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.

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.

Surface plasmon resonance is a powerful technique for label-free and real-time characterization of molecular interactions at interfaces. However, the detection of small molecules still remains a challenge. Here, we report on the direct detection of a low molecular weight compound by its receptor presented as a monolayer. Moreover, the signal observed is more than twice the expected mass-weighted response. To establish the origin of the signal enhancement, we present herein a theoretical model that simulates the maximal SPR response by taking into account the aptamer conformational change. We demonstrated that the thickness layer variation is not the only parameter to be considered. We highlighted that the conformational transition of the aptamer also induces a deviation of the refractive index increment (RII) of the target /aptamer complex from the sum of the RII of individual entities. This non-additivity of the RII significantly contributes to the magnitude of the signal. We also propose the prediction of the maximal SPR response in function of the penetration depth, the ratio of the mass-weighted RII of the partners, the sensing layer thickness and the correction of the complex RII. This model provides new insights in parameters to be considered for analysis of SPR signals.

Multilayer films are formed using host-guest interaction between two derivatized chitosans, one, with beta-cyclodextrin cavities and the other with adamantyl moieties.

Aptamers have emerged as promising biorecognition elements for the development of biosensors. The present work focused on the direct detection, by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D), of a low molecular weight (LMW) compound (less than 200 Da) with an aptamer receptor presented as an oriented monolayer on surface. These techniques are powerful for label-free, real-time characterization and quantification of molecular interactions at interfaces. Herein, we analyzed the influence of aptamer surface density on the recognition properties. A decrease of the surface 2 concentration was shown to improve the affinity for the target due to a higher kinetic association constant that could be explained by a limitation of the steric hindrance of the aptamer on the surface. An aptamer folding is produced upon recognition of the LMW target that gives rise to the modification of the layer on the surface. This induces a displacement of water acoustically coupled to the sensing layer, a thickness layer variation and a deviation of the refractive index increment (RII) of the target/aptamer complex from the sum of the RII of individual entities. We also demonstrated that the recognition signal was still detectable for low aptamer density (lower than 1 pmol.cm-2).

The investigation of recognition events between carbohydrates and proteins, especially the understanding of how spatial factors and binding avidity are correlated, remains a great interest for glycobiology. In this context we have investigated by nanogravimetry (QCM-D) and surface plasmon resonance (SPR), the kinetics and thermodynamics of the interaction between concanavalin A (Con A) and various neoglycopeptide ligands of low molecular weight. Regioselectively addressable functionalized templates (RAFT) have been used as scaffolds for the design of multivalent neoglycopeptides bearing thiol or biotin functions for their anchoring on transducer surfaces. Although these multivalent neoglycopeptide ligands cannot span multiple binding sites within the same Con A protein, they have increased activities relative to their monovalent counterpart. Our results emphasize that the multivalent RAFT ligands function by clustering several lectins, which leads to enhanced affinities.

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular-like G-quadruplex motif 1 (parallel G-quadruplex conformation), an intramolecular G-quadruplex 2, and a duplex DNA 3 have been designed and developed. The method is based on the concept of template-assembled synthetic G-quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G-quadruplex conformation. Various known G-quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a pi-stacking binding mode showed a higher binding affinity for intermolecular-like G-quadruplexes 1, whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2. In addition, the present method has also provided information about the selectivity of ligands for G-quadruplex DNA over the duplex DNA. A numerical parameter, termed the G-quadruplex binding mode index (G4-BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G-quadruplex 1 against intramolecular G-quadruplex 2. The G-quadruplex binding mode index (G4-BMI) of a ligand is defined as follows: G4-BMI=K(D)(intra)/K(D)(inter), where K(D)(intra) is the dissociation constant for intramolecular G-quadruplex 2 and K(D)(inter) is the dissociation constant for intermolecular G-quadruplex 1. In summary, the present work has demonstrated that the use of parallel-constrained quadruplex topology provides more precise information about the binding modes of ligands.

A series of nine Ni(II) salophen complexes involving one, two, or three alkyl-imidazolium side-chains was prepared. The lengths of the side-chains were varied from one to three carbons. The crystal structure of one complex revealed a square planar geometry of the nickel ion. Fluorescence resonance energy transfer melting of G-quadruplex structures in the presence of salophen complex were performed. The G-quadruplex DNA structures were stabilized in the presence of the complexes, but a duplex DNA was not. The binding constants of the complexes for parallel and antiparallel G-quadruplex DNA, as well as hairpin DNA, were measured by surface plasmon resonance. The compounds were selective for G-quadruplex DNA, as reflected by equilibrium dissociation constant KD values in the region 0.1-1 μM for G-quadruplexes and greater than 2 μM for duplex DNA. Complexes with more and shorter side-chains had the highest binding constants. The structural basis for the interaction of the complexes with the human telomeric G-quadruplex DNA was investigated by computational studies: the aromatic core of the complex stacked over the last tetrad of the G-quadruplex with peripherical cationic side chains inserted into opposite grooves. Biochemical studies (telomeric repeat amplification protocol assays) indicated that the complexes significantly inhibited telomerase activity with IC50 values as low as 700 nM; the complexes did not significantly inhibit polymerase activity.