Giulio Oliviero

A biofunctional polymeric coating for microcantilever molecular recognition

An innovative route to activate silicon microcantilevers (MCs) for label free molecular recognition is presented. The method consists in coating the underivatized MCs with a functional ter-polymer based on N,N-dimethylacrylamide (DMA) bearing N-acryloyloxysuccinimide (NAS) and 3-(trimethoxysilyl)propyl-methacrylate (MAPS), two functional monomers that confer to the polymer the ability to react with nucleophilic species on biomolecules and with glass silanols, respectively. The polymer was deposited onto MCs by dip coating. Polymer coated MCs were tested in both static and dynamic modes of actuation, featuring detection of DNA hybridization as well as protein/protein interaction. In the dynamic experiments, focused on protein detection, the MCs showed an average mass responsivity of 0.4 Hz/pg for the first resonant mode and of 2.5 Hz/pg for the second resonant mode. The results of the static experiments, dedicated to DNA hybridization detection, allowed for direct estimation of the DNA duplex formation energetics, which resulted fully consistent with the nominal expected values. These results, together with easiness and cheapness, high versatility, and excellent stability of the recognition signal, make the presented route a reliable alternative to standard SAM functionalization (for microcantilevers (MCs) and for micro-electro-mechanical systems (MEMS) in general).

Nanomechanical Recognition of N-Methylammonium Salts

Turning molecular recognition into an effective mechanical response is critical for many applications ranging from molecular motors and responsive materials to sensors. Herein, we demonstrate how the energy of the molecular recognition between a supramolecular host and small alkylammonium salts can be harnessed to perform a nanomechanical task in a univocal way. Nanomechanical Si microcantilevers (MCs) functionalized by a film of tetra-phosphonate cavitands were employed to screen as guests the compounds of the butylammonium chloride series 1-4, which comprises a range of low molecular weight (LMW) molecules (molecular mass < 150 Da) that differ from each other by one or a few N-methyl groups (molecular mass 15 Da). The cavitand surface recognition of each individual guest drove a specific MC bending (from a few to several tens of nanometer), disclosing a direct, label-free, and real-time mean to sort them. The complexation preferences of tetraphosphonate cavitands toward ammonium chloride guests 1-4 were independently assessed by isothermal titration calorimetry. Both direct and displacement binding experiments concurred to define the following binding order in the alkylammonium series: 2 > 3 ≈ 1 ≫ 4. This trend is consistent with the number of interactions established by each guest with the host. The complementary ITC experiments showed that the host-guest complexation affinity in solution is transferred to the MC bending. These findings were benchmarked by implementing cavitand-functionalized MCs to discriminate sarcosine from glycine in water.

Protein thin film machines

We report the first example of microcantilever beams that are reversibly driven by protein thin film machines fueled by cycling the salt concentration of the surrounding solution. We also show that upon the same salinity stimulus the drive can be completely reversed in its direction by introducing a surface coating ligand. Experimental results are throughout discussed within a general yet simple thermodynamic model.

On the difference of equilibrium constants of DNA hybridization in bulk solution and at the solid-solution interface.

The origin of the difference between the equilibrium (affinity) constants of ligand–receptor binding in bulk solution and at a solid‐solution interface is discussed in terms of Gibbsian interfacial thermodynamics. It results that the difference is determined by the surface work that the ligand–receptor interaction spends to accommodate surface binding, and in turn that the value of the surface equilibrium constant (strongly) depends on the surface that confines the event. This framework consistently describes a wide set of experimental observations of DNA surface hybridization, correctly predicting that within the surface work window for DNA hybridization, that ranges from −90 to 75 kJmol−1, the ratio between surface and bulk equilibrium constants ranges from 10−16 to 1013, spanning 29 orders of magnitude. Copyright

Molecular recognition by contact angle: proof of concept with DNA hybridization.

A molecular recognition reaction supported by a solid-phase assay drives a specific change in the solid-solution interfacial tension. This prompts contact angle (CA) analysis, being a straightforward route to evaluate this property, to play the unedited role of label-free probe of the reaction. The concept is proven by the successful recognition of DNA hybridization and is further supported by the agreement between the results and the underpinning thermodynamics.

On the thermodynamics of biomolecule surface transformations.

Biological surface science is receiving great and renewed attention owing the rising interest in applications of nanoscience and nanotechnology to biological systems, with horizons that range from nanomedicine and biomimetic photosynthesis to the unexpected effects of nanomaterials on health and environment. Biomolecule surface transformations are among the fundamental aspects of the field that remain elusive so far and urgently need to be understood to further the field. Our recent findings indicate that surface thermodynamics can give a substantial contribution toward this challenging goal. In the first part of the article, we show that biomolecule surface transformations can be framed by a general and simple thermodynamic model. Then, we explore its effectiveness by addressing some typical cases, including ligand-receptor surface binding, protein thin film machines, nanomechanical aspects of the biomolecule-nanoparticle interface and nanomechanical biosensors.

Advances in Parallel Screening of Drug Candidates

In the hit to lead process, a drug candidate is selected from a set of potential leads by screening its binding with potential targets. This review focuses on the lead identification assays that employ a bio-chemical or bio-physical test to detect molecular recognition events between proteins and small molecules in a parallel format. These tests require either the lead or the target immobilization followed by incubation with the set of potential interaction partners and detection of a signal related to the target-ligand binding. In the first part of the review the different detection strategies amenable for drug screening are discussed. In the second part, a review of immobilization approaches for leads or targets, allowing the parallel screening of arrays of molecules, is presented.