Peptide Aptamers in Label-Free Protein Detection - American

Mar 25, 2009 - Therapeutics, Leeds Institute of Molecular Medicine, St. James's University Hospital, Beckett Street, Leeds LS9 7TF. The early detectio...
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Anal. Chem. 2009, 81, 3314–3320

Peptide Aptamers in Label-Free Protein Detection: 2. Chemical Optimization and Detection of Distinct Protein Isoforms Jason J. Davis,*,† Jan Tkac,†,§ Rachel Humphreys,† Anthony T. Buxton,‡ Tracy A. Lee,‡ and Paul Ko Ferrigno‡ Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, and Section of Experimental Therapeutics, Leeds Institute of Molecular Medicine, St. James’s University Hospital, Beckett Street, Leeds LS9 7TF The early detection and diagnosis of cancer lies central to successful treatment and improved patient outcome. Current techniques are limited by the nature of the biological receptor and the assays available. This paper reports the use of novel biological probes, peptide aptamers, in detecting cyclin-dependent protein kinases (CDKs) whose activity is important in proliferating and cancerous cells. We describe, specifically, the optimization of an orientated peptide aptamer surface and its utilization in establishing a highly specific, low-nanomolar sensitive, detection protocol for the active form of CDK2. In comparing target binding affinity of two different aptamers (pep6 and pep9), both constructed through the insertion of peptide sequences into the surface of a scaffold protein, one was observed to be consistently more effective. Significantly, the pep9 aptamers were able to detect subtle changes in the conformation of CDK2 associated with activation of its catalytic activity that may be caused by the phosphorylation of a single amino acid (threonine 160). A typical response toward the inactive form of CDK2 was in the range of 0.5-2% of the binding of the active form of CDK2 in the concentration range from 2 to 20 nM. Although antibodies are occasionally able to recognize conformations in their targets, this is the first time that a nonantibody protein probe has been used to detect an active protein isoform. Because peptide aptamers are usually raised against full-length proteins, this raises the possibility that peptide aptamers will be able to extend the repertoire of probes that recognize protein conformations, post-translational modifications (PTMs), or conformations stabilized by PTMs. The widespread use of DNA microarray technology for monitoring gene expression has generated valuable insight into various physiological states, analyzing clustered gene expression and revealing coregulated gene networks. An analysis of gene expression is not, however, sufficient to provide a knowledge of * To whom correspondence should be addressed. Fax: 044 (1865) 275914. Phone: 044 (1865) 275914. † University of Oxford. ‡ Leeds Institute of Molecular Medicine. § Permanent address: Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 812 37 Bratislava, Slovak Republic.

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protein abundance and activity or cellular processes.1 The detection and quantification of protein biomarkers in biological samples lies central to proteomics, drug design, disease diagnosis, and therapeutic development.2-4 However, the generation of viable protein detection platforms is challenging. Proteins demonstrate a staggering variety of chemistries, affinities, and specificities, and in many cases, there is requirement for multimerization, interaction with other proteins, or post-translational modification for activity or specific binding to be significant. There is no simple process for the amplification of proteins (like PCR in case of DNA), and prokaryotic expression and purification is neither facile nor does it guarantee functional integrity. The inherent instability of many proteins is a concern in microarray shelf life, and the need to control surface-bound orientation (and thereby maximize the fraction of immobilized protein able to bind target analyte) is demanding.5-8 Though the interaction of protein with a solid surface is, of course, well-studied, this is a highly variable interaction for which a molecular-level of understanding is only just emerging.9 In many cases, it appears that initial interactions are predominantly nonspecific, hydrophobic, and reversible. Over time, the protein may unfold and trigger more hydrophobic interactions and, ultimately, irreversible adsorption.10,11 These unfolding interactions are associated with the acquirement of a free energy minimum and, often, a “lateral spreading” of the protein structure.12,13 The designed chemisorption of proteins on surfaces introduces not only more control of recognition efficacy but also the potential for increased molecular stability. Simulated (1) Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol. Cell. Biol. 1999, 19, 1720–1730. (2) Mor, G.; Visintin, I.; Lai, Y.; Zhao, H.; Schwartz, P.; Rutherford, T.; Yue, L.; Bray-Ward, P.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7677– 7682. (3) Kingsmore, S. F.; Patel, D. D. Curr. Opin. Biotechnol. 2003, 14, 74–81. (4) Brian, B. H. Proteomics 2003, 3, 2116–2122. (5) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55–63. (6) LaBaer, J.; Ramachandran, N. Curr. Opin. Chem. Biol. 2005, 9, 14–19. (7) Kwon, Y.; Han, Z.; Karatan, E.; Mrksich, M.; Kay, B. K. Anal. Chem. 2004, 76, 5713–5720. (8) Sapsford, K. E.; Liron, Z.; Shubin, Y. S.; Ligler, F. S. Anal. Chem. 2001, 73, 5518–5524. (9) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110–115. (10) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3–30. (11) Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Langmuir 2003, 19, 1861–1872. (12) Kondo, A.; Oku, S.; Higashitani, K. J. Colloid Interface Sci. 1991, 143, 214– 221. (13) Buijs, J.; Hlady, V. J. Colloid Interface Sci. 1997, 190, 171–181. 10.1021/ac802513n CCC: $40.75  2009 American Chemical Society Published on Web 03/25/2009

and experimental investigations of the effects of surface tethering on both protein fold and these dynamic interfacial interactions have suggested the possibility of large effects, some of which appear to lead to increased protein stability over the solution-phase form.14 The first protein microarrays were built on the most available and well-characterized biorecognition elementssantibodies. Unfortunately, antibodies do not function well in the microarray format, because typically only a small fraction (20%) specifically recognize the target protein, with cross-reactivity with other analytes in the microarray format negatively influencing detection selectivity.15,16 Current antibody-based assays are commonly based on sandwich formats in which antigen binding to the immobilized antibody is detected through the use of a secondary, labeled, antibody.17,18 Though sensitive, this method is laborious (multiple incubation and washing steps are typically required) and often requires a specifically labeled secondary antibody for every antigen of interest. The direct electrochemical or optical tagging of analytes may make the assay more facile but brings with it significant concern about the degree to which biorecognition is compromised through the use of a “modified” analyte. For example, many fluorophores are hydrophobic and may induce conformational change on association with a protein secondary structure.19 Labeling protocols can also be time-consuming and may lead to high background signals. Alternative protein-receptive molecules are thus of considerable interest. Peptide Aptamer Arrays. DNA/RNA aptamers have been recently used for formation of protein microarrays20-22 and exhibit good chemical/thermal stability and limited batch variation during production. Labeling protocols are flexible, and binding properties can be changed on demand. Despite these strengths, only a limited number (∼20) of protein-recognizing DNA/RNA aptamers are currently known,23 and those assembled into microarray format have exhibited significant sensitivity toward cations.24 Peptide aptamers are defined as peptide recognition moieties that are presented in, and conformationally constrained by, an engineered non-antibody scaffold.25 Engineered scaffold proteins for specific biorecognition events include anticalins modeled on lipocalin structures, trinectins derived from a fibronectin III domain, and affibody molecules, which are engineered from the Z domain of protein A.26 The tuneable detection, high-density surface immobilization, and robust structural characteristics of (14) Friedel, M.; Baumketner, A.; Shea, J. E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 8396–8401. (15) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2001, 2, Research0004. (16) Michaud, G. A.; Salcius, M.; Zhou, F.; Bangham, R.; Bonin, J.; Guo, H.; Snyder, M.; Predki, P. F.; Schweitzer, B. I. Nat. Biotechnol. 2003, 21, 1509– 1512. (17) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760–1763. (18) Predki, P. F. Curr. Opin. Chem. Biol. 2004, 8, 8–13. (19) Kirby, R.; Cho, E. J.; Gehrke, B.; Bayer, T.; Park, Y. S.; Neikirk, D. P.; McDevitt, J. T.; Ellington, A. D. Anal. Chem. 2004, 76, 4066–4075. (20) Balamurugan, S.; Obubuafo, A.; Soper, S. A.; McCarley, R. L.; Spivak, D. A. Langmuir 2006, 22, 6446–6453. (21) LeFloch, F.; Ho, H. A.; Leclerc, M. Anal. Chem. 2006, 78, 4727–4731. (22) Radi, A.-E.; Sa´nchez, J. L. A.; Baldrich, E.; O’Sullivan, C. K. J. Am. Chem. Soc. 2006, 128, 117–124. (23) Collett, J. R.; Cho, E. J.; Ellington, A. D. Methods 2005, 37, 4–15. (24) Jiang, Y.; Fang, X.; Bai, C. Anal. Chem. 2004, 76, 5230–5235. (25) Colas, P.; Cohen, B.; Jessen, T.; Grishina, I.; McCoy, J.; Brent, R. Nature 1996, 380, 548–550. (26) Binz, H. K.; Amstutz, P.; Pluckthun, A. Nat. Biotechnol. 2005, 10, 1257– 1268.

engineered scaffold proteins, called here generically “peptide aptamers”, offer much in the design of microarrays.27 Unlike antibodies, which are generally organism-produced, post-translationally modified, conformationally fragile, and lose activity when surface confined, peptide aptamers are robust (e.g., melting temperature greater than 70 °C for the stefin A triple mutant (STM) scaffold used here28) and can be selected from large libraries via phage display or, in our approach, yeast two hybrid screens. The unique advantage of the latter is that it allows peptide aptamers routinely to recognize conformational “epitopes” rather than linear amino acid sequences which are favored by antibodies. Scaffold proteins are chosen for their inherent stability and generally lack post-translational modification. As engineered proteins, peptide aptamers can be further altered (if necessary) for enhanced surface stability. Finally, the relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers should greatly simplify microarray production. It is thus theoretically possible to avoid the considerable problem presented by protein-protein variability by using a single scaffold to present a large range of interaction surfaces to solution. The number of peptide aptamers known so far outranks the number of DNA/RNA aptamers. We have reported the development of a novel scaffold molecule called STM, based on human stefin A, an intracellular inhibitor of cathepsins.28 STM possesses three sites, distant from each other in the primary sequence of the protein yet adjacent in the folded protein and naturally used by stefin A to bind to target proteins. These offer the possibility of creating larger and more complex interaction surfaces for improved affinity and specificity. A major distinction between STM and affibodies is that STM has been engineered to lack detectable affinity for all intracellular human proteins,28 which should decrease background binding and hence increase signal-to-noise in any microarray format. This is likely to be of key importance in reducing background binding of irrelevant proteins when analyzing complex mixtures such as serum or plasma. Surface plasmon resonance (SPR) is a robust technology offering a real-time analysis of biological recognition in a variety of media. It is, specifically, sensitive to changes in the index of refraction at or near the surface of a metallic film. Being an in situ technique suitable for the label-free kinetic and thermodynamic analysis of interactions between analytes and artificial receptive surfaces, SPR sensorgrams are readily utilized in optimizing the formation and regeneration of a recognition layer.29-32 In recent work, we described the generation and SPR assaying characteristics of the STM scaffold protein adlayer.29 Orientational control was achieved through the chemisorption of an engineered surface cysteine residue to an underlying gold surface with concurrent presentation of the scaffold recognition surface to solution. We used this system to establish an SPR assay with excellent operational stability, a limit of detection of 1 nM (150 ng mL-1), and to determine the affinity constant of (27) Ladner, R. C.; Ley, A. C. Curr. Opin. Biotechnol. 2001, 12, 406–410. (28) Woodman, R.; Yeh, J. T. H.; Laurenson, S.; Ko Ferrigno, P. J. Mol. Biol. 2005, 352, 1118–1133. (29) Davis, J. J.; Tkac, J.; Laurenson, S.; Ko Ferrigno, P. Anal. Chem. 2007, 79, 1089–1096. (30) Homola, J. Chem. Rev. 2008, 108, 462–493. (31) Cooper, M. A. Nat. Rev. Drug Discovery 2002, 1, 515–528. (32) Malmqvist, M. Nature 1993, 361, 186–187.

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Figure 1. Peptide aptamers used in this work. (A) Amino acid composition (single-letter code) of the STM scaffold and its amino terminal extension highlighting some functional features relevant to this work. The first 61 amino acids, in uppercase letters, derive from the pET30a(+) expression vector (Novagen) with some modifications (ref 29). The hexahistidine sequence, the cysteine residue inserted to allow orientated immobilization on gold, and the StrepII tag (ref 57) are each underlined. The following 98 amino acids, given in lowercase letters, are the sequence of the STM scaffold itself. Amino acids highlighted in red represent changes made compared to the parent stefin A protein (ref 28). Peptide inserts that create peptide aptamers are into the “ngp” sequence toward the C-terminal end of STM. The cloning strategy used results in the duplication of the “gp” residues so that peptide inserts are always flanked with a glycine-proline dipeptide. (B) Effects of peptide insertions on the physical properties of peptide aptamers. Peptide sequences from Colas et al. (ref 25) are given in the single-letter amino acid code. Predicted MW ) predicted molecular weight, in daltons. Predicted pI ) predicted isoelectric point. Predictions were calculated using the algorithm at http://www.iut-arles.up.univ-mrs.fr/w3bb/d_abim/.

Scheme 1. Surface Activation of the Surface via PDEA Protocola

a The activated esters generated within mixed self-assembled monolayer (SAM) layers (not shown) are modified with PDEA. The generated reactive disulfide moieties are subsequently coupled to the free thiols of a ligand. The immobilization protocol, applicable across a broad pH range, facilitates a scaffold orientation with the peptide insert exposed to the solution.

interaction of STM for a cognate antibody. We present here an extension of this work in which peptide aptamers based on the scaffold (Figure 1) have been immobilized on a modified gold electrode in the generation of a specific target protein assay. We have screened 10 different scaffold immobilization protocols in maximizing recognition activity (analyte response per unit of immobilized scaffold) while suppressing nonspecific signal generation. In so doing, a calibratable assay with 2 nM limit of detection (LOD) for the CDK2 cancer marker has been established. The aptamer assay is, moreover, capable of sensitively distinguishing between two different forms of the target. In all assays orientational control was achieved by surface cysteine coupling to either a bare gold surface or a preprepared PDEA ([2-(2-pyridinyldithio)ethaneamine]) adlayer (Scheme 1). The latter method is known to be an effective, nondestructive, and 3316

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homogeneous immobilization protocol applicable across a broad pH range.17,33-36 In contrast to maleimido coupling protocols, protein immobilization on PDEA layers is chemically or electrochemically reversible and, accordingly, of considerable interest from a “smart release” perspective.37-39 EXPERIMENTAL SECTION Materials and Reagents. 11-Mercaptoundecanoic acid (C10-COOH), 16-mercaptohexadecanoic acid (C15-COOH), 6-mercaptohexanol (C6-OH), EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), NHS (N-hydroxysuccinimide), cysteamine, and BSA (bovine serum albumin) were obtained from Sigma-Aldrich (Poole, Dorset, U.K.). An OEG-OH thiol (1mercapto-11-undecyl)tri(ethylene glycol), 99+% laboratory grade) was purchased from Asemblon (Seattle, WA). An OEG-COOH thiol (HS(CH2)10(OCH2CH2)3OCH2COOH, >95%) was purchased from Prochimia Surfaces Sp. (Sopot, Poland). PDEA was obtained from Biacore (GE Healthcare, U.K.). The active form of CDK2 (a complex of phosphorylated CDK2 and cyclin A2) was purchased from Cell Signaling Technology Inc. (Danvers, U.S.A.). The inactive form of CDK2 (without phosphorylation and not complexed with cyclin A2 protein) was provided by Abnova Corp. (Walnut, U.S.A.). All other chemicals used were of highest purity available without further purification. (33) Lofas, S.; Johnsson, B.; Edstrom, A.; Hansson, A.; Lindquist, G.; Hillgren, R.; Stigh, L. Biosens. Bioelectron. 1995, 10, 813–822. (34) Brogan, K. L.; Schoenfisch, M. H. Langmuir 2005, 21, 3054–3060. (35) Renberg, B.; Shiroyama, I.; Engfeldt, T.; Nygren, P.-A.; Karlstroem, A. E. Anal. Biochem. 2005, 341, 334–343. (36) Vostiar, I.; Tkac, J.; Mandenius, C.-F. Anal. Chem. 2005, 342, 152–159. (37) Schlecht, U.; Nomura, Y.; Bachmann, T.; Karube, I. Bioconjugate Chem. 2002, 13, 188–193. (38) You, Y.; Hong, C.; Pan, C. J. Phys. Chem. C 2007, 111, 16161–16166. (39) Moore, E. J.; Curtin, M.; Ionita, J.; Maguire, A. R.; Ceccone, G.; Galvin, P. Anal. Chem. 2007, 79, 2050–2057.

Construction of Prokaryotic Expression Plasmids. The vector pET30a+CYS+ was constructed using a QuickChange mutagenesis kit (Stratagene) to introduce a single cysteine residue between the amino-terminal hexahistidine (HIS6-tag) and the multiple cloning site. The STM or peptide aptamer open reading frames were subcloned into pET30a+ and pET30a+CYS+ vectors in the EcoRI restriction site to create STMpET30a+ and Cys+STM-pET30a+ plasmids to express STM (the empty scaffold) and peptide aptamers without or with an amino-terminal cysteine residue. Expression of Recombinant STM-CYS+ Protein, Pep6 and Pep9. STM-Cys+ (pI of 5.04, Mw of 18 kDa), pep6 (pI of 5.73, Mw of 20 kDa), and pep9 (pI of 5.06, Mw of 21 kDa; see Figure 1 for further details) were expressed as HIS6-tag fusion proteins in BL21(DE3)pLysS Escherichia coli cells. The cells containing STM-pET30a+Cys+ plasmids were grown to midlog phase at 37 °C and induced with 1 mM isopropyl β-thiogalactopyranoside at 37 °C for 3 h. Cells were harvested by centrifugation and lysed for 20 min at room temperature with BugBuster primaryamine-free bacterial lysis reagent (Novagen, Nottingham, U.K.) including protease inhibitors (Roche, Lewes, U.K.). The lysate was centrifuged at 4000g for 30 min and incubated with nickel nitrilotriacetic acid (Ni-NTA) resin (Qiagen, Crawley, U.K.) for 2 h at 4 °C. The Ni-NTA resin was washed with 30 vol of wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 7.4). HIS6-tagged fusion protein was eluted from the resin with elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole, pH 7.4). The eluted protein was exchanged into 50 mM sodium phosphate buffer (PB) using PD-10 protein desalting columns (GE Healthcare). Protein purification was analyzed both by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with antihuman cystatin A monoclonal antibody (AbCam, Cambridge, U.K.). SPR Assays. For all measurements, a double-channel SPR reader was used (Eco Chemie, Netherlands). The instrument is equipped with a cuvette, and a gold sensor disk (diameter 17 mm) is mounted to the optical lens through index-matched oil. An autosampler is used to inject or remove test solutions, and measurements were typically carried out with a volume of 45 µL. Measurements were performed with mixing, where the solution was continuously aspirated and dispensed at a flow rate of 16.7 µL s-1. A gold chip (Windsor Scientific Ltd., U.K.) was treated with a hot piranha (1 + 3 mixture of concentrated H2SO4 plus concentrated H2O2; handle with special care) for 2 min, washed with deionized water and then with EtOH (absolute), and left for no longer than 3 min before being inserted into the doublechannel SPR reader. The SPR measurements were carried out using 10 mM phosphate pH 7.4 (PB) as a running buffer. Immobilization Protocol 1 (Chemisorption on Bare Gold). Scaffold protein (10 µM in PB) was mixed with a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) gel (TCEP immobilized on a dextran matrix, Pierce) and allowed to react for 45-60 min at room temperature to selectively reduce any interprotein disulfide bonds. The protein was then centrifuged (1000g for 1 min), and the supernatant collected and used immediately. Backfilling with OEG-OH thiol (5 µL of 1 mM OEG-OH added into 40 µL PB giving final concentration of 110 µM sonicated for 20-30

s prior injection) was then carried out for 20 min prior to extensive surface washing with running buffer. Both immobilization of the STM protein and back-filling were performed in situ, with the gold chip mounted in the spectrometer. Immobilization Protocol 2 (PDEA Protocol). PDEA selfassembled monolayers (SAMs) were prepared either as single components from 1 mM ethanolic solution or as mixed layers with a coadsorbate in ethanolic solution (in situ for 60 min under stirred conditions). The carboxylic group was subsequently activated by a 15 min injection of a 1:1 mixture of 0.2 M EDC and 0.05 M NHS in deionized water into the cell followed by 40 mM PDEA in 0.1 M borate pH 8.5 (15 min). The surface was then washed with PB until a stable baseline was established. Protein immobilization was typically carried out from a 1 µM solution in PB for 45 min and followed by PB washing to a stable signal. Finally, active PDEA was blocked using cystamine (50 mM in 1 M NaCl and 0.1 M sodium acetate pH 4.0, stirred solution for 15 min) and the surface rewashed. Specific binding on this STM-Cys+ surface was investigated by injection of 60 nM monoclonal antiSTM antibody (Abcam, Cambridge, U.K.) and nonspecific binding by injection of 60 nM BSA (Sigma) solution. Immobilization of peptide aptamers (pep6 and pep9) was carried out with the same PDEA immobilization protocol, but using lower protein concentration (0.5 µM). Binding properties of immobilized peptide aptamers were investigated using 10 nM of active CDK2 and 10 nM of inactive CDK2 proteins. To determine detection limits and dynamic range, experiments were repeated using varying concentrations (from 2 to 20 nM and 5 to 50 nM for the active and inactive forms of CDK2, respectively). A typical assay protocol consisted of a 10 min association phase and 5 min dissociation phase induced by injection of plain PB into the cell. The signal was read at the end of dissociation phase and converted into the surface coverage using the relationship 122 mdeg ) 1 ng mm-2.29 Atomic Force Microscopy. Ambient tapping mode atomic force microscopy (TMAFM) imaging was carried out with a Nanowizard microscope (JPK, Ltd.) in conjunction with the integrated JPK Nanowizard SPM control software. A gold chip 11 × 11 mm2 (Arrandee) was flame-annealed before cleaning with freshly prepared piranha (3:1 concd H2SO4/35% H2O2) for 10 min, washed with deionized water, and then with absolute EtOH. A freshly cleaned chip was immersed in 1:100 OEGCOOH/OEG-OH mixture of thiols (both 1 mM in absolute EtOH) for 3 h before washing with absolute EtOH and then by PB. The chip was then incubated with 0.04 M EDC and 0.01 M NHS (ratio 1:1) for 1 h before washing in PB. Then it was incubated in 15 mM PDEA for 20 min before washing with running buffer and finally in 500 nM STM-Cys+ protein in PB overnight at 4 °C, then washed in running buffer before being imaged. Rotated monolithic silicon probes with a symmetric tip, attached to triangular cantilevers 115-135 µm in length (Budget Sensors Tap300), were operated at resonances in the 275-330 kHz range (drive 400-1500 mV). Height, amplitude, and phase data were simultaneously collected. The JPK Nanowizard is linearized in all three dimensions using a closed-loop feedback system which ensures precise positioning in all three axes allowing high-resolution images to be obtained with fewer scans which minimizes possible contamination Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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Table 1. Interface Composition, Immobilized Scaffold Coverage, and Target Protein Recognition Characteristicsa carboxylic acid (A) 1 2 3 4 5 6 7 8 9 10

bare Au C10COOH C10COOH C10COOH C15COOH C15COOH C10COOH C10COOH OEG-COOH OEG-COOH

STM-Cys+ (∼18 kDa) alcohol (B)

ratioA/B

OEG-OH

N/A 100:0 1:10 1:100 1:10 1:100 1:10 1:100 1:10 1:100

OEG-OH OEG-OH OEG-OH OEG-OH C6-OH C6-OH OEG-OH OEG-OH

mdeg 289 ± 47 169 ± 27