Protein−Diazonium Adduct Direct Electrografting onto SPRi-Biochip

Jul 2, 2009 - A direct protein immobilization method for surface plasmon resonance imaging (SPRi) gold chip arraying is exposed. The biomolecule elect...
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Protein-Diazonium Adduct Direct Electrografting onto SPRi-Biochip Benjamin P. Corgier,† Sophie Bellon,‡ Marielle Anger-Leroy,‡ Loic J. Blum,† and Christophe A. Marquette*,† †

Laboratoire de G enie Enzymatique et Biomol eculaire, Institut de Chimie et Biochimie Mol eculaires et Supramol eculaires Universit e Lyon 1, CNRS 5246 ICBMS B^ atiment CPE, 43, bd du 11 novembre 1918, 69622 Villeurbanne, Cedex, France, and ‡GenOptics SA, Centre Scientifique, Plateau du Moulon, B^ atiment 503, BP05, 91401 Orsay, Cedex, France Received March 3, 2009. Revised Manuscript Received May 19, 2009 A direct protein immobilization method for surface plasmon resonance imaging (SPRi) gold chip arraying is exposed. The biomolecule electroaddressing strategy, previously demonstrated by our team on carbon surfaces, is here valuably involved and adapted to create a straightforward and efficient protein immobilization process onto SPRi-biochips. The proteins, modified with an aryl-diazonium adduct, are addressed to the SPRi chip surface through the electroreduction of the aryl-diazonium. The biomolecule deposition was followed through SPRi live measurements during the electrografting process. A specially designed setup enabled us to directly observe the mass increasing at the sensor surface while the proteins were electrografted. A pin electrospotting method, allowing the achievement of distinct sensing layers on gold SPRi-biochips, was used to generate microarray biochips. The integrity of the immobilized proteins and the specificity of the detection, based on antigen/antibody interactions, were demonstrated for the detection of specific antibodies and ovalbumin. The SPRi detection limit of ovalbumin using the electroaddressing of anti-ovalbumin IgG was compared with two other immobilization procedures, cystamine-glutaraldehyde selfassembled monolayer and pyrrole, and was found to be a decade lower than these ones (100 ng/mL, i.e., 2 nM).

Introduction The principle of protein biochip is based on interactions between an immobilized biomolecule and a “target” biomolecule in solution. A large majority of the systems are based on immunoglobulin interactions1 and require the labeling of a fragile sensing element,2 usually a secondary antibody. Interestingly, surface plasmon resonance (SPR), first described 40 years ago,3 enables in real time4 the measurement of binding events without labeling of the detected target biomolecules. Moreover, the arising of SPR imaging5 (SPRi) in the 1980s has opened the path to the multiplexed SPR detection. Usually, SPRi based detections provide a lower sensitivity than classical SPR (ppm vs ppb limit of detection) but with the strong advantage of simultaneous multiple event investigation in an array format. As for all analytical systems, the immobilization method involved has, as an absolute requirement, to maintain the native structure and function of the immobilized entities.6-8 The wide heterogeneity of structure, stability, and reactivity of the proteins is then a real challenge to develop standard immobilization procedures for SPRi based analysis.6 Several technologies were developed based on thiol chemistry to functionalize the SPR gold substrate prior to the immobilization of the sensing elements.7 This process enabled the development of SPRi systems based on *To whom correspondence should be addressed. E-mail: christophe. [email protected].

(1) Marquette, C. A.; Blum, L. J. Biosens. Bioelectron. 2006, 21(8), 1424–1433. (2) Hempen, C.; Karst, U. Anal. Bioanal. Chem. 2006, 384(3), 572–583. (3) Kretschmann, E.; Raether, H. Z. Naturforsch., A: Astrophys., Phys. Phys. Chem. 1968, 23a(12), 2135–2138. (4) Boozer, C.; Kim, G.; Cong, S. X.; Guan, H. W.; Londergan, T. Curr. Opin. Biotechnol. 2006, 17(4), 400–405. (5) Rothenhausler, B.; Knoll, W. Nature 1988, 332, (6165), 615-618. (6) Yuk, J. S.; Ha, K. S. Exp. Mol. Med. 2005, 37(1), 1–10. (7) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121(35), 8044–8051. (8) Wegner, G. J.; Lee, H. J.; Corn, R. M. Anal. Chem. 2002, 74(20), 5161–5168.

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oligonucleotide arrays,11-14 peptide arrays,8 low-molecular weight protein arrays,9 and carbohydrate arrays.10 The immobilization through dextran molecules11 was also extensively used in an attempt to preserve the biomolecules’ native conformation.12 Nevertheless, recent studies comparing the DNA immobilization using the dextran based technology and the mercaptoundecanoic acid chemistry have shown that the first suffers of a lack of reproducibility.13 Additional technologies were also developed, taking advantage of the electropolymerization of pyrrole derivatives.14,15 Insoluble films of pyrrole-oligonucleotide,16 pyrrolepeptide,17 or pyrrole-protein18,19 were demonstrated as suitable for SPRi measurements. In this paper, we present a straightforward immobilization method based on aryl-diazonium modification of biomolecules in an effort to improve the protein microarray SPRi detection capabilities. The obtained immobilized biomolecules present the advantage of being in close contact with the SPR chip, without the presence of additional polymers (polypyrrole) or large anchoring (9) Lee, H. J.; Nedelkov, D.; Corn, R. M. Anal. Chem. 2006, 78(18), 6504–6510. (10) Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am. Chem. Soc. 2003, 125(20), 6140–6148. (11) Tombelli, S.; Mascini, M.; Turner, A. P. F. Biosens. Bioelectron. 2002, 17(11-12), 929–936. (12) Lofas, S.; Johnsson, B. J. Chem. Soc., Chem. Commun. 1990, 21(1), 1526– 1528. (13) Mannelli, I.; Courtois, V.; Lecaruyer, P.; Roger, G.; Millot, M. C.; Goossens, M.; Canva, M. Sens. Actuators, B 2006, 119(2), 583–591. (14) Asavapiriyanont, S.; Chandler, G. K.; Gunawardena, G. A.; Pletcher, D. J. Electroanal. Chem. 1984, 177(1-2), 229. (15) Diaz, A. F.; Kanazawa, K. K.; Gardini, G. P. J. Chem. Soc., Chem. Commun. 1979, 14, 635–636. (16) Guedon, P.; Livache, T.; Martin, F.; Lesbre, F.; Roget, A.; Bidan, G.; Levy, Y. Anal. Chem. 2000, 72(24), 6003–6009. (17) Cherif, B.; Roget, A.; Villiers, C. L.; Calemczuk, R.; Leroy, V.; Marche, P. N.; Livache, T.; Villiers, M. B. Clin. Chem. 2006, 52(2), 255–262. (18) Wolowacz, S. E.; Hin, B.; Lowe, C. R. Anal. Chem. 1992, 64(14), 1541–1545. (19) Grosjean, L.; Cherif, B.; Mercey, E.; Roget, A.; Levy, Y.; Marche, P. N.; Villiers, M. B.; Livache, T. Anal. Biochem. 2005, 347(2), 193–200.

Published on Web 07/02/2009

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molecules (dextran thiol). The achieved SPRi signals might then be improved. This procedure was described as a direct electroaddressing of biomolecules. The immobilization of proteins,20,21 DNA probes,22,23 or enzyme24 was described on graphite materials. This strategy stands on the electrochemical properties of aryldiazonium salts,25,26 leading to the formation of a covalent bond between the aryl residue and the electrode surface. Recently, the electrografting of diazonium-biomolecule adducts (both DNA probes and proteins) was also profitably performed at the surface of gold thin films electrodeposited on screen-printed carbon electrode microarrays.27 First, the direct electrografting of proteins on top gold plated SPRi glass chips is presented. The sensing layers generated are then studied for SPRi detection. The analytical performances of the biosensing system are evaluated in terms of specificity, sensitivity, and limit of detection. The behavior of the SPRi using electrografted protein was studied using immobilized rabbit and human IgG, directly detected with anti-IgG antibodies. Active anti-ovalbumin antibodies electrodeposited onto the SPRi system were then used for the labeless detection of free ovalbumin.

Experimental Section Reagents. 4-Aminophenylacetic acid (4-carboxymethylaniline; CMA), bovine serum albumin (BSA), 4-bromobenzene diazonium (BrDz), N-hydroxysuccinimide (NHS), N,N0 -dicyclohexyl-carbodiimide (DCC), anti-rabbit IgG antibodies developed in mouse, human IgG, and ovalbumin were purchased from Sigma-Aldrich (France). Sodium nitrite and Veronal (diethylmalonylurea sodium) were obtained from Prolabo (France). Immunoglobulins from rabbit serum (rabbit IgG) were supplied by Life Line Lab (Pomezia, Italy). Anti-ovalbumin antibodies developed in rabbit were purchased from Tebu (France).

Functionalization of Proteins and Electrografting on SPRi-Biochip. Rabbit-IgG, anti-ovalbumin antibodies, and human IgG were functionalized with an aniline derivative (CMA) according to the protocol described previously.27,28,33 Briefly, 10 mg of 4-carboxymethylaniline (CMA) was activated in the presence of 20.6 mg of DCC and 11.5 mg of NHS in 1 mL of DMSO for 30 min under stirring. Then 50 μL of the activated CMA was added to 500 μL of a 5 mg/mL protein solution in 0.1 M carbonate buffer, pH 11. This solution was left to react overnight at 4 °C before being purified and concentrated to 5 mg/mL under centrifugation using Microcon YM-3 (Millipore, USA). The obtained retentate was recovered in d.d. water and stored at -20 °C. The functionalized proteins were diazotated in an aqueous solution of 20 mM HCl, 20 mM NaNO2 for 20 min under stirring in ice prior to the immobilization. It is important to note that even if the presented method has been validated using both antibodies (20) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Arango, D. C.; Brozik, S. M. Biosens. Bioelectron. 2008, 23(6), 757–764. (21) Corgier, B. P.; Marquette, C. A.; Blum, L. J. J. Am. Chem. Soc. 2005, 127(51), 18328–18332. (22) Harper, J. C.; Polsky, R.; Wheeler, D. R.; Dirk, S. M.; Brozik, S. M. Langmuir 2007, 23(16), 8285–8287. (23) Corgier, B. P.; Laurent, A.; Perriat, P.; Blum, L. J.; Marquette, C. A. Angew. Chem., Int. Ed. 2007, 46(22), 4108–4110. (24) Polsky, R.; Harper, J. C.; Dirk, S. M.; Arango, D. C.; Wheeler, D. R.; Brozik, S. M. Langmuir 2007, 23(2), 364–366. (25) Barbier, B.; Pinson, J.; Desarmot, G.; Sanchez, M. J. Electrochem. Soc. 1990, 137(6), 1757–1764. (26) Delamar, M.; Hitmi, R.; Pinson, J.; Saveant, J. M. J. Am. Chem. Soc. 1992, 114(14), 5883–5884. (27) Corgier, B. P.; Li, F.; Blum, L. J.; Marquette, C. A. Langmuir 2007, 23(16), 8619–8623. (28) Szunerits, S.; Knorr, N.; Calemczuk, R.; Livache, T. Langmuir 2004, 20(21), 9236–9241.

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and enzyme (peroxidase),23 the effect of such a treatment on the protein integrity might be carefully taken into consideration. A two-electrode setup using a batch SPRi cell was used during the protein electrografting live recording (Figure 1A). The batch cell was composed of a 200 μL Teflon chamber positioned on the top of the SPRi-biochip. A 1 mm diameter platinum electrode was immersed in this cell and used as a counter/pseudoreference electrode. Here, the deposition protocol was a cyclic voltammetry method described previously:21 three voltammograms were performed from 0 to -2 V at a scan rate of 200 mV/s. In order to generate protein arrays, the freshly diazotated proteins were electrografted to the gold SPRi-biochip (GenOptics) surface using a manual pin electrospotting technique (Figure 1B).16 Briefly, the tip of the pin was approached in close contact with the gold surface and a potential pulse was generated between the gold layer (working electrode) and the counter electrode (located in the tip filled with the freshly diazotated proteins). This electrical pulse was set to -2.5 V applied during 2 s. CMA-protein immobilization on the gold surface in the absence of electrical pulse was observed. The two well-known effects that explain the formation of spots without electroaddressing are (i) the adsorption of protein on gold materials and (ii) the reactivity of aryl-diazonium toward gold. Every gold surface was blocked with hexanethiol solution prior to use, in order to reduce this non-electroaddressed protein immobilization and to focus on the electrografting of CMA-proteins.

SPRi Interaction Monitoring on SPRi-Plex (GenOptics). All interaction studies were performed in 10 mM phosphatebuffered saline (PBS) on SPRi-biochip presaturated with PBS containing 1% BSA. The samples were injected in the 6 μL flow cell using a 500 μL injection loop. A constant 50 μL/min flow rate was maintained during the entire experiment using a syringe pump. A regeneration step was performed following each sample injection by injecting a chaotropic solution composed of 0.1 M glycine, pH 2. The optical setup has been detailed before.16 Briefly, light was shone on the reverse side of the SPRi-biochip, which was constructed of a high optical index glass prism coated with thin layers of chromium and gold (2 and 50 nm, respectively) (Figure 1C). Changes in light reflectivity attributable to antigens or antibodies interacting with the immobilized antibodies were recorded by using a 12-bit charge-coupled device (CCD) camera. Sequential images were recorded at 1.5 s intervals, and binding kinetics were monitored by SPRi-view software (GenOptics). The measured values of reflectivity variation were converted into protein quantities per surface unit (in pg/mm2) using a calibration of the SPRi detection/apparatus and based on the following formula: Γ ¼

ΔRLzc SP, R ðDn=DCÞ

ð1Þ

where ΔR is the reflectivity variation in percent, Lzc = 1.02  10-4 mm (penetration depth of the plasmon wave), ∂n/∂C=1.9  10-10 mm3/pg, and SP,R =2.25  103%/RIU (sensitivity of the SPR in percent per refractive index unit).

Results and Discussion Modifying SPRi-biochips with aryl-diazonium molecules is believed to be a promising technology to achieve high quality multiplexed SPRi sensors in array format. Since such an approach has never been used before, our first concern was to confirm the actual possibility of the cathodic electrografting of aryl-diazonium cation onto the 50 nm gold film of the SPRi-biochip. Therefore, a setup for the live SPRi recording of the electrografting events has been built in order to directly follow the aryldiazonium mass deposition during the electroaddressing process. An electrochemical cell has been adapted on the top of the SPRi Langmuir 2009, 25(16), 9619–9623

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Figure 1. (A) Protein electrografting live recording setup. (B) Pin electrospotting deposition of diazonium-protein adducts.16 (C) SPRi measurement principle: the flowing solution is processing over the sensing layers in a 6 μL Teflon chamber.

cell, giving the possibility to concomitantly perform electrochemical and SPRi measurements (Figure 1A). This kind of SPRi coupling was previously published for the scanning electrochemical microscopy writing of polymer onto a SPRi chip, but without any live recording possibilities.28 A first result obtained using this setup is presented in Figure 2A. Here, the electrografting of a basic aryl-diazonium molecule was evidenced. The 4-bromobenzendiazonium (BrDz) was electroaddressed, and the SPRi signal was followed at the same time. The first voltammogram displays a well-defined peak at -1.7 V (red square in Figure 2A), representative of the aryldiazonium reduction using gold electrode, whereas the disappearance of the reduction peak within the second and the third voltammograms evidences the electrode surface fouling. This phenomenon is typical of the aryl grafting onto the gold surface during the electrochemical process.29,30 Moreover, while the three consecutive cyclic potential scans from 0 to -2 V (200 mV/s) were applied, a clear increasing of the SPRi signal (reflectivity change) occurred. This fact proves the actual molecule deposition at the surface of the SPRi-biochip under the electroreduction of the basic aryl-diazonium residue. As expected, the first potential scan generated the highest mass deposition, with a 10% reflectivity change. The following second and third scans only generated a 1.5% and 0.5% reflectivity change, respectively, because of the surface coverage. A similar behavior was observed using protein-diazonium adducts (Figure 2B). In that case, rabbit IgGs modified with 4-carboxymethylaniline (CMA) were diazotated (20 mM HCl, 20 mM NaNO2) at a concentration of 100 μg/mL and their electrografting followed using the live SPRi recording setup. As described above for the crude aryl-diazonium residue in solution, the first voltammogram generated the highest reflectivity change (5%) and the three scans altogether generated a total electrodeposition reflectivity change of 7.7%. As for the crude diazonium, the majority of the diazonium entities reacted during the first electroreductive scan. (29) Lyskawa, J.; Belanger, D. Chem. Mater. 2006, 18(20), 4755–4763. (30) Laforgue, A.; Addou, T.; Belanger, D. Langmuir 2005, 21(15), 6855–6865.

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Figure 2. Electrografting live recording onto SPRi-biochip in the presence of (A) bromobenzene diazonium 5 mM or (B) rabbit IgG-CMA 100 μg/mL.

These reflectivity changes were converted into protein coverage using the SPRi-view software (see the Experimental Section), giving a coverage of 1027 pg/mm2. Approximating the immobilized IgG (150 kDa) to a 5 nm radius globular protein, a theoretical maximum coverage of 3160 pg/mm2 can be calculated. A good correlation between these two densities was then found, evidencing the efficiency of the electrografting procedure. As a control experiment, rabbit IgG-diazonium adducts were immobilized in the absence of potential scan. The reflectivity change curve is represented in blue in Figure 2B. As can be seen, a very low 0.5% reflectivity (69 pg/mm2) change was recorded in these conditions, evidencing the necessary electroreduction of the diazonium to obtain an efficient immobilization. The interaction between the rabbit IgG immobilized at the surface of these SPRi-biochips and the injected anti-rabbit IgG was then monitored using the SPRi-Plex. Seven injections, separated by a regeneration step (glycine 0.1 M, pH 2), of increasing anti-rabbit IgG concentrations, from 34 to 48 μg/mL, were DOI: 10.1021/la900762s

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performed, and the corresponding interaction curves recorded. Figure 3A presents a difference image obtained following the injection of 17 μg/mL anti-rabbit IgG, together with its quantification. The reflectivity variation was changing with the spotted rabbit IgG concentration. Indeed, the reflectivity variation increased with the spotting concentration to reach a maximum for 8 μg/mL and then decreased for spots electrografted using 20 μg/mL rabbit IgG, which suffered steric hindrances and led to weaker signals. Particular attention also had to be given to the control spots (absence of immobilized proteins) on which no detectable signal was observed, evidencing the low nonspecific binding of the system. As another key result, Figure 3B presents the reflectivity change obtained onto spots of nonaddressed or nondiazotated 8 μg/mL rabbit IgG-diazonium adduct (onto a distinct SPRi-biochip). In these latter cases, the reflectivity changes were found to be only 8% of the change observed on spots of addressed 8 μg/mL rabbit IgG-diazonium adduct. This result points out the essential need of the electroreduction in order to graft the antibodies. The sensorgram obtained from the spots prepared using the optimum rabbit IgG concentration (8 μg/mL) was exploited to generate an anti-rabbit IgG calibration curve. The reflectivity variations were converted into protein density using the Genoptics software (using eq 1), and the calibration curve obtained is presented in Figure 3C. A clear dose-response behavior was observed with the anti-rabbit IgG injected. Moreover, a really satisfying low interspot variation of 4% was found which evidences the very good reproducibility of the electrografting process. The open circle curve of Figure 3C presents the SPRi signal obtained onto nonspecific spots composed of addressed human IgG-CMA. As can be seen, the anti-rabbit IgG did not bind to these nonspecific sensing layers. As an essential experiment to study the integrity of the immobilized CMA modified antibodies, the detection of ovalbumin by immobilized IgG against ovalbumin was evaluated. This model was used to evaluate the integrity of proteins following pin electrospotting. Indeed, such a capture assay involves the binding capacity of the immobilized antibodies and will be highly sensitive to every loss of protein integrity during the immobilization process. The achievement of a specific signal will then be directly related to the preservation of the antibodies’ reactivity toward their specific target. For that purpose, polyclonal anti-ovalbumin antibodies were modified with CMA and used to generate matrices of 30 sensing layers using three different anti-ovalbumin concentrations (30, 60, and 120 μg/mL) and anti-rabbit IgG antibodies as the nonspecific binding control. SPRi measurements were performed on the entire SPRi-biochip following injections of different ovalbumin concentrations. Within the tested spotting concentrations, the stronger SPRi signals were obtained on anti-ovalbumin-CMA spots electrodeposited at 120 μg/mL concentration. It is important to point out the difference in the concentration necessary to produce optimum rabbit IgG spots (8 μg/mL) and the optimum concentration for anti-ovalbumin spots (120 μg/mL). This difference is related to the reactivity variation, from protein to protein, according to CMA covalent grafting, which led to a lower electroaddressing efficiency of the different biomolecules. The Genoptics software (using eq 1) was used to convert reflectivity variations into quantity of fixed protein per surface unit. The ovalbumin calibration curve obtained is presented in Figure 3D and is characterized by a detection limit of 100 ng/mL (2 nM) (signal(31) Mercier, K.; Anger-Leroy, M. Antibody/Protein interaction on a biochip functionalized with a Cystamine /Glutaraldehyde layer, biochip robustness. http:// www.genoptics-spr.com/doc/File/Application%20Note/2322-Cyst_Glut% 20Ac_Prot%20robustness.pdf (25 February).

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Figure 3. (A) Difference image and (B) quantification of the reflectivity changes obtained following the injection of anti-rabbit IgG at a concentration of 17 μg/mL. (C) Anti-rabbit IgG calibration curve obtained using the reflectivity variation of 8 μg/mL spots (n=4) of rabbit IgG (b) and human IgG (open circles). (D) Quantification of the SPRi detection of free ovalbumin (n=4) using anti-ovalbumin spots (9) and rabbit IgG spots (open squares). Each injection was separated by a regeneration step (glycine 0.1 M, pH 2).

to-noise of 3) and a detection ranging over 4 decades. This detection limit was found to be 1 decade lower than those obtained with other surface chemistries used on SPRi-biochips, such as cystamineglutaraldehyde self-assembled monolayer31,32 and pyrrole.33 This interesting analytical capacity can be attributed to the sensing layer itself. Actually, the electrografting of diazonium-antibodies produces a sensing layer that is in the highest closeness to the surface, compared to other techniques. We suppose that this particular characteristic brings optimal sensitivity through SPRi detections.

Conclusions The electroaddressing of protein-diazonium adducts was shown to be an efficient technology to prepare SPRi chips in an array format. The developed method allows the rapid production of multiple spots of protein on the SPRi-biochip with an excellent spatial addressing and a robust link between the biomolecule and the gold surface. First, the pair rabbit IgG/anti-rabbit IgG was used as interaction model to prove both the quality of the obtained spots and the specificity of the measured interactions. As a matter of fact, the spots achieved using the developed electrografting procedure were reproducible and enabled the detection of specific interactions through SPRi measurements. Second, anti-ovalbumin antibodies were also immobilized using the electrografting procedure, and they were directly involved in a capture assay for which the integrity of the (32) Mercier, K.; Anger-Leroy, M. Reproducibility of Antibody/Protein Interactions on Cystamine/Glutaraldehyde Functionalized Biochip. http://www. genoptics-spr.com/doc/File/Application%20Note/2321-cyst-glu%20Ac_prot% 20reproducibility.pdf (25 February). (33) Vollmer, N.; Anger-Leroy, M. Biochip Robustness of Pyrrolated-Conjugate Antibody/Antigen Interaction. http://www.genoptics-spr.com/doc/File/ Application%20Note/1322-PPy,%20Ac-prot,%20robustesse.pdf (25 February).

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immobilized entity is a crucial parameter. In this case, interesting performances were obtained for the detection of free ovalbumin with a detection limit of 100 ng/mL (2 nM). Therefore, the fixation of the protein on the gold plated surface appears to have very low effect on the protein integrity, even if the electrografting process is presumed to be performed under stringent conditions for biomolecules and especially proteins (pH 2.5). Thus, in a parallel study34 in which monoclonal antibodies were used, a special approach had to be used, using addressed protein (34) Marquette, C.; Bouteille, F.; Corgier, B.; Degiuli, A.; Blum, L. Anal. Bioanal. Chem. 2009, 393(4), 1191–1198.

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A, in order to obtain orientated and active immobilized antibodies. The developed pin electrografting technology involving biomolecule-diazonium adducts opens then a new path for the arraying of biomolecules at the surface of conducting materials while avoiding the time-consuming chemical prefunctionalization of surfaces (alkenethiol, dextran, or streptavidin/biotin modification). Supporting Information Available: Example of two injection/regeneration cycles. This material is available free of charge via the Internet at http://pubs.acs.org.

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