Anal. Chem. 2003, 75, 2117-2122
Reactive Polymer Coatings: A First Step toward Surface Engineering of Microfluidic Devices Jo 1 rg Lahann,† Mercedes Balcells,‡,§ Hang Lu,† Teresa Rodon,† Klavs F. Jensen,† and Robert Langer*,†
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Harvard-Massachusetts Institute of Technology Biomedical Engineering Department, Cambridge, Massachusetts 02139, and Chemical Engineering Department, Institut Quı´mic de Sarria` , Barcelona, Spain
We report fabrication, characterization, and use of microfluidic analysis devices containing surface-immobilized cell-capturing molecules. Amino-terminated biotin ligands are immobilized onto the luminal surface of a microdevice and effectively support self-assembly of proteins, antibodies, and mammalian cells. For this purpose, chemical vapor deposition (CVD) polymerization is used to functionalize PDMS-made microfluidic devices with poly[para-xylylene carboxylic acid pentafluorophenolester-copara-xylylene]. The resulting reactive coating shows excellent adhesion when deposited in thin films (∼100 nm, and the distribution of the pentafluorophenol ester groups is reasonably uniform within the microchannel inner surface, as examined by fluorescence microscopy. The utility of these devices for cell-based bioassays is demonstrated by monitoring the concentration-dependent effect of the disintegrin echistatin on cell adhesion. The described assay format could be relevant to clinical research in various fields, including angiogenesis research. Miniaturized cell assays are of interest in the evaluation of pharmacologically active molecules, including molecules that affect cell proliferation and adhesion. Driven by major advances in microfluidic biosystems, such as the development of micro total analysis systems (µTAS),1 microfabricated cell sorters,2 microseparators for DNA3 and proteins,4,5 and cell-based assays,6 the microfabrication of biologically meaningful microenvironments is within the scope of recent activities.7 Bioassays that exploit miniaturized formats are intrinsically advantageous (in part * To whom correspondence should be addressed. Fax: 617-258-8827. E-mail:
[email protected]. † Massachusetts Institute of Technology. ‡ Harvard-Massachusetts Institute of Technology Biomedical Engineering Department. § Institut Quı´mic de Sarria`. (1) Berg, A.; Olthius, W.; Bergveld, P. In Micro Total Analysis Systems 2000; Kluiver Academics: Dordrecht, 2000. (2) Fu, A. Y.; Spence, C.; Scherer, F. H.; Arnold, F. H.; Quake, S. R. Nat. Biotechnol. 1999, 17, 1109-1111. (3) Effenhauser, C. S.; Bruin, J. M.; Paulus, A.; Ehrat, M. Anal. Chem. 1997, 69, 3451-3457. (4) Mao, H.; Yang, T.; Cremer P. S. Anal. Chem. 2002, 74, 379-385. (5) Chen, S. H.; Sung, W. C.; Lee, G. B.; Lin, Z. Y.; Chen, P. W.; Liao, P. C. Electrophoresis 2001, 22, 3972-3977. (6) Li, P.; Harrison, D. J. Anal. Chem. 1997, 69, 1564-1568. (7) Tien, J.; Chen, C. S. IEEE Eng. Med. Biol. 2002, 21, 95-98. 10.1021/ac020557s CCC: $25.00 Published on Web 04/02/2003
© 2003 American Chemical Society
because of their small sample volumes and massively parallel processing), but they heavily rely on defined surface characteristics, such as wettability, surface topology, and interfacial charge distribution. Therefore, defined and stable surface properties along with the capability to immobilize active biomolecules in the luminal surfaces of the microfluidic devices are keys to their use as analytical tools. Often in the past, microfluidic devices have been made of silicon or glass,8 but these materials may not be the first choice for many microfluidic applications, especially in biology or medicine.9 Several properties of silicon and glass could limit their use in microfluidic devices, including (i) limited biocompatibility, (ii) intrinsic stiffness, (iii) unfavorable geometry, and (iv) incompatibility with soft materials needed, for example, for the incorporation of valves.10 PDMS is often discussed as an alternative because of its favorable mechanical properties11 and its straightforward manufacturing by rapid prototyping.12 However, PDMS is hydrophobic and allows nonspecific protein adhesion.13 The absence of functional groups at the PDMS surface prevents covalent immobilization of proteins, enzymes, or antibodies. PDMS also suffers from the lack of defined and constant surface properties under ambient conditions.14 The high surface-to-volume ratios in microfluidic devices imply that slight inhomogeneities in the surface can cause device malfunction.15 Although several approaches have been described for surface modification of PDMS-based microfluidic devices, such as plasma treatment,12 silanization of previously oxidized PDMS,16 polymer grafting,17 adsorption of polyelectrolytes,18 adsorption of detergents or quaternary amines,13 and precoating with proteins,19 modified (8) Harrison, D. J.; Manz, A.; Fan Z. H.; Luedi, H.; Widmer H. M. Anal. Chem. 1992, 64, 1908-1919. (9) Quake, S. R.; Scherer A. Science 2000, 290, 1536-1540. (10) Unger, M. A.; Chou, H.-P.; Thorsen, T.; Scherer, A.; Quake, S. R. Science 2000, 288, 113-116. (11) Johnson, T. J.; Ross S.; Gaitan M.; Locascio L. E. Anal. Chem. 2001, 73, 3656-3661. (12) Duffy, D. C.; MacDonald, J. C.; Schueller, O. J. A.; Whitesides, G. A. Anal. Chem. 1998, 70, 4974-4984. (13) Ocvirk, G.; Munroe, M.; Tang, T.; Oleschuk, R.; Westra, K.; Harrison, D. J. Electrophoresis 2000, 21, 107-115. (14) Linder, V.; Verpoorte, E.; Thormann, W.; de Rooij, N. F.; Sigrist, H. Anal. Chem. 2001, 73, 4181-4189. (15) Li, Y.; Phohl, T.; Kim, J. H.; Yasa, M.; Wen, Z.; Kim, M. W.; Safina, C. R. Biomed. Microdevices 2001, 3, 239-244. (16) Grzybowski, B. A.; Haag, R.; Bowden, N.; Whitesides, G. M. Anal. Chem. 1998, 70, 4645-4652. (17) Hu, S.; Ren, X.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. Anal. Chem. 2002, 74, 4117-4123.
Analytical Chemistry, Vol. 75, No. 9, May 1, 2003 2117
Figure 1. Chemical vapor deposition polymerization of reactive coatings, such as PPX-PPF.
PDMS surfaces slowly recover their original hydrophobicity.14 Although surface modification of glass substrates via silane chemistry has been well-established, simple well-defined surface modification protocols for polymers, such as PDMS, are still being developed.20 The deposition of thin polymer films to establish chemically defined interfaces offers a unique way to overcome these limitations. Functionalized poly(p-xylylene)s are currently under investigation for protein attachment21-23 or for patterning of polymer brushes.24 Thin film deposition is usually conducted by CVD polymerization, a room temperature process that requires no catalyst, solvent, or initiator. Ideally, no byproducts are created.25 Recently, this approach was extended by deposition of reactive coatings (cf. Figure 1), that is, poly(p-xylylene carboxylic acid pentafluorophenolester-co-p-xylylene)26 (PPX-PPF) and poly(p-xylylene-2,3-dicarboxylic acid anhydride).27 Without the need for further activation, the high chemical reactivity of their functional groups supported conversion with biological ligands or proteins and was used for surface patterning using microcontact printing.28 In this work, we report the deposition of PPX-PPF on the luminal surface of a polymer-based microfluidic device. We had two objectives: First, to develop a simple procedure for protein immobilization within polymer-based lab-on-the-chip devices, such as PDMS-based microfluidic devices; second, to demonstrate the usefulness of these devices for screening of pharmacologically relevant compounds in cell-based assays. (18) Barker, S. L.; Tarlov, M. J.; Canavan, H.; Hickman, J. J.; Locascio, L. E. Anal. Chem. 2000, 72, 4899-4903. (19) Yang, T.; Jung, S.; Mao, H.; Cremer, P. S. Anal. Chem. 2001, 73, 165-169. (20) Henry, A. C.; Tutt, T. J.; Galloway, M.; Davidson, Y.; McWorter, C. S.; Soper, S. A.; McCarley, R. L. Anal. Chem. 2000, 72, 5331-5337. (21) Lahann, J.; Plu ¨ ster, W.; Klee, D.; Ho¨cker, H. Biomaterials 2001, 22, 817826. (22) Lahann, J.; Klee, D.; Ho ¨cker, H. Macromol. Rapid Commun. 1998, 19, 441444. (23) Lahann, J.; Langer, R. Macromolecules 2002, 35, 4380-4386. (24) Lahann, J.; Langer, R. Macromol. Rapid Commun. 2001, 22 (12), 968971. (25) Greiner, A. Trends Polym. Sci. 1997, 5, 12-16. (26) Lahann, J.; Choi, I. S.; Lee, J.; Jensen, K. F.; Langer, R. Angew. Chem., Int. Ed. Engl. 2001, 40, 3166-3168. (27) Lahann, J.; Balcells, M.; Rodon, T.; Lee, J.; Choi, I. S.; Jensen, K. F.; Langer, R. Langmuir 2002, 18, 3632-3638. (28) Lahann, J.; Langer, R. Polym. Preprints 2001, 42, 113-115.
2118
Analytical Chemistry, Vol. 75, No. 9, May 1, 2003
Figure 2. Schematic drawing that describes the bonding of CVDcoated PDMS to a glass slide.
EXPERIMENTAL SECTION Device Fabrication. The microfluidic devices were designed using Freehand 9.0 (Macromedia, San Francisco, CA), and printed on high-resolution emulsion transparencies (Pageworks, Cambridge, MA). The pattern was subsequently transferred using standard photolithography onto a layer of photopatternable epoxy SU-8-50 (Micro Chem Corp., Newton, MA) that was spin-coated on a silicon wafer. The SU-8-50 was then hard-baked at 105 °C and developed in propylene glycol methyl ether acetate. The negative master was silanized with tridecafluoro-1,1,2,2,-tetrahydrooctyl-1-trichlorosilane (UCT, Bristol, PA) to prevent PDMS from adhering to the surfaces during molding. The PDMS prepolymer (Essex Brownell, Trerose, PA) was molded on the negative master, cured for 2 h, and peeled off to form the devices. The reactive coating was then deposited onto the PDMS substrate (see the section below). Because the reactive coating could be oxidized in oxygen plasma, bonding of the microchannel to glass substrate was achieved by oxidizing only the surfaces outside the lumen of the channel and bonding with epoxy (cf. Figure 2). The oxidation facilitates the spreading of the epoxy on the surfaces and also strengthens the bond. Next, to provide fluidic access, holes were bored in the PDMS, and polypropylene tubing (VWR) was inserted. Liquids were supplied to the microfluidic device via syringes connected to the tubing. Precursor Synthesis and CVD Polymerization. [2.2]Paracyclophane pentafluorophenol ester (PPF) was synthesized via a three-step synthesis described in detail elsewhere.26 All products were characterized by infrared, nuclear magnetic resonance spectroscopy (1H and 13C), and mass spectrometry. The spectroscopic data were found to be in accordance with literature data.26 When used as precursor for CVD, the purity of PPF was above
98.5%, as determined by gas chromatography. PPX-PPF was obtained from PPF by CVD polymerization using an installation consisting of a sublimation zone, a pyrolysis zone, and a deposition chamber.21 A defined amount of starting material was placed in the sublimation zone, and the microfluidic device was fixed on the sample holder at a temperature below 15 °C. The pressure was adjusted to 0.12 mbar, and the pyrolysis zone was heated to 600 °C. Subsequently, starting material was slowly sublimed by increasing the temperature of the sublimation zone to 230 °C. Polymerization resulted in deposition of transparent polymer films on the substrate. Surface Characterization. XPS data were recorded on flat PDMS samples using an Axis Ultra X-ray photoelectron spectrometer (Kratos Analyticals) equipped with an AlKR X-ray source. Pass energy was 160 eV for survey spectra and 10 eV for highresolution spectra. All spectra were calibrated in reference to the unfunctionalized aliphatic carbon at a binding energy of 285.0 eV. Spectra were recorded on flat PDMS samples coated with PPXPPF. Spectroscopic ellipsometry was carried out for a coated silicon substrate on a variable angle spectroscopic ellipsometer (J. A. Woollam Inc.) using a Cauchy model for curve fitting. Thickness was detected on silicon slides (1 cm2) that were coated in parallel to the microfluidic devices. For fluorescence microscopy, samples were rinsed well with PBS and examined with a HFX-DX fluorescence microscope (Nikon, Japan) and a computeraided picture capturing system (IP-spectrum software). Film stability was examined by incubation of a flat PDMS substrate coated with the reactive coating in an aqueous PBS buffer (pH 7.4) for 7 days at room temperature. After drying the sample with a stream of nitrogen, adhesion of the reactive coating was verified by pressing a 1 cm2 of Scotch tape onto the polymer coating. After subsequent peeling off, the sample was examined by optical microscopy. Microfluidic Immunoassay. Microchannels coated with PPXPPF were filled with a solution of amino-terminated biotin (1 mM, dimethylformamide/ethanol, (10:90 v/v)) for 60 min. Biotin-coated samples were incubated in sterile Petri dishes with washing buffer (PBS buffer (pH 7.4) containing 0.1% (w/v) bovine albumin and 0.02% (v/v) Tween 20) for 30 min and with a solution of streptavidin (10 mM, Pierce, Rockford, IL) or fluorescein-labeled streptavidin (10 mM, Pierce) for another 60 min (all steps in PBS buffer containing 0.1% (w/v) bovine serum albumin and 0.02% (v/v) Tween 20). The microchannels were then rinsed three times with washing buffer (PBS, 0.02% (v/v) Tween 20) and exposed for 120 min to a solution of biotin-conjugated human anti-R5integrin (HAI, 6 µg/mL, Pharmingen, San Diego, CA). Microfluidic Cell Assay. Bovine aortic endothelial cells (BAEC) were purchased from Cell Systems, WA, and cultured as described elsewhere.30 Cells in passage 4 were used for attachment assays in the microchannels at 105 cell/mL in serum-free Dulbecco’s modified Eagle’s medium (DMEM) containing variable concentrations of echistatin for 4 h of seeding time. All experiments were carried out in triplicate. All values are reported as the mean standard deviation ((SD). Statistical analysis was performed by single factor ANOVA for repeated measures (29) Gorham, W. F.; Yeh, Y. L. J. Org. Chem. 1969, 34, 2366-2370. (30) Rosenthal, A. M.; Gotlieb, A. I. In Cell Cultue Techniques in Heart and Vessel Research; Piper, I., Ed.; Springer-Verlag: Berlin, 1990; pp 117-129.
followed by a paired t test. Values of P < 0.05 (1-tailed analysis) were considered significant. RESULTS AND DISCUSSION The reactive coating has several chemical features that make it a promising candidate for surface modification of microfluidic devices enabling surface engineering; specifically, (i) it establishes a chemical interface with high reactivity for primary amino groups, while preventing the underlying PDMS from swelling; (ii) aminoterminated biotin ligands substitute the pentafluorophenol groups within seconds, forming chemically stable amide bonds;31 and (iii) the poly(p-xylylene) backbone accounts for chemical inertness and insolubility. Affinity-capturing surfaces that interact specifically with biomolecules or cell receptors may be engineered using this approach (cf. Figure 3). The test format allows evaluation of potential lead compounds that target surface-expressed cell receptors. In the event of a tight interaction with the cell receptor, binding ability to the affinity-capturing microchannel surfaces is inhibited, resulting in reduced cell adhesion. The microscopic format of the device allows for monitoring of the drug activity in the microchannel using fluorescence-based techniques. Surface Modification of the PDMS Device. The microfluidic devices were fabricated using rapid-prototyping techniques.32 Changes in the original design or implementation of additional features were uncomplicated and allowed for accommodation of more complex layouts. The precursor PPF was synthesized from [2.2]paracyclophane via three-step synthesis.26 Reactive coating PPX-PPF was homogeneously deposited on the substrates by means of CVD polymerization. The CVD process was adapted from a procedure of Gorham, which is commercially used to produce solvent and pinhole-free coatings.29 In the CVD process, the dimer [2.2]paracyclophane is transferred into a pyrolysis zone after its sublimation. Control of polymerization parameters allows selective cleavage of the C-C single bonds, resulting in the corresponding quinodimethanes.33,34 Reaction conditions must be controlled to avoid decomposition of the functional groups under the conditions of quantitative conversion into quinodimethanes. In this study, purified PPF was sublimated under a reduced pressure of 0.12 mbar at temperatures above 230 °C. Sublimated PPF was then transferred to the pyrolysis zone, which was heated to 600 °C. Subsequently, p-quinodimethanes were transferred to a cooler deposition chamber where they spontaneously polymerized. During the polymerization, the temperature of the PDMS substrate was kept below 15 °C. Thicknesses of the PPX-PPF films were determined to be between 90 and 150 nm using spectroscopic ellipsometry. When synthesized under these conditions, the chemical composition of PPX-PPF was in good accordance with previously reported values, as determined by X-ray photoelectron spectroscopy27 (Table 1). No silicon signals were detected, implying homogeneous coating of the substrate. Deposited in thin films, the reactive coating showed excellent adhesion properties on the PDMS substrate, and there were no signs of (31) Yang, Z.; Belu, A. M.; Liebmann-Vinson, A.; Sugg, H.; Chilkoti, A. Langmuir 2000, 16, 7482-7492. (32) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. Engl. 1998, 37, 550575. (33) Simon, P.; Mang, S.; Hasenhindl, A.; Gronski, W.; Greiner, A. Macromolecules 1998, 31, 8775-8780. (34) Schmidt, C.; Stuempfen, V.; Wendorff, J. H.; Hasenhindl, A.; Gronski, W.; Ishaque, M.; Greiner, A. Acta Polymer. 1998, 49, 232-235.
Analytical Chemistry, Vol. 75, No. 9, May 1, 2003
2119
Figure 3. Schematic representation of the surface modification steps that lead to a microdevice with a biologically active surface. PDMS is first modified with a reactive coating, which is then used to bind biotin ligands and to self-assemble streptavidin. Biotin-labeled HAI is then bound to the modified PDMS surface and used to study cell surface receptor activity. Table 1. Chemical Composition of PPX-PPF-Coated PDMS in Atom-%, as Determined by XPS
BE [eV] calcd found storeda a
C-C
C-CO-O
C-O
C-CO-O
C-F
π f π*
CdO
C-O
C-F
Si
285.0 46.0 49.1 48.0
285.7 3.1 2.3 2.5
286.7 5.3 2.7 3.2
288.9 3.0 3.9 3.2
290.6 16.2 16.4 16.3
291.7 1.5 1.9
532.5 3.5 3.0 3.3
533.9 5.2 2.9 3.5
686.9 17.7 18.2 18.1
101.8 0 0 0
Surface stored under argon atmosphere for 3 months.
delaminating during the subsequent chemical procedures, as verified by optical microscopy. Furthermore, reactive coatings were stable in a dry air atmosphere for several weeks26 and insoluble in common solvents, such as dimethylformamide, chloroform, acetone, ethanol, or aqueous solutions, implying that the coating is homogeneous. Immobilization of Biomolecules. The adhesion of cells is a process essential to organogenesis, development, wound healing, angiogenesis, and tissue remodeling.35 Cell attachment to the extracellular matrix is primarily mediated by integrins, a widely expressed family of heterodimeric cell surface receptors. Synthetic molecules have been shown to modulate cell adhesion,36 whereas antibody blockage of the R5-integrin, a constituent of the Fn receptor in endothelial cells (EC), was recently shown to inhibit up-regulation of PGI2 production of EC cultured on Fn-coated tissue culture plates and to reduce cell adhesion.37 Other studies suggest the R5β1-integrin plays a crucial role in angiogenesis, resulting in tumor growth in vivo.38 By creating an integrincapturing surface within the inner lumen of a microchannel, cells can be studied within a functional microdevice. (35) Garcia, A. J.; Boettinger, D. Biomaterials 1999, 20, 2427-2433. (36) Rollins, B. J. Blood 1997, 90, 909-928. (37) Balcells, M.; Edelman, E. R. J. Cell. Physiol. 2002, 191, 155-161.
2120
Analytical Chemistry, Vol. 75, No. 9, May 1, 2003
For this purpose, amino-derived biotin ligands were covalently confined to the surface via formation of amide bonds.39 This ligand was chosen because it undergoes nearly quantitative conversion with surface-bound pentafluorophenol ester groups.31 Streptavidin was then used as a bivalent linker that allows orientationally defined confinement of the biologically active molecules. To examine the immobilization of biotin ligands within the microchannel, we allowed fluorescein-conjugated streptavidin to bind to the biotin-modified surface. Figure 4 shows a PDMS microchannel coated with PPX-PPF and modified with biotin, which was incubated with fluorescein-conjugated streptavidin for 60 min. Despite the brighter center region that may be attributed to nonuniform illumination, the distribution of fluorescence is reasonably uniform over the entire microchannel. The enhanced fluorescence at both borders of the channel indicates that streptavidin also was bound to the channel walls. These findings are in agreement with the outcome of an earlier study that focused on CVD modification of two-dimensional substrates.27 (38) Kim, S.; Bell, K.; Mousa, S. A.; Varner, J. A. Am. J. Pathol. 2000, 156, 13451362. (39) Hyun, J.; Zhu, Y.; Liebmann-Vinson, A.; Beebe, T. P.; Chilkoti, A. Langmuir 2001, 17, 6358-6367.
Figure 4. Fluorescence micrograph of the microfluidic device coated with PPX-PPF after immobilization of amino-terminated biotin and selfassembly of fluorescein-conjugated streptavidin.
To further use this method for immobilization of biotinylated biomolecules, the above-described procedure was slightly modified by using purified streptavidin instead of fluorescein-conjugated streptavidin. The use of streptavidin as a linker generates a universal platform for further attachment of biotin-conjugated proteins, since streptavidin has two pairs of binding sites on opposite faces. Streptavidin shows high-affinity binding for biotin (KD ) 10-14 M).40 Therefore, one pair of binding sites is used to link the protein to the biotin-coated surface, leaving two binding sites on the opposite face for further assembly of relevant biomolecules, such as enzymes, antibodies, polysaccharides, or polynucleotides, which were shown to undergo biotinylation.41 Thus, a microdevice with a biotin-presenting surface modification represents a platform with wide compatibility with existing bioassays. In our specific application, surface-confinement of biotinconjugated antibody HAI was achieved by incubating the microchannel with a mixture of albumin and HAI for 2 h at room temperature. It should be noted, at this point, that the biotinylation of HAI selectively targeted the heavy-chain of HAI to enable optimal exposure of cell-binding moieties. Biological Activity Monitored by a Cell-Based Assay. We proposed a cell-based adhesion assay to assess the in vitro activity of echistatin, a potent disintegrin and cell adhesion inhibitor. The basic assay layout is described in Figure 5. Immobilization of an antibody that specifically captures R5-integrin (HAI) to a biotinmodified, PPF-PPX-coated microdevice was used to confine endothelial cells inside the microfluidic system (Figure 3). Streptavidin served as a linker that selectively bound to both surface-confined biotin and biotin-labeled HAI. For cell immobilization, a suspension of BAEC in serum-free DMEM was then filled in the microchannels presenting R5integrin-capturing molecules (anti-HAI) bound throughout the luminal surface. We used this assay layout to study the pharmacological activity of echistatin, a small protein known to interact with the VAT-2 receptor.42 Cell suspensions in serum-free DMEM (40) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055-2060. (41) Savage, D.; Mattson, G.; Desai, S.; Neilander, G.; Morgensen, S.; Conklin, E. Avidin-Biotin Chemistry: A Handbook; Pierce Chemical Company: Rockford, IL, 1994. (42) Dennis, M. S.; Henzel, W. J.; Pitti, R. M.; Lipari, M. T.; Napier, M. A.; Deisher, T. A.; Bunting, S.; Lazarus, R. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2471-2475.
Figure 5. Schematic representation of the cell assay format used to study cell adhesion events in response to cell adhesion inhibitor candidates (e.g., echistatin).
containing increasing concentrations of echistatin were perfused into the modified microchannels. After 4 h of seeding time, nonadherent cells were separated by thorough rinsing, and the dose-dependent activity of echistatin was assessed by counting attached endothelial cells under the contrast microscope (Figure 6). Fn-coated tissue culture polystyrene (physisorbed fibronectin) was used as reference surface. The minimum concentration of echistatin for biological activity was determined to be 0.1 µg/mL (18.4 µM) and was equal for both anti-HIA-immobilized and Fn-coated surfaces. Above this minimum, inhibition of cell adhesion increased logarithmically (r2 ) 0.962) with the concentration of the disintegrin. For higher concentrations, the inhibition was higher on the antibody-coated surfaces than on the reference surfaces, revealing a higher selectivity of the antibody-immobilized surfaces toward EC adhesion. These results provide first evidence of the application of the above-described systems as cell-based biosensors for chemical or biological agents4 or microfluidic cell assays.43 CONCLUSIONS The need for increased throughput in drug-discovery screening while reducing development and operating costs is continuing to drive the development of microfluidic assays. However, broad use of polymer-based microfluidic devices is restricted by the lack of defined surface modification protocols. The aim of this study was to establish a general, but simple protocol for preparation of polymer-based microfluidic devices with defined and chemically reactive interfaces. We deposited submicrometer thin reactive coatings on the interior surface of microfluidic devices (prior to assembly) to provide defined and chemically reactive interfaces. These reactive coatings can be compatible with complex biological features, because they represent a designable interlayer stable under the conditions of the bioassay. Although demonstrated for a PDMS-based microfluidic device, the substrate-independent nature of CVD polymerization makes the procedure equally applicable to other polymer-based microfluidic devices (and glass or silicon). (43) Takayama, S.; Ostuni, E.; LeDuc, P.; Naruse, K.; Ingber, D. E.; Whitesides, G. M. Nature 2001, 411, 1016.
Analytical Chemistry, Vol. 75, No. 9, May 1, 2003
2121
Figure 6. Dose-dependent activity of the cell adhesion inhibitor echistatin. Echistatin’s biological activity is based on tight binding to R5integrin and decreases cell adhesion. (A) BAEC cells were preincubated for 4 h in a serum-free DMEM suspension containing echistatin of various concentrations and were then transferred into a microchannel decollated with anti-HAI. Echistatin binding activity was studied as a function of inhibition of the cell’s ability to adhere to the antibody-coated microchannel. Physisorbed fibronectin was used as reference. Values are reported as the mean ( SD, and statistical analysis was performed by single-factor ANOVA for repeated measures followed by a paired t test. Values of P < 0.05 (1-tailed analysis) were considered significant. (B) Representative micrograph of a device filled with BAEC that were preincubated with buffer without echistatin. (C) Representative micrograph of a device filled with BAEC that was preincubated with buffer with the highest echistatin concentration of 33 µg/mL (6 µM).
The resulting reactive coating was then used for immobilization and self-assembly of a cascade of biological ligands, proteins, and cells. By developing a microfluidic device with a biotin-presenting interior surface, a wide range of bioassays can potentially be conducted in a microfluidic format. We chose to illustrate a cellbased microfluidic assay and conducted dose-dependent studies on cellular interactions with cell adhesion mediators. While overcoming restrictions associated with conventional PDMS-based microfluidic devices, the methodology retains PDMSintrinsic advantages, for example, processing by rapid prototyping, broad availability, and low costs. The variability in functional groups that can be prepared by CVD polymerization allows the application-driven surface engineering of microfluidic devices. Reactive functional groups,23 such as those in PPX-PPF, enable immobilization of a wide variety of biomolecules, amino- or
2122 Analytical Chemistry, Vol. 75, No. 9, May 1, 2003
carboxylic acid groups that could control surface charges and electro-osmotic flows, and alkyl groups that could provide hydrophobic interfaces for electrochromatographic applications. ACKNOWLEDGMENT This work was supported by the Fonds der Chemischen Industrie and the National Institutes of Health. We further thank Prof. H. Ho¨cker and PD. Dr. D. Klee, RWTH Aachen, Germany for the use of their custom-built installation for CVD polymerization. M. Balcells thanks Prof. Elazer Edelman for his support (N.I.H. HL60407 and GM49039). Received for review February 13, 2003. AC020557S
September
9,
2002.
Accepted
