Bioconjugate Chem. 2005, 16, 245−249
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Preparation of a Gradient Biotinylated Polyethylene Surface To Bind Streptavidin-FITC Moon Suk Kim,†,§ Kwang Su Seo,‡,§ Gilson Khang,‡ and Hai Bang Lee*,† Nanobiomaterials Laboratory, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong, Daejeon 305-600, Korea, and Department of Polymer/Nano Science and Technology, Chonbuk National University, 664-14, Duckjin, Jeonju 561-756, Korea. Received June 16, 2004; Revised Manuscript Received February 1, 2005
A novel biotinylated gradient surface was designed to bind a streptavidin-FITC. The gradient polyethylene (PE) surface was prepared by a corona treatment with a power increase along the PE length, and successively, biotinylated gradient PE surface was prepared by the reaction of polyethylenimine (PEI) and biotin. Surface properties of the gradient PE surface changed according to PE length and chemical modification. The biotinylated gradient PE surface provided a continuous gradient of streptavidin-FITC through binding of avidin-biotin. The luminosity of fluorescence that corresponds to a streptavidin-bound FITC on the biotinylated gradient PE surface increased gradually on the surface along the PE length.
INTRODUCTION
Scheme 1
When polymer materials are introduced in in vivo, they can act as a substrate for attaching extracellular matrix (ECM) proteins or cells but they simultaneously induce immunogenic responses (1-4). To prevent or decrease immunogenic responses, recent development of polymer materials has focused on the design of biomimetic materials that are able to recognize ECM or specific cell (5-8). The surface modification of materials was expected to be particularly useful in the design of biomimetic materials modified by bioactive molecules with biomolecular recognizable properties (9, 10). The introduction of the bioactive molecules was usually performed by either physical or chemical reaction (11-16). The bioactive molecule-modified biomaterials can recognize the targetable receptor molecules that are present under biological conditions. The interactions between bioactive molecules and targetable receptors are also important for the design of biomimetic materials, because the evaluation of biomimetic materials can be affected by binding efficiency. The interactions are dependent on several factors such as the affinity between bioactive molecules and targetable receptors, the density of bioactive molecules, and the spatial distribution of bioactive molecules (17-19). Thus, the design of biomimetic materials that can selectively detect or identify targetable biomolecules as well as minimize nonspecific binding of biomolecules was a challenging subject; in addition, the designed biomimetic materials may offer the potential of biomedical applications such as biomolecular interactions (2022), cell motility (23-25), and diagnostic studies (26, 27). However, the surface modification has some limitations. Since the surface modification has been performed on a uniform surface, the evaluation of the behavior of * Corresponding author. E-mail:
[email protected], tel: 8242-860-7220, fax: 82-42-861-4151). † Korea Research Institute of Chemical Technology. ‡ Chonbuk National University. § M. S. Kim and K. S. Seo contributed equally to this paper.
bioactive molecules on the biomaterial has been tediously conducted through several experiments. A gradient surface is the surface on which a gradually varying chemical composition exists along its length. If
Figure 1. Plot of (A) water contact angle versus length treated by corona and (B) fluorescence intensity versus length treated by corona.
10.1021/bc049860l CCC: $30.25 © 2005 American Chemical Society Published on Web 02/23/2005
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Figure 2. AFM images at the position (A) 0.5 cm, (B) 2.5 cm, and (C) 4.5 cm after corona treatment.
a bioactive molecule is introduced on a gradient surface, the gradient bioactive molecule on the gradient surface can illustrate various results through a single experiment. This can be a powerful tool to evaluate the ability of bioactive molecules (28-30). However, there were few studies on the interaction between a gradient surface with a bioactive molecule and a targetable biomolecule. The aim of our research was to develop a simple applicable method using gradient surfaces for biomedical applications. We present here the first approach for design of a novel gradient polyethylene (PE) surface by an incorporation of biotin and the binding of streptavidin-FITC on the biotinylated gradient surface. RESULTS AND DISCUSSION
Corona discharge treatment is one of the useful methods for introducing functional groups and/or roughness onto a polymer surface. We recently reported the preparation of a gradient surface by radiofrequency corona
discharge with knife-type electrodes and the applications as biomaterials (31, 32). In this work, the chemical modification after corona discharge treatment was carried out as shown in Scheme 1. In the first step, the hydrocarbon backbone on the PE surface was activated to the carbon radicals form by the corona discharge treatment. The activated carbon radicals in the presence of oxygen provided a number of functional groups such as the hydroxyl group, ether, ketone, aldehyde, carboxylic acid, and carboxylic ester on the PE surface. Since the corona power gradually increased along the PE length, the density of functional groups gradually increased. To examine the surface property after corona treatment, the gradient PE surface was examined by water contact angle, Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy, atomic force microscopy (AFM), and electron spectroscopy for chemical analysis (ESCA). The water contact angle of untreated PE surface was 99°, indicating a complete hydrophobic
Communications
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Figure 3. ESCA spectra of (A) untreated PE, (B) corona-treated PE, and (C) PEI-modified PE at the position 4.5 cm. (Left: carbon 1s core level spectra, Right: survey scan spectra).
surface property. As shown in Figure 1A, the water contact angle of PE surface decreased gradually from 99° to 45-50° along the PE length with increasing corona power, indicating the gradual wettability change from completely hydrophobic to extremely hydrophilic. FTIRATR spectra showed a peak assignable to the CdO functional group at 1735 cm-1 and gradual peak intensity increase as the corona power increased (33). Figure 2 shows AFM images along the PE length after corona treatment. The PE surface exhibited a gradient roughness increase from 10 to 100 nm as corona power increases. The chemical structure changes of the corona-treated PE surface were also investigated by ESCA (Figure 3). The untreated PE surface exhibited only a broad C1s peak at a binding energy of 285 eV (Figure 3A). After corona discharge treatment, new peaks assignable to
ketone or aldehyde groups, hydroxyl or ether groups, and carboxylic acid or ester groups were observed at 287.9, 286.3 and 289.1 eV, respectively (Figure 3B). We confirmed the formation of various functional groups, especially the carboxylic acid group that can serve as reaction site on the PE surface. The carboxylic acid on the surface may chemically react with a functional group such as NH2. Polyamine compounds that have long spacers can be used for immobilization of the surface and consequently allow flexible and multifunctional sites on the surface in a biological environment. Thus chemical modification of the surface using polyamine was performed. First, the carboxylic group on the corona-treated PE surface was activated by N-hydroxysuccinimide (NHS) and EDC (Nethyl-N′-(3-dimethylaminopropyl)carbodiimide). The activated carboxylic group reacted with the amine com-
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Scheme 2
pounds such as 1,9-nonanediamine (ND) or polyethylenimine (PEI). In FTIR-ATR, the peak assignable to the CdO functional group almost disappeared at 1735 cm-1, while a new peak was observed at around 1586 cm-1 after reaction with ND or PEI. The ESCA spectrum of PEI modified on the gradient PE surface is shown in Figure 3C. A gradual increase in height of the C1s shoulder at 288.2 eV due to the C-N species within the PEI was observed along the PE length. In addition, 1s of nitrogen was observed at 399 eV in the survey scan spectrum (Figure 3C), and the intensity also gradually increased along the PE length, implying a nitrogen content increase on the PE surface (Table S1) (34). The results of FTIR-ATR and ESCA indicated that amine compounds were gradually modified on the gradient PE surface. Among several binding systems, the binding affinity of the avidin-biotin interaction (KD ∼10-15 M) is one of the highest to occur in nature as noncovalent bonding (35, 36). Thus, we choose the binding system of avidinbiotin to design a gradient PE surface with a bioactive molecule. The amine-modified gradient PE surface was reacted with biotin that was preactivated by NHS and EDC. The new signal appeared at 1725 cm-1 after the reaction of biotin in FTIR-ATR spectrum (33). In the subsequent step, the gradient biotinylated PE surface was incubated in a solution of streptavidin-FITC (Scheme 2). The binding of the streptavidin-FITC to the gradient biotinylated PE surfaces was characterized by fluoresence microscopy. The fluorescence microscope image, which corresponds to a streptavidin-bound FITC bonded on the gradient biotinylated PE surfaces, is shown in Figure 4
Figure 4. Change of fluorescence images on the gradient PE surface from 0 to 5 cm after the binding of streptavidin-FITC.
(37). The luminosity of fluorescence increased gradually on the surface when corona power for the surface increased along the length (38). Plotting of the length treated by corona versus the fluorescence intensity of the streptavidin-FITC reveals a gradient as shown in Figure 1B (39). The longer the length treated by corona, the smaller the water contact angle value and the stronger the fluorescence intensity , which could well explain the gradient PE surface. In conclusion, we prepared a gradient PE surface using a corona treatment with power increase along the PE length and biotinylated gradient PE surface through the reaction of PEI and biotin. The biotinylated gradient PE surface provided a continuous gradient of streptavidinFITC through recognizing their binding partners from
solution. We believe that this method will be useful in the study of the analysis of functional bioactive molecules on the gradient surface through one experiment because the designed gradient PE surface is experimentally simpler than using a uniform surface. ACKNOWLEDGMENT
We would like to thank KMOICE (N11-A08-1402-051-3) for funding. Supporting Information Available: Experimental procedures, Table S1, and Figures S1-S6. This material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Metzger, D. W. (2002) Immune responses to tissue-engineered extracellular matrix used as a bioscaffold. Ann. N. Y. Acad. Sci. 961, 335-336. (2) Vogel, V., and Baneyx, G. (2003) The tissue engineeting puzzle: a molecular perspective. Annu. Rev. Biomed. Eng. 5, 441-463. (3) Sarraf, C. E., Harris, A. B., McCulloch, A. D., and Eastwood, M. (2003) Heart valve and arterial tissue engineering. Cell Prolif. 36, 241-254. (4) Rihova, B. (2000) Immunocompatibility and biocompatibility of cell delivery systems. Adv. Drug Delivery Rev. 42, 65-80. (5) Prokop, A. (2001) Bioartificial pancreas: materials, devices, function, and limitations. Diabetes Technol. Ther. 3, 431449. (6) Shin, H., Jo, S., and Mikos, A. G. (2003) Biomimetic materials for tissue engineering. Biomaterials 24, 4353-4364. (7) Courtney, J. M., Zhao, X. B., Qian, H., and Sharma, A. (2003) Modification of polymer surfaces: optimization of approaches. Perfusion 18, 33-39. (8) Reyes, C. D., and Garcia, A. J. (2003) Engineering integrinspecific surfaces with a triple-helical collagen-mimetic peptide. J. Biomed. Mater. Res. 65, 511-523. (9) Zhi, Z. L., Morita, Y., Hasan, Q., and Tamiya, E. (2003) Micromachining microcarrier-based biomolecular encoding for miniaturized and multiplexed immunoassay. Anal. Chem. 75, 4125-4131. (10) Quirk, R. A., Chan, W. C., Davies, M. C., Tendler, S. J., and Shakesheff, K. M. (2001) Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lactic acid). Biomaterials 22, 865-872. (11) Bures, P., Huang, Y., Oral, E., and Peppas, N. A. (2001) Surface modifications and molecular imprinting of polymers in medical and pharmaceutical applications. J. Controlled Release 72, 25-33. (12) Brash, J. L. (2000) Exploiting the current paradigm of blood-material interactions for the rational design of bloodcompatible materials. J. Biomater. Sci. Polym. Ed. 11, 11351146. (13) Ryu, D. D., and Nam, D. H. (2000) Recent progress in biomolecular engineering. Biotechnol. Prog. 16, 2-16.
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