Dissecting the Carbohydrate Specificity of the Anti-HIV-1 2G12

Article pubs.acs.org/Langmuir

Dissecting the Carbohydrate Specificity of the Anti-HIV‑1 2G12 Antibody by Single-Molecule Force Spectroscopy Elena Martines,† Isabel García,†,§ Marco Marradi,†,§ Daniel Padro,‡ and Soledad Penadés*,†,§ †

Laboratory of Glyconanotechnology, Biofunctional Nanomaterials Unit, ‡Molecular Imaging Unit, CIC biomaGUNE, and Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Paseo de Miramón 182, 20009 San Sebastian, Spain

§

S Supporting Information *

ABSTRACT: Broadly neutralizing anti-HIV-1 monoclonal antibody 2G12 exclusively targets a conserved cluster of high-mannose oligosaccharides present on outer viral envelope glycoprotein gp120. This characteristic makes the otherwise immunogenically “silent” glycan shield of gp120 a tempting target for drug and vaccine design. However, immune responses against carbohydrate-based mimics of gp120 have failed to provide immunization against HIV-1 infection, highlighting the need to understand the molecular events that determine immunogenicity better. In this work, the unbinding kinetics of the gp120−2G12 (k0 = 0.002 ± 0.09 s−1, x⧧ = 1.5 ± 1.2 nm), Man4−2G12 (k0 = 0.35 ± 0.32 s−1, x⧧ = 0.6 ± 0.2 nm), and Man5−2G12 interactions were measured by single-molecule force spectroscopy. To our knowledge, this is the first single-molecule study aimed at dissecting the carbohydrate−antibody recognition of the gp120−2G12 interaction. We were able to confirm crystallographic models that show both the binding of the linear Man4 arm to 2G12 and also the multivalent gp120 glycan binding to 2G12. These results demonstrate that single-molecule force spectroscopy can be successfully used to dissect the molecular mechanisms underlying immunity.

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INTRODUCTION Recent advances in the discovery of new potent broadly neutralizing antibodies against the human immunodeficiency virus type 1 (HIV-1)1 and promising results from the Thai human clinical trial2 are bringing back optimism in the quest for an effective antibody-based vaccine against HIV-1. Nevertheless, despite nearly three decades of searching for a vaccine, the specific immune components sufficient to protect against HIV-1 are not known.3 Several broadly neutralizing antibodies have been proposed as the basis for designing protective mechanisms against HIV-1 in recent years.4,5 Among them, monoclonal antibody (mAb) 2G12 was the first carbohydratetargeting antibody to be discovered. 2G12 recognizes the mannose-rich glycan shield of gp120 with high affinity6,7 (Figure 1a) and has an unusual structure that involves a domain swap between the two heavy chains8 that allows the formation of an additional nonconventional carbohydrate binding site. The major target of 2G12 is the undecasaccharide Man9(GlcNAc)2 (Figure 1 b), which exposes terminal arms Manα1-2Manα1-2Manα1-3Man (Man4 or D1 arm, Figure 1c) and Manα1-2Manα1-3[Manα1-2Manα1-6]Man (Man5 or D2D3 arm, Figure 1d). Crystallographic data have shown that antibody combining sites are occupied by Manα1-2Man residues from the D1 arm of the antigen whereas the secondary binding site interacts with the D2 arms.8 On the basis of these data, a molecular model for the gp120−2G12 interaction has been proposed in which four separate Man9(GlcNAc)2 entities © 2012 American Chemical Society

mediate the binding of gp120 to 2G12, where the unusually high affinity of the interaction is due to the recognition of a multivalent array of oligomannose residues by an equivalent array of antibody binding sites that match the spacing of the individual sugars within the oligomannose cluster on gp120.8 Later, ELISA assays and crystal structures of 2G12 in complex with various portions of Man9(GlcNAc)2 indicated that the D3 arm of the antigen is also capable of binding to 2G129 whereas the binding of the D2D3 arm has also been observed by NMR.10,11 Accordingly, Man4 and Man5 derivatives were identified as effective inhibitors as Man9 at inhibiting the gp120−2G12 interaction.9,12 Since then, several multivalent constructs mimicking the high-mannose cluster of gp120 have been developed in the attempt to find a synthetic, carbohydrate-based vaccine that would produce 2G12-like antibodies.13−17 Unfortunately, attempts to elicit a humoral response to synthetic carbohydrate constructs have proven unsuccessful so far,18−20 calling for further investigations of the molecular mechanisms underlying carbohydrate-based immunity against HIV-1. The kinetics of the gp120−2G12 interaction and the influence o f ind ivid ual o ligomanno side arms of Man9(GlcNAc)2 such as the Man4 arm and the Man5 arm Received: August 29, 2012 Revised: November 22, 2012 Published: November 30, 2012 17726

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glycoprotein gp120. By measuring the dissociation kinetics of the antibody with the whole glycoprotein and with two individual oligomannose arms of Man9(GlcNAc)2, namely, the linear Man4 arm and the branched Man5 arm, we gathered direct evidence at the single-molecule level that the gp120− 2G12 bond has a longer lifetime than the D1−2G12 bond. These results can be correlated to previous findings in which several carbohydrate−antibody bonds are responsible for the full multivalent gp120−2G12 interaction and suggest that the low conformational flexibility of the oligomannose cluster can ensure structural specificity with respect to the recognition of HIV-1 by mAb 2G12.

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MATERIALS AND METHODS

Materials. Commercially available analytical-grade materials were used. The synthons were of the highest available purity. α-Maleimidoω-carboxy-succinimidyl ester poly(ethylene glycol) (mal-PEG-NHS, MW = 2950 Da), α-hydroxy-ω-carboxy poly(ethylene glycol) (OHPEG-COOH, MW = 3300 Da), and α-ω-disuccinimidyl ester poly(ethylene glycol) (NHS-PEG-NHS, MW = 3000 Da) were purchased from Iris Biotech (Germany). (3-Aminopropyl)triethoxysilane (APTES), triethanolamine (TEA), and methyl α-Dmannoside were purchased from Sigma (Spain). The synthesis of the oligomannosides was carried out as previously reported.12,40 Man5-like and Man4-like synthetic oligomannosides as depicted in Figure 1c,d were used. Monoclonal antibody 2G12 (145 kDa)41 was kindly supplied by Dr. D. Katinger (Polymun Scientific, Vienna, Austria). Recombinant glycosylated gp120 from an HIV-1 CN54 clone (repository reference ARP683, 120 kDa) was obtained from the Programme EVA Centre for AIDS Reagents, NIBSC, U.K., supported by the EC FP6/7 Europrise Network of Excellence, AVIP and NGIN Consortia, and the Bill and Melinda Gates GHRC-CAVD Project and was donated by Prof. Ian Jones (Reading University, U.K.). AFM Probe Functionalization. The AFM probes were cleaned by dipping for 10 min in chloroform and then for 5 min in a piranha solution (H2SO4/H2O2, 70:30 v/v at 50 °C), rinsed with a copious amount of deionized filtered water and ethanol, and dried under a flow of N2. The cleaned AFM probes were immediately immersed in a 1.5% v/v solution of APTES in dry toluene. A custom-made Teflon holder ensured that the tips were facing downward and far from the bottom of the container in order to avoid the deposition of aggregates on the tip. After 2 h, the silanized probes were washed with toluene, ethanol, water, and PBS (pH 7.4). Subsequently, the probes were immersed in a dry DMSO solution of mal-PEG-NHS (3 mg/mL), NHS-PEG-NHS (15 mg/mL), or OH-PEG-NHS (15 mg/mL) for oligomannoside, gp120, and control functionalizations, respectively. After 3 h, the malmodified probes were washed with DMSO, dried under a flow of N2, and immersed in a PBS solution of Man4 (1 mg/mL) or Man5 (1 mg/ mL) at room temperature overnight. For gp120 conjugation, NHSmodified probes were incubated with a 50 μg/mL solution of gp120 in PBS (37 °C, 1 h), washed with PBS, and incubated with TEA (0.5%, 1 h). After being rinsed with PBS, the functionalized probes were not allowed to dry out before the measurements and were used immediately or stored at 4 °C in PBS for up to 24 h. The probefunctionalization protocol was validated by performing control experiments, during which NHS-modified AFM probes were functionalized with 5-mercaptopentyl α-D-mannopyranoside (ManC5SH), concanavalin, and antibodies and analyzed by XPS, TOF-SIMS, and fluorescence microscopy (Supplementary Methods in SI). Gold Chip Functionalization. The gold surface of a CM5 sensor chip (GE Healthcare) was carefully extracted from the chip and used to immobilize 2G12 on carboxymethylated dextran (MW = 500 000 Da) covalently linked to the gold surface through epoxy alkanethiols. Activated carboxyl groups on the dextran reacted covalently with the amines of the antibody, whereas the surrounding dextran minimized nonspecific adhesion during AFM measurements. The detailed chip functionalization strategy is reported in the Supporting Information. Our force measurements confirm that dextran chips can be efficiently

Figure 1. (a) Schematic illustration of mAb 2G12 binding to glycoprotein gp120 on the outer envelope of HIV-1. (b−d) Chemical structure and schematic representation of (b) Man9(GlcNAc)2, (c) synthetic Man4, and (d) synthetic Man5 glycosides used in this work.

have been measured by NMR10,15 and SPR methods14,15,21 but never by single-molecule experiments. The atomic force microscope (AFM) has been widely used to measure the kinetic parameters of a variety of receptor−ligand interactions at the single-molecule level, including carbohydrate−carbohydrate,22−24 carbohydrate−protein,25−29 and antibody−antigen interactions.30−36 In immunology, single-molecule force spectroscopy (SMFS) has been used to study the gp120−CD4 bond on live cells37 and the interaction of whole viruses with their receptors.38,39 In this work, SMFS has been applied to dissect the interaction between mAb 2G12 and its viral antigen on 17727

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used for single-molecule force spectroscopy measurements as previously shown by Stevens et al.42 SMFS Measurements. SMFS measurements were carried out with a commercial AFM instrument (Nanowizard II, JPK, Berlin) at room temperature using soft triangular silicon nitride cantilevers (MLCT, Veeco) for the Man4−2G12 and Man5−2G12 measurements and using rectangular silicon nitride cantilevers (OMCL-TR400PSA, Olympus) for the gp120−2G12 measurements. The spring constants of every AFM cantilever were individually determined by the thermal noise method.43 Force−distance curves were recorded in 0.2 μm filtered PBS (pH 7.4). Between 400 and 2000 force−distance curves per velocity were acquired under identical conditions by raster scanning the tip over a 5 μm × 5 μm area. The tip was allowed to interact with the surface for 0.1 s with a force of 100−150 pN. The retraction velocities ranged from 100 to 2000 nm/s and were cycled in order to distribute tip wearing effects over all of the loading rates. Tip wearing effects appeared with increasing tip velocity and acquisition time. The specificity of the Man4−2G12, Man5−2G12, and gp120− 2G12 interactions was verified by blocking with 500 mM methyl α-Dmannoside in PBS at the end of the experiment. Negative controls were performed by functionalizing the tip with hydroxyl-terminated PEG linkers (HO-PEG-NHS, hereafter referred to as PEG-OH) and measuring their interaction with 2G12-functionalized chips. Data Analysis. The data were analyzed with the JPK data processing software. All rupture events were fitted with the freely jointed chain (FJC) polymer stretching model, which yielded the polymer contour length, Kuhn length, instantaneous loading rate, and unbinding force at rupture. When the force curves showed more than one rupture event, only the last rupture event was considered in order to avoid the possible convolution of several parallel ruptures. Initially, histograms showing the distributions of all of the contour lengths of the FJC-selected ruptures were plotted for each velocity, which allowed us to identify a contour length peak between 10 and 80 nm for the Man4−2G12 and Man5−2G12 measurements and between 10 and 60 nm for the gp120−2G12 measurements. The ruptures occurring within these contour length bandwidths were further filtered to include only stretchings with Kuhn lengths of between 0.5 and 0.9 nm to include PEG-related stretchings only (average Kuhn length 0.7 nm) and plotted as force histograms. The most probable forces of interaction were determined by fitting the force histograms to a Gaussian model. The data were then analyzed according to the stochastic Bell−Evans model of a forced dissociation under an external load.44,45

Figure 2. Schematic illustration of the experimental setup (not to scale) showing a dextran chip functionalized with 2G12 interacting with PEGylated AFM chips functionalized with (a) the linear tetramannoside Man4 arm of Man9(GlcNAc)2, (b) the branched pentasaccharide Man 5 arm of Man 9 (GlcNAc) 2 , and (c) Man9(GlcNAc)2 present on the whole recombinant gp120 protein.

(inset a1). The normal distributions of the contour lengths suggest that the observed unbinding events were specific (Figure 3a, inset a2) because they showed defined peaks at 31− 41 nm. These values are higher than the expected stretched length of 24 nm for 3000 Da PEG molecules, and we attribute the discrepancy to some degree of stretching from the dextran chains as previously demonstrated.42 The low but nonnegligible binding probabilities (Pb = 7−18% across loading rates) suggest that single-molecule interactions were observed.46 The specific force distributions also exhibited a prominent peak (Figure 3a) that shifted to higher forces with higher velocities (Table S1), as expected for specific ligand− receptor unbinding. When PEG-OH was attached to the tip and interacted with 2G12, no binding was detectable. Moreover, the addition of 500 mM methyl α-D-mannoside to the measuring buffer decreased the Man4−2G12 binding probability to a residual 0.1%, thus proving the specificity of the interaction. Having observed that the unbinding forces became stronger with increasing loading rates, the Man4−2G12 unbinding events were analyzed with the Bell−Evans model44,45 for slip bonds, which predicts a shift of the most frequent rupture force F (as defined by the maximum in the force distribution) with larger loading rates according to eq 1.34

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RESULTS AND DISCUSSION A schematic representation of our experimental system is illustrated in Figure 2. We used thiol-ending Man4 and Man5 ligands to immobilize the oligosaccharides at the tip functionalized by maleimide using poly(ethylene glycol) (PEG) spacers. Fully glycosilated gp120 protein was covalently attached to Nhydroxy-succinimide (NHS)-functionalized tips using its Lys residues for peptidic coupling. Freshly prepared thiol Man4 and Man5 ligands were used in each experiment, and previous treatment with dithiothreitol (DTT) was performed to minimize the dimerization by S−S bond formation. Control functionalization experiments confirmed the validity of the immobilization strategy for Man4, Man5, and gp120 on the AFM tips, where XPS, TOF-SIMS, and fluorescence microscopy analyses confirmed the presence of mannose and protein residues on the AFM probes (Figures S1−S5 in the Supporting Information (SI)). mAb 2G12 was immobilized on carboxymethylated dextran, and this procedure is described in detail in the SI (chip functionalization strategy). The full list of specific contour length and force histograms of the SMFS experiments between Man4 and 2G12 can be found in the SI (Table S1). Representative force−distance curves of the Man4−2G12 interaction are shown in Figure 3a

F=

kBT ⧧

x

⎡ x⧧r ⎤ F ⎥ ln⎢ ⎢⎣ k 0kBT ⎥⎦

(1)

where k0 is the unstressed thermal off-rate, kBT and rF denote the thermal energy and the instantaneous loading rate, respectively, and x⧧ is the reaction length representing the distance between the minimum of the binding potential and the transition state that separates bound from free states. In a dynamic force spectroscopy spectrum, the measured F values are plotted against the respective loading rates in a semi17728

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Figure 4. Dynamic force spectra of the gp120−2G12 unbinding. Solid and dashed lines represent the best fits of each spectrum to eq 1. The statistical error of the unbinding force was estimated to be the width of the distribution divided by the square root of the number of unbinding events.34 The instantaneous loading rate rF was calculated as rF = kckts/ (kc + kt), with kc being the spring constant of cantilever, kt being the instantaneous spring constant of tether at rupture, and s being the probe velocity.48

Table 1. Kinetic Parameters of the Unbinding of 2G12 with Man4 and gp120 Showing Kinetic Off-Rate k0 and Reaction Length x⧧, with the Errors Calculated by the Propagation of Uncertainty

Figure 3. SMFS results and the analysis of (a) the specific Man4− 2G12 interaction and (b) the specific gp120−2G12 interaction. (a) Representative force distribution histogram measured by SMFS between Man4 and 2G12 at 317 pN/s (n = 72). Inset a1 shows representative force−distance curves, where only the last rupture was considered to be specific (Materials and Methods). The corresponding FJC fit is shown in Figure S6a. Inset a2 shows the corresponding rupture distance distribution. (b) Representative force distribution histogram measured by SMFS between gp120 and 2G12 at 391 pN/s (n = 45). Inset b1 shows representative force−distance curves. The corresponding FJC fit is shown in Figure S6b. Only the last rupture events were considered to be specific. Inset b2 shows the corresponding rupture distance distribution. Solid lines over histograms are Gaussian fits.

gp120 Man4

k0 (s−1)

x⧧ (nm)

0.002 ± 0.09 0.35 ± 0.32

1.5 ± 1.2 0.6 ± 0.2

Man5−2G12 interaction,10 which might result in unbinding forces that are too low or too infrequent to be detected by our experimental setup. Low binding probabilities were again observed for the gp120−2G12 interaction (Pb = 5−9%), with defined contour length peaks at 21−27 nm that are coherent with the expected stretched length of 24 nm for 3000 Da PEG molecules (Figure 3b, inset b2). The full list of specific force and rupture distance histograms of the force spectroscopy experiments between gp120 and 2G12 can be found in Table S1. An analysis of the rupture forces between gp120 and 2G12 also revealed a peak in the force histograms (Figure 3b), with peak values shifting to higher forces with higher velocities. The addition of 500 mM methyl α-D-mannoside to the measuring buffer decreased the gp120−2G12 binding probability to a residual 0.2%, thus proving the specificity of the interaction. The Bell−Evans model was also fitted to the gp120−2G12 dynamic force spectrum; however, the slope of the linear fit was very low in this case. Therefore, it cannot be excluded that equilibrium unbinding was observed.49 In fact, extrapolating to zero force yielded kinetic parameters with a very high error (k0 = 0.002 ± 0.09 s−1, x⧧ = 1.5 ± 1.2 nm) (Table 1). Thermal offrates on the order of ∼10−3 s−1 have been previously measured by SMFS for the unbinding of antigens from antibodies such as anti-HSA,33 antifluorescein scFv,34 anti-GCN4 scFv,31 antiMUC1 scFv,36 and antilysozyme Fv.30 However, although reaction lengths longer than 1 nm are not common, and Marshall et al.28 have found x⧧ = 2.41 nm for the unbinding of P-selectin-sPSGL-1, which, interestingly, is another protein− glycoprotein interaction like gp120−2G12. We applied the analytical model from Gomez-Casado et al.49 to determine if the Man4−2G12 bond can be modeled as a

logarithmic way. The reaction length x⧧ is then obtained from the slope of the linear fit of the data by the Bell−Evans model, and the unstressed thermal off-rate k0 can be deduced by linearly extrapolating the experimental data to zero external force. Figure 4 shows the dynamic force spectra of the Man4−2G12 and gp120−2G12 interactions. The off-rate and reaction length of the Man4−2G12 interaction (Table 1) are both higher than measured by force spectroscopy for other carbohydrate−lectin (k0 = 0.17 s−1, x⧧ = 0.27 nm)25 and selectin−glycoprotein (k0 = 0.2 s−1, x⧧ = 0.14 nm)27 interactions. A remarkable exception to this trend is the L-selectin−PSGL-1 bond (k0 = 3 s−1, x⧧ = 0.4 nm).47 Instead, the single sLeX−E-selectin and sLeX−P-selectin bonds measured by SMFS have shown kinetic parameters similar to those of the Man4−2G12 bond (k0 = 0.3 s−1, x⧧ = 0.5 nm; k0 = 0.3 s−1, x⧧ = 0.45 nm),29 which establishes an interesting similarity between different single carbohydrate− protein interactions. In the case of the Man5−2G12 interaction, the binding probabilities across loading rates dropped to 0.4−1.3%. Therefore, unlike previous reports,9,10 we did not detect any significant Man5−2G12 interaction because its binding probability fell in the same range as nonspecific binding. The discrepancy could be due to the low affinity (2.9 mM) of the 17729

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might “dilute” the immune response to multivalent carbohydrate anti-HIV vaccine candidates18 because antibodies raised against different cluster conformations would not be as effective at targeting HIV-1. These force spectroscopy results exemplify structural specificity by showing that although 2G12 binds part of the oligomannoside cluster with a shorter lifetime, only exact matching with the full oligomannoside cluster will lead to a persistent bond. In turn, this suggests that anti-HIV-1 monoclonal antibodies raised against flexible constructs might not have the exact geometrical requirements to bind with a long lifetime to gp120 in vivo, although they might still be capable of binding with short-bond survival rates. Therefore, our results demonstrate the need to consider both chemical and structural specificity when designing carbohydrate-based synthetic vaccine candidates.

binding subunit of the full antibody−antigen interaction gp120−2G12. This model was successfully used to predict the valency and the kinetics of the interaction between synthetic multivalent assemblies. In the case of a divalent interaction, k 0 ‐ divalent =

2 × k 0 ‐ monovalent Keq ‐ monovalent × Ceff

(2)

where the thermal off-rate of the monovalent interaction (k0‑monovalent), the binding constant of the monovalent interaction (Keq‑monovalent), and the local effective concentration of molecules at the tip (Ceff) have been used to predict the thermal off-rate of the divalent interaction k0‑divalent. The local effective concentration is Ceff =

3 2ANR rms

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CONCLUSIONS Single-molecule force spectroscopy has been successfully applied to measure the lifetimes of the bonds among antiHIV-1 monoclonal antibody 2G12, fully glycosylated outer viral envelope protein gp120, and its linear Man4 arm. The branched Man5 arm did not show any significant interaction with 2G12, but the linear Man4 arm interacted with unbinding parameters similar to those of previously measured carbohydrate−protein interactions. The interaction of 2G12 with gp120 was longerlived than with Man4, and it was shown that analytical predictions agree with a model where at least three Man4 branches interact with 2G12. Although further detailed investigations and a dissection of hierarchical multivalent interactions are needed, these experiments confirm that single-molecule force spectroscopy is a powerful tool for relating immunity to molecular properties. These results also suggest that the design of next-generation fully synthetic multivalent carbohydrate-based vaccines should mimic not only the chemical but also the structural properties of the antigenic clusters.

(3)

where A is the surface area covered by one PEG-attached antigen, N is Avogadro’s number, and Rrms is the root-meansquare end-to-end distance of the PEG linker (MW = 3000 kDa).50 If we assume k0‑monovalent to be the Man4−2G12 thermal off-rate and Keq‑monovalent to be the affinity of Man4−gp120 as measured by STD NMR (Keq‑monovalent ≅ 2.5 × 103 M−1 10), then the predicted k0‑divalent would be ∼0.03 s−1 and k0‑trivalent would be ∼0.006 s−1. We find remarkable agreement between the latter value and our fitted thermal off-rate for the gp120− 2G12 interaction, which is coherent with the multivalent binding configuration that was revealed by the crystal structure of Fab 2G12 bound to Man9(GlcNAc)2.8 In fact, the crystal structure showed a rigid (i.e., with low conformational flexibility) cluster of up to four Man9(GlcNAc)2 molecules bound to the carbohydrate-recognition domain of the 2G12 antibody, where the linear Man4 arm occupies the antibody combining sites. Therefore, our results seem to confirm that at least three Man4−2G12 bonds are responsible for the full multivalent gp120−2G12 interaction. There are obvious limitations to this analysis. First, the analytical model by Gomez-Casado et al. has been verified with a well-controlled chemical system, and the gp120−2G12 interaction has a hierarchical structure, where partial binding could be achieved by one or more Man9(GlcNAc)2 molecules or by individual moieties of Man9(GlcNAc)2.9 Moreover, the uncertainty in the gp120−2G12 thermal dissociation rate is high as a result of the low slope of the linear Bell-model fit. Nevertheless, these results show that with appropriate molecular systems SMFS can be successfully used to dissect the complex interactions involved in immunity. Moreover, we have proven that although the linear Man4 arm is capable of individual binding to 2G12 the full gp120−2G12 antibody−antigen interaction has a longer lifetime as expected for multivalent interactions. Multivalency is key in several natural recognition processes51 as a binding mechanism that can be rapidly modulated by regulating an assembly of low-affinity interactions. Multivalency can lead to a significant enhancement in binding strength for weakly binding ligands such as protein−carbohydrate systems.52 The binding of multivalent clusters needs defined geometrical matching, as is the case for 2G12 recognizing oligomannose chains on gp120 at a spacing of about 35 Å.8 Therefore, structural matching in addition to chemical specificity is added to the recognition process.53 It has indeed previously been suggested that high conformational flexibility in the oligomannose arms of synthetic Man9(GlcNAc)2 clusters

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ASSOCIATED CONTENT

S Supporting Information *

Control experiments to test the probe functionalization strategy. XPS and TOF-SIMS spectra. Fluorescence imaging of AFM probes after secondary immunostaining. Chip functionalization strategy. Contour length distributions. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was funded by the Ministry of Science and Innovation (MICINN, grants CTQ2008-04638 and CTQ201127268), the European Union (grant CHAARM, Health-F32009-242136), and the Department of Industry of the Basque Country (grant Etortek 2009). We thank Dr. R. Richter and Prof. B. Ackhremitchev for discussions, Dr. D. Katinger for providing 2G12, and Dr. C. Serra (CACTI, University of Vigo) for help with XPS and TOF-SIMS measurements. 17730

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