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Straightforward Protein Immobilization on Sylgard 184 PDMS Microarray Surface Kevin A. Heyries, Christophe A. Marquette,* and Loı¨c J. Blum Laboratoire de Ge´ nie Enzymatique et Biomole´ culaire, Institut de Chimie et Biochimie Mole´ culaires et Supramole´ culaires, UniVersite´ Lyon 1 - CNRS 5246 ICBMS, Baˆ timent CPE, 43, Bd du 11 NoVembre 1918, 69622 Villeurbanne, Cedex, France ReceiVed January 4, 2007 In this work, a straightforward technique for protein immobilization on Sylgard 184 is described. The method consists of a direct transfer of dried protein/salt solutions to the PDMS interface during the polymer curing. Such non-conventional treatment of proteins was found to have no major negative consequence on their integrity. The mechanisms of this direct immobilization were investigated using a lysine modified dextran molecule as a model. Clear experimental results suggested that both chemical bounding and molding effect were implicated. As a proof of concept study, three different proteins were immobilized on a single microarray (Arachis hypogaea lectin, rabbit IgG, and human IgG) and used as antigens for capture of chemiluminescent immunoassays. The proteins were shown to be easily recognized by their specific antibodies, giving antibody detection limits in the fmol range.
Introduction The past decade has witnessed a fast expansion of micro fabricated devices and especially biochips and integrated microarrays. These developments, concomitant with the microfluidic and microdevice growth, have pushed technology developers to find new materials to overcome the main disadvantagesscost and technical requirement (clean room)s of glass and silicon, traditionally used for biochips fabrication.1 Among a lot of proposed polymeric materials (PMMA, PTFE, FPE, and PDMS),2 poly(dimethyl)siloxane (PDMS), and particularly one of its elastomeric derivatives (Sylgard 184) rapidly became the most popular, thanks to its chemical and physical properties.3 Indeed, numerous applications in the field of medical and microengineering4 became possible because of the PDMS low toxicity, possible processing in standard laboratory conditions, low curing temperature, optical transparency, and cost effectiveness.5 However, despite these advantages, major drawbacks exist when working with bare PDMS which are its native highly hydrophobic properties, its relative permeability to solvent,6,7 and its biofouling tendency, leading to nonspecific adsorption * Corresponding author. E-mail:
[email protected]. (1) Sia, S. K.; Whitesides, G. M. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis 2003, 24 (21), 35633576. (2) Becker, H.; Locascio, L. E. Polymer microfluidic devices. Talanta 2002, 56 (2), 267-287. (3) Ng, J. M. K.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Components for integrated poly(dimethylsiloxane) microfluidic systems. Electrophoresis 2002, 23, 3461-3473. (4) Colas, A.; Curtis, J. Silicone biomaterials: history and chemistry & medical application of silicone, Academic Press ed.; Elsevier: The Netherlands, 2004; p 864. (5) Lottersy, J. C.; Olthuis, W.; Veltink, P. H.; Bergveld, P. The mechanical properties of the rubber elastic polymer polydimethylsiloxane for sensor applications. J. Micromech. Microeng. 1997, 7, 145-147. (6) Duineveld, P. C.; Lilja, M.; Johansson, T.; Inganas, O. Diffusion of solvent in PDMS elastomer for micromolding in capillaries. Langmuir 2002, 18 (24), 9554-9559. (7) Muzzalupo, R.; Ranieri, G. A.; Golemme, G.; Drioli, E. Self-diffusion measurements of organic molecules in PDMS and water in sodium alginate membranes. J. Appl. Polym. Sci. 1999, 74 (5), 1119-1128.
of proteins in many biomaterials applications.8,9 Regarding its convenience for biochip and microarray developments, the main negative aspect of PDMS is its chemical inertness8 which critically lowered the possibilities of immobilizing biomolecules directly on PDMS structures (microfluidic components). Numerous propositions have then been made to introduce reactive chemical functions on PDMS surfaces. Abundant examples were described based on oxygen plasma exposure of PDMS10 or to a lesser extent ozone exposure and UV treatment11 to generate surface silanol groups, allowing classical silane surface chemistry. These modifications were shown to be limited in time due to buried PDMS chains migration leading to hydrophobic recovery.12,13 Other interesting approaches were proposed based on UV graft polymerization,14 silanization of oxidized PDMS,15,16 phospholipid bilayer modifications,17,18 or polyelectrolytes multilayers (8) Mata, A.; Fleischman, A. J.; Roy, S. Characterization of Polydimethylsiloxane (PDMS) Properties for Biomedical Micro/Nanosystems. Biomed. MicrodeVices 2005, 7, 281-293. (9) Linder, V.; Verpoorte, E.; Thormann, W.; de Rooij, N. F.; Sigrist, H. Surface Biopassivation of Replicated Poly(dimethylsiloxane) Microfluidic Channels and Application to Heterogeneous Immunoreaction with On-Chip Fluorescence Detection. Anal. Chem. 2001, 73, 4161-4169. (10) Ye, H.; Gu, Z.; Gracias, D. H. Kinetics of Ultraviolet and Plasma Surface Modification of Poly(dimethylsiloxane) Probed by Sum Frequency Vibrational Spectroscopy. Langmuir 2006, 22 (4), 1863-1868. (11) Efimenko, K.; Wallace, W. E.; Genzer, J. Surface Modification of Sylgard184 Poly(dimethyl siloxane) Networks by Ultraviolet and Ultraviolet/Ozone Treatment. J. Colloid Interface Sci. 2002, 254 (2), 306-315. (12) Hillborga, H.; Anknerc, J. F.; Geddea, U. W.; Smithd, G. D.; Yasudae, H. K.; Wikstrom, K. Crosslinked polydimethylsiloxane exposed to oxygen plasma studied by neutron reflectometry and other surface specific techniques. Polymer 2000, 41, 6851-6863. (13) Hillborg, H.; Tomczak, N.; Olah, A.; Schonherr, H.; Vancso, G. J. Nanoscale Hydrophobic Recovery: A Chemical Force Microscopy Study of UV/ Ozone-Treated Cross-Linked Poly(dimethylsiloxane). Langmuir 2004, 20 (3), 785-794. (14) Hu, S.; Ren, X.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. Surface Modification of Poly(dimethylsiloxane) Microfluidic Devices by Ultraviolet Polymer Grafting. Anal. Chem. 2002, 74 (16), 4117-4123. (15) Papra, A.; Bernard, A.; Juncker, D.; Larsen, N. B.; Michel, B.; Delamarche, E. Microfluidic Networks Made of Poly(dimethylsiloxane), Si, and Au Coated with Polyethylene Glycol for Patterning Proteins onto Surfaces. Langmuir 2001, 17, 4090-4095. (16) Sui, G.; Wang, J.; Lee, C. C.; Lu, W.; Lee, S. P.; Leyton, J. V.; Wu, A. M.; Tseng, H. R. Solution-Phase Surface Modification in Intact Poly(dimethylsiloxane) Microfluidic Channels. Anal. Chem. 2006, 78 (15), 5543-5551. (17) Mao, H.; Yang, T.; Cremer, P. S. Design and Characterization of Immobilized Enzymes in Microfluidic Systems. Anal. Chem. 2002, 74, 379-385.
10.1021/la070018o CCC: $37.00 © 2007 American Chemical Society Published on Web 03/14/2007
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depositions.19,20 Photoinduced polymer grafting21 or photografting polymer using benzophenone22 were also used to create an intermediate layer on PDMS surfaces. All of these methods suffer from several drawbacks which for the most critical are the chemical instability of the surface modification obtained and the lack of a simple process for the immobilization of biomolecules. Developing such direct modification of PDMS surfaces with biomolecules, in a microarray format, has been the main objective of our group. Thus, a method was proposed for the direct entrapment in PDMS of micro (120-1 µm)23,24 and nano (330-50 nm)25,26 beads, bearing biological molecules such as enzymes, antibodies, oligonucleotides, and peptides. The beads were then spotted and dried on a 3D master, covered with Sylgard 184, cured, and recovered, after peeling off, as spots of beads entrapped at the surface of the bare PDMS. We propose herein to push forward this methodology to demonstrate the possibility of functionalizing the PDMS surface by direct entrapment of biomolecules. Thus, the present work will demonstrate the surface incorporation, during the PDMS curing, of molecules as small as 3000 Da (dextran polymer) or as fragile as proteins (antibodies). The mechanism of this immobilization will be studied, and a model will be proposed based on experimental evidence. Morphological studies through atomic force microscopy of the spots obtained in different conditions will also be proposed and discussed according to the analytical signal measured.
Experimental Section Materials. Arachis hypogaea lectin (from peanut), anti-Arachis hypogaea lectin antibodies developed in rabbit, human IgG, luminol (3-aminophthalhydrazide), and peroxidase-labeled streptavidin were purchased from Sigma (France). Peroxidase-labeled polyclonal anti-human Ig(G, A, M) antibodies developed in goat and peroxidase-labeled polyclonal anti-rabbit IgG(H+L) antibodies developed in mouse were supplied by Jackson ImmunoResearch (USA). Biotin-labeled dextran (3 and 500 kDa, lysine fixable) and biotin-labeled dextran (3 kDa) were obtained from Molecular Probes (The Netherlands). Immunoglobulins from rabbit serum (rabbit IgG) were obtained from Life Line Lab (Pomezia, Italy). The PDMS precursor and curing agent (Sylgard 184) were supplied by Dow Corning (France). All buffers and aqueous solutions were made with distilled, demineralized water. (18) Yang, T.; Baryshnikova, O. K.; Mao, H.; Holden, M. A.; Cremer, P. S. Investigations of Bivalent Antibody Binding on Fluid-Supported Phospholipid Membranes: The Effect of Hapten Density. J. Am. Chem. Soc. 2003, 125 (16), 4779-4784. (19) Makamba, H.; Hsieh, Y.-Y.; Sung, W.-C.; Chen, S.-H. Stable Permanently Hydrophilic Protein-Resistant Thin-Film Coatings on Poly(dimethylsiloxane) Substrates by Electrostatic Self-Assembly and Chemical Cross-Linking. Anal. Chem. 2005, 77, 3971-3978. (20) Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S. Dynamic Coating Using Polyelectrolyte Multilayers for Chemical Control of Electroosmotic Flow in Capillary Electrophoresis Microchips. Anal. Chem. 2000, 72, 5939-5944. (21) Goda, T.; Konno, T.; Takai, M.; Moro, T.; Ishihara, K. Biomimetic phosphorylcholine polymer grafting from polydimethylsiloxane surface using photo-induced polymerization. Biomaterials 2006, 27 (30), 5151-5160. (22) Wang, Y.; Lai, H.-H.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L. Covalent Micropatterning of Poly(dimethylsiloxane) by Photografting through a Mask. Anal. Chem. 2005, 77, 7539-7546. (23) Marquette, C. A.; Blum, L. J. Direct immobilization in poly(dimethylsiloxane) for DNA, protein and enzyme fluidic biochips. Anal. Chim. Acta 2004, 506 (2), 127-132. (24) Marquette, C. A.; Blum, L. J. Conducting elastomer surface texturing: a path to electrode spotting: Application to the biochip production. Biosens. Bioelectron. 2004, 20 (2), 197-203. (25) Marquette, C. A.; Degiuli, A.; Imbert-Laurenceau, E.; Mallet, F.; Chaix, C.; Mandrand, B.; Blum, L. J. Latex bead immobilisation in PDMS matrix for the detection of p53 gene point mutation and anti-HIV-1 capsid protein antibodies. Anal. Bioanal. Chem. 2005, 381 (5), 1019-1024. (26) Marquette, C. A.; Cretich, M.; Blum, L. J.; Chiari, M. Protein microarrays enhanced performance using nanobeads arraying and polymer coating. Talanta 2006, in press, corrected proof.
Figure 1. Overview of the technique highlighting the four main steps leading to the achievement of protein spots directly entrapped at the PDMS interface.
Biochip Preparation (Figure 1). The biochips were prepared by arraying 1.3 nL drops of spotting solutions with a BioChip Arrayer BCA1 (Perkin-Elmer). Spotting solutions were prepared, when not mentioned, in carbonate buffer 0.1 M pH 9. Proteins (human IgG, rabbit IgG, and peanut lectin) spotting solutions were prepared at 1 mg/mL. Biotin modified dextran spotting solutions were prepared to contain a constant concentration of biotin of 232 µmol/L. Each array was composed of 16 spots (identical or not, diameter ) 150 µm) that were deposited on the surface of a 3D Teflon master composed of 24 rectangular structures (w ) 5 mm, l ) 5 mm, h ) 1 mm). Teflon was chosen as the deposition material according to its hydrophobicity and its convenience for 3D micromachining. After spotting, the drops were dried, and the arrays were transferred to the PDMS interface, by pouring a mixture of precursor and curing agent (10:1) onto the Teflon substrate and curing for 20 min at 90 °C. Peeling off the PDMS polymer then terminated the biochip preparation. Prior to any further use, the biochips were saturated with VBSTA (Veronal buffer 30 mM, NaCl 0.2 M, pH 8.5 with addition of tween 20 0.1% v/v and BSA 1% w/v) for 20 min at 37 °C. Immobilized Molecules Detection. The immobilized molecules (i.e., biotinylated dextran, Arachis hypogaea lectin, rabbit IgG, or human IgG) were detected through chemiluminescent labeling using peroxidase-labeled streptavidine, anti-lectin,
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Table 1. Analytical Characteristics of Rabbit IgG Microarrays Prepared Using Different Immobilization Procedures immobilization method
signala (SD)
latex 1 µm26 silica 330 nm26 direct entrapment of 10-15 nm proteins
20420 a.u. (9.4%) 8748 a.u. (13.5%) 20240 a.u. (8.2%) (Supporting Information 1)
LOD,b ng/mL 100 50 10
a Calculated from three microarrays incubated with 1 µg/mL of antirabbit IgG. b Limit of detection (LOD) calculated for a signal-to-noise ratio of 3.
peroxidase-labeled anti-rabbit, or anti-human IgG, respectively. The different labeled proteins were incubated (20 µL) on the saturated microarrays for 1 h at 37 °C and the excess reagents washed out with a 20 min incubation in VBS (Veronal buffer 30 mM, NaCl 0.2 M, pH 8.5). The microarrays were then placed in the CCD camera’s (Las1000 Plus, Intelligent Dark Box II, FUJIFILM) measurement chamber for light integration for 10 min (measuring solution: VBS containing in addition 220 µM of luminol, 200 µM of p-iodophenol and 500 µM of hydrogen peroxide). The numeric micrographs obtained were quantified with a FUJIFILM image analysis program (Image Gauge 3.122).
Results The immobilization of biomolecules and particularly proteins has been one of the major targets of our group for the last 15 years.27 The last 3 years have been particularly devoted to the development of innovative immobilization methods, compatible with the spotting technology widely used for microarrays. Thus, technological solutions for spotting beads bearing protein were proposed based on the entrapment of those beads at the PDMS/ air interface.23-26 In the present work, we highlight an interesting phenomenon leading to the transfer to the elastomer/air interface of proteins not preimmobilized on carrier beads. Table 1 summarizes the results obtained with previously described microarrays prepared with 1 µm latex beads bearing rabbit IgG or 330 nm silica beads bearing rabbit IgG and with the actual free rabbit IgG system. The three different microarrays were prepared using a similar protocol (i.e., spotting, drying, molding of PDMS, curing, and peeling off) and incubated with peroxidaselabeled anti-rabbit IgG antibodies. Surprisingly, the direct entrapment was found to be more effective than the beads-based format previously developed. These results suggest that lowering the size of the entrapped entity, from a 1 µm bead to 10-15 nm immunoglobulin protein,28,29 does not fail the immobilization of accessible rabbit IgGs. The analytical signal obtained is then really convincing with high chemiluminescent intensities obtained with a relatively low SD value, giving the best limit of detection (LOD) of the three systems. Since rabbit IgG/anti-rabbit IgG are model proteins with wellknown high affinity and stability,30,31 weaker recognition systems (27) Blum, L. J.; Coulet, P. R. Biosensor Principles and Applications; Marcel Dekker: New York, 1991; p 357. (28) Godoy, S.; Chauvet, J. P.; Boullanger, P.; Blum, L. J.; Girard-Egrot, A. P. New Functional Proteo-glycolipidic Molecular Assembly for Biocatalysis Analysis of an Immobilized Enzyme in a Biomimetic Nanostructure. Langmuir 2003, 19 (13), 5448-5456. (29) Godoy, S.; Violot, S.; Boullanger, P.; Bouchu, M.-N.; Leca-Bouvier, B. D.; Blum, L. J.; Girard-Egrot, A. P. Kinetics Study of Bungarus fasciatus Venom Acetylcholinesterase Immobilised on a Langmuir-Blodgett Proteo-Glycolipidic Bilayer. ChemBioChem 2005, 6 (2), 395-404. (30) Deshpande, S. S. Enzyme Immunoassays, Chapman & Hall ed.; ITP: New York, 1996; p 464. (31) The Immunoassay Handbook, 3rd ed.; Elsevier: The Netherlands, 2005; p 930.
Figure 2. Proposed mechanisms for the interactions between protein and PDMS during the elastomer curing step.
such as human IgG/anti-human IgG and peanut allergen (Arachis hypogaea lectin)/anti-allergen were studied. In every case, a very good recognition of the immobilized protein was experienced, with no possibility of removing the immobilized entities, even in very harsh conditions. Indeed, proteins/PDMS microarrays were submitted to a vigorous washing under stirring in 100 mL of VBSTA buffer for 18 h. Protein spots morphologies before and after immersion were compared using optical microscopy, and no major change was noticed. Moreover, the variation of the immobilized protein reactivity before and after immersion was found to be 10%. The proteins were then firmly immobilized at the PDMS interface. The effect of the polymerization process (drying and heating 20 min at 90 °C), which submits proteins to denaturing conditions, was investigated. Indeed, these uncommon conditions are not supposed to be compatible with the proteins used for immunodetection. However, protein drying for immunoassay developments has been already extensively used, particularly within the micro-contact printing field,32,33 demonstrating the protein stability following such treatment.34 To evaluate the effect of the curing step (90 °C) on the integrity of the dried proteins, microarrays were prepared by polymerizing PDMS at room temperature (25 ( 2°C) onto protein spots for 48 h. The analytical performances of these microarrays were found to compare well with the ones prepared at 90 °C, evidencing the low effect of the elevated temperature on the subsequent immobilized antigenantibody recognition. Different mechanisms could be involved in the direct immobilization of accessible proteins during the PDMS curing (Figure 2). First a molding effect, comparable to the key/lock couples observed within the molecular imprinting research field.35-37 Then, hydrophobic interactions, as shown by Bartzoka (32) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Printing Patterns of Proteins. Langmuir 1998, 14 (9), 2225-2229. (33) Arjan, P. Q.; Elisabeth, P.; Sven, O. Recent advances in microcontact printing. Anal. Bioanal. Chem. 2005, 381 (3), 591-600. (34) LaGraff, J. R.; Chu-LaGraff, Q. Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments. Langmuir 2006, 22 (10), 4685-4693. (35) Turner, N. W.; Jeans, C. W.; Brain, K. R.; Allender, C. J.; Hlady, V.; Britt, D. W. From 3D to 2D: A Review of the Molecular Imprinting of Proteins. Biotechnol. Prog. 2006, in press. (36) Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O’Mahony, J.; Whitcombe, M. J. Molecular imprinting science and technology: a survey of the literature for the years up to and including 2003. J. Mol. Recognit. 2006, 19 (2), 106-180. (37) Marty, J. D.; Mauzac, M. Molecular imprinting: State of the art and perspectives. Microlithography - Molecular Imprinting; Springer: Berlin, 2005; Vol. 172, pp 1-35.
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Figure 3. Effect of different amino acids on the chemiluminescent signals obtained from microarrays composed of directly immobilized lysine-modified biotinylated dextran. The immobilized dextran was detected through peroxidase-labeled streptavidin 1 µg/mL (30 min, 37 °C).
and co-worker,38,39 could also be involved between proteins and uncured PDMS. Finally, covalent bindings between the protein and the polymer could occur while PDMS is curing, mainly through poisoning of the Kardstedt catalyst40-42 by the amino or thiol groups of the protein lateral chains (as lone pair electron donors; Supporting Information 2). Dextran chains bearing biotin and lysine residues were chosen as model molecules to study this direct entrapment. The presence of the accessible immobilized molecule was then evidenced using peroxidase labeled streptavidine and chemiluminescent imaging. 500 kDa and 3 kDa dextran chains were thus successfully immobilized at the PDMS/air interface. The very large size difference between the two polymers did not appear to critically influence the immobilization efficiency, proving that molecules as small as 3000 Da could be trapped and accessible at the elastomer surface. Within the dextran chains used, only the amino group of the lysine lateral chains could be involved in a chemical reaction with the Kardstedt catalyst during the Sylgard 184 curing. As a control experiment, dextran chains not bearing any lysine residues were spotted and transferred to the elastomer. The chemiluminescent signal obtained was then 45% of the initial signal, demonstrating the implication of the amino group in the immobilization process but also evidencing the molding effect implicated in 45% of the immobilization efficiency. Further studies were performed to complete this theoretical immobilization mechanism. 3 kD dextran molecules bearing lysine residues were spotted in the presence of different concentrations of free amino acids: glycine, lysine, and cysteine (Figure 3). Glycine, with only its R-amino group, was found to (38) Bartzoka, V.; Brook, M. A.; McDermott, M. R. Silicone-Protein Films: Establishing the Strength of the Protein-Silicone Interaction. Langmuir 1998, 14 (7), 1892-1898. (39) Bartzoka, V.; Brook, M. A.; McDermott, M. R. Protein-Silicone Interactions: How Compatible Are the Two Species? Langmuir 1998, 14 (7), 1887-1891. (40) Perutz, S.; Kramer, E. J.; Baney, J.; Hui, C. Y.; Cohen, C. Investigation of adhesion hysteresis in poly(dimethylsiloxane) networks using the JKR technique. J. Polym. Sci. Part B: Polym. Phys. 1998, 36 (12), 2129-2139. (41) Quirk, R. P.; Kim, H.; Polce, M. J.; Wesdemiotis, C. Anionic Synthesis of Primary Amine Functionalized Polystyrenes via Hydrosilation of Allylamines with Silyl Hydride Functionalized Polystyrenes. Macromolecules 2005, 38, 78957906. (42) Katsuhiko Kishi, T. I.; Ozono, M.; Tomita, I.: Endo, T. Development and application of a latent hydrosilylation catalyst. IX. Control of the catalytic activity of a platinum catalyst by polymers bearing amine moieties. J. Polym. Sci. Part A: Polym. Chem. 2000, 38 (5), 804-809.
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Figure 4. Atomic force microscopy (NT-MDT, tapping mode) images of spots of lysine modified biotinylated dextran obtained in water (A) or in 0.1 M carbonate buffer, pH 9 (B). The arrows indicate the edge of each spot. The straight lines correspond to the presented profiles.
have little effect on the immobilization of the dextran molecules, even at high concentration (50 mM). On the contrary, lysine and cysteine were shown to inhibit strongly and with a dose dependence relation the immobilization of the biotinylated polymer. These results are in agreement with the involvement of a poisoning of the Kardstedt catalyst in the immobilization process. A drastic effect of the cysteine, which is known as a very efficient Kardstedt catalyst poison,43 was observed. Indeed, 78% and 40% of the immobilization of the 3 kDa dextran was inhibited by the presence of the maximum concentration of cysteine and lysine, respectively. The 60% of remaining immobilization in the presence of 50 mM lysine could then be attributed to the others proposed mechanisms (i.e., molding effect and hydrophobic interactions). Regarding the poor hydrophobicity of the dextran backbone, hydrophobic interactions were believed to be minimum. Potential interactions through the carbohydrate moiety of the lysine-dextran were then also considered. Thus, immobilization inhibition tests were performed by spotting dextran in the presence of maltose (a subunit of dextran). No effect on the lysinedextran immobilization was observed for maltose concentrations up to 100 mM. According to the results presented above, two mechanisms appeared to be preponderant in the actual lysine-dextran immobilization on PDMS: chemical bounding through poisoning of the Kardstedt catalyst by the primary amine of the lysine residue and a molding effect, similar to a key/lock mechanism. Our previous works on bead assisted protein immobilization on microarrays25,26 have demonstrated the usefulness of increasing the specific area of the spots. Indeed, increasing this area while keeping constant the geometrical one enables the immobilization of a higher amount of proteins per spot and then the increase of the microarray performances. Herein, since the proteins are spotted and transferred directly to the PDMS interface, an original way to increase this specific area has been to use spot solutions with relatively high salt concentration. Indeed, the salt charged protein (43) Marian, M.; Winter, H. H. Relaxation patterns of endlinking polydimethylsiloxane near the gel point. Polym. Bull. 1998, 40 (2), 267-274.
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a concentration of 1 mg/mL in carbonate buffer (pH 9) and transferred to the PDMS interface. The spotting pattern appeared on the upper part of Figure 5. Arachis hypogaea lectin, rabbit IgG, and human IgG were spotted as eight replicas. Bovine serum albumin (BSA) was used as a negative control for all of the tested antibodies (i.e., anti-rabbit IgG, anti-human IgG, and antilectin). As can be seen (Figure 5), absolutely no nonspecific signal was detected outside of the area delimited by the specific protein spots. Classical dose response curves can be observed (Supporting Information 1) from each range of antibody concentrations tested, giving detection limits of 20 ng/mL for anti-rabbit IgG and anti-human IgG and 10 ng/mL for antilectin. These concentrations correspond, in the 20 µL incubation volume, to amounts of antibody in the fmol range (2.6 and 1.3 fmol), which are considered low enough for most of the major immunoassay applications.31,44
Conclusion
Figure 5. Spotting pattern and chemiluminescent image of the microarray prepared by spotting Arachis hypogaea lectin, rabbit IgG, and human IgG in 0.1M carbonate buffer (pH 9).
solutions crystallize during the drying step, leading to highly textured surfaces. Thus, during the PDMS pouring and drying steps, these surfaces were used as a master to produce PDMS replica entrapping proteins, having a high specific area. Two examples of this technique are illustrated by the AFM images of protein spots obtained in pure water and in the presence of carbonate buffer 0.1 M (Figure 4). The two spots obviously exhibit very different surfaces as evidenced by the spot profiles. Calculated from the AFM images, the specific area increasing between both spots was found to be 1 order of magnitude. This difference of the surface geometry has a direct repercussion on the chemiluminescent signal obtained from those spots. Indeed, a more than 200% increase of the signal was observed when carbonate was added to the spotting solution, and this was irrespective of the buffer pH used (7, 9, and 11). This enhancement of the signal is then not linked to an increase of the reactivity of the lysine chains amino group at high pH but to the actual increase of the specific area of the spot. In order to fully characterize the analytical possibilities of the developed microarray, three different proteins were spotted at
In summary, we have developed a new approach to directly modify Sylgard 184 surfaces with spots of protein and demonstrated its use for microarray-based immunoassays. The mechanisms of this direct protein immobilization have been investigated, and clear experimental results suggested that both chemical bonding and molding effects were implicated in the observed phenomenon. Chemical bonding between the protein and the PDMS elastomer structure was believed to be mainly due to a poisoning of the Kardstedt catalyst used during the polymer curing. This poisoning was demonstrated to be related, with reference to previous works,38,40,41 to the presence of free amino or thiol groups in the immobilized molecules. Moreover, one interesting point is that Si-H functions have been reported to be sensitive to hydrolysis45 and, regarding the influence of nucleophilic groups such as primary amino groups, it could be postulated that interactions could also occurr between protein lateral chains and Si-H functions. Future work includes the integration of such microarrays in microfluidic systems, thanks to the use of PDMS as immobilization support. Acknowledgment. Published with the support of the European Commission, Sixth Framework Program, Information Society Technologies. NANOSPAD (No. 016610). Supporting Information Available: Dose response curves and the kardstedt catalyst reaction cycle. This material is available free of charge via the Internet at http://pubs.acs.org. LA070018O (44) Wu, A. H. B. A selected history and future of immunoassay development and applications in clinical chemistry. Clin. Chim. Acta 2006, 369 (2), 119-124. (45) Brook, M. A. Silicon in Organic, Organometallic, and Polymer Chemistry; John Wiley & Sons: New York, 2000.
