Stable Permanently Hydrophilic Protein-Resistant Thin-Film Coatings

Guodong Sui, Jinyi Wang, Chung-Cheng Lee, Weixing Lu, Stephanie P. Lee, Jeffrey V. Leyton, Anna M. Wu, and Hsian-Rong Tseng. Analytical Chemistry 2006...
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Anal. Chem. 2005, 77, 3971-3978

Stable Permanently Hydrophilic Protein-Resistant Thin-Film Coatings on Poly(dimethylsiloxane) Substrates by Electrostatic Self-Assembly and Chemical Cross-Linking Honest Makamba, Ya-Yu Hsieh, Wang-Chou Sung, and Shu-Hui Chen*

Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan

Poly(dimethylsiloxane) (PDMS) is a biomaterial that presents serious surface instability characterized by hydrophobicity recovery. Permanently hydrophilic PDMS surfaces were created using electrostatic self-assembly of polyethyleneimine and poly(acrylic acid) on top of a hydrolyzed poly(styrene-alt-maleic anhydride) base layer adsorbed on PDMS. Cross-linking of the polyelectrolyte multilayers (PEMS) by carbodiimide coupling and covalent attachment of poly(ethylene glycol) (PEG) chains to the PEMS produced stable, hydrophilic, protein-resistant coatings, which resisted hydrophobicity recovery in air. Attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy revealed that the thin films had excellent chemical stability and resisted hydrophobicity recovery in air over 77 days of measurement. The spectra also showed a dense coverage for PEG dialdehyde and excellent resistance to protein adsorption from undiluted rat serum. Atomic force microscopy revealed dense coverage with PEG dialdehyde and PEG diamine. Contact angle measurements showed that all films were hydrophilic and that the PEG dialdehyde-topped thin film had a virtually constant contact angle (∼20°) over the five months of the study. Electrokinetic analysis of the coatings in microchannels always exposed to air also gave good protein separation and constant electroosmotic flow during the five months that the measurements were done. We expect that the stable, hydrophilic, protein-resistant thin-film coatings will be useful for many applications that require long-term surface stability. Historically, poly(dimethylsiloxane) (PDMS) has been used in medicine for making devices such as implants,1,2 catheters, pacemaker encapsulants, and ocular lenses.3 More recently, the use of PDMS has been extended to the fabrication of microfluidic chips.4-7 The application of PDMS has been driven by its good properties, which include low toxicity and flexibility. Despite the * To whom correspondence should be addressed. E-mail: shchen@ mail.ncku.edu.tw. (1) Lahey, F. H. Ann. Surg. 1946, 124, 1027-1028. (2) De Nicola, R. R. J. Urol. 1950, 63 (1), 168-172. (3) Dow Corning Corp. Implant Information Booklet; 1998. (4) Qin, D.; Xia, Y.; Whitesides, G. M. Adv. Mater. 1996, 8, 917-919. 10.1021/ac0502706 CCC: $30.25 Published on Web 05/20/2005

© 2005 American Chemical Society

many advantages that PDMS has, its applications in microfluidics and medicine have been problematic because PDMS is highly hydrophobic. Even when the surface is made hydrophilic, PDMS gradually reverts to the hydrophobic state due to surface rearrangements. The surface instability of PDMS has not been fully addressed to date. Various approaches have been used to modify PDMS surfaces for various applications. Modification procedures include exposure to energy sources such as plasma,9,10 corona discharge,11 and ultraviolet light,12 polyelectrolyte multilayers (PEMS),13 radiationinduced graft polymerization,14,15 silanization,16 atom-transfer radical polymerization,17,18 chemical vapor deposition,19 cerium(IV) catalysis,20,21 phospholipid bilayer modification,22-24 and more recently, sol-gel modifications.7 Most of these approaches do not solve the problem of hydrophobicity recovery via the silica-like layer or deformations since the hydrophilic groups are directly (5) Duffy, D. C.; McDonald, J. C.; Schuller, O. J. A. Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984. (6) Masuda, S.; Washizu, M.; Nanba, T. IEEE Trans. Ind. Appl. 1989, 25, 762737. (7) Roman, G. T.; Hlaus, T.; Bass, K. J.; Seelhammer, T. G.; Culbertson, C. T. Anal. Chem. In press. (8) Decher, G. Science 1997, 277, 1232-1237. (9) Fritz, J. L.; Owen, M. J. J. Adhes. 1995, 54, 33-45. (10) Lai, J. Y.; Lin, Y. Y.; Denq, Y. L.; Shyu, S. S.; Chen, J. K. J. Adhes. Sci. Technol. 1996, 10, 231-242. (11) Efimenko, K.; Wallace, W. E.; Genzer, J. J. Colloid Interface Sci. 2002, 254, 306-315. (12) Hillborg, H.; Gedde, U. W. Polym. Sci. 1998, 39, 1991-1998. (13) Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S. Anal. Chem. 2000, 72, 5939-5944. (14) Hu, S.; Ren, X.; Backman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. Anal. Chem. 2002, 74, 4117-4123. (15) Hu, S.; Ren, X.; Backman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. Anal. Chem. 2004, 74, 1865-1870. (16) Papra, A.; Bernard, A.; Juncker, D.; Larsen, N. B.; Michel, B.; Delamarche, E. Langmuir 2001, 17, 4090-4095. (17) Xiao, D.; Zhang, H.; Wirth, M. Langmuir 2002, 18, 9971-9976. (18) Xiao, D.; Le, T. V.; Wirth, M. J. Anal. Chem. 2004, 76, 2055-2061. (19) Lahann, J.; Balcelis, M.; Hang, L.; Rodon, T.; Jensen, K. F.; Langer, T. Anal. Chem. 2003, 75, 2117-2122. (20) Slentz, B. E.; Penner, N. A.; Regnier, F. E. J. Chromatogr., A 2002, 948, 225-233. (21) Sung, W. C.; Huang, S. Y.; Liao, P. C.; Lee, G. B.; Li, C. W.; Chen, S. H. Electrophoresis 2003, 24, 3648-3654. (22) Yang, T.; Jung, S.; Hang, L.; Mao, H.; Cremer, P. S. Anal. Chem. 2001, 73, 165-169. (23) Yang, T.; Baryshnikova, O. K.; Mao, H.; Holden, M. A.; Cremer, P. S. J. Am. Chem. Soc. 2003, 125, 4779-4784. (24) Mao, H.; Yang, T.; Cremer, P. S. Anal. Chem. 2002, 74, 379-385.

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attached to the PDMS surface and thus exposed to the migrating groups from the bulk. The silica-like layer is produced on the PDMS surface during exposure to energy sources such as plasma or corona discharge. The deformations result because of the differences in elasticity between the brittle silica-like layer and the flexible PDMS bulk.25 The modification via sol-gel chemistry could suffer less from hydrophobicity recovery since the silica particles produced are homogeneously distributed throughout the PDMS matrix.7 Based on the reported data, however, the increase of hydrophilicity through sol-gel modifications is relatively small since the contact angle was reduced by only 17%. Moreover, the resistance to protein adsorption for PDMS-SiO2 material could still be poor due to the silica-like surface. Here we perform electrostatic layer-by-layer electrostatic selfassembly (ELBL) of polyelectrolytes with chemical cross-linking on the PDMS surface as a way of imparting lasting hydrophilicity to the surface. ELBL is a strikingly simple method that allows creation of nanoscale structures by alternate adsorption of polyanions and polycations on virtually any substrate to produce polyelectrolyte multilayers (PEMS).26 The ELBL technique has not found widespread application yet, but potential applications,26-30 including repair of damaged blood vessels,30 have been reported. PEMS coatings, however, are known to bleed, causing a quick deterioration of performance with time. Here, the PEMS are chemically cross-linked to retain long-term stability. Moreover, in most of the works on ELBL, there has been no attempt to top the PEMS with neutral hydrophilic polymers to make the PEMS more versatile. Here, the PEMS are covalently topped with poly(ethylene glycol) (PEG) to resist protein adsorption. In our method, the PEMS run parallel to the surface, thus protecting the attached PEG from direct exposure to the hydrophobic groups. The attached PEG imparts protein resistance to the thin films, which is crucial in devices such as blood containers, dialyzers, protein separation, and vascular grafts. While a lot of proteinresistant coatings have been reported on many surfaces,31-41 very few protein-resistant coatings on PDMS have been reported. The (25) Owen, M. J.; Smith, P. J. J. Adhes. Sci. Technol. 1994, 8, 1063-1075. (26) Farhat, T. R.; Schlenoff, J. B. Electrochem. Solid-State Lett. 2002, 5 (4), B13-B15. (27) Hiller, J. R.; Mendelsohn, J. D.; Rubner, M. F. Nat. Mater. 2002, 1, 59-63. (28) Park, M. K.; Deng, S.; Advincula, R. C. J. Am. Chem. Soc. 2004, 126, 1372313731. (29) Mamedov, A. A.; Kotov, N. A.; Prato, M.; Guldi, D. M.; Wicksted, J. P.; Hirsch, A. Nat. Mater. 2002, 1, 190-194. (30) Thierry, B.; Francoise, M. W.; Merhi, Y.; Tabrizian, M. J. Am. Chem. Soc. 2004, 126, 7494-7495. (31) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436. (32) Hernwerth, S.; Eck, W.; Reihardt, S.; Grunze. J. Am. Chem. Soc. 2003, 125, 9359-9366. (33) Bearinger, J. P.; Terrettaz, S.; Michel, R.; Tirelli, N.; Vogel, H.; Textor, M.; Hubbell, J. A. Nat. Mater. 2003, 2, 259-264. (34) Ruiz-Taylor, L. A.; Martin, T. L.; Witte, Z. K.; Indermuhle, P.; Nock. S.; Wagne, P. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 852-857. (35) Groll, J.; Amirgoulova, E. V.; Ameringer, T.; Heyes, C. D.; Rocker, C.; Nienhaus, G. U.; Moller, M. J. Am. Chem. Soc. 2004, 126, 4234-4239. (36) Dalsin, J. L.; Hu, B. H.; Lee, B. P.; Messersmith, P. B. J. Am. Chem. Soc. 2003, 125, 4253-4258. (37) Bergstrom, K.; Osterberg, E.; Holmberg, K.; Riggs, J. A.; Van Alstine, J. M.; Schuman, T. A.; Burns, N. L.; Harris, J. M. Colloids Surf. A 1993, 77, 159-169. (38) Osterberg, E.; Bergstrom, K.; Holmberg, K.; Schuman, T. P.; Riggs, J. A.; Burns, N. L.; Harris, J. M. J. Biomed. Mater. Res. 1995, 29, 741-747. (39) Holland, N. B.; Qiu, Y.; Ruegsegger, M.; Marchant, R. E. Nature 1998, 392, 799-801.

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durability of medical devices made of plastics such PDMS is still questionable, and better methods of modifying these surfaces are still needed to improve reliability.42 The permanently hydrophilic surfaces we created could also be useful for commercial PDMS microfluidic devices and stable nondenaturing surfaces for immobilization of cell receptors and enzymes. EXPERIMENTAL SECTION Materials. PDMS oligomer, Sylgard 184 and curing agent were acquired from Dow Corning. PEG diamine, MW 6000, was obtained from Rapp Polymere. PEG dicarboxylate, MW 6000, was obtained from Fluka Chemika, and PEG dialdehyde, MW 3400, was obtained from Nektar Therapeutics. Potassium sulfate was obtained from J.T. Baker. The SDS gel for protein separations was obtained from Beckman Coulter. Cy3 was obtained from Amersham Biosciences. All other chemicals were obtained from Sigma-Aldrich. Substrate Preparation and Modification. The PDMS Sylgard 184 oligomer was mixed with curing agent in the ratio of 10:1 to form a PDMS prepolymer mixture and degassed in a vacuum. The degassed PDMS mixture was then added dropwise to glass substrates (1 cm × 1 cm), which had been cleaned by sonication in a 50/50 acetone/deionized (DI) water mixture and dried in the oven. The PDMS mixture was allowed to spread over the surface of each glass substrate. The glass substrates coated with the PDMS mixture (henceforth called PDMS substrates) were placed in the over at 60 °C for at least 4 h to allow polymerization of the PDMS. All modifications of the PDMS substrates were performed with the substrates in polystyrene dishes. The PDMS substrates were oxidized in an oxygen plasma for 60 s in a Harrick plasma cleaner/ sterilizer with radio frequency level set at high. The PDMS substrates were then left for 9 h to recover a little hydrophobicity. Subsequently, the oxidized PDMS substrates were exposed to a solution of hydrolyzed poly(styrene-alt-maleic anhydride) (hPSMA), MW 350 000, 0.25% (w/v) and left overnight. The h-PSMA was obtained by hydrolyzing PSMA using the method used by Whitesides’s group.43 The unbound h-PSMA was rinsed with DI water (2 × 150 mL). The h-PSMA-coated PDMS substrates were then coated with branched polyethyleneimine (PEI), MW 750 000, 1% (w/v) in DI water by exposing them to the PEI solution for 1 h. Subsequently, the PDMS substrates were coated with poly(acrylic acid) (PAA), MW 100 000, 0.25% (w/v) in DI water by exposing them to the PAA solution for 1 h as well. The sequence of polyelectrolyte coatings was repeated to obtain the desired number of PEMS. Between the PEI and PAA exposures, the substrates were washed with DI water (2 × 150 mL). The polyelectrolyte layers were cross-linked by carbodiimide coupling, which forms amide bonds between PEI and PAA layers. Carbodiimide cross-linking of the PEMS was done by submerging the PDMS substrates in a solution of 30 mg/mL/1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) and 5 mg/mL N-hydroxysuccinimide (NHS) (EDC/NHS solution) for (40) Piehler, J.; Brecht, A.; Geckeler, K. E.; Gauglitz, G. Biosens. Bioelectron. 1996, 11, 579-590. (41) Metzke, M.; Bai, J. Z.; Guan, Z. J. Am. Chem. Soc. 2003, 125, 7760-7761. (42) Uyama, Y.; Kato, K.; Ikada, Y. Adv. Polym. Sci. 1998, 137, 1-39. (43) Stroock, A. D.; Kane, R. S.; Weck, M.; Metallo, S. J.; Whitesides, G. M. Langmuir 2003, 19, 2466-2472.

one night at 4 °C. For attaching PEG chains to PEMS, a similar procedure was used. PEG diamine was attached to PEMS topped with PAA while PEG dicarboxylate was attached to PEMS topped with PEI. To attach PEG diamine or PEG dicarboxylate to the PEMS, the functionalized PEG solid was dissolved in EDC/NHS solution at 1 mg/mL. Next, this solution was poured onto the PDMS substrates with non-cross-linked PEMS and left to react overnight at ∼4 °C. This means that cross-linking of the PEMS occurred at the time of the attachment of the PEG chains. PEG dialdehyde was attached to PEI-topped PEMS by reductive amination. A 1 mg/mL aliquot of PEG dialdehyde was dissolved in a pH 7.4 buffer solution made up of phosphate (1 mM) and potassium sulfate (0.6 M). Subsequently, sodium cyanoborohydride (2.5 mg/mL) was dissolved in the buffer solution containing the PEG dialdehyde. This solution was then poured onto the PDMS substrates coated with non-cross-linked PEMS. The reaction was carried out at 60 °C overnight. After the attachment of PEG aldehyde to the PEI top layer, the substrate was washed with DI water (2 × 150 mL) and further exposed to EDC/NHS for one night to cross-link the PEMS below the PEG aldehyde. After modification, the PDMS substrates were dried in an oven at 60 °C for 1 h under a flow of nitrogen and kept dry in a desiccator in the dark at room temperature. Protein Adsorption. Plasma oxidized and thin-film modified PDMS substrates were exposed to 100 ppm proteins dissolved in phosphate (20 mM at pH 7.4) buffer solution for 1 h. They were then washed with phosphate buffer (100 mL) and DI water (2 × 150 mL) and dried in nitrogen before analysis. For protein adsorption under more rigorous conditions, the substrates were exposed to undiluted rat serum. Before exposure to rat serum, the atomic percentages of the surfaces were determined by X-ray photoelectron spectroscopy (XPS). Subsequently, the substrates were exposed to undiluted rat serum with a protein concentration of 48 mg/mL (48 000 ppm) for 1 h. They were then washed with phosphate buffer and DI water as above and dried under nitrogen before a second XPS analysis. Three measurements were done for serum samples and the averages taken. Surface Characterization of the PDMS Substrates. Attenuated total reflection Fourier transform infrared spectroscopy (ATRFT-IR) spectroscopy measurements were performed on the modified PDMS substrates using a Perkin-Elmer GX spectrometer, and the spectra were an average of 50 scans at a resolution of 4 cm-1. The PDMS background spectrum was subtracted since it interferes with the PEG signals in some regions of the IR spectrum. X-ray photoelectron spectroscopy was done on a Sigma VG Scientific spectrometer at a pressure of 1.6 × 10-9 Torr with a monochromatized Al KR source at an energy value of 1486.68 eV with an X-ray spot size of 300 µm. A flood gun was used to reduce charging of the surface. Spectra were referenced to the C 1s binding energy of 285 eV. Peak fitting was performed using VG Sigma software. Tapping mode atomic force microscopy (AFM) measurements were performed on a Digital Instruments Nanoscope IIIa microscope at a scan rate of 0.5 Hz using an etched silicon probe. Contact angle measurements were done on a homemade instrument equipped with a JVC CCD camera. Imaging was controlled by Matrox Inspector 4.1 program, and the computer was fitted with a Matrox Videocatch Card. A 20-µL

droplet of DI water was used for each measurement of the contact angle. The contact angle was measured three times for each surface and the average taken. Microchip Fabrication and Modification. The PDMS microchannels used had a cross-sectional area of 20 µm × 20 µm as shown in Figure S6A (Supporting Information; not drawn to scale). The PDMS piece with the channel pattern was made by replica molding on a PDMS master as described previously.21 The formed microchip was left for 9 h in the air. Subsequently, the channels were flushed with 0.25% (w/v) h-PSMA and left for one night. After h-PSMA coating, the channels were washed with DI water using vacuum pump for ∼5 min. The PEI and PAA were then flushed alternately into the channels. Each polyelectrolyte was left for 1 h and then flushed out with vacuum pump. The unbound polyelectrolyte was removed by flushing with DI water using a vacuum pump for ∼5 min. The DI water was also removed from the channels by vacuum pump before flushing with the next polyelectrolyte. Cross-linking was performed overnight as with the substrates. After the thin films were cross-linked, treated with PEG, or both, the channels were always kept dry except during electrokinetic measurements. Electrokinetic Studies and Electrophoretic Separations. Control of electroosmotic flow (EOF) measurements and electrophoretic separation was done using the LabVIEW program (National Instruments, Austin, TX). Two high-voltage power supplies from Betan (Hicksville, NY) were used. The EOF was measured by the current monitoring method at pH 4.44 After conditioning the chip with 10 mM phosphate buffer at pH 4 for 40 min, reservoir A was filled with 1 mM phosphate buffer at pH 4 (Figure S6A in Supporting Information). A voltage of -500 V was applied to the electrode in reservoir A while keeping the electrode in reservoir D grounded. As the 1 mM buffer filled the channel, driven by the EOF, the current decreased until it reached a constant value. The time taken for the current to reach a constant value was noted. The EOF was calculated by the equation, EOF ) L2/tv, where L (in centimeters) is the length between the two electrodes, t (seconds) is the time taken by the current to reach the constant lower value, and v (in volts) is the applied voltage. After measurements, the channels were washed with DI water and dried by flowing nitrogen through them. The microchannels were kept dry in the dark to reduce oxidation that may occur to the PEG chains. Electrophoretic separations were performed using the gated injection method.45 Channel conditioning was performed for 40 min. After filling the microchannels with separation buffer, the sample solution was placed in reservoir A. A voltage of -1 kV was applied to reservoir A and -1.2 kV was applied to reservoir B with reservoirs C and D grounded. For injection, -1 kV was applied to reservoir A for 5 s while reservoir B was floating. For separation, -1.2 kV was reapplied to reservoir B and -1 kV to reservoir A with reservoirs C and D grounded. The Cy3-labeled protein mixture containing avidin, bovine serum albumin, and lysozyme were separated and detected by laser-induced fluorescence detection. Detection was done at a position of 2.5 cm from the channel cross. The optical system used for excitation and (44) Huang, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 1837-1838. (45) Jacobson, S. C.; Koutny, L. B.; Hergenroder, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107-1113.

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Figure 1. ATR-FT-IR spectra showing amide peak intensity with number of layers. (A) Non-cross-linked h-PSMA/(PEI/PAA)2PEI/PEG dicarboxylate; (B) h-PSMA/PEI; (C) h-PSMA/(PEI/PAA); (D) h-PSMA/(PEI/PAA)2; (E) h-PSMA/(PEI/PAA)3. (F) h-PSMA/(PEI/PAA)4PEI. All PEMS except (A) were cross-linked.

fluorescence detection is exactly the one used in a previous publication.46 RESULTS AND DISCUSSION Surface Modification and Characterization. The oxidized substrates or channels were exposed to the h-PSMA solution 9 h after oxidation to allow a little hydrophobicity recovery. The h-PSMA then strongly interacted with the hydrophobic PDMS bulk material by the benzene rings while the carboxylic groups remained on the surface of the PDMS for cross-linking with polyelectrolytes. The PEI/PAA layers on top of the h-PSMA layer produced PEMS, which were then cross-linked by carbodiimide coupling. The formation of cross-linked PEMS was monitored by measuring the peak intensity of the ATR-FT-IR amide signals around 1650 (carbonyl amide stretch) and 1550 cm-1 (N-H amide bend) on the PDMS substrates. Figure 1 shows the peak intensity with increasing number of layers. The amide peak intensity increases with increase in layer number. For the PEMS topped with PAA (spectra D and E), there is a signal around 1700 cm-1 from the residual carboxylic groups on PAA that remain after cross-linking. For all PEMS topped with PEI, the amide peak at 1650 cm-1 was much larger than the peak at 1550 cm-1 probably due to the branching of PEI, which allows it to react with more carboxylic groups on the PAA layers further below it. This gives a larger rise for the peak at 1650 cm-1 of spectrum F. In fact, spectrum F shows that no or very few carboxylic groups remain on the PEI-topped layers (Figure 1). We observed this for all other PEMS topped with PEI. For the PEMS topped with PAA, however, the peaks at 1650 cm-1 and at 1550 cm-1 were almost the same height (Figure 1). A plot of the N-H amide peak intensity at 1550 cm-1 against number of layers is almost linear (Figure S1 Supporting Information). (46) Lin, C. C.; Lee, G. B.; Chen, S. H. Electrophoresis 2002, 23, 3550-3557.

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The PEMS topped with either PEI or PAA offer possibilities of immobilizing other polymers with useful specific properties on PDMS, imparting versatility to the surface. Most workers on PEMS have not explored the possibility of performing further reactions on top of the PEMS; instead they have incorporated the polymers within the PEMS themselves.47,48 While this is advantageous for simple organic molecules, it may not be the same for proteins, whose properties may be significantly altered when embedded in PEMS. For PEMS topped with PAA, PEG diamine MW 6000 was attached, while for a PEMS topped with PEI, PEG dicarboxylic acid MW 6000 and PEG dialdehyde of MW 3400 were used. PEG dicarboxylate and PEG diamine were attached by carbodiimide coupling to the top PEI and PAA polyelectrolyte layers, respectively. PEG dialdehyde was attached to the top PEI layer by reductive amination. ATR-FT-IR was also used to monitor PEG immobilization on the PEMS. Figure S2 (Supporting Information) shows the effect of attaching PEG diamine to an h-PSMA/ (PEI/PAA)4 thin film. It can be seen that there is not much change in the amide signals after the attachment of PEG diamine. There is a slight increase in the amide carbonyl stretch signal at 1650 cm-1 after the immobilization of PEG diamine. There is also a slight decrease in the carboxylic acid carbonyl stretch at 1733 cm-1 when PEG diamine is immobilized. This shows that the PEG diamine uses up some of the carboxylic groups on the PAA top layer. Figure S2 also shows that PEG diamine does not react with all the carboxylic groups on the PAA top layer because the peak at 1733 cm-1 is still visible, probably due to interchain repulsions. The attachment of PEG dicarboxylate and PEG dialdehyde to h-PSMA/(PEI/PAA)4/PEI film is shown in Figure 2. No detectable change in the spectrum was observed for the attachment of PEG dicarboxylate. For the attachment of PEG dialdehyde, there (47) Jessel, N.; Atalar, F.; Lavalle, P.; Mutterer, J.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Ogier, J. Adv. Mater. 2003, 15, 692-695. (48) Wu, A.; Yoo, D.; Lee, J. K.; Rubner, M. F. J. Am. Chem. Soc. 1999, 121, 4883-4891.

Figure 2. ATR-FT-IR spectra showing the effect of immobilizing PEG dicarboxylate 6000 and PEG dialdehyde 3400 on h-PSMA/(PEI/PAA)4PEI PEM thin film. (A) h-PSMA/(PEI/PAA)4PEI; (B) h-PSMA/(PEI/PAA)4PEI/PEG dicarboxylate; (C) h-PSMA/(PEI/PAA)4PEI/PEG dialdehyde 3400.

was a large reduction in the amide signals, which can be attributed to the utilization of some of the reactive amine groups in the PEMS during the attachment of PEG dialdehyde by reductive amination (Figure 2C). The PEG dialdehyde-coated film showed strong PEG signals. A medium-sized peak at 963 cm-1 is attributed to the PEG CH2 rocking vibrations. A strong sharp peak at 1107 cm-1 and a weaker one at 1147 cm-1 represent C-O-C stretching of the PEG chains24 (Figure 2C). The ether CH2 twisting modes can be seen around 1243 and 1281 cm-1. The band at 1349 cm-1 is for the ether CH2 wagging mode, while the signal at 1467 cm-1 represents alkyl CH2 scissoring.31 There is a band around 1716 cm-1, which can be associated mainly with a carbonyl stretch from residual aldehyde groups from PEG dialdehyde. The strong band with a maximum at 2883 cm-1 and with a shoulder at 2945 cm-1 is attributed to the PEG CH2 symmetric and asymmetric stretches respectively31 (Figure 2 inset). Hydroxyl groups produced by the aldol condensation show a broad weak signal between 3200 and 3650 cm-1 (Figure S3C Supporting Information). The spectra (Figure S3A and B Supporting Information) show the amine stretch at 3294 cm-1 due to PEI on the thin films h-PSMA/(PEI/ PAA)4/PEI and h-PSMA/(PEI/PAA)4/PEI/PEG dicarboxylate, respectively. This signal due to the amine stretch disappears when PEG dialdehyde is immobilized (Figure S3C Supporting Information). The dense surface coverage of PEG dialdehyde is due to the aldol condensation,49,50 which occurs during the reductive amination reaction. The PEG aldehyde chains polymerize by the aldol reaction, forming a dense PEG layer within the upper spaces of the PEMS. Such dense surface coverage is usually seen with PEG self-assembled monolayers on gold or silver substrates.32,33 The attachment of the PEG chains was also followed by XPS. For all the coatings, the XPS survey spectra showed the presence (49) Kingshott, P.; Thissen, T.; Griesser, H. J. Biomaterials 2002, 23, 20432056. (50) Margel, S.; Rembaum, A. Macromolecules 1980, 13, 19-24.

of the Si signal from the PDMS substrate, which means that the film thickness was within the 10-nm sampling depth of the XPS instrument. The thickness of the coatings could not be measured because of the surface roughness. We used a maximum of 4.5 bilayers, which would be ∼6 nm in thickness when using a literature value (1.2 nm) for a PEI-FITC/PAA bilayer (ref 1, Supporting Information). Also, the reduction of the amide signal (Figure 2C) after attachment of PEG dialdehyde shows that the PEG dialdehyde polymerizes within the upper spaces in the PEMS, using up some of the primary amine groups on the PEMS. This means that the attachment of PEG dialdehyde would not increase the film thickness that much. Figure 3 shows the XPS C 1s high-resolution spectra for the attachment of PEG dialdehyde to the PEM film h-PSMA/(PEI/PAA)4PEI. Figure 3B shows the effect of immobilization of PEG dialdehyde on h-PSMA/(PEI/ PAA)4PEI (Figure 3A). There is a dramatic increase in the percentage of the ether peak around 287-eV (30 ( 2% increase) and the increase is ascribed to the dense PEG layer. For PEG dicarboxylate immobilization, the carbon spectrum was very similar to that for h-PSMA/(PEI/PAA)4PEI as shown in the overlaid spectra in Figure S4 (Supporting Information) for comparison. The oxygen high-resolution spectrum gives an increase of 4.5 ( 0.25% for the peak at 533 eV after PEG dicarboxylate immobilization (figure not shown). The dense surface coverage of PEG dialdehyde is also due to the collapse of the PEG chains at 60 °C (the cloud point temperature of PEG), which reduces chain repulsions, resulting in higher grafting to the amine groups on the surface.49 The attachment of PEG diamine to the PEM h-PSMA/(PEI/PAA)4 did not result in a noticeable change in the high-resolution carbon spectrum. The oxygen high-resolution spectra, however, give a 10 ( 3% increase in the ether oxygen peak at 533 eV after immobilization of PEG diamine on h-PSMA/(PEI/PAA)4. Figure S5A and B (Supporting Information) shows sample spectra for the immobilization of PEG Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Figure 3. High-resolution XPS carbon spectra showing the effect of immobilizing PEG dialdehyde on the film h-PSMA/(PEI/PAA)4/PEI. (A) C 1s spectrum for h-PSMA/(PEI/PAA)4/PEI; (B) C 1s spectrum for h-PSMA/(PEI/PAA)4/PEI/PEG dialdehyde 3400.

diamine to h-PSMA/(PEI/PAA)4. The spectral data show that PEG dicarboxylate has low graft density, and this can be explained by the low percentage of primary amines on PEI (25%). Contact angle measurements were performed on some of the thin films. The contact angle of a water droplet on the h-PSMA/ (PEI/PAA)4/PEI thin film was measured to be 42.4°. Upon attachment of PEG dicarboxylate onto the h-PSMA/(PEI/PAA)4/ PEI film, the contact angle went down to 5.5°. Immobilization of PEG dialdehyde also resulted in a big reduction of the contact angle from 42.4° to 17.4° and then stabilized at 20°, which is much smaller than the value, 90.2°, reported for sol-gel modified surfaces.7 PEG diamine on h-PSMA/(PEI/PAA)4 also resulted in a noticeable change in the contact angle (53.4° to 32°). The surprisingly low contact angle obtained after PEG dicarboxylate immobilization can be attributed to the surface chemical heterogeneity,51,52 which results from the sparse distribution of the immobilized PEG chains. The influence of surface heterogeneity on the contact angle however is still not yet fully understood.51,52 Tapping mode AFM was used to study the surface morphology of the cross-linked thin films on PDMS substrates. The threedimensional (3D) 2 µm × 2 µm images for the coatings h-PSMA/ (PEI/PAA)2/PEI, h-PSMA/(PEI/PAA)2PEG diamine, and h-PSMA/ (PEI/PAA)2/PEI/PEG dialdehyde are shown in Figure 4A-C, respectively. Figure 4A shows the 3D image for the coating (51) Adamson, A. W.; Ghast, A. P. Physical Chemistry of Surfaces, 6th ed; John Wiley & Sons Inc: New York, 1997; p 357. (52) Leger, L.; Joanny, J. F. Rep. Prog. Phys. 1992, 431-486.

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h-PSMA/(PEI/PAA)2/PEI with a root-mean-square roughness (rms) value of 6.7 nm. The coating shows clusters that result from the cross-linked layers. Sparsely distributed clusters are known to occur for most polyelectrolyte films with such few layers.53-56 The 5 µm × 5µm 3D and 2D images for h-PSMA/(PEI/PAA) are shown in Figure S6A and B (Supporting Information) and 5 µm × 5µm 3D and 2D images for h-PSMA/(PEI/PAA)2/PEI (taken at a different position from the Image in Figure 4A) are shown in Figure S6C and D (Supporting Information). Evident are the small clusters that cover the underlying deformations (indicated by arrows) on the PDMS substrate. The clusters get larger as the number of layers increases (compare Figures S6B and S6D), similar to what has been observed in the literature.53,55 The deformations may also occur during the curing of the PDMS material. The bridging of the deformations by cross-linked PEMS8 helps in protecting the attached polymer from direct exposure to migrating hydrophobic groups. Figure 4B shows the 3D image for the coating h-PSMA/(PEI/PAA)2PEG diamine (rms 9.94). The image shows the formation of large, extensive features, which are associated with the PEG chains. This image suggests that the PEG diamine chains cover most of the PAA-topped PEM surface. Figure 4C shows the 3D image for the coating h-PSMA/(PEI/ PAA)2/PEI/PEG dialdehyde with rms value 11.3. The image shows that there is complete coverage of the surface by the polymerized PEG chains. This also confirms the ATR-FT-IR and XPS data. The ridge near the edge in Figure 4C may be due to the movements that could have occurred during the polymerization of the PDMS material. The sparsely immobilized PEG dicarboxylate is shown in Figure S6E and S6F (Supporting Information). Stability of the Thin-Film Modified Surfaces. The biggest problem with PDMS is that of surface instability. To our knowledge, there has been one real attempt to produce stable PDMS surfaces.57 We used ATR-FT-IR, XPS, contact angle, and electrokinetic measurements to study the stability of the cross-linked thin films. For ATR-FT-IR, the peak intensity of h-PSMA/(PEI/ PAA)4/PEI/PEG dialdehyde was monitored over a period of time. Figure S7A (Supporting Information) shows that the PEG signals between 900 and 1500 cm-1 are virtually unchanged during the 77 days of measurement. Figure S7B shows that the aldehyde and amide signals of the same h-PSMA/(PEI/PAA)4/PEI/PEG dialdehyde have the same trend, indicating that both the PEG top layer and the amide cross-links below the PEG layer are stable. For the period of the measurements, the PEG chains did not show any signs of degradation for which they are known.58,59 The unchanged ATR-FT-IR signals of the coatings taken over a period of time indicate that the thin films are not sinking into the PDMS. The spectra measurements were not repeated beyond 77 days due to the availability of the instruments, but the stability of the modification is expected to be longer based on the contact angle (53) Lowman, G. M.; Buratto, S. K. Thin Solid Films 2002, 405, 135-140. (54) Caruso, F.; Furlong, D. N.; Ichinose, I.; Kunitake, K. Langmuir 1998, 14, 4559-4565. (55) Therry, B.; Winnik, F. M.; Merhi, Y.; Silver, J.; Tabrizian, M. Biomacromolecules 2003, 4, 1564-1571. (56) Lobo, R. F. M.; Pereira-da-Silva, M. A.; Raposo, M.; Faria, R. M.; Oliveira, O. N. Nanotechnology, 2003, 14, 101-108. (57) Genzer, J.; Efimenko, K. Science 2000, 290, 2130-2133. (58) Brach, D. W.; Wheeler, B. C.; Brewer, G. J.; Leckband, D. E. Biomaterials 2001, 22, 1035-1047. (59) Sharma, S.; Johnson, R. W.; Desai, T. A. Langmuir 2004, 20, 348-356.

Figure 4. Tapping mode 3D images for some of the coatings on PDMS. (A) h-PSMA/(PEI/PAA)2/PEI, rms 6.7 nm, z-scale 120 nm. (B) h-PSMA/ (PEI/PAA)2/PEG diamine 6000, rms 9.94 nm, z-scale 150 nm. (C) h-PSMA/(PEI/PAA)2/PEI/PEG dialdehyde 3400, rms 11.3 nm, z-scale 200 nm.

and EOF measurements. The wettability of the modified surfaces was monitored by measuring changes in the contact angle with time. Figure S8 (Supporting Information) shows that the PEMS topped with PEG dialdehyde maintain a virtually constant contact angle for nearly five months. The other two coatings h-PSMA/ (PEI/PAA)4/PEG diamine and h-PSMA/(PEI/PAA)4PEI are slightly less constant than the PEG dialdehyde-topped thin films. We attribute this to contamination, because when the samples were washed with DI water and dried under nitrogen, the contact angles were reduced to almost the original value and for PEG diamine, the slight rise may also be due to the degradation of the PEG chains. The PEG dialdehyde coating seems to resist contamination since it was not washed with DI water during the measurements. The contact angle data, however, show that all the thin films are generally stable and have long-lasting hydrophilicity. To our knowledge, this is the first time that hydrophilic coatings on PDMS show such high stability in air over such a long period. XPS data for trends in the atomic percentages over time are shown for the films h-PSMA/(PEI/PAA)4/PEI, h-PSMA/(PEI/ PAA)4/PEI/PEG dicarboxylate, and h-PSMA/(PEI/PAA)4/PEI/ PEG dialdehyde (Table S1 Supporting Information). The carbon content was almost constant during the 77 days of measurement showing no signs of hydrophobicity attack. Examination of the high-resolution spectra of the thin-film coating h-PSMA/(PEI/ PAA)4/PEI/PEG dialdehyde showed that the ether peak from PEG at ∼287 eV was 55.8% of the total peak area on day 1 of the measurements, and on day 77 of the measurements, the ether peak was 55% of the total peak area (figures not shown). This indicates stability of the polymerized PEG dialdehyde chains. This also agrees with the ATR-FT-IR data shown in Figure S7. The coatings h-PSMA/(PEI/PAA)4/PEI/PEG dicarboxylate and h-PSMA/(PEI/PAA)4/PEI showed similar consistency in the percentage of functional groups. While degradation of the polymerized PEG dialdehyde is not excluded, many PEG groups should be oxidized before any detectable degradation can be seen in the spectra. The electrokinetic stability was tested over a period of five months by EOF measurements. EOF tests were performed at pH 4 on chips with the following cross-linked thin-film coatings: (1) h-PSMA/(PEI/PAA)2/PEI/PEG dicarboxylate, (2) h-PSMA/(PEI/ PAA)2/PEI/PEG dialdehyde, and (3) h-PSMA/(PEI/PAA)2/PEI. As shown in Figure S9, the EOF of the PEG dicarboxylate coating had a near-constant value of -1.2 × 10-4 cm2 v-1 s-1 for five months. The PEG dialdehyde coating also showed similar stability

with an EOF of around -2.5 × 10-4 cm2 v-1 s-1. The EOF value of the chip without PEG also had a constant value of around -3.23 × 10-4 cm2 v-1s- I . The higher EOF values for channels without PEG can be easily interpreted in terms of the available surface charge. The covalent binding of PEG dicarboxylate chains uses up some of the available amine groups of the PEI layer in forming amide bonds, which reduces the number of nitrogens that can be protonated. This is another proof for the binding of PEG dicarboxylate to the PEMS. To our knowledge, this is the first time that surface coatings on PDMS channels show electrokinetic stability over such a long period of time. In previous modifications, the modified channels were stable for about 45 h14 and 3 weeks.18 The EOF data show that the thin-film coatings produced in this work are permanently hydrophilic. The stability is owed to the cross-linking. Mechanically strong, homogeneous composites made of PEI, PAA, and single-walled nanotubes deposited as multilayers have been produced in Kotov’s group using amide and glutaraldehyde cross-linking, showing that ELBL coupled with cross-linking are powerful tools for making tough polymeric structures.29 From the EOF data, it can also be concluded that the coated films neither sink into the PDMS bulk nor delaminate from the PDMS substrate under exposure to liquid flow for long periods. This may be useful for medical devices that deliver fluids. Protein Adsorption on Modified Substrates and Electrophoretic Separation on the Modified Chip. Protein adsorption was quantified by subtracting the atomic percentage of nitrogen in unexposed substrate from the percentage after exposing the substrate, according to the XPS data. For substrates coated with PEG diamine and PEG dialdehyde, there was no detectable change in the nitrogen percentage after exposure to 100 ppm protein solutions (figure not shown). We then subjected the substrates to undiluted rat serum containing 48 mg/mL (48 000 ppm) proteins. Figure 5 shows the percentage change in nitrogen after exposure to undiluted rat serum. Oxidized PDMS had the highest increase in nitrogen percentage increase (Figure 5A). The coatings h-PSMA/(PEI/PAA)4/PEG diamine and h-PSMA/(PEI/ PAA)4/PEI/PEG dialdehyde showed increases in nitrogen of 0.8 and 0.6%, respectively. These represent 87 and 90% reduction in protein adsorption, respectively, when compared to oxidized PDMS. To our knowledge, such a high resiatnace to protein has not been previously reported on modified PDMS surfaces. A 95% reduction was obtained for gold surfaces coated with PEG copolymers after exposure to human serum (53 mg/mL protein) using surface plasmon resonance detection.33 The resistance could Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Figure 5. Changes in nitrogen percentage after exposure to undiluted rat serum. (A) Oxidized PDMS. (B) h-PSMA/(PEI/PAA)4/ PEI. (C) h-PSMA/(PEI/PAA)4/PEI/PEG dicarboxylate 6000. (D) h-PSMA/ (PEI/PAA)4/PEG diamine 6000. (E) h-PSMA/(PEI/PAA)4/PEI/PEG dialdehyde 3400.

be useful for making nonbiofouling surfaces on PDMS medical devices. The PDMS microchannels modified with the coating h-PSMA/ (PEI/PAA)2/PEI/PEG dialdehyde was tested for the separation of three proteins. Figure S10A and B (Supporting Information) shows chip schematic and the electropherogram, respectively. It is noticeable that all three proteins were well separated and no appreciable peak tailing or band broadening was noticed, indicating that no significant protein adsorption occurred. All the peaks had an efficiency of at least 56 000 N/m (theoretical plates per meter), which is similar to other separations reported in the literature.60 CONCLUSIONS We have shown for the first time that truly stable, hydrophilic coatings on PDMS surfaces can be achieved using simple ELBL (60) Dou, Y.-H.; Bao, N.; Xu, J.-J.; Meng, F.; Chen, H.-Y. Electrophoresis 2004, 25, 3024-3031.

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and chemical cross-linking. The thin-film coatings show high resistance to hydrophobicity recovery in air, and when topped with PEG chains, they show strong protein resistance in undiluted rat serum. The strong PEG peaks in the XPS and IR spectra show that the density of some of the PEG coatings produced in this work can be as high as that seen on surfaces such as gold. Also the amine and carboxylic groups in the thin films can allow for the immobilization of important biomolecules. This stability is also important for medical devices made of PDMS. For medical devices, cross-linkable biocompatible polyelectrolytes30,55 can be used before attaching nonpolyelectrolyte polymers. We have also shown for the first time that it is possible to have constant EOF on PDMS channels for months. The long-term stability and reproducibility of the coating could open ways for commercial PDMS microfluidic chips with hydrophilic surfaces. ACKNOWLEDGMENT This work was supported by the National Science Council of Taiwan. The authors also thank the Advanced Semiconductor Engineering Group (ASE INC), Kaohsiung, Taiwan, and the Center for Micro/Nano Technology Research, National Cheng Kung University, Tainen, Taiwan, for equipment access and technical support. SUPPORTING INFORMATION AVAILABLE ATR-FT-IR and XPS spectra, AFM images for some of the coatings, contact angle trends, EOF trends schematic for the microchip, electropherogram for protein separation, and table showing atomic percentages. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review February 14, 2005. Accepted March 9, 2005. AC0502706