Langmuir 2008, 24, 10187-10195
10187
Reactive Epoxy-Functionalized Thin Films by a Pulsed Plasma Polymerization Process Benjamin Thierry,* Marek Jasieniak, Louis C. P. M. de Smet,† Krasimir Vasilev, and Hans J. Griesser Ian Wark Research Institute, UniVersity of South Australia, Mawson Lakes Campus, Mawson Lakes, Adelaide, SA 5095, Australia ReceiVed April 11, 2008. ReVised Manuscript ReceiVed July 1, 2008 A novel plasma functionalization process based on the pulsed plasma polymerization of allyl glycidyl ether is reported for the generation of robust and highly reactive epoxy-functionalized surfaces with well-defined chemical properties. Using a multitechnique approach including X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), infrared spectroscopy (FT-IR), atomic force microscopy (AFM) and ellipsometry, the effect of the plasma deposition parameters on the creation and retention of epoxy surface functionalities was characterized systematically. Under optimal plasma polymerization conditions (duty cycle: 1 ms/20 ms and 1 ms/200 ms), reactive uniform films with a high level of reproducibility were prepared and successfully used to covalently immobilize the model protein lysozyme. Surface derivatization was also carried out with ethanolamine to probe for epoxy groups. The ethanolamine blocked surface resisted nonspecific adsorption of lysozyme. Lysozyme immobilization was also done via microcontact printing. These results show that allyl glycidyl ether plasma polymer layers are an attractive strategy to produce a reactive epoxy functionalized surface on a wide range of substrate materials for biochip and other biotechnology applications.
1. Introduction The introduction of reactive epoxy groups on the surface of solid substrate materials is of high interest in the fields of biomedical engineering and biotechnology. Epoxides can be coupled with amines, sulfhydryls, and other nucleophiles, which can be utilized to covalently immobilize biomacromolecules such as DNA (e.g., 5′-aminoalkylated oligonucleotides), enzymes and proteins (Scheme 1). Epoxy-functionalized biochips are readily available and widely used in the design of DNA and antibody/ protein microarrays.1,2 The suitability of epoxy surfaces for immobilization of biomolecules has been well established in protein array technology, although some proteins may benefit from the increased stereospecificity of oriented attachment schemes provided by specific substrates (e.g., nickel-mediated affinity binding of His-tagged proteins). The wide chemical reactivity of epoxy-containing substrates also offers the possibility of multipoint, covalent attachment with different nucleophiles placed on the surface of proteins and enzymes, potentially providing an efficient protection against conformational changes of the protein induced by any distorting agent.3 The presence of a high density of reactive epoxy groups is considered to be a key element toward the multipoint attachment and subsequent stabilization of biomacromolecules. A common epoxidation agent is 3-glycidoxypropyltrimethoxysilane. Hydrolysis of the silane moieties drives the attachment of organosilanes to glass/silica substrates resulting in siloxane bonds at the surface. Excessive polymerization in the solvent * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: +61 8 8302 3689. Fax: +61 8 8302 3683. † Current address: Department of Chemical Technology DelftChemTech, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands. (1) Angenendt, P.; Glokler, J.; Murphy, D.; Lehrach, H.; Cahill, D. J. Anal. Biochem. 2002, 309, 253–60. (2) Preininger, C.; Sauer, U. Sens. Actuator B-Chem. 2003, 90, 98–103. (3) Mateo, C.; Grazu, V.; Palomo, J. M.; Lopez-Gallego, F.; Fernandez-Lafuente, R.; Guisan, J. M. Nat. Protocols 2007, 2, 1022–1033.
Scheme 1. Pulsed Plasma Epoxidation of Surfaces and Bioconjugation of Proteins
phase and incomplete monolayer formation are, however, often observed and significantly reduce the robustness and reproducibility of organosilanes-based epoxidation processes. The lack of stability resulting from hydrolysis of the siloxane bonds is also an issue in many biomedical applications. Other conventional epoxidation methods involve the use of epoxy resins (e.g., ARChip Epoxy). Experimental studies indicate the need for better reproducibility and homogeneity of biochips in order to reduce autofluorescence and background fluorescence and increase the immobilization capacity and hybridization efficiency.1,2,4,5 The pulsed plasma polymerization of glycidyl methacrylate has been proposed recently as an alternative toward the preparation of well-defined and reproducible epoxy-rich thin films.6,7 Plasma polymerization processes present many advantages for the surface modification of biomedical micro- and nanodevices: under (4) Lee, C. Y.; Harbers, G. M.; Grainger, D. W.; Gamble, L. J.; Castner, D. G. J. Am. Chem. Soc. 2007, 129, 9429–9438. (5) Mahajan, S.; Kumar, P.; Gupta, K. C. Anal. Biochem. 2006, 353, 299–301. (6) Tarducci, C.; Kinmond, E. J.; Badyal, J. P. S.; Brewer, S. A.; Willis, C. Chem. Mater. 2000, 12, 1884–1889. (7) Harris, L. G.; Schofield, W. C. E.; Badyal, J. P. S. Chem. Mater. 2007, 19, 1546–1551.
10.1021/la801140u CCC: $40.75 2008 American Chemical Society Published on Web 08/05/2008
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optimized conditions, plasma thin films can be readily polymerized on a wide range of substrates from radio frequency (RF) plasma as highly adherent, pinhole-free, and conformal coatings.8,9 In addition, refinement of the plasma processes enables creation of well-controlled patterns7,10-13 and chemical gradients.14,15 A wide range of bio(macro)molecules have been immobilized onto plasma functionalized surfaces.8,9,16-19 Retention of the monomer’s reactive groups, such as epoxy, amine, carboxyl, hydroxyl, is critical for the optimal functionalization of the substrate and the stable introduction of reactive groups on the surface. Low power plasmas can be used to achieve a high degree of monomer retention and to produce thin films, which bear high structural resemblance to that of the parent monomer. The use of plasma discharges pulsed in the millisecond time scale is preferable to continuous wave (CW) plasma operation in order to achieve such low power plasmas. Equivalent power as low as 0.1 W can be produced in pulsed RF processes. Equivalent power is defined as
P ) Pinput × DC where Pinput is the input power and DC is the duty cycle, defined as time plasma on/(time plasma on + time plasma off). In addition, conventional polymerization reaction pathways can occur during the duty cycle off period, enhancing the retention of the desired functional groups. A wide range of plasma films with high degree of functionality retention have been reported.6,20-26 In this work, we report the pulsed plasma polymerization of a novel monomer, allyl glycidyl ether (AGE), and conduct a systematic, multitechnique characterization of the plasma polymerization parameters. Compared to the previously used monomer glycidyl methacrylate,6,7 AGE offers the advantages of a higher vapor pressure and hence improved ease of plasma polymerization and process control, as well as faster deposition rate. AGE plasma polymer films also may be more resistant to UV degradation, which is well-known to affect acrylate polymers (8) Siow, K. S.; Britcher, L.; Kumar, S.; Griesser, H. J. Plasma Process. Polym. 2006, 3, 392–418. (9) Forch, R.; Chifen, A. N.; Bousquet, A.; Khor, H. L.; Jungblut, M.; Chu, L. Q.; Zhang, Z.; Osey-Mensah, I.; Sinner, E. K.; Knoll, W. Chem. Vapor Depos. 2007, 13, 280–294. (10) Thissen, H.; Hayes, J. P.; Kingshott, P.; Johnson, G.; Harvey, E. C.; Griesser, H. J. Smart Mater. Struct. 2002, 11, 792–799. (11) Goessl, A.; Garrison, M. D.; Lhoest, J. B.; Hoffman, A. S. J. Biomater. Sci.-Polym. Ed. 2001, 12, 721–738. (12) Favia, P.; Sardella, E.; Gristina, R.; d’Agostino, R. Surf. Coat. Technol. 2003, 169, 707–711. (13) Bretagnol, F.; Ceriotti, L.; Valsesia, A.; Sasaki, T.; Ceccone, G.; Gilliland, D.; Colpo, P.; Rossi, F. Nanotechnology 2007, 18, 135303–135308. (14) Whittle, J. D.; Barton, D.; Alexander, M. R.; Short, R. D. Chem. Commun. 2003, 1766–1767. (15) Zelzer, M.; Majani, R.; Bradley, J. W.; Rose, F.; Davies, M. C.; Alexander, M. R. Biomaterials 2008, 29, 172–184. (16) Zhang, Z. H.; Feng, C. L. Appl. Surf. Sci. 2007, 253, 8915–8922. (17) Lopez, L. C.; Gristina, R.; Ceccone, G.; Rossi, F.; Favia, P.; d’Agostino, R. Surf. Coat. Technol. 2005, 200, 1000–1004. (18) Thierry, B.; Winnik, F. M.; Mehri, Y.; Silver, J.; Tabrizian, M. Biomaterials 2004, 25, 3895–905. (19) Hartley, P. G.; McArthur, S. L.; McLean, K. M.; Griesser, H. J. Langmuir 2002, 18, 2483–2494. (20) van Os, M. T.; Menges, B.; Foerch, R.; Vancso, G. J.; Knoll, W. Chem. Mater. 1999, 11, 3252–3257. (21) Choukourov, A.; Biederman, H.; Slavinska, D.; Hanley, L.; Grinevich, A.; Boldyryeva, H.; Mackova, A. J. Phys. Chem. B 2005, 109, 23086–23095. (22) Rinsch, C. L.; Chen, X. L.; Panchalingam, V.; Eberhart, R. C.; Wang, J. H.; Timmons, R. B. Langmuir 1996, 12, 2995–3002. (23) Mackie, N. M.; Castner, D. G.; Fisher, E. R. Langmuir 1998, 14, 1227– 1235. (24) Fraser, S.; Short, R. D.; Barton, D.; Bradley, J. W. J. Phys. Chem. B 2002, 106, 5596–5603. (25) Voronin, S. A.; Zelzer, M.; Fotea, C.; Alexander, M. R.; Bradley, J. W. J. Phys. Chem. B 2007, 111, 3419–3429. (26) Voronin, S. A.; Bradley, J. W.; Fotea, C.; Zelzer, M.; Alexander, M. R. J. Vac. Sci. Technol. A 2007, 25, 1093–1097.
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including methyl methacrylate plasma polymers.27 The effect of the duty cycle and RF input power on the retention of epoxy functionalities on the surface of allyl glycidyl ether plasma polymers (AGEpp) was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR). The growth, stability and surface morphology of the epoxy-rich thin films were studied using ellipsometry and atomic force microscopy (AFM). AGEpp coatings were also derivatized with ethanolamine to probe for epoxy functionalities. Finally, the covalent immobilization of a model protein, lysozyme, was investigated.
2. Experimental Section 2.1. Materials. Silicon wafers were cut from 100 mm diameter wafer disks obtained from Micro Materials and Research Cons. Pty Ltd. (Australia). Prior to plasma deposition, substrates were cleaned with Piranha solution: concentrated sulfuric acid was poured into a beaker and hydrogen peroxide (30% aqueous solution) slowly added at a 2:1 (v/v) ratio of sulfuric acid:hydrogen peroxide. The samples were immersed in the Piranha solution for 20 min before being washed with a copious amount of MilliQ water and rapidly blown dry in a stream of filtered nitrogen gas. (Warning: care must be taken as the reaction is highly exothermic and reacts strongly with carboneous materials). Allyl glycidyl ether (>99%) was obtained from Sigma-Aldrich, and used as received (Warning: Many epoxides are suspected carcinogens and appropriate care should be exercised in their use). Lysozyme was from chicken egg white (Sigma-Aldrich). Polydimethylsiloxane (PDMS) elastomers (SYLGARD 184, Dow Corning, USA) were used according to the manufacturer’s indication. Ultrapure water used in the experiments was obtained from a Millipore water treatment system (organic content less than 5 ppb). All other chemicals were analytical grade. 2.2. Plasma Polymerization. The plasma polymerization of the allyl glycidyl ether monomer was carried out in a custom-built reactor described elsewhere.28,29 Briefly, the reactor chamber is defined by a glass cylinder with a height of 35 cm and a diameter of 17 cm. The gas pressure was monitored via a Pirani gauge and controlled by needle valves (BOC Edwards). Clean silicon wafer samples were placed on the lower electrode (diameter, 9.5 cm), with the upper electrode U-shaped (distance between the electrodes: 12.5 cm). The parameters for the plasma polymerization of the monomer were varied to achieve a systematic characterization of the effect of the duty cycle and plasma RF input power. A typical base pressure prior to the introduction of the monomer was 1 × 10-3 mbar. An initial monomer pressure of 0.21 mbar was used. 2.3. Characterization. X-Ray Photoelectron Spectroscopy. XPS analyses were conducted using a Kratos AXIS Ultra DLD X-ray photoelectron spectrometer with a monochromatic Al KR X-ray source (h
