lavalle, P.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C. Langmuir 2001, 17, 7414-7424. (11) Knight, C.; Wierzbicki, A.; Laursen, R. A.; Zhang, W. Cryst. Growth Des. 2001, 1, 429-438. (12) Bonekamp, B. C.; van der Schee, H. A.; Lyklema, J. Croat. Chem. Acta 1983, 56, 695-704. (13) Pagac, E.; Tilton, R. D.; Prieve, D. C. Langmuir 1998, 14, 51065112. (14) Latour, R. A.; Trembley, S. D.; Tian, Y.; Lickfield, G. C.; Wheeler, A. P. J. Biomed. Mater. Res. 2000, 49, 58-65. (15) Ding, X.; Henrichs, S. M. Mar. Chem. 2002, 77, 225-237. (16) Bonekamp, B. C., Ph.D. Thesis, Agricultural University, Wageningen, Netherlands, 1984. (17) Rustemeier, O.; Killmann, E. J. Colloid Interface Sci. 1997, 190, 360-370.
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surface, including bovine serum albumin,18 immunoglobulin G,19 and fibrinogen.20 The effects of pH, salt concentration, chain length, and surface charge density have been investigated previously for the adsorption of PL to polystyrene microspheres.16,21 The adsorption isotherms in those studies have been measured by using spectrophotometric detection of PL concentrations in colloids before and after centrifuge separation of polystyrene microspheres. It was found that the adsorption behavior changed dramatically as the solution pH was changed to cause the helix-coil transition of the PL molecules in bulk solution. Under neutral and acidic conditions, the mass quantity of PL adsorbed did not depend on chain length, indicating the PL molecules adsorbs in a flat configuration on the surface. However, at high pH, when PL takes on a helical form, the mass quantity of PL adsorbed was found to increase as the PL chain length increased, suggesting that the molecule is adsorbed end-on on the surface. Experiments were also done with varying salt concentration. Observations were consistent with the model that PL adsorption is affected by the helix-coil transition in solution: at high pH, PL is not charged and exhibits a helical structure; as the pH is lowered to
