Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Straightforward Protein Immobilization on Sylgard 184 PDMS
                                           Microarray Surface
B Langmuir                                                                                                                ...
Protein Immobilization on Sylgard 184                                                                                     ...
D Langmuir                                                                                                                ...
Protein Immobilization on Sylgard 184              PAGE EST: 4.8                                                          ...
Upcoming SlideShare
Loading in …5

Straightfoward Protein Immobilization...


Published on

Published in: Technology, Business
  • Be the first to comment

  • Be the first to like this

Straightfoward Protein Immobilization...

  1. 1. Straightforward Protein Immobilization on Sylgard 184 PDMS Microarray Surface Kevin A. Heyries, Christophe A. Marquette,* and Loıc J. Blum ¨ Laboratoire de Genie Enzymatique et Biomoleculaire, Institut de Chimie et Biochimie Moleculaires et ´ ´ ´ Supramoleculaires, UniVersite Lyon 1 - CNRS 5246 ICBMS, Batiment CPE, 43, Bd du 11 NoVembre ´ ´ ˆ 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 of proteins in many biomaterials applications.8,9 Regarding its convenience for biochip and microarray developments, the main The past decade has witnessed a fast expansion of micro negative aspect of PDMS is its chemical inertness8 which critically fabricated devices and especially biochips and integrated lowered the possibilities of immobilizing biomolecules directly microarrays. These developments, concomitant with the mi- on PDMS structures (microfluidic components). Numerous crofluidic and microdevice growth, have pushed technology propositions have then been made to introduce reactive chemical developers to find new materials to overcome the main functions on PDMS surfaces. Abundant examples were described disadvantagesscost and technical requirement (clean room)s based on oxygen plasma exposure of PDMS10 or to a lesser of glass and silicon, traditionally used for biochips fabrication.1 extent ozone exposure and UV treatment11 to generate surface silanol groups, allowing classical silane surface chemistry. These Among a lot of proposed polymeric materials (PMMA, PTFE, modifications were shown to be limited in time due to buried FPE, and PDMS),2 poly(dimethyl)siloxane (PDMS), and par- PDMS chains migration leading to hydrophobic recovery.12,13 ticularly one of its elastomeric derivatives (Sylgard 184) rapidly Other interesting approaches were proposed based on UV graft became the most popular, thanks to its chemical and physical polymerization,14 silanization of oxidized PDMS,15,16 phospho- properties.3 Indeed, numerous applications in the field of medical lipid bilayer modifications,17,18 or polyelectrolytes multilayers and microengineering4 became possible because of the PDMS low toxicity, possible processing in standard laboratory conditions, (8) Mata, A.; Fleischman, A. J.; Roy, S. Characterization of Polydimethyl- low curing temperature, optical transparency, and cost effective- siloxane (PDMS) Properties for Biomedical Micro/Nanosystems. Biomed. MicrodeVices 2005, 7, 281-293. ness.5 (9) Linder, V.; Verpoorte, E.; Thormann, W.; de Rooij, N. F.; Sigrist, H. However, despite these advantages, major drawbacks exist Surface Biopassivation of Replicated Poly(dimethylsiloxane) Microfluidic Chan- nels and Application to Heterogeneous Immunoreaction with On-Chip Fluorescence when working with bare PDMS which are its native highly Detection. Anal. Chem. 2001, 73, 4161-4169. hydrophobic properties, its relative permeability to solvent,6,7 (10) Ye, H.; Gu, Z.; Gracias, D. H. Kinetics of Ultraviolet and Plasma Surface Modification of Poly(dimethylsiloxane) Probed by Sum Frequency Vibrational and its biofouling tendency, leading to nonspecific adsorption Spectroscopy. Langmuir 2006, 22 (4), 1863-1868. (11) Efimenko, K.; Wallace, W. E.; Genzer, J. Surface Modification of Sylgard- 184 Poly(dimethyl siloxane) Networks by Ultraviolet and Ultraviolet/Ozone * Corresponding author. E-mail: Treatment. J. Colloid Interface Sci. 2002, 254 (2), 306-315. (1) Sia, S. K.; Whitesides, G. M. Microfluidic devices fabricated in poly- (12) Hillborga, H.; Anknerc, J. F.; Geddea, U. W.; Smithd, G. D.; Yasudae, (dimethylsiloxane) for biological studies. Electrophoresis 2003, 24 (21), 3563- H. K.; Wikstrom, K. Crosslinked polydimethylsiloxane exposed to oxygen plasma 3576. studied by neutron reflectometry and other surface specific techniques. Polymer (2) Becker, H.; Locascio, L. E. Polymer microfluidic devices. Talanta 2002, 2000, 41, 6851-6863. 56 (2), 267-287. (13) Hillborg, H.; Tomczak, N.; Olah, A.; Schonherr, H.; Vancso, G. J. (3) Ng, J. M. K.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Components Nanoscale Hydrophobic Recovery: A Chemical Force Microscopy Study of UV/ for integrated poly(dimethylsiloxane) microfluidic systems. Electrophoresis 2002, Ozone-Treated Cross-Linked Poly(dimethylsiloxane). Langmuir 2004, 20 (3), 23, 3461-3473. 785-794. (4) Colas, A.; Curtis, J. Silicone biomaterials: history and chemistry & medical (14) Hu, S.; Ren, X.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. application of silicone, Academic Press ed.; Elsevier: The Netherlands, 2004; Surface Modification of Poly(dimethylsiloxane) Microfluidic Devices by Ultra- p 864. violet Polymer Grafting. Anal. Chem. 2002, 74 (16), 4117-4123. (5) Lottersy, J. C.; Olthuis, W.; Veltink, P. H.; Bergveld, P. The mechanical (15) Papra, A.; Bernard, A.; Juncker, D.; Larsen, N. B.; Michel, B.; Delamarche, properties of the rubber elastic polymer polydimethylsiloxane for sensor E. Microfluidic Networks Made of Poly(dimethylsiloxane), Si, and Au Coated applications. J. Micromech. Microeng. 1997, 7, 145-147. with Polyethylene Glycol for Patterning Proteins onto Surfaces. Langmuir 2001, (6) Duineveld, P. C.; Lilja, M.; Johansson, T.; Inganas, O. Diffusion of solvent 17, 4090-4095. in PDMS elastomer for micromolding in capillaries. Langmuir 2002, 18 (24), (16) Sui, G.; Wang, J.; Lee, C. C.; Lu, W.; Lee, S. P.; Leyton, J. V.; Wu, A. 9554-9559. M.; Tseng, H. R. Solution-Phase Surface Modification in Intact Poly(dimeth- (7) Muzzalupo, R.; Ranieri, G. A.; Golemme, G.; Drioli, E. Self-diffusion ylsiloxane) Microfluidic Channels. Anal. Chem. 2006, 78 (15), 5543-5551. measurements of organic molecules in PDMS and water in sodium alginate (17) Mao, H.; Yang, T.; Cremer, P. S. Design and Characterization of membranes. J. Appl. Polym. Sci. 1999, 74 (5), 1119-1128. Immobilized Enzymes in Microfluidic Systems. Anal. Chem. 2002, 74, 379-385. 10.1021/la070018o CCC: $37.00 © xxxx American Chemical Society Published on Web 03/14/2007 PAGE EST: 4.8
  2. 2. B Langmuir Heyries et al. 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 modi- fication 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 strepta- vidin 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) anti- bodies developed in mouse were supplied by Jackson Immuno- Research (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 Figure 1. Overview of the technique highlighting the four main rabbit serum (rabbit IgG) were obtained from Life Line Lab steps leading to the achievement of protein spots directly entrapped (Pomezia, Italy). The PDMS precursor and curing agent (Sylgard at the PDMS interface. 184) were supplied by Dow Corning (France). All buffers and aqueous solutions were made with distilled, demineralized water. Biochip Preparation (Figure 1). The biochips were prepared by arraying 1.3 nL drops of spotting solutions with a BioChip (18) Yang, T.; Baryshnikova, O. K.; Mao, H.; Holden, M. A.; Cremer, P. S. Arrayer BCA1 (Perkin-Elmer). Spotting solutions were prepared, Investigations of Bivalent Antibody Binding on Fluid-Supported Phospholipid Membranes: The Effect of Hapten Density. J. Am. Chem. Soc. 2003, 125 (16), when not mentioned, in carbonate buffer 0.1 M pH 9. Proteins 4779-4784. (human IgG, rabbit IgG, and peanut lectin) spotting solutions (19) Makamba, H.; Hsieh, Y.-Y.; Sung, W.-C.; Chen, S.-H. Stable Permanently were prepared at 1 mg/mL. Biotin modified dextran spotting Hydrophilic Protein-Resistant Thin-Film Coatings on Poly(dimethylsiloxane) Substrates by Electrostatic Self-Assembly and Chemical Cross-Linking. Anal. solutions were prepared to contain a constant concentration of Chem. 2005, 77, 3971-3978. biotin of 232 µmol/L. Each array was composed of 16 spots (20) Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S. Dynamic Coating (identical or not, diameter ) 150 µm) that were deposited on the Using Polyelectrolyte Multilayers for Chemical Control of Electroosmotic Flow in Capillary Electrophoresis Microchips. Anal. Chem. 2000, 72, 5939-5944. surface of a 3D Teflon master composed of 24 rectangular (21) Goda, T.; Konno, T.; Takai, M.; Moro, T.; Ishihara, K. Biomimetic structures (w ) 5 mm, l ) 5 mm, h ) 1 mm). Teflon was chosen phosphorylcholine polymer grafting from polydimethylsiloxane surface using photo-induced polymerization. Biomaterials 2006, 27 (30), 5151-5160. as the deposition material according to its hydrophobicity and (22) Wang, Y.; Lai, H.-H.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, its convenience for 3D micromachining. After spotting, the drops N. L. Covalent Micropatterning of Poly(dimethylsiloxane) by Photografting through were dried, and the arrays were transferred to the PDMS interface, a Mask. Anal. Chem. 2005, 77, 7539-7546. (23) Marquette, C. A.; Blum, L. J. Direct immobilization in poly(dimethyl- by pouring a mixture of precursor and curing agent (10:1) onto siloxane) for DNA, protein and enzyme fluidic biochips. Anal. Chim. Acta 2004, the Teflon substrate and curing for 20 min at 90 °C. Peeling off 506 (2), 127-132. the PDMS polymer then terminated the biochip preparation. Prior (24) Marquette, C. A.; Blum, L. J. Conducting elastomer surface texturing: a path to electrode spotting: Application to the biochip production. Biosens. to any further use, the biochips were saturated with VBSTA Bioelectron. 2004, 20 (2), 197-203. (Veronal buffer 30 mM, NaCl 0.2 M, pH 8.5 with addition of (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 tween 20 0.1% v/v and BSA 1% w/v) for 20 min at 37 °C. the detection of p53 gene point mutation and anti-HIV-1 capsid protein antibodies. Immobilized Molecules Detection. The immobilized mol- Anal. Bioanal. Chem. 2005, 381 (5), 1019-1024. ecules (i.e., biotinylated dextran, Arachis hypogaea lectin, rabbit (26) Marquette, C. A.; Cretich, M.; Blum, L. J.; Chiari, M. Protein microarrays enhanced performance using nanobeads arraying and polymer coating. Talanta IgG, or human IgG) were detected through chemiluminescent 2006, in press, corrected proof. labeling using peroxidase-labeled streptavidine, anti-lectin,
  3. 3. Protein Immobilization on Sylgard 184 Langmuir C Table 1. Analytical Characteristics of Rabbit IgG Microarrays Prepared Using Different Immobilization Procedures immobilization signala (SD) LOD,b method ng/mL latex 1 µm26 20420 a.u. (9.4%) 100 silica 330 nm26 8748 a.u. (13.5%) 50 direct entrapment 20240 a.u. (8.2%) 10 of 10-15 nm (Supporting Information 1) proteins a Calculated from three microarrays incubated with 1 µg/mL of anti- rabbit 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 Figure 2. Proposed mechanisms for the interactions between protein 30 mM, NaCl 0.2 M, pH 8.5). and PDMS during the elastomer curing step. The microarrays were then placed in the CCD camera’s (Las- 1000 Plus, Intelligent Dark Box II, FUJIFILM) measurement such as human IgG/anti-human IgG and peanut allergen (Arachis chamber for light integration for 10 min (measuring solution: hypogaea lectin)/anti-allergen were studied. In every case, a very VBS containing in addition 220 µM of luminol, 200 µM of good recognition of the immobilized protein was experienced, p-iodophenol and 500 µM of hydrogen peroxide). The numeric with no possibility of removing the immobilized entities, even micrographs obtained were quantified with a FUJIFILM image in very harsh conditions. Indeed, proteins/PDMS microarrays analysis program (Image Gauge 3.122). were submitted to a vigorous washing under stirring in 100 mL of VBSTA buffer for 18 h. Protein spots morphologies before Results and after immersion were compared using optical microscopy, The immobilization of biomolecules and particularly proteins and no major change was noticed. Moreover, the variation of the has been one of the major targets of our group for the last 15 immobilized protein reactivity before and after immersion was years.27 The last 3 years have been particularly devoted to the found to be 10%. The proteins were then firmly immobilized at development of innovative immobilization methods, compatible the PDMS interface. with the spotting technology widely used for microarrays. Thus, The effect of the polymerization process (drying and heating technological solutions for spotting beads bearing protein were 20 min at 90 °C), which submits proteins to denaturing conditions, proposed based on the entrapment of those beads at the PDMS/ was investigated. Indeed, these uncommon conditions are not air interface.23-26 In the present work, we highlight an interesting supposed to be compatible with the proteins used for immu- phenomenon leading to the transfer to the elastomer/air interface nodetection. However, protein drying for immunoassay develop- of proteins not preimmobilized on carrier beads. Table 1 ments has been already extensively used, particularly within the summarizes the results obtained with previously described micro-contact printing field,32,33 demonstrating the protein microarrays prepared with 1 µm latex beads bearing rabbit IgG stability following such treatment.34 To evaluate the effect of the or 330 nm silica beads bearing rabbit IgG and with the actual curing step (90 °C) on the integrity of the dried proteins, free rabbit IgG system. The three different microarrays were microarrays were prepared by polymerizing PDMS at room prepared using a similar protocol (i.e., spotting, drying, molding temperature (25 ( 2°C) onto protein spots for 48 h. The analytical of PDMS, curing, and peeling off) and incubated with peroxidase- performances of these microarrays were found to compare well labeled anti-rabbit IgG antibodies. Surprisingly, the direct with the ones prepared at 90 °C, evidencing the low effect of the entrapment was found to be more effective than the beads-based elevated temperature on the subsequent immobilized antigen- format previously developed. antibody recognition. These results suggest that lowering the size of the entrapped Different mechanisms could be involved in the direct im- entity, from a 1 µm bead to 10-15 nm immunoglobulin protein,28,29 mobilization of accessible proteins during the PDMS curing does not fail the immobilization of accessible rabbit IgGs. The (Figure 2). First a molding effect, comparable to the key/lock analytical signal obtained is then really convincing with high couples observed within the molecular imprinting research chemiluminescent intensities obtained with a relatively low SD field.35-37 Then, hydrophobic interactions, as shown by Bartzoka value, giving the best limit of detection (LOD) of the three systems. Since rabbit IgG/anti-rabbit IgG are model proteins with well- (32) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; known high affinity and stability,30,31 weaker recognition systems Biebuyck, H. Printing Patterns of Proteins. Langmuir 1998, 14 (9), 2225-2229. (33) Arjan, P. Q.; Elisabeth, P.; Sven, O. Recent advances in microcontact (27) Blum, L. J.; Coulet, P. R. Biosensor Principles and Applications; Marcel printing. Anal. Bioanal. Chem. 2005, 381 (3), 591-600. Dekker: New York, 1991; p 357. (34) LaGraff, J. R.; Chu-LaGraff, Q. Scanning force microscopy and (28) Godoy, S.; Chauvet, J. P.; Boullanger, P.; Blum, L. J.; Girard-Egrot, A. fluorescence microscopy of microcontact printed antibodies and antibody P. New Functional Proteo-glycolipidic Molecular Assembly for Biocatalysis fragments. Langmuir 2006, 22 (10), 4685-4693. Analysis of an Immobilized Enzyme in a Biomimetic Nanostructure. Langmuir (35) Turner, N. W.; Jeans, C. W.; Brain, K. R.; Allender, C. J.; Hlady, V.; Britt, 2003, 19 (13), 5448-5456. D. W. From 3D to 2D: A Review of the Molecular Imprinting of Proteins. (29) Godoy, S.; Violot, S.; Boullanger, P.; Bouchu, M.-N.; Leca-Bouvier, B. Biotechnol. Prog. 2006, in press. D.; Blum, L. J.; Girard-Egrot, A. P. Kinetics Study of Bungarus fasciatus Venom (36) Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, Acetylcholinesterase Immobilised on a Langmuir-Blodgett Proteo-Glycolipidic N.; Nicholls, I. A.; O’Mahony, J.; Whitcombe, M. J. Molecular imprinting science Bilayer. ChemBioChem 2005, 6 (2), 395-404. and technology: a survey of the literature for the years up to and including 2003. (30) Deshpande, S. S. Enzyme Immunoassays, Chapman & Hall ed.; ITP: J. Mol. Recognit. 2006, 19 (2), 106-180. New York, 1996; p 464. (37) Marty, J. D.; Mauzac, M. Molecular imprinting: State of the art and (31) The Immunoassay Handbook, 3rd ed.; Elsevier: The Netherlands, 2005; perspectives. Microlithography - Molecular Imprinting; Springer: Berlin, 2005; p 930. Vol. 172, pp 1-35.
  4. 4. D Langmuir Heyries et al. 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). Figure 4. Atomic force microscopy (NT-MDT, tapping mode) and co-worker,38,39 could also be involved between proteins and 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 uncured PDMS. Finally, covalent bindings between the protein the edge of each spot. The straight lines correspond to the presented and the polymer could occur while PDMS is curing, mainly profiles. through poisoning of the Kardstedt catalyst40-42 by the amino or thiol groups of the protein lateral chains (as lone pair electron have little effect on the immobilization of the dextran molecules, donors; Supporting Information 2). even at high concentration (50 mM). On the contrary, lysine and Dextran chains bearing biotin and lysine residues were chosen cysteine were shown to inhibit strongly and with a dose as model molecules to study this direct entrapment. The presence dependence relation the immobilization of the biotinylated of the accessible immobilized molecule was then evidenced using polymer. These results are in agreement with the involvement peroxidase labeled streptavidine and chemiluminescent imaging. of a poisoning of the Kardstedt catalyst in the immobilization 500 kDa and 3 kDa dextran chains were thus successfully process. immobilized at the PDMS/air interface. The very large size A drastic effect of the cysteine, which is known as a very difference between the two polymers did not appear to critically efficient Kardstedt catalyst poison,43 was observed. Indeed, 78% influence the immobilization efficiency, proving that molecules and 40% of the immobilization of the 3 kDa dextran was inhibited as small as 3000 Da could be trapped and accessible at the by the presence of the maximum concentration of cysteine and elastomer surface. lysine, respectively. The 60% of remaining immobilization in Within the dextran chains used, only the amino group of the the presence of 50 mM lysine could then be attributed to the lysine lateral chains could be involved in a chemical reaction others proposed mechanisms (i.e., molding effect and hydrophobic with the Kardstedt catalyst during the Sylgard 184 curing. As interactions). Regarding the poor hydrophobicity of the dextran a control experiment, dextran chains not bearing any lysine backbone, hydrophobic interactions were believed to be minimum. residues were spotted and transferred to the elastomer. The Potential interactions through the carbohydrate moiety of the chemiluminescent signal obtained was then 45% of the initial lysine-dextran were then also considered. Thus, immobilization signal, demonstrating the implication of the amino group in the inhibition tests were performed by spotting dextran in the presence immobilization process but also evidencing the molding effect of maltose (a subunit of dextran). No effect on the lysine- implicated in 45% of the immobilization efficiency. dextran immobilization was observed for maltose concentrations Further studies were performed to complete this theoretical up to 100 mM. immobilization mechanism. 3 kD dextran molecules bearing According to the results presented above, two mechanisms lysine residues were spotted in the presence of different appeared to be preponderant in the actual lysine-dextran concentrations of free amino acids: glycine, lysine, and cysteine immobilization on PDMS: chemical bounding through poisoning (Figure 3). Glycine, with only its R-amino group, was found to of the Kardstedt catalyst by the primary amine of the lysine (38) Bartzoka, V.; Brook, M. A.; McDermott, M. R. Silicone-Protein Films: residue and a molding effect, similar to a key/lock mechanism. Establishing the Strength of the Protein-Silicone Interaction. Langmuir 1998, 14 Our previous works on bead assisted protein immobilization (7), 1892-1898. (39) Bartzoka, V.; Brook, M. A.; McDermott, M. R. Protein-Silicone on microarrays25,26 have demonstrated the usefulness of increasing Interactions: How Compatible Are the Two Species? Langmuir 1998, 14 (7), the specific area of the spots. Indeed, increasing this area while 1887-1891. keeping constant the geometrical one enables the immobilization (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. of a higher amount of proteins per spot and then the increase of J. Polym. Sci. Part B: Polym. Phys. 1998, 36 (12), 2129-2139. the microarray performances. Herein, since the proteins are spotted (41) Quirk, R. P.; Kim, H.; Polce, M. J.; Wesdemiotis, C. Anionic Synthesis of Primary Amine Functionalized Polystyrenes via Hydrosilation of Allylamines and transferred directly to the PDMS interface, an original way with Silyl Hydride Functionalized Polystyrenes. Macromolecules 2005, 38, 7895- to increase this specific area has been to use spot solutions with 7906. relatively high salt concentration. Indeed, the salt charged protein (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 (43) Marian, M.; Winter, H. H. Relaxation patterns of endlinking polydim- A: Polym. Chem. 2000, 38 (5), 804-809. ethylsiloxane near the gel point. Polym. Bull. 1998, 40 (2), 267-274.
  5. 5. Protein Immobilization on Sylgard 184 PAGE EST: 4.8 Langmuir E 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 anti- lectin). 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 anti- lectin. 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 In summary, we have developed a new approach to directly modify Sylgard 184 surfaces with spots of protein and dem- onstrated 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 Figure 5. Spotting pattern and chemiluminescent image of the microarray prepared by spotting Arachis hypogaea lectin, rabbit amino or thiol groups in the immobilized molecules. Moreover, IgG, and human IgG in 0.1M carbonate buffer (pH 9). one interesting point is that Si-H functions have been reported to be sensitive to hydrolysis45 and, regarding the influence of solutions crystallize during the drying step, leading to highly nucleophilic groups such as primary amino groups, it could be textured surfaces. Thus, during the PDMS pouring and drying postulated that interactions could also occurr between protein steps, these surfaces were used as a master to produce PDMS lateral chains and Si-H functions. replica entrapping proteins, having a high specific area. Future work includes the integration of such microarrays in Two examples of this technique are illustrated by the AFM microfluidic systems, thanks to the use of PDMS as immobiliza- images of protein spots obtained in pure water and in the presence tion support. of carbonate buffer 0.1 M (Figure 4). The two spots obviously exhibit very different surfaces as evidenced by the spot profiles. Acknowledgment. Published with the support of the European Calculated from the AFM images, the specific area increasing Commission, Sixth Framework Program, Information Society between both spots was found to be 1 order of magnitude. This Technologies. NANOSPAD (No. 016610). difference of the surface geometry has a direct repercussion on the chemiluminescent signal obtained from those spots. Indeed, Supporting Information Available: Dose response curves and a more than 200% increase of the signal was observed when the kardstedt catalyst reaction cycle. This material is available free of carbonate was added to the spotting solution, and this was charge via the Internet at 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 LA070018O of the lysine chains amino group at high pH but to the actual increase of the specific area of the spot. (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. In order to fully characterize the analytical possibilities of the (45) Brook, M. A. Silicon in Organic, Organometallic, and Polymer Chemistry; developed microarray, three different proteins were spotted at John Wiley & Sons: New York, 2000.