Straightforward Protein Immobilization on Sylgard 184 PDMSMicroarray 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 etSupramole´culaires, UniVersiteLyon 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 methodconsists of a direct transfer of dried protein/salt solutions to the PDMS interface during the polymer curing. Suchnon-conventional treatment of proteins was found to have no major negative consequence on their integrity. Themechanisms 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 proofof concept study, three different proteins were immobilized on a single microarray (Arachis hypogaealectin, rabbitIgG, and human IgG) and used as antigens for capture of chemiluminescent immunoassays. The proteins were shownto 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 microfabricated devices and especially biochips and integratedmicroarrays. These developments, concomitant with the mi-crofluidic and microdevice growth, have pushed technologydevelopers to find new materials to overcome the maindisadvantagesscost and technical requirement (clean room)sof 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 par-ticularly one of its elastomeric derivatives (Sylgard 184) rapidlybecame the most popular, thanks to its chemical and physicalproperties.3 Indeed, numerous applications in the field of medicaland microengineering4 became possible because of the PDMSlow toxicity, possibleprocessing instandard laboratoryconditions,low curing temperature, optical transparency, and cost effective-ness.5

However, despite these advantages, major drawbacks existwhen working with bare PDMS which are its native highlyhydrophobic properties, its relative permeability to solvent,6,7

and its biofouling tendency, leading to nonspecific adsorption

of proteins in many biomaterials applications.8,9 Regarding itsconvenience for biochip and microarray developments, the mainnegative aspect of PDMS is its chemical inertness8which criticallylowered the possibilities of immobilizing biomolecules directlyon PDMS structures (microfluidic components). Numerouspropositions have then been made to introduce reactive chemicalfunctions on PDMS surfaces. Abundant examples were describedbased on oxygen plasma exposure of PDMS10 or to a lesserextent ozone exposure and UV treatment11 to generate surfacesilanol groups, allowing classical silane surface chemistry. Thesemodifications were shown to be limited in time due to buriedPDMS chains migration leading to hydrophobic recovery.12,13

Other interesting approaches were proposed based on UV graftpolymerization,14 silanization of oxidized PDMS,15,16phospho-lipid bilayer modifications,17,18 or polyelectrolytes multilayers

* Corresponding author. E-mail: christophe.marquette@univ-lyon1.fr.(1) Sia, S. K.; Whitesides, G. M. Microfluidic devices fabricated in poly-

(dimethylsiloxane) for biological studies.Electrophoresis2003, 24 (21), 3563-3576.

(2) Becker, H.; Locascio, L. E. Polymer microfluidic devices.Talanta2002,56 (2), 267-287.

(3) Ng, J. M. K.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Componentsfor integrated poly(dimethylsiloxane) microfluidic systems.Electrophoresis2002,23, 3461-3473.

(4) Colas, A.; Curtis, J.Silicone biomaterials: history and chemistry & medicalapplication of silicone, Academic Press ed.; Elsevier: The Netherlands, 2004;p 864.

(5) Lottersy, J. C.; Olthuis, W.; Veltink, P. H.; Bergveld, P. The mechanicalproperties of the rubber elastic polymer polydimethylsiloxane for sensorapplications.J. Micromech. Microeng.1997, 7, 145-147.

(6) Duineveld, P. C.; Lilja, M.; Johansson, T.; Inganas, O. Diffusion of solventin PDMS elastomer for micromolding in capillaries.Langmuir2002, 18 (24),9554-9559.

(7) Muzzalupo, R.; Ranieri, G. A.; Golemme, G.; Drioli, E. Self-diffusionmeasurements of organic molecules in PDMS and water in sodium alginatemembranes.J. Appl. Polym. Sci.1999, 74 (5), 1119-1128.

(8) Mata, A.; Fleischman, A. J.; Roy, S. Characterization of Polydimethyl-siloxane (PDMS) Properties for Biomedical Micro/Nanosystems.Biomed.MicrodeVices2005, 7, 281-293.

(9) Linder, V.; Verpoorte, E.; Thormann, W.; de Rooij, N. F.; Sigrist, H.Surface Biopassivation of Replicated Poly(dimethylsiloxane) Microfluidic Chan-nels and Application to Heterogeneous Immunoreaction with On-Chip FluorescenceDetection.Anal. Chem.2001, 73, 4161-4169.

(10) Ye, H.; Gu, Z.; Gracias, D. H. Kinetics of Ultraviolet and Plasma SurfaceModification of Poly(dimethylsiloxane) Probed by Sum Frequency VibrationalSpectroscopy.Langmuir2006, 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/OzoneTreatment.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 plasmastudied by neutron reflectometry and other surface specific techniques.Polymer2000, 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 Ultra-violet 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 Coatedwith Polyethylene Glycol for Patterning Proteins onto Surfaces.Langmuir2001,17, 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(dimeth-ylsiloxane) Microfluidic Channels.Anal. Chem.2006, 78 (15), 5543-5551.

(17) Mao, H.; Yang, T.; Cremer, P. S. Design and Characterization ofImmobilized Enzymes in Microfluidic Systems.Anal. Chem.2002, 74, 379-385.

10.1021/la070018o CCC: $37.00 © xxxx American Chemical SocietyPAGE EST: 4.8Published on Web 03/14/2007

depositions.19,20Photoinduced polymer grafting21or photograftingpolymer using benzophenone22 were also used to create anintermediate layer on PDMS surfaces.

All of these methods suffer from several drawbacks which forthe most critical are the chemical instability of the surfacemodification obtained and the lack of a simple process for theimmobilization of biomolecules. Developing such direct modi-fication of PDMS surfaces with biomolecules, in a microarrayformat, has been the main objective of our group. Thus, a methodwas proposed for the direct entrapment in PDMS of micro (120-1µm)23,24 and nano (330-50 nm)25,26 beads, bearing biologicalmolecules such as enzymes, antibodies, oligonucleotides, andpeptides. The beads were then spotted and dried on a 3D master,covered with Sylgard 184, cured, and recovered, after peelingoff, as spots of beads entrapped at the surface of the bare PDMS.

We propose herein to push forward this methodology todemonstrate the possibility of functionalizing the PDMS surfaceby direct entrapment of biomolecules. Thus, the present workwill demonstrate the surface incorporation, during the PDMScuring, of molecules as small as 3000 Da (dextran polymer) oras fragile as proteins (antibodies).

The mechanism of this immobilization will be studied, and amodel will be proposed based on experimental evidence.Morphological studies through atomic force microscopy of thespots obtained in different conditions will also be proposed anddiscussed according to the analytical signal measured.

Experimental SectionMaterials. Arachis hypogaealectin (from peanut), anti-Arachis

hypogaealectin antibodies developed in rabbit, human IgG,luminol (3-aminophthalhydrazide), and peroxidase-labeled strepta-vidin were purchased from Sigma (France). Peroxidase-labeledpolyclonal anti-human Ig(G, A, M) antibodies developed in goatand 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, lysinefixable) and biotin-labeled dextran (3 kDa) were obtained fromMolecular Probes (The Netherlands). Immunoglobulins fromrabbit serum (rabbit IgG) were obtained from Life Line Lab(Pomezia, Italy). The PDMS precursor and curing agent (Sylgard184) were supplied by Dow Corning (France). All buffers andaqueous 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 BioChipArrayer 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 solutionswere prepared at 1 mg/mL. Biotin modified dextran spottingsolutions were prepared to contain a constant concentration ofbiotin of 232 µmol/L. Each array was composed of 16 spots(identical or not, diameter) 150µm) that were deposited on thesurface of a 3D Teflon master composed of 24 rectangularstructures (w ) 5 mm,l ) 5 mm,h ) 1 mm). Teflon was chosenas the deposition material according to its hydrophobicity andits convenience for 3D micromachining. After spotting, the dropswere dried, and the arrays were transferred to the PDMS interface,by pouring a mixture of precursor and curing agent (10:1) ontothe Teflon substrate and curing for 20 min at 90°C. Peeling offthe PDMS polymer then terminated the biochip preparation. Priorto any further use, the biochips were saturated with VBSTA(Veronal buffer 30 mM, NaCl 0.2 M, pH 8.5 with addition oftween 20 0.1% v/v and BSA 1% w/v) for 20 min at 37°C.

Immobilized Molecules Detection.The immobilized mol-ecules (i.e., biotinylated dextran,Arachis hypogaealectin, rabbitIgG, or human IgG) were detected through chemiluminescentlabeling using peroxidase-labeled streptavidine, anti-lectin,

(18) Yang, T.; Baryshnikova, O. K.; Mao, H.; Holden, M. A.; Cremer, P. S.Investigations of Bivalent Antibody Binding on Fluid-Supported PhospholipidMembranes: 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 PermanentlyHydrophilic 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 CoatingUsing Polyelectrolyte Multilayers for Chemical Control of Electroosmotic Flowin Capillary Electrophoresis Microchips.Anal. Chem.2000, 72, 5939-5944.

(21) Goda, T.; Konno, T.; Takai, M.; Moro, T.; Ishihara, K. Biomimeticphosphorylcholine polymer grafting from polydimethylsiloxane surface usingphoto-induced polymerization.Biomaterials2006, 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 througha Mask.Anal. Chem.2005, 77, 7539-7546.

(23) Marquette, C. A.; Blum, L. J. Direct immobilization in poly(dimethyl-siloxane) for DNA, protein and enzyme fluidic biochips.Anal. Chim. Acta2004,506 (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 forthe 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 microarraysenhanced performance using nanobeads arraying and polymer coating.Talanta2006, in press, corrected proof.

Figure 1. Overview of the technique highlighting the four mainsteps leading to the achievement of protein spots directly entrappedat the PDMS interface.

B Langmuir Heyries et al.

peroxidase-labeled anti-rabbit, or anti-human IgG, respectively.The different labeled proteins were incubated (20µL) on thesaturated microarrays for 1 h at 37°C and the excess reagentswashed out with a 20 min incubation in VBS (Veronal buffer30 mM, NaCl 0.2 M, pH 8.5).

The microarrays were then placed in the CCD camera’s (Las-1000 Plus, Intelligent Dark Box II, FUJIFILM) measurementchamber for light integration for 10 min (measuring solution:VBS containing in addition 220µM of luminol, 200 µM ofp-iodophenol and 500µM of hydrogen peroxide). The numericmicrographs obtained were quantified with a FUJIFILM imageanalysis program (Image Gauge 3.122).

Results

The immobilization of biomolecules and particularly proteinshas been one of the major targets of our group for the last 15years.27 The last 3 years have been particularly devoted to thedevelopment of innovative immobilization methods, compatiblewith the spotting technology widely used for microarrays. Thus,technological solutions for spotting beads bearing protein wereproposed based on the entrapment of those beads at the PDMS/air interface.23-26In the present work, we highlight an interestingphenomenon leading to the transfer to the elastomer/air interfaceof proteins not preimmobilized on carrier beads. Table 1summarizes the results obtained with previously describedmicroarrays prepared with 1µm latex beads bearing rabbit IgGor 330 nm silica beads bearing rabbit IgG and with the actualfree rabbit IgG system. The three different microarrays wereprepared using a similar protocol (i.e., spotting, drying, moldingof PDMS, curing, and peeling off) and incubated with peroxidase-labeled anti-rabbit IgG antibodies. Surprisingly, the directentrapment was found to be more effective than the beads-basedformat previously developed.

These results suggest that lowering the size of the entrappedentity, from a 1µm bead to 10-15 nm immunoglobulin protein,28,29

does not fail the immobilization of accessible rabbit IgGs. Theanalytical signal obtained is then really convincing with highchemiluminescent intensities obtained with a relatively low SDvalue, giving the best limit of detection (LOD) of the three systems.

Since rabbit IgG/anti-rabbit IgG are model proteins with well-known high affinity and stability,30,31weaker recognition systems

such as human IgG/anti-human IgG and peanut allergen (Arachishypogaealectin)/anti-allergen were studied. In every case, a verygood recognition of the immobilized protein was experienced,with no possibility of removing the immobilized entities, evenin very harsh conditions. Indeed, proteins/PDMS microarrayswere submitted to a vigorous washing under stirring in 100 mLof VBSTA buffer for 18 h. Protein spots morphologies beforeand after immersion were compared using optical microscopy,and no major change was noticed. Moreover, the variation of theimmobilized protein reactivity before and after immersion wasfound to be 10%. The proteins were then firmly immobilized atthe PDMS interface.

The effect of the polymerization process (drying and heating20 min at 90°C), which submits proteins to denaturing conditions,was investigated. Indeed, these uncommon conditions are notsupposed to be compatible with the proteins used for immu-nodetection. However, protein drying for immunoassay develop-ments has been already extensively used, particularly within themicro-contact printing field,32,33 demonstrating the proteinstability following such treatment.34To evaluate the effect of thecuring step (90°C) on the integrity of the dried proteins,microarrays were prepared by polymerizing PDMS at roomtemperature (25( 2°C) onto protein spots for 48 h. The analyticalperformances of these microarrays were found to compare wellwith the ones prepared at 90°C, evidencing the low effect of theelevated temperature on the subsequent immobilized antigen-antibody recognition.

Different mechanisms could be involved in the direct im-mobilization of accessible proteins during the PDMS curing(Figure 2). First a molding effect, comparable to the key/lockcouples observed within the molecular imprinting researchfield.35-37Then, hydrophobic interactions, as shown by Bartzoka

(27) Blum, L. J.; Coulet, P. R.Biosensor Principles and Applications; MarcelDekker: 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 BiocatalysisAnalysis of an Immobilized Enzyme in a Biomimetic Nanostructure.Langmuir2003, 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 VenomAcetylcholinesterase Immobilised on a Langmuir-Blodgett Proteo-GlycolipidicBilayer. ChemBioChem2005, 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.

(32) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.;Biebuyck, H. Printing Patterns of Proteins.Langmuir1998, 14 (9), 2225-2229.

(33) Arjan, P. Q.; Elisabeth, P.; Sven, O. Recent advances in microcontactprinting. Anal. Bioanal. Chem.2005, 381 (3), 591-600.

(34) LaGraff, J. R.; Chu-LaGraff, Q. Scanning force microscopy andfluorescence microscopy of microcontact printed antibodies and antibodyfragments.Langmuir2006, 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 scienceand 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 andperspectives.Microlithography - Molecular Imprinting; Springer: Berlin, 2005;Vol. 172, pp 1-35.

Table 1. Analytical Characteristics of Rabbit IgG MicroarraysPrepared Using Different Immobilization Procedures

immobilizationmethod

signala (SD) LOD,b

ng/mL

latex 1µm26 20420 a.u. (9.4%) 100silica 330 nm26 8748 a.u. (13.5%) 50direct entrapmentof 10-15 nmproteins

20240 a.u. (8.2%)(Supporting Information 1)

10

a Calculated from three microarrays incubated with 1µg/mL of anti-rabbit IgG.b Limit of detection (LOD) calculated for a signal-to-noiseratio of 3.

Figure 2. Proposed mechanisms for the interactions between proteinand PDMS during the elastomer curing step.

Protein Immobilization on Sylgard 184 Langmuir C

and co-worker,38,39could also be involved between proteins anduncured PDMS. Finally, covalent bindings between the proteinand the polymer could occur while PDMS is curing, mainlythrough poisoning of the Kardstedt catalyst40-42 by the aminoor thiol groups of the protein lateral chains (as lone pair electrondonors; Supporting Information 2).

Dextran chains bearing biotin and lysine residues were chosenas model molecules to study this direct entrapment. The presenceof the accessible immobilized molecule was then evidenced usingperoxidase labeled streptavidine and chemiluminescent imaging.500 kDa and 3 kDa dextran chains were thus successfullyimmobilized at the PDMS/air interface. The very large sizedifference between the two polymers did not appear to criticallyinfluence the immobilization efficiency, proving that moleculesas small as 3000 Da could be trapped and accessible at theelastomer surface.

Within the dextran chains used, only the amino group of thelysine lateral chains could be involved in a chemical reactionwith the Kardstedt catalyst during the Sylgard 184 curing. Asa control experiment, dextran chains not bearing any lysineresidues were spotted and transferred to the elastomer. Thechemiluminescent signal obtained was then 45% of the initialsignal, demonstrating the implication of the amino group in theimmobilization process but also evidencing the molding effectimplicated in 45% of the immobilization efficiency.

Further studies were performed to complete this theoreticalimmobilization mechanism. 3 kD dextran molecules bearinglysine residues were spotted in the presence of differentconcentrations of free amino acids: glycine, lysine, and cysteine(Figure 3). Glycine, with only itsR-amino group, was found to

have little effect on the immobilization of the dextran molecules,even at high concentration (50 mM). On the contrary, lysine andcysteine were shown to inhibit strongly and with a dosedependence relation the immobilization of the biotinylatedpolymer. These results are in agreement with the involvementof a poisoning of the Kardstedt catalyst in the immobilizationprocess.

A drastic effect of the cysteine, which is known as a veryefficient Kardstedt catalyst poison,43was observed. Indeed, 78%and 40% of the immobilization of the 3 kDa dextran was inhibitedby the presence of the maximum concentration of cysteine andlysine, respectively. The 60% of remaining immobilization inthe presence of 50 mM lysine could then be attributed to theothers proposed mechanisms (i.e., molding effect and hydrophobicinteractions). Regarding the poor hydrophobicity of the dextranbackbone,hydrophobic interactionswerebelieved tobeminimum.Potential interactions through the carbohydrate moiety of thelysine-dextran were then also considered. Thus, immobilizationinhibition tests were performed by spotting dextran in the presenceof maltose (a subunit of dextran). No effect on the lysine-dextran immobilization was observed for maltose concentrationsup to 100 mM.

According to the results presented above, two mechanismsappeared to be preponderant in the actual lysine-dextranimmobilization on PDMS: chemical bounding through poisoningof the Kardstedt catalyst by the primary amine of the lysineresidue and a molding effect, similar to a key/lock mechanism.

Our previous works on bead assisted protein immobilizationon microarrays25,26have demonstrated the usefulness of increasingthe specific area of the spots. Indeed, increasing this area whilekeeping constant the geometrical one enables the immobilizationof a higher amount of proteins per spot and then the increase ofthemicroarrayperformances.Herein, since theproteinsarespottedand transferred directly to the PDMS interface, an original wayto increase this specific area has been to use spot solutions withrelatively high salt concentration. Indeed, the salt charged protein

(38) Bartzoka, V.; Brook, M. A.; McDermott, M. R. Silicone-Protein Films:Establishing the Strength of the Protein-Silicone Interaction.Langmuir1998, 14(7), 1892-1898.

(39) Bartzoka, V.; Brook, M. A.; McDermott, M. R. Protein-SiliconeInteractions: How Compatible Are the Two Species?Langmuir1998, 14 (7),1887-1891.

(40) Perutz, S.; Kramer, E. J.; Baney, J.; Hui, C. Y.; Cohen, C. Investigationof 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 Synthesisof Primary Amine Functionalized Polystyrenes via Hydrosilation of Allylamineswith Silyl Hydride Functionalized Polystyrenes.Macromolecules2005,38, 7895-7906.

(42) Katsuhiko Kishi, T. I.; Ozono, M.; Tomita, I.: Endo, T. Development andapplication of a latent hydrosilylation catalyst. IX. Control of the catalytic activityof a platinum catalyst by polymers bearing amine moieties.J. Polym. Sci. PartA: Polym. Chem.2000, 38 (5), 804-809.

(43) Marian, M.; Winter, H. H. Relaxation patterns of endlinking polydim-ethylsiloxane near the gel point.Polym. Bull.1998, 40 (2), 267-274.

Figure 3. Effect of different amino acids on the chemiluminescentsignals obtained from microarrays composed of directly immobilizedlysine-modified biotinylated dextran. The immobilized dextran wasdetected through peroxidase-labeled streptavidin 1µg/mL (30 min,37 °C).

Figure 4. Atomic force microscopy (NT-MDT, tapping mode)images of spots of lysine modified biotinylated dextran obtained inwater (A) or in 0.1 M carbonate buffer, pH 9 (B). The arrows indicatethe edge of each spot. The straight lines correspond to the presentedprofiles.

D Langmuir Heyries et al.

solutions crystallize during the drying step, leading to highlytextured surfaces. Thus, during the PDMS pouring and dryingsteps, these surfaces were used as a master to produce PDMSreplica entrapping proteins, having a high specific area.

Two examples of this technique are illustrated by the AFMimages of protein spots obtained in pure water and in the presenceof carbonate buffer 0.1 M (Figure 4). The two spots obviouslyexhibit very different surfaces as evidenced by the spot profiles.Calculated from the AFM images, the specific area increasingbetween both spots was found to be 1 order of magnitude. Thisdifference of the surface geometry has a direct repercussion onthe chemiluminescent signal obtained from those spots. Indeed,a more than 200% increase of the signal was observed whencarbonate was added to the spotting solution, and this wasirrespective of the buffer pH used (7, 9, and 11). This enhancementof the signal is then not linked to an increase of the reactivityof the lysine chains amino group at high pH but to the actualincrease of the specific area of the spot.

In order to fully characterize the analytical possibilities of thedeveloped microarray, three different proteins were spotted at

a concentration of 1 mg/mL in carbonate buffer (pH 9) andtransferred to the PDMS interface. The spotting pattern appearedon the upper part of Figure 5.Arachis hypogaealectin, rabbitIgG, and human IgG were spotted as eight replicas. Bovine serumalbumin (BSA) was used as a negative control for all of thetested antibodies (i.e., anti-rabbit IgG, anti-human IgG, and anti-lectin). As can be seen (Figure 5), absolutely no nonspecificsignal was detected outside of the area delimited by the specificprotein spots. Classical dose response curves can be observed(Supporting Information 1) from each range of antibodyconcentrations tested, giving detection limits of 20 ng/mL foranti-rabbit IgG and anti-human IgG and 10 ng/mL for anti-lectin. These concentrations correspond, in the 20µL incubationvolume, to amounts of antibody in the fmol range (2.6 and 1.3fmol), which are considered low enough for most of the majorimmunoassay applications.31,44

Conclusion

In summary, we have developed a new approach to directlymodify Sylgard 184 surfaces with spots of protein and dem-onstrated its use for microarray-based immunoassays. Themechanisms of this direct protein immobilization have beeninvestigated, and clear experimental results suggested that bothchemical bonding and molding effects were implicated in theobserved phenomenon. Chemical bonding between the proteinand the PDMS elastomer structure was believed to be mainlydue to a poisoning of the Kardstedt catalyst used during thepolymer curing. This poisoning was demonstrated to be related,with reference to previous works,38,40,41to the presence of freeamino or thiol groups in the immobilized molecules. Moreover,one interesting point is that Si-H functions have been reportedto be sensitive to hydrolysis45 and, regarding the influence ofnucleophilic groups such as primary amino groups, it could bepostulated that interactions could also occurr between proteinlateral chains and Si-H functions.

Future work includes the integration of such microarrays inmicrofluidic systems, thanks to the use of PDMS as immobiliza-tion support.

Acknowledgment. Published with the support of the EuropeanCommission, Sixth Framework Program, Information SocietyTechnologies. NANOSPAD (No. 016610).

Supporting Information Available: Dose response curves andthe kardstedt catalyst reaction cycle. This material is available free ofcharge via the Internet at http://pubs.acs.org.

LA070018O

(44) Wu, A. H. B. A selected history and future of immunoassay developmentand applications in clinical chemistry.Clin. Chim. Acta2006, 369(2), 119-124.

(45) Brook, M. A.Silicon in Organic, Organometallic, and Polymer Chemistry;John Wiley & Sons: New York, 2000.

Figure 5. Spotting pattern and chemiluminescent image of themicroarray prepared by spottingArachis hypogaealectin, rabbitIgG, and human IgG in 0.1M carbonate buffer (pH 9).

Protein Immobilization on Sylgard 184 PAGE EST: 4.8 LangmuirE