Influence of Surface Chemistry on Host/Guest Interactions: A Model Study on Redox-Sensitive ß-Cyclodextrin/Ferrocene Complexes.

While host/guest interactions are widely used to control molecular assembly on surfaces, quantitative information on the effect of surface chemistry on their efficiency is lacking. To address this question, we combined electrochemical characterization with quartz crystal microbalance with dissipation monitoring to study host/guest interactions between surface-attached ferrocene (Fc) guests and soluble ß-cyclodextrin (ß-CD) hosts. We identified several parameters that influence the redox response, ß-CD complexation ability, and repellent properties of Fc monolayers, including the method of Fc grafting, the linker connecting Fc with the surface, and the diluting molecule used to tune Fc surface density. The study on monovalent ß-CD/Fc complexation was completed by the characterization of multivalent interactions between Fc monolayers and ß-CD-functionalized polymers, with new insights being obtained on the interplay between the surface chemistry, binding efficiency, and reversibility under electrochemical stimulus. These results should facilitate the design of well-defined functional interfaces and their implementation in stimuli-responsive materials and sensing devices.

Determinants of Superselectivity─Practical Concepts for Application in Biology and Medicine.

Multivalent interactions are common in biological systems and are also widely deployed for targeting applications in biomedicine. A unique feature of multivalent binding is "superselectivity". Superselectivity refers to the sharp discrimination of surfaces (e.g., on cells or cell compartments) by their comparative surface densities of a given receptor. This feature is different from the conventional "type" selectivity, which discriminates surfaces by their distinct receptor types. In a broader definition, a probe is superselective if it converts a gradual change in any one interaction parameter into a sharp on/off dependency in probe binding.This Account describes our systematic experimental and theoretical efforts over the past decade to analyze the determinants of superselective binding. It aims to offer chemical biologists, biophysicists, biologists, and biomedical scientists a set of guidelines for the interpretation of multivalent binding data, and design rules for tuning superselective targeting. We first provide a basic introduction that identifies multiple low-affinity interactions and combinatorial entropy as the minimal set of conditions required for superselective recognition. We then introduce the main experimental and theoretical tools and analyze how salient features of the multivalent probes (i.e., their concentration, size, ligand valency, and scaffold type), of the surface receptors (i.e., their affinity for ligands, surface density, and mobility), and of competitors and cofactors (i.e., their concentration and affinity for the ligands and/or receptors) influence the sharpness and the position of the threshold for superselective recognition.Emerging from this work are a set of relatively simple yet quantitative data analysis guidelines and superselectivity design rules that apply to a broad range of probe types and interaction systems. The key finding is the scaling variable xS which faithfully predicts the influence of the surface receptor density, probe ligand valency, receptor-ligand affinity, and competitor/cofactor concentrations and affinities on superselective recognition. The scaling variable is a simple yet versatile tool to quantitatively tune the on/off threshold of superselective probes. We exemplify its application by reviewing and reinterpreting literature data for selected biological and biomedical interaction systems where superselectivity clearly is important.Our guidelines can be deployed to generate a new mechanistic understanding of multivalent recognition events inside and outside cells and the downstream physiological/pathological implications. Moreover, the design rules can be harnessed to develop novel superselective probes for analytical purposes in the life sciences and for diagnostic/therapeutic intervention in biomedicine.

Controlling Superselectivity of Multivalent Interactions with Cofactors and Competitors.

Moieties that compete with multivalent interactions or act as cofactors are common in living systems, but their effect on multivalent binding remains poorly understood. We derive a theoretical model that shows how the superselectivity of multivalent interactions is modulated by the presence of cofactors or competitors. We find that the role of these participating moieties can be fully captured by a simple rescaling of the affinity constant of the individual ligand-receptor bonds. Theoretical predictions are supported by experimental data of the membrane repair protein annexin A5 binding to anionic lipid membranes in the presence of Ca2+ cofactors and of the extracellular matrix polysaccharide hyaluronan (HA) binding to CD44 cell surface receptors in the presence of HA oligosaccharide competitors. The obtained findings should facilitate understanding of multivalent recognition in biological systems and open new routes for fine-tuning the selectivity of multivalent nanoprobes in medicinal chemistry.

Thermoresponsive Fluorescence Switches Based on Au@pNIPAM Nanoparticles.

Despite numerous studies emphasizing the plasmonic impact on fluorescence, the design of a dynamic system allowing on-demand fluorescence switching in a single nanostructure remains challenging. The reversibility of fluorescence switching and the versatility of the approach, in particular its compatibility with a wide range of nanoparticles and fluorophores, are among the main experimental difficulties. In this work, we achieve reversible fluorescence switching by coupling metal nanoparticles with fluorophores through stimuli-responsive organic linkers. As a proof of concept, we link gold nanoparticles with fluorescein through thermoresponsive poly(N-isopropylacrylamide) at a tunable grafting density and characterize their size and optical response by dynamic light scattering, absorption, and fluorescence spectroscopies. We show that the fluorescence emission of these hybrid nanostructures can be switched on-demand using the thermoresponsive properties of poly(N-isopropylacrylamide). The described system presents a general strategy for the design of nanointerfaces, exhibiting reversible fluorescence switching via external control of metal nanoparticle/fluorophore distance.

Photo-/Electroinduced Irreversible Isomerization of 2,2'-Azobispyridine Ligands in Arene Ruthenium(II) Complexes.

Novel arene RuII complexes containing 2,2'-azobispyridine ligands were synthesized and characterized by using 1 H and 13 C NMR spectroscopy, UV/vis spectroscopy, electrochemistry, DFT calculations and single-crystal X-ray diffraction. Z-configured complexes featuring unprecedented seven-membered chelate rings involving the nitrogen atom of both pyridines were isolated and were shown to undergo irreversible isomerization to the corresponding E-configured five-membered chelate complexes in response to light or electrochemical stimulus.

Self-Assembled Monolayers of Redox-Active 4d-4f Heterobimetallic Complexes.

In this work, we report the preparation of functional interfaces incorporating heterobimetallic systems consisting in the association of an electroactive carbon-rich ruthenium organometallic unit and a luminescent lanthanide ion (Ln = Eu3+ and Yb3+). The organometallic systems are functionalized with a terminal hexylthiol group for subsequent gold surface modification. The formation of self-assembled monolayers (SAMs) with these complex molecular architectures are thoroughly demonstrated by employing a combination of different techniques, including infrared reflection absorption spectroscopy, ellipsometry, contact angle, and cyclic voltammetry measurements. The immobilized heterobimetallic systems show fast electron-transfer kinetics and, hence, are capable of fast electrochemical response. In addition, the characteristic electrochemical signals of the SAMs were found to be sensitive to the presence of lanthanide centers at the bipyridyl terminal units. A positive shift of the potential of the redox signal is readily observed for lanthanide complexes compared to the bare organometallic ligand. This effect is equally observed for preformed complexes and on-surface complexation. Thus, an efficient ligating recruitment of europium and ytterbium cations at gold-modified electrodes is demonstrated, allowing for an easy electrochemical detection of the lanthanide ions along with an alternative preparative method of SAMs incorporating lanthanide cations compared to the immobilization of the preformed complex.

Bacteria-Based Production of Thiol-Clickable, Genetically Encoded Lipid Nanovesicles.

Despite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Herein, a microbial platform for high-yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients, or detergents) and their straightforward surface functionalization, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria-based proteoliposomes are an attractive eco-friendly alternative that can outperform currently used liposomes.

Multivalent Recognition at Fluid Surfaces: The Interplay of Receptor Clustering and Superselectivity.

The interaction between a biological membrane and its environment is a complex process, as it involves multivalent binding between ligand/receptor pairs, which can self-organize in patches. Any description of the specific binding of biomolecules to membranes must account for the key characteristics of multivalent binding, namely, its unique ability to discriminate sharply between high and low receptor densities (superselectivity), but also for the effect of the lateral mobility of membrane-bound receptors to cluster upon binding. Here we present an experimental model system that allows us to compare systematically the effects of multivalent interactions on fluid and immobile surfaces. A crucial feature of our model system is that it allows us to control the membrane surface chemistry, the properties of the multivalent binder, and the binding affinity. We find that multivalent probes retain their superselective binding behavior at fluid interfaces. Supported by numerical simulations, we demonstrate that, as a consequence of receptor clustering, superselective binding is enhanced and shifted to lower receptor densities at fluid interfaces. To translate our findings into a simple, predictive tool, we propose an analytical model that enables rapid predictions of how the superselective binding behavior is affected by the lateral receptor mobility as a function of the physicochemical characteristics of the multivalent probe. We believe that our model, which captures the key physical mechanisms underpinning multivalent binding to biological membranes, will greatly facilitate the rational design of nanoprobes for the superselective targeting of cells.

Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin.

Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs). Stability and kinetics of SAv binding are characterized by quartz crystal microbalance with dissipation monitoring, while the residual valency of immobilized SAv is quantified using spectroscopic ellipsometry by monitoring binding of biotinylated probes. Purpose-designed SAv constructs having controlled valencies (mono-, di-, trivalent in terms of biotin-binding sites) are studied to rationalize the results obtained on regular (tetravalent) SAv. We find that divalent interaction of SAv with biotinylated surfaces is a strict requirement for stable immobilization, while monovalent attachment is reversible and, in the case of SLBs, leads to the extraction of biotinylated lipids from the bilayer. The surface density and lateral mobility of biotin, and the SAv surface coverage are all found to influence the average orientation and residual valency of SAv on a biotinylated surface. We demonstrate how the residual valency can be adjusted to one or two biotin binding sites per immobilized SAv by choosing appropriate surface chemistry. The obtained results provide means for the rational design of surface-confined supramolecular architectures involving specific biointeractions at tunable valency. This knowledge can be used for the development of well-defined bioactive coatings, biosensors and biomimetic model systems.

Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes.

Aptamers have emerged as promising biorecognition elements in the development of biosensors. The present work focuses on the application of quartz crystal microbalance with dissipation monitoring (QCM-D) for the enantioselective detection of a low molecular weight target molecule (less than 200 Da) by aptamer-based sensors. While QCM-D is a powerful technique for label-free, real-time characterization and quantification of molecular interactions at interfaces, the detection of small molecules interacting with immobilized receptors still remains a challenge. In the present study, we take advantage of the aptamer conformational changes upon the target binding that induces displacement of water acoustically coupled to the sensing layer. As a consequence, this phenomenon leads to a significant enhancement of the detection signal. The methodology is exemplified with the enantioselective recognition of a low molecular weight model compound, L-tyrosinamide (L-Tym). QCM-D monitoring of L-Tym interaction with the aptamer monolayer leads to an appreciable signal that can be further exploited for analytical purposes or thermodynamics studies. Furthermore, in situ combination of QCM-D with spectroscopic ellipsometry unambiguously demonstrates that the conformational change induces a nanometric decrease of the aptamer monolayer thickness. Since QCM-D is sensitive to the whole mass of the sensing layer including water that is acoustically coupled, a decrease in thickness of the highly hydrated aptamer layer induces a sizable release of water that can be easily detected by QCM-D.

Designing multivalent probes for tunable superselective targeting.

Specific targeting is common in biology and is a key challenge in nanomedicine. It was recently demonstrated that multivalent probes can selectively target surfaces with a defined density of surface binding sites. Here we show, using a combination of experiments and simulations on multivalent polymers, that such "superselective" binding can be tuned through the design of the multivalent probe, to target a desired density of binding sites. We develop an analytical model that provides simple yet quantitative predictions to tune the polymer's superselective binding properties by its molecular characteristics such as size, valency, and affinity. This work opens up a route toward the rational design of multivalent probes with defined superselective targeting properties for practical applications, and provides mechanistic insight into the regulation of multivalent interactions in biology. To illustrate this, we show how the superselective targeting of the extracellular matrix polysaccharide hyaluronan to its main cell surface receptor CD44 is controlled by the affinity of individual CD44-hyaluronan interactions.

Quartz crystal microbalance with dissipation monitoring and spectroscopic ellipsometry measurements of the phospholipid bilayer anchoring stability and kinetics of hydrophobically modified DNA oligonucleotides.

Decorating lipid bilayers with oligonucleotides has great potential for both fundamental studies and applications, taking advantage of the membrane properties and the specific Watson-Crick base pairing. Here, we systematically studied the binding of DNA oligonucleotides with the frequently used hydrophobic anchors cholesterol, stearyl, and distearyl to supported lipid bilayers made of dioleoylphosphatidylcholine (DOPC) by quartz crystal microbalance with dissipation monitoring and spectroscopic ellipsometry (SE). All three anchors were found to incorporate well into DOPC lipid membranes, yet only the distearyl-based anchor remained stable in the bilayer when it was rinsed. The unstable anchoring of the cholesterol- and stearyl-based oligonucleotides can, however, be stabilized by hybridization of the oligonucleotides to complementary DNA modified with a second hydrophobic anchor of the same type. In all cases, the incorporation into the lipid bilayer was found to be limited by mass transport, although micelle formation likely reduced the effective concentration of available oligonucleotides in some samples, leading to substantial differences in binding rates. Using a viscoelastic model to determine the thickness of the DNA layer and elucidating the surface coverage by SE, we found that at equal bulk concentrations double-stranded DNA constructs attached to the lipid bilayer establish a layer that is thicker than that of single-stranded oligonucleotides, whereas the DNA surface densities are similar. Shortening the length of the oligonucleotides, on the other hand, does alter both the thickness and surface density of the DNA layer. This indicates that at the bulk oligonucleotide concentrations employed in our experiments, the packing of the oligonucleotides is not affected by the anchor type, but rather by the length of the DNA. The results are useful for material and biomedical applications that require efficient linking of oligonucleotides to lipid membranes.

Superselective targeting using multivalent polymers.

Despite their importance for material and life sciences, multivalent interactions between polymers and surfaces remain poorly understood. Combining recent achievements of synthetic chemistry and surface characterization, we have developed a well-defined and highly specific model system based on host/guest interactions. We use this model to study the binding of hyaluronic acid functionalized with host molecules to tunable surfaces displaying different densities of guest molecules. Remarkably, we find that the surface density of bound polymer increases faster than linearly with the surface density of binding sites. Based on predictions from a simple analytical model, we propose that this superselective behavior arises from a combination of enthalpic and entropic effects upon binding of nanoobjects to surfaces, accentuated by the ability of polymer chains to interpenetrate.

A new configurational bias scheme for sampling supramolecular structures.

We present a new simulation scheme which allows an efficient sampling of reconfigurable supramolecular structures made of polymeric constructs functionalized by reactive binding sites. The algorithm is based on the configurational bias scheme of Siepmann and Frenkel and is powered by the possibility of changing the topology of the supramolecular network by a non-local Monte Carlo algorithm. Such a plan is accomplished by a multi-scale modelling that merges coarse-grained simulations, describing the typical polymer conformations, with experimental results accounting for free energy terms involved in the reactions of the active sites. We test the new algorithm for a system of DNA coated colloids for which we compute the hybridisation free energy cost associated to the binding of tethered single stranded DNAs terminated by short sequences of complementary nucleotides. In order to demonstrate the versatility of our method, we also consider polymers functionalized by receptors that bind a surface decorated by ligands. In particular, we compute the density of states of adsorbed polymers as a function of the number of ligand-receptor complexes formed. Such a quantity can be used to study the conformational properties of adsorbed polymers useful when engineering adsorption with tailored properties. We successfully compare the results with the predictions of a mean field theory. We believe that the proposed method will be a useful tool to investigate supramolecular structures resulting from direct interactions between functionalized polymers for which efficient numerical methodologies of investigation are still lacking.

Reversible control over molecular recognition in surface-bound photoswitchable hydrogen-bonding receptors: towards read-write-erase molecular printboards.

The synthesis of an anthracene-bearing photoactive barbituric acid receptor and its subsequent grafting onto azide-terminated alkanethiol/Au self-assembled monolayers by using an Cu(I) -catalyzed azide-alkyne reaction is reported. Monolayer characterization using contact-angle measurements, electrochemistry, and spectroscopic ellipsometry indicate that the monolayer conversion is fast and complete. Irradiation of the receptor leads to photodimerization of the anthracenes, which induces the open-to-closed gating of the receptor by blocking access to the binding site. The process is thermally reversible, and polarization-modulated IR reflection-absorption spectroscopy indicates that photochemical closure and thermal opening of the surface-bound receptors occur in 70 and 100 % conversion, respectively. Affinity of the open and closed surface-bound receptor was characterized by using force spectroscopy with a barbituric-acid-modified atomic force microscope tip.

Redox strategy for reversible attachment of biomolecules using bifunctional linkers.

Soft attachment of streptavidin to ß-cyclodextrin-modified pegylated SAMs was efficiently performed in a reversible and repetitive way via orthogonal bifunctional linkers involving streptavidin-biotin recognition and redox-driven multivalent host-guest (ß-cyclodextrin-ferrocene) interactions.

Electrochemically controlled adsorption of Fc-functionalized polymers on beta-CD-modified self-assembled monolayers.

This work presents an in situ study of the adsorption/desorption behavior of ferrocene(Fc)-functionalized linear polymers on a gold surface covered with beta-cyclodextrin(beta-CD)-modified self-assembled monolayers (SAMs). The characterization of binary SAMs obtained with HS-(CH(2))(11)-EG(6)-N(3) and HS-(CH(2))(11)-EG(4)-OH (EG, ethylene glycol) was performed using a quartz crystal microbalance with dissipation monitoring (QCM-D), cyclic voltammetry, and contact angle measurements. The functionalization of SAMs with beta-CD was made via the "click" reaction between the beta-CD monoalkyne derivative and azide groups exhibited by SAMs. The formation of the host-guest complex between SAM-beta-CD and Fc-derivatized polymers (chitosan (CHI) and poly(allylamine hydrochloride) (PAH)) was studied by QCM-D. The viscoelastic model of Voinova was used to fit QCM-D curves recorded during the adsorption and electrochemically controlled desorption of CHI-Fc and PAH-Fc on SAM-beta-CD. Using QCM-D coupled to cyclic voltammetry, we demonstrated that CHI-Fc and PAH-Fc can be successfully deposited on a SAM-beta-CD-coated gold surface forming a stable multivalent inclusion complex between Fc moieties of polymer and beta-CD cavities of SAM. We also showed that all specifically attached polymer chains can be detached from the SAM-beta-CD-coated gold surface by applying an electric field.