Supramolecularly Oriented Immobilization of Proteins Using Cucurbit

Article pubs.acs.org/Langmuir

Supramolecularly Oriented Immobilization of Proteins Using Cucurbit[8]uril Arántzazu González-Campo,† Melanie Brasch,† Dana A. Uhlenheuer,∥ Alberto Gómez-Casado,† Lanti Yang,† Luc Brunsveld,∥ Jurriaan Huskens,*,† and Pascal Jonkheijm*,† †

Molecular Nanofabrication Group, Department of Science and Technology, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE, Enschede, Netherlands ∥ Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven, Netherlands S Supporting Information *

ABSTRACT: A supramolecular strategy is used for oriented positioning of proteins on surfaces. A viologen-based guest molecule is attached to the surface, while a naphthol guest moiety is chemoselectively ligated to a yellow fluorescent protein. Cucurbit[8]uril (CB[8]) is used to link the proteins onto surfaces through specific charge-transfer interactions between naphthol and viologen inside the CB cavity. The assembly process is characterized using fluorescence and atomic force microscopy, surface plasmon resonance, IR-reflective absorption, and X-ray photoelectron spectroscopy measurements. Two different immobilization routes are followed to form patterns of the protein ternary complexes on the surfaces. Each immobilization route consists of three steps: (i) attaching the viologen to the glass using microcontact chemistry, (ii) blocking, and (iii) either incubation or microcontact printing of CB[8] and naphthol guests. In both cases uniform and stable fluorescent patterns are fabricated with a high signal-to-noise ratio. Control experiments confirm that CB[8] serves as a selective linking unit to form stable and homogeneous ternary surface-bound complexes as envisioned. The attachment of the yellow fluorescent protein complexes is shown to be reversible and reusable for assembly as studied using fluorescence microscopy.

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INTRODUCTION

An ideal starting point for such surface-anchored protein assemblies is the macrocyclic host molecule cucurbit[8]uril (CB[8]),7 which has a cavity capable of forming 1:2 complexes with peptide motifs.8 For example, proteins genetically encoded with an N-terminal FGG motif can be assembled into a homodimer via 2-fold binding of the peptide motif within CB[8].8b CB[8] is also known to accommodate stable 1:1:1 ternary complexes with an electron-deficient supramolecular guest molecule such as methyl viologen (MV) and an appropriately corresponding electron-rich guest such as alkoxynaphthalene, as shown in Scheme 1.9,10 Formation of such ternary complexes is characterized by host−guest-assisted charge transfer (CT) interactions with binding constants of 1011−1013 M−2.9,10 CB[8] can therefore tie together two different building blocks to form a supramolecular assembly. As a result, CB[8] has recently been applied in constructing in solution molecular machines,11 polymeric assemblies,12 and peptide8a,13 and protein assemblies.8b,14 Using CB[8] in combination with redox-active guest moieties, will contribute to developing novel tailor-made routes to dynamically

Supramolecular positioning of proteins at surfaces has widened the scope of protein technological applications, allowing for the design and development of dynamic protein biochips.1 Adopting supramolecular chemistry is an attractive strategy to modulate biomimetic functions in solution.2,3 Immobilization approaches based on biological and synthetic supramolecular motifs have been reported in recent years;4 however, it remains challenging to design supramolecular protein conjugates with functional groups that are able to reversibly immobilize proteins onto solid supports.5 The recent report by Grunwald et al. accomplishes an autoinhibition strategy in which a surfacebound multivalent chelator head is tethered with an intramolecular ligand that can initially compete with and inhibit binding of His-tagged proteins while binding was activated by a photoreaction separating the intramolecular ligand from the multivalent chelator head.5c While the above-mentioned dynamic supramolecular protein immobilization strategies are documented in the literature, functional attachments of proteins to solid supports through elements that are responsive to electrochemical stimuli is still required and an important target for dynamic substrates for cell-responsive studies when using proteins.6 © 2012 American Chemical Society

Received: October 7, 2012 Revised: November 6, 2012 Published: November 7, 2012 16364

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Scheme 1. Chemical Structures of Synthesized Compounds and Protein: MV2+-NH2 1, YFP-naphthol 2, Lis-naphthol 3, and CB[8]

Scheme 2. Functionalization of Silica Particles and Formation of the Ternary Complex on the Particlesa

a

See text for details. The last step is a mild sonication step to loosen the particles from the surface. The final particle is represented as a cartoon.

consequently released via chemical reduction,15c in this report we reversibly immobilize and pattern proteins. This is not a trivial step, as it is well-known that constructing protein arrays requires more steps and is more complex than the generation of DNA and peptide microarrays, in particular, owing to the sensitive nature of proteins, which often results in (partial) denaturation upon chemical treatment and immobilization.1,16 The choice of the immobilization method can seriously affect the three-dimensional structure and orientation of the protein.1,16 In addition, no information is currently available on the details of orienting proteins on MV-modified surfaces using CB[8] in combination with microcontact printing. Therefore, we have studied in detail the supramolecular patterning of proteins using the CB[8]-mediated complexation

immobilize proteins and access to controllable assembly by a stimulus. We believe that this strategy provides a new avenue to construct dynamic functional interfaces, e.g. for responsive cell−interface studies.6 Here we report the site-specific immobilization of proteins to solid surfaces through a ternary complex mediated by CB[8], motivated by recent work on the use of CB[8] to graft supramolecular polymers, colloids, and short peptides onto Au surfaces.6,15 While CB[7] has been used for stable protein immobilization,4d,e in this work we report the first use of CB[8]-mediated assembled monolayers for the supramolecular assembly of proteins. Although N-terminal tryptophan pentapeptides were selectively immobilized onto a viologenfunctionalized gold surface with CB[8] ternary complexes and 16365

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Figure 1. Fluorescence microscopy images of ternary complexes on silica particles formed by (a) CB[8] capturing surface-bound 1 and YFPnaphthol 2 from solution with (b) a zoom-in image with (c) corresponding bright field image. (d) Bright-field and fluorescence microscopy (inset) image of control assembly experiment using YFP without naphthol (more control experiments can be found in the Supporting Information).

We first investigated the possibility of assembling YPFnaphthol 2 onto the surface of microparticles using CB[8] as the linker to surface-bound viologen. To this end, an aqueous suspension of SiO2-microparticles of 4.74 μm in diameter on average were spin-coated (1000 rpm, 20 s) onto an oxidized chip to ease the handling of the particles during functionalization of the surfaces with amino-functionalized viologen 1 (Scheme 2). After 30-min exposure to oxygen plasma, vaporized TPEDA (N-([3-(t riethoxysilyl)propyl]ethylenediamine) was reacted with the OH groups of the surface of the particles, resulting in the formation of aminoterminated particles. Then after rinsing, incubation followed in a solution of glutaric anhydride to yield carboxylic acid terminated-particles. Subsequently, the carboxylic acid groups of the particles were NHS-activated (N-hydroxysuccinimide) in a MES buffer (pH = 5.6) and, after rinsing, directly incubated for 2 h in a deoxygenated solution of 1 using a carbonate buffer (pH = 9.2).17,20 Copious rinsing with buffer was followed by the removal of the particles from the surface by slight sonication for 2 min in acetone. These MV2+-functionalized silica particles were deposited onto a freshly oxidized glass slide and immersed for 1 h in a 10 μM PBS solution of YFPnaphthol 2 and 75 μM CB[8] in PBS buffer for 2 h. The CB[8]-controlled protein assembly on the particles was studied using fluorescence microscopy (Figure 1). The recorded bright field and fluorescence microscopy images show intense emission from all particles, only in the presence of all three supramolecular components (Figure 1a). Control experiments using YFP without the naphthol moiety (Figure 1b) or when CB[8] was left out (Figure S3c, Supporting Information) showed only particles that were not fluorescent. These results indicate that the protein assembly on the particles occurs as envisioned through the CB[8]-mediated association of the naphthol tag with the viologen capped particles and that these guest moieties do not lead to unspecific binding or protein interactions. The observation made by fluorescence microscopy were confirmed by IR experiments on the functionalized particles, showing the characteristic signals of CB[8], which correspond to respectively CO and C−N stretch vibrations (Figure S7a, Supporting Information). Subsequently, the protein assembly on glass biochip surfaces was investigated. Aiming to use fluorescence spectroscopy to monitor the supramolecular assembly of the protein on the surface, glass surfaces are favored over gold for generating protein chips to avoid quenching of the emission (here of lissamine or YFP) to the surface as commonly observed in the case of gold surfaces.1 In addition, microcontact chemistry21

between viologen-modified glass biochips, which is the most commonly used substrate in biotechnological research,1 and naphthol-modified proteins.

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RESULTS AND DISCUSSION We have chosen yellow fluorescent protein (YFP) as a model protein for immobilization onto surfaces. YFP was chosen as it allows the assembly process to be followed using fluorescence spectroscopy to specifically visualize the interaction between the surface and the protein. This will provide direct information on the assembly process as well as on the integrity of the protein, in contrast to optical techniques that visualize the host−guest assembly in the CB host cavity.14 In order to demonstrate the versatility of the immobilization of the reversible supramolecular bioconjugates, the immobilization to both particles and chip surfaces were each investigated. The viologen-based guest molecules (Scheme 1) were synthesized from commercially available bipyridine (Supporting Information). MV was obtained by slow addition of methyl iodide to 4,4′-bipyridine. The monomethylated product was further reacted with 3-bromopropylamine to give an asymmetric substituted viologen derivative,17,18 which was coupled to appropriately functionalized glass chips (vide infra). The naphthalene-based guest molecule (Scheme 1) was synthesized from commercially available 2-naphthalenol to which methyl-6bromohexanoate was coupled (Scheme S1, Supporting Information). After deprotection, the carboxylic acid was reacted with a Boc- and StBu-protected cysteine, which was first modified with a tetraethylene glycol spacer to create distance between the protein and the surface. After purification using column chromatography, the resulting compound was Boc-deprotected, and subsequently, shortly prior to protein ligation, also the StBu disulfide was removed by addition of TCEP. Following standard protocols C-terminal thioesterderivatized YFP was expressed, purified,19 and ligated to the cysteine-modified naphthol guest molecule in the presence of 2mercaptoethanesulfonate overnight to give complete conversion. Subsequently, the functional YFPs were purified from any remaining small molecule reagents by centrifugal filtration. The purity of these functionalized YFP proteins (2) was confirmed by LC−MS (Figures S1 and S2, Supporting Information). Furthermore, 6-(2-naphthol)hexanoic acid was reacted with tetraethyleneglycol diamine and subsequently with lissamine sulfonyl chloride to give a naphthol-functionalized fluorescent dye 3 (Schemes 1 and S2, Supporting Information). This dye was used for studying and optimizing the surface assembly and patterning. 16366

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Scheme 3. Two Immobilization Routes Are Followed for Patterning Complexes on Glass Chipsa

a

See text for details.

was employed as a viable approach to fabricate arrays of protein complexes at the chip surface (Scheme 3), which are of interest in nanotechnology and materials science.1 In the case of the CB[8]-induced attachment of naphtholYFP to viologen particles (vide supra), a one-step incubation of the viologen particles was carried out in a solution containing both CB[8] as well as naphthol-YFP (vide supra). This strategy contrasts with previous reports on CB[8]-mediated immobilization on surfaces.15 For example, the immobilization of naphthol-terminated colloids onto viologen-terminated surfaces was carried out in a stepwise fashion by Scherman et al.; i.e., first the host CB[8] was assembled onto the pre-immobilized viologen motifs and then these viologen−CB[8] surfaces were immersed into a solution of naphthol colloids or tryptophan peptides.15c,d Another strategy was reported in the case of the growing of poly(pseudorotaxane)s on surfaces by Kim et al.15b In this case, a substrate was immersed in a solution containing the CB[8]−viologen complex and subsequently this solution was exchanged for a solution containing guest molecules.15a,b We performed SPR experiments on gold surfaces, which are compatible with SPR read-out, to investigate whether the chosen assembly route leads to differences in stability of the complexes at the surface. SPR was used to measure the change in reflectivity upon assembly at the surface, which is used as a qualitative read-out of the average surface coverage. To this end, SAMs on gold were obtained through the so-called mixed assembly of 3-mercaptopropionic acid (MPA) and 1-(1-decyl6-thiol)-1-methyl-4,4-bipyridinium.15a Although such types of SAMs are generally less ordered, the strong repulsive interactions between bipyridyl dications is favorably diminished.22 Three SPR sensograms were recorded on mixed MPA/ MV2+-monolayers (Figure 2a,b,d), and one SPR sensogram was measured on a mixed monolayer of MPA/MV2+−CB[8] (Figure 2c). CB[8] and 2-naphthol were used at 75 and 10 μM, respectively. The SPR experiments confirm the specific interaction between naphthol guests only in the presence of CB[8], while the assemblies are equilibrating between surfacebound and dissolved species when buffer flowed over the surfaces, in agreement with thermodynamic expectations.9 Furthermore, under these conditions, a monolayer, which was made by overnight immersion of a slide into a mixture of MV2+−CB[8] complexes, led to a higher naphthol surface coverage (Figure 2c) when compared to the sequential surface incubation of CB[8] and naphthol (Figure 2b), whereas the simultaneous surface incubation of CB[8] and naphthol yields the highest naphthol coverage (Figure 2a) by favorably making use of the high ternary association constant. Therefore, in the following experiments the latter assembly protocol (simultaneous incubation) will be employed.

Figure 2. SPR sensograms of (a) flowing over a MV2+ monolayer first with PBS buffer and then a solution consisting of a mixture of CB[8] and 2-naphthol (75 μM/10 μM); (b) flowing over a MV2+ monolayer first with PBS buffer, then a solution consisting of only CB[8] (75 μM), and subsequently a solution consisting of only 2-naphthol (10 μM); (c) a monolayer, made by dipping a slide into a preassembled mixture of MV2+/CB[8], was, after drying, immersed into first buffer and then a solution of 2-naphthol (10 μM); and (d) flowing over a MV2+ monolayer first with PBS buffer and then a solution of 2naphthol (10 μM). Squares indicate switching of the flow back to PBS buffer.

To pattern naphthol-YFP 2 onto glass, TPEDA-activated glass was used to react with N,N-carbonyldiimidazole (CDI) to form imidazolide (IM) monolayers on glass as previously described.23 Two immobilization routes were followed to form patterns of the ternary complexes on the glass surfaces, as displayed in Scheme 3. Each immobilization route consisted of three steps: (i) attaching the viologen to the glass, (ii) blocking, and (iii) either simultaneous incubation or printing with CB[8] and naphthol guests. In the first step of both immobilization routes reactive microcontact printing (μCP) was employed to form either patterned (Scheme 3 top) or full (Scheme 3 bottom) monolayers of methyl viologen. To this end, nonoxidized PDMS (polydimethylsiloxane) stamps, either flat or with 50 μm dots, were inked for 30 s with an aqueous solution of 2.5 mM of 1 and placed into contact with the IM monolayers for 1 h. After removal of the stamps and rinsing, the MV2+terminated surfaces were taken to the second step, which is similar for both immobilization routes. In this step, remaining reactive areas were blocked by immersion into a solution of either 2-aminoethanol (100 mM) or amino-polyethyleneglycol (1 mM) (see for more details Scheme S3 and Figure S4, Supporting Information). Subsequently, in the third step of the upper immobilization route, printed patterns of 1 were immersed in a mixture of 2 or 3/CB[8], whereas following the lower immobilization route, the third step involved another 16367

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printing step. In the latter case a PDMS stamp (with either lines or dots) was inked for 30 s with a mixture of 2 or 3/CB[8] and brought in contact with the surface for 15 min. In the experiments, in the case of the assembly on the particles, 10 μM solutions of naphthol guests and 75 μM CB[8] were used (vide supra). Whereas in both immobilization routes a 10 μM solution of naphthol-lissamine was used as well, naphthol-YFP was diluted to 1 μM in the lower immobilization route while in the upper immobilization route also a 10 μM solution of naphthol-YFP was used. By using the lower concentration in the case of the lower immobilization route almost no aggregates of the proteins were observed on the patterns. This observation is presumably related to the inking of the stamp, which is prone to unwanted protein aggregation, and this can be simply reduced by lowering the ink concentration. After the removal of the stamp and washing of the chips, the surfaces were inspected by fluorescence microscopy (Figure 3a−d). Fluorescent patterns of YFP-naphthol 2 or lissaminenaphthol 3 clearly show a sharp and uniform contrast between the protein areas (dots or lines) and the background. A series of test printing experiments following the upper immobilization route was performed in which the ink solution of 1 was diluted with different amounts of co-inks, aminoethanol, or NH2− PEG-OMe. Using a ratio of 1:1000 and 1:10 respectively resulted in optimal uniformity of the fluorescent patterns, while the signal to background ratio was 10:1 vs 17:1, respectively. Control experiments (Figure 3) show that no patterns were detected when the MV2+ surfaces were incubated without CB[8] or with guests that lacked the naphthol moiety. Also no fluorescent patterns were observed when either CB[7] or surfaces lacking MV2+ were used. We can thus conclude that CB[8] serves as a selective linking unit to form stable and homogeneous ternary surface-bound complexes between naphthol-modified proteins and MV2+-modified glass surfaces as envisioned. Similar results were obtained (Figure 3d) when following the lower immobilization route in Scheme 3. Additionally, after prolonged shaking the fluorescent patterns of the supramolecular complexes in a PBS solution were reexamined by fluorescence microscopy (Figure S5, Supporting Information). No significant difference was detected between intensity profiles of cross sections of the fluorescence microscopy images before and after shaking, indicating stable protein complexation to the glass surface. Further characterization of the protein patterns on glass surfaces was done using tapping mode atomic force microscopy in liquid (AFM). The AFM image (Figure 3e) shows a height difference (4.7 nm, height profile is given in Figure S6 of the Supporting Information) between the YFP lines and the background, which is consistent with the length of the YFP barrel plus linker. The formation of CB[8]-mediated protein immobilization onto the glass surface was additionally confirmed using IRreflective absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS) measurements. IRRAS spectra show the disappearance of the C−H stretch vibration of the IM-moiety at 3025 cm−1,23 while new bands at 2968, 2927, 2850, and 1265 cm−1 appeared upon attachment of MV2+ to the glass surface (Figure S7b,c, Supporting Information). The C1s region in the XPS spectra of IM monolayers comprises four peaks after deconvolution with intense bands at 284.8 eV (C−C) and 286.1 eV (C−C−N) and less intense bands at 286.7 eV (N−C−N) and 288.9 eV, which is indicative of the urea bond (Figure S8, Supporting Information). Further

Figure 3. (a) Fluorescence microscopy image of lissamine-naphthol in ternary complex following the upper immobilization route (Scheme 3); the inset shows the result when leaving out CB[8]. Two fluorescence images of YFP-naphthol in ternary complex following the upper immobilization route (Scheme 3); insets show images of controls when (b) YFP was without a naphthol tag and (c) no CB[8] was used. In the procedure yielding images in part b aminoethanol was used while for those shown in part c amino-polyethyleneglycol was used as co-ink and blocking reagent. (d) Fluorescence images of YFPnaphthol in ternary complex following the lower immobilization route (Scheme 3); insets show results when using YFP without naphthol. (e) Tapping mode AFM in liquid height image of patterned YFP-naphthol in ternary complexes on glass following the lower immobilization route (Scheme 3).

functionalization of the surface with MV2+ intensifies the C− C band as expected, while upon incubating the MV2+ surface with a solution containing both naphthol guest and CB[8] an increase of the relative contribution of the urea band was observed. Also, the band at 287.5 eV of the C−C−2N motif in CB appeared in agreement with the literature.24 XP spectra revealed that the elemental C/N ratio changed in agreement with the expected molecular composition. Concomitant with the changes in XPS upon complexing CB and naphthol guests, characteristic signals of CB[8] appeared at 1737 and 1470 cm−1, which correspond to respectively CO and C−N stretch vibrations,24 while a significant increase in the protein’s 16368

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Figure 4. Fluorescence images of (a) patterns of Lis- and YFP-naphthol (inset) that (b) disappeared after reduction with Zn in both cases and (c) images after air oxidation and reincubation with CB[8] and Lis- and YFP-naphthol (inset). The patterns were fabricated following the lower immobilization route as depicted in Scheme 3.

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amide signals was detected (Figure S7b,c, Supporting Information). When the MV2+ monolayer was incubated with YFP without naphthol or a monolayer lacking the methyl viologen, no CB signals were detected. These results from IR and XPS indicate that CB[8] links the naphthol guest to the surface-bound viologen and is in agreement with the results from fluorescence microscopy experiments. As a next step, the reversibility of the protein assembly was explored on glass surfaces. To this end, we decided to adopt the reported dissociation of the ternary complex upon electrochemical reduction of viologen as was previously reported and characterized by cyclic voltammetry.25 However, since our reversibility experiments will be performed on glass slides, we examined the glass surfaces (Figure 4) and particles (Figure S9, Supporting Information) at different stages by fluorescence microscopy instead of cyclic voltammetry.25 After formation of stable, fluorescent patterns, the disassembly of the complex was carried out by one-electron reduction of the MV2+ monolayers by incubating the glass slides in a suspension of activated Zn powder in PBS.12a,26 The fluorescence images of the patterns, before and after reduction of the MV2+ monolayer were recorded (Figure 4a,b) showing the complete disappearance of the patterns, which indicates that the ternary complexes were disassembled. The same results were obtained in the case of the particles (Figure S9, Supporting Information). In an attempt to reinstall the patterns, the monolayers were shortly exposed to air and subsequently immersed in a fresh solution of CB[8] and naphthol guests. After incubation, the slides were rinsed with buffer and water and dried before imaging (Figure 4c). The emission intensity profiles recorded have the same intensity as compared to the start. This sequence of experiments could be repeated several times, indicating that those surfaces can be reused for supramolecular assembly. Taken together these results indicate that monolayers of viologen are suitable for the release of naphthol-tagged proteins, reversibly linked to the surface using the supramolecular host molecule CB[8].

ASSOCIATED CONTENT

S Supporting Information *

Experimental details such as general methods and materials; synthetic details of compounds, particles, stamps, and monolayers; and characterization details of the particle and glass surfaces, including figures of reference experiments. 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] (J.H.), [email protected] (P.J.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank St-ERC (259183-PJ, 204554-LB), Agaur-Beatriu de Pinós (AGC). This research forms part of the Project P4.02 Superdices of the research program of the BioMedical Materials Institute, cofunded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation.

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REFERENCES

(1) Jonkheijm, P.; Weinrich, D.; Schroeder, H.; Niemeyer, C. M.; Waldmann, H. Chemical strategies for generating protein biochips. Angew. Chem., Int. Ed. 2008, 47, 9618−47. (2) For reviews see: (a) Aida, T.; Meijer, E. W.; Stupp, S. I. Functional supramolecular polymers. Science 2012, 335, 813−7. (b) Fenske, T.; Korth, H.-G.; Mohr, A.; Schmuck, C. Advances in switchable supramolecular nanoassemblies. Chem.Eur. J. 2012, 18, 738−55. (c) Uhlenheuer, D. A.; Petkau, K.; Brunsveld, L. Combining supramolecular chemistry with biology. Chem. Soc. Rev. 2010, 39, 2817−26. (d) Sacca, B.; Niemeyer, C. M. DNA origami: The art of folding DNA. Angew. Chem., Int. Ed. 2012, 51, 58−66. (3) For recent examples see: (a) Jun, H. W.; Yuwono, V.; Paramonov, S. E.; Hartgerink, J. D. Enzyme-mediated degradation of peptide−amphiphile nanofiber networks. Adv. Mater. 2005, 17, 2612− 17. (b) Nalluri, S. K. M.; Voskuhl, J.; Bultema, J. B.; Boekema, E. J.; Ravoo, B. J. Light-responsive capture and release of DNA in a ternary supramolecular complex. Angew. Chem., Int. Ed. 2011, 50, 9747−51. (c) Park, K. M.; Lee, D.-W.; Sarkar, B.; Jung, H.; Kim, J.; Ko, Y. H.; Lee, K. E.; Jeon, H.; Kim, K. Reduction-sensitive, robust vesicles with a non-covalently modifiable surface as a multifunctional drug-delivery platform. Small 2010, 13, 1430−41. (d) Lee, D.-W.; Park, K. M.; Banerjee, M.; Ha, S. H.; Lee, T.; Suh, K.; Paul, S.; Jung, H.; Kim, J.; Selvapalam, N.; Ryu, S. H.; Kim, K. Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host−guest binding pair. Nat. Chem. 2011, 3, 154−9. (e) Kim, C.; Agasti, S. S.; Zhu, Z.; Isaacs, L.; Rotello, V. M. Recognition-mediated activation of therapeutic gold nanoparticles inside living cells. Nat. Chem. 2010, 2, 962−66. (f) Liu, Y.; Wang, H.; Kamei, K. I.; Yan, M.; Chen, K.-J.; Yuan, Q.; Shi, L.; Lu, Y.; Tseng, H.-R. Delivery of intact transcription factor by using self-assembled supramolecular nanoparticles. Angew. Chem., Int. Ed. 2011, 50, 3058−62. (g) Meyer, R.; Niemeyer, C. M.

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CONCLUSIONS The capability to employ CB[8] to chemoselectively and siteselectively bind naphthol-functionalized proteins into patterns on surfaces and on particles through a ternary supramolecular complex has been presented for the first time. Optimized protocols have been developed for the successful immobilization onto methyl viologen-functionalized surfaces yielding strong and effective protein immobilization in uniform protein monolayers. Reversibility has been demonstrated by reducing the viologen functionalities. CB[8] offers a novel tailor-made route to dynamically immobilize proteins and, in combination with redox-active guest moieties, access to controllable assembly by a stimulus.4h We believe that this strategy provides a new avenue to construct dynamic functional interfaces, e.g., for responsive cellinterface studies.6 16369

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