Paolo Bergese

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).

Residual matrix from different separation techniques impacts exosome biological activity

Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales.

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.

Cavitand‐Grafted Silicon Microcantilevers as a Universal Probe for Illicit and Designer Drugs in Water

The direct, clean, and unbiased transduction of molecular recognition into a readable and reproducible response is the biggest challenge associated to the use of synthetic receptors in sensing. All possible solutions demand the mastering of molecular recognition at the solid-liquid interface as prerequisite. The socially relevant issue of screening amine-based illicit and designer drugs is addressed by nanomechanical recognition at the silicon-water interface. The methylamino moieties of different drugs are all first recognized by a single cavitand receptor through a synergistic set of weak interactions. The peculiar recognition ability of the cavitand is then transferred with high fidelity and robustness on silicon microcantilevers and harnessed to realize a nanomechanical device for label-free detection of these drugs in water.

Colorimetric Nanoplasmonic Assay To Determine Purity and Titrate Extracellular Vesicles

Extracellular Vesicles (EVs) - cell secreted vesicles that carry rich molecular information of the parental cell and constitute an important mode of intercellular communication - are becoming a primary topic in translational medicine. EVs (that comprise exosomes and microvesicles/microparticles) have a size ranging from 40 nm to 1 μm and share several physicochemical proprieties, including size, density, surface charge, and light interaction, with other nano-objects present in body fluids, such as single and aggregated proteins. This makes separation, titration, and characterization of EVs challenging and time-consuming. Here we present a cost-effective and fast colorimetric assay for probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between colloidal gold nanoplasmonics, nanoparticle-protein corona, and nanoparticle-membrane interaction. The assay hits a limit of detection of protein contaminants of 5 ng/μL and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the nanoparticle-protein corona in analytical chemistry.

Quantifying the nanomachinery of the nanoparticle-biomolecule interface.

A study is presented of the nanomechanical phenomena experienced by nanoparticle-conjugated biomolecules. A thermodynamic framework is developed to describe the binding of thrombin-binding aptamer (TBA) to thrombin when the TBA is conjugated to nanorods. Binding results in nanorod aggregation (viz. directed self-assembly), which is detectable by absorption spectroscopy. The analysis introduces the energy of aggregation, separating it into TBA-thrombin recognition and surface-work contributions. Consequently, it is demonstrated that self-assembly is driven by the interplay of surface work and thrombin-TBA recognition. It is shown that the work at the surface is about -10 kJ mol(-1) and results from the accumulation of in-plane molecular forces of pN magnitude and with a lifetime of <1 s, which arises from TBA nanoscale rearrangements fuelled by thrombin-directed nanorod aggregation. The obtained surface work can map aggregation regimes as a function of different nanoparticle surface conditions. Also, the thermodynamic treatment can be used to obtain quantitative information on surface effects impacting biomolecules on nanoparticle surfaces.

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.

Cavitands Endow All-Dielectric Beads With Selectivity for Plasmon-Free Enhanced Raman Detection of Nε-Methylated Lysine.

SiO2/TiO2 microbeads (T-rex) are promising materials for plasmon-free surface-enhanced Raman scattering (SERS), offering several key advantages in biodiagnostics. In this paper we report the combination of T-rex beads with tetraphosphonate cavitands (Tiiii), which imparts selectivity toward Nε-methylated lysine. SERS experiments demonstrated the efficiency and selectivity of the T-rex-Tiiii assays in detecting methylated lysine hydrochloride (Nε-Me-Lys-Fmoc) from aqueous solutions, even in the presence of the parent Lys-Fmoc hydrochloride as interferent. The negative results obtained in control experiments using TSiiii ruled out any other form of surface recognition or preferential physisorption. MALDI-TOF analyses on the beads exposed to Nε-Me-Lys-Fmoc revealed the presence of the Tiiii•Nε-Me-Lys-Fmoc complex. Raman analyses based on the intensity ratio of Nε-Me-Lys-Fmoc and cavitand-specific modes resulted in a dose-response plot, which allowed for estimating the concentration of Nε-methylated lysine from initial solutions in the 1 × 10(-3) to 1 × 10(-5) M range. These results can set the basis for the development of new Raman assays for epigenetic diagnostics.

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

Exploiting Surface Plasmon Resonance (SPR) Technology for the Identification of Fibroblast Growth Factor-2 (FGF2) Antagonists Endowed with Antiangiogenic Activity

Angiogenesis, the process of new blood vessel formation, is implicated in various physiological/pathological conditions, including embryonic development, inflammation and tumor growth. Fibroblast growth factor-2 (FGF2) is a heparin-binding angiogenic growth factor involved in various physiopathological processes, including tumor neovascularization. Accordingly, FGF2 is considered a target for antiangiogenic therapies. Thus, numerous natural/synthetic compounds have been tested for their capacity to bind and sequester FGF2 in the extracellular environment preventing its interaction with cellular receptors. We have exploited surface plasmon resonance (SPR) technique in search for antiangiogenic FGF2 binders/antagonists. In this review we will summarize our experience in SPR-based angiogenesis research, with the aim to validate SPR as a first line screening for the identification of antiangiogenic compounds.