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Langmuir 2005, 21, 8572-8575
Control of Attachment of Bovine Serum Albumin to Pulse Plasma-Polymerized Maleic Anhydride by Variation of Pulse Conditions Shasha Liu,† Margarida M. L. M. Vareiro,† Stuart Fraser,‡ and A. Toby A. Jenkins*,† Department of Chemistry, University of Bath, Bath BA2 7AY, U.K., and Department of Engineering Materials, Mappin Street, University of Sheffield, Sheffield, S1 3JD, U.K. Received June 2, 2005. In Final Form: July 11, 2005 This letter describes how the irreversible attachment of bovine serum albumin (BSA) to films of plasmapolymerized maleic anhydride can be measured by an indirect antibody-binding assay and how this attachment appears to be strongly affected by the polymerization conditions. Surface plasmon resonance (SPR) was used to follow the binding of the antibody, anti-bovine serum albumin (aBSA), to proteinmodified plasma-polymerized maleic anhydride films. It was found that BSA could be irreversibly bound to polymers made under pulse plasma conditions, but BSA did not bind to polymers made under continuous wave conditions. Moreover, the degree of antibody binding, which is directly related to the quantity of BSA on the polymer, correlated with the plasma duty cycle (ton/ton + toff): lower duty cycle pulse plasma conditions gave greater BSA attachment. We speculate that BSA is being covalently bound to the polymer via the reaction of amine groups on lysine residues in BSA with the retained anhydride group functionality in the polymer.
Introduction The attachment of cells to surfaces is a protein-mediated process. Fibronectin and vitronectin are, for example, involved in the attachment of endothelial cells to polystyrene. In many cases, the attachment and growth of cells on surfaces is a desirable outcome, for example, when culturing human tissue for transplant.1 However, in some cases cell growth on surfaces can be disadvantageous, for example, the growth of blood clots around vascular grafts, resulting in recipients of such grafts having to take daily blood-thinning drugs such as Warfarin.2 One approach to preventing such clot formation might be to prevent the attachment of cells by blocking the surface with serum albumin.3 Serum albumin has been previously used to prevent cell adhesion.4 One difficulty, however, of artificial implants in high flow environments is that serum albumin can be quickly washed away by, for example, blood flow. A possible solution to this problem is to covalently bind serum albumin to the surface of devices such as stents or catheters prior to implantation. Covalently bound protein may remain attached and suppress cell attachment in a high-pressure flow environment such as human arteries. Maleic anhydride (MA) films have properties that make them of particular interest for surface modification. They can be formed in such a way that anhydride group functionality is retained, which can then be used for further chemical modification.5 They swell in water to form what * To whom correspondence should be addressed. E-mail:
[email protected]. † University of Bath. ‡ University of Sheffield. (1) Bullett, N. A.; Whittle, J. D.; Short, R. D.; Douglas, C. W. I. J. Mater. Chem. 2003, 15, 1546-1553. (2) Chester, J. F. Ann. Coll. Surg. H. K. 2002, 6, 97-101. (3) Ying, P.; Yu, Y.; Jin, G.; Tao, Z. Colloids Surf., B: Biointerfaces 2003, 32, 1-10. (4) Taborelli, M.; Eng, L.; Descouts, P.; Ranieri, J. P.; Bellamkonda, R.; Aebischer, P. J. Biomed. Mater. Res. 1995, 29, 707-714. (5) Ryan, M. E.; Hynes, A. M.; Badyal, J. P. S. Chem. Mater. 1996, 8, 37-42.
is probably a polyelectrolyte film;6 they can be formed using plasma polymerization; and their chemical functionality can be tuned by pulsing the input power. Ryan et al. described the first detailed study of the radio frequency (plasma)-induced polymerization of maleic anhydride in 1996.5 They showed that variations in applied power and the duty cycle could be used to vary the chemical functionality of plasma-polymerized maleic anhydride films. Importantly, it was found that at low equivalent power (Peq ) Pmax(ton/ton + toff)) XPS and FTIR measurements revealed the retention of the maleic anhydride ring in the film. They concluded that the retention of the anhydride group was dependent on both the equivalent power and the duty cycle. Maleic anhydride films have been studied by a number of investigators who have looked at the adsorption of lipid bilayers7 and the attachment of amino-functionalized alkyl chains8 and the polymerization of styrene on plasma-polymerized maleic anhydride films.9 Recent work by Roucoules et al. has shown that short-chain allylamine can be coupled to pulse plasmapolymerized maleic anhydride and subsequent DielsAlder chemistry can be performed on the grafted alkene group functionality.10 Many other groups have used SPR as a method of quantifying the binding of proteins to surfaces and methods by which surfaces can be made resistant to protein adsorption. A good example is the work of Mar et al.,11 who produced plasma films of triethylene glycol dimethyl ether and used SPR to measure the adsorption of bovine serum albumin.11 Related work by Zhang et al. (6) Schiller, S.; Hu, J.; Jenkins, A. T. A.; Timmons, R. B.; SanchezEstrada, F. S.; Knoll, W.; Fo¨rch, R. Chem. Mater. 2002, 14, 235-242. (7) Jenkins, A. T. A.; Hu, J.; Wang Y. Z.; Schiller, S.; Fo¨rch, R.; Knoll, W. Langmuir 2000, 16, 6381-6384. (8) Jacobsen, V.; Menges, B.; Fo¨rch, R.; Mittler, S.; Knoll, W. Thin Solid Films 2002, 409, 185-193. (9) Teare, D. O. H.; Schofield, W. C. E.; Roucoules, V.; Badyal, J. P. S. Langmuir 2003, 19, 2398-2403. (10) Roucoules, V.; Fail, C. A.; Schofield, W. C. E.; Teare, D. O. H.; Badyal, J. P. S. Langmuir 2005, 21, 1412-1415. (11) Mar, M. N.; Ratner, B. D.; Sinclar, S. Y. Sens. Actuators, B 1999, 54, 125.
10.1021/la051449e CCC: $30.25 © 2005 American Chemical Society Published on Web 08/09/2005
Letters
Langmuir, Vol. 21, No. 19, 2005 8573
Figure 1. (a) Immobilization of proteins by covalent coupling of lysine residues with retained MA functional groups in polymer. (b) Determination of polymer-bound BSA by SPR measurement of anti-BSA binding.
on plasma-polymerized di(ethylene glycol) monovinyl ether films showed that polymerization conditions (plasma duty cycle) could be used to control the binding of fibrinogen, BSA, and immunoglobulin.12 In this work, we investigated the attachment of BSA to plasma-polymerized maleic anhydride films formed on modified gold surfaces. The effect of the plasma duty cycle on the degree of protein attachment was investigated by measuring the binding of antibody aBSA on the proteinmodified surfaces following protein binding and subsequent washing of the surface with detergent to remove nonspecifically adsorbed protein using SPR. SPR is now used routinely to study protein-protein or ligand-protein interactions on surfaces.13 It provides a label-free, quantitative, real-time method of following the attachment of proteins to surfaces and by the fitting of data can provide information on the association constant of, for example, protein-antibody conjugates.14 Methods and Materials Plasma Polymerization. A home-built reactor, based on the design of R. D. Short, University of Sheffield, was used. It consisted of a 30-cm-long, 10-cm-diameter glass tube (QVF) and stainless steel flanges.1 A coaxial power systems amplifier supplied 13.56 MHz radio frequency (rf) power, which could be attenuated via a pulse control unit. Oxygen, for reactor cleaning, was fed in via a gas control valve on one of the flanges. Maleic anhydride monomer vapor was introduced via a Young’s tap. An Edwards RV3 pump was used to create a vacuum down to 2 × 10-3 mbar. This reactor is more fully described in ref 1. The rf power from the amplifier is connected through a matching unit (coaxial power systems) to a coil of solid copper wire (diameter 1.5 mm). The stainless steel flanges are connected to the earth. The matching unit contains an inductor and variable capacitor for matching to the reactor impedance, thus ensuring minimal reflected power from the reactor back to the amplifier (
