SERS-based nanobiosensing for ultrasensitive detection of the p53 tumor suppressor

Background: One of the main challenges in biomedicine is improvement of detection sensitivity to achieve tumor marker recognition at a very low concentration when the disease is not significantly advanced. A pivotal role in cancer defense is played by the p53 tumor suppressor, therefore its detection with high sensitivity may contribute considerably to early diagnosis of cancer. In this work, we present a new analytical method based on surface-enhanced Raman spectroscopy which could significantly increase the sensitivity of traditional bioaffinity techniques. p53 molecules were anchored to gold nanoparticles by means of the bifunctional linker 4-aminothiophenol (4-ATP). The characteristic vibrational bands of the p53-4-ATP nanoparticle system were then used to identify the p53 molecules when they were captured by a recognition substrate comprising a monolayer of azurin in molecules possessing significant affinity for this tumor suppressor. The Raman signal enhancement achieved by 4-ATP-mediated crosslinking of p53 to 50 nm gold nanoparticles enabled detect of this protein at a concentration down to 5 x 10(-13) M

Biomolecule recognition using piezoresistive nanomechanical force probes

Highly sensitive sensors are one of the enabling technologies for the biomarker detection in early stage diagnosis of pathologies. We have developed a self-sensing nanomechanical force probe able for detecting the unbinding of single couples of biomolecular partners in nearly physiological conditions. The embedding of a piezoresistive transducer into a nanomechanical cantilever enabled high force measurement capability with sub 10-pN resolution. Here, we present the design, microfabrication, optimization, and complete characterization of the sensor. The exceptional electromechanical performance obtained allowed us to detect biorecognition specific events underlying the biotin-avidin complex formation, by integrating the sensor in a commercial atomic force microscope.This work has been supported by the Spanish Ministry of Science and Innovation through projects NANOSELECT-CSD2007-00041(Consolider-Ingenio 2010) and TEC2011-23600, by the European Union through the COST ACTION TD1002 and partly supported by the PRIN-MIUR Project No. 2009 WPZM4S and by AIRC (Grant IG10412.)Peer reviewe

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53

Interaction of human hemoglobin and semi-hemoglobins with the Staphylococcus aureus hemophore IsdB: a kinetic and mechanistic insight

Among multidrug-resistant bacteria, methicillin-resistant Staphylococcus aureus is emerging as one of the most threatening pathogens. S. aureus exploits different mechanisms for its iron supply, but the preferred one is acquisition of organic iron through the expression of hemoglobin (Hb) receptors. One of these, IsdB, belonging to the Isd (Iron-Regulated Surface Determinant) system, was shown to be essential for bacterial growth and virulence. Therefore, interaction of IsdB with Hb represents a promising target for the rational design of a new class of antibacterial molecules. However, despite recent investigations, many structural and mechanistic details of complex formation and heme extraction process are still elusive. By combining site-directed mutagenesis, absorption spectroscopy, surface plasmon resonance and molecular dynamics simulations, we tackled most of the so far unanswered questions: (i) the exact complex stoichiometry, (ii) the microscopic kinetic rates of complex formation, (iii) the IsdB selectivity for binding to, and extracting heme from, α and β subunits of Hb, iv) the role of specific amino acid residues and structural regions in driving complex formation and heme transfer, and (v) the structural/dynamic effect played by the hemophore on Hb

Distribution of conformational states as common source of g-and A-strain in the ESR spectra of proteins and glasses

Additional experimental evidence on the structural and dynamical similarity between proteins and glasses is provided by the analysis conducted on the ESR spectra, at different temperatures, of some copper proteins and of some Cu++-doped glasses formed by water and a second component (NaOH, DMSO, alcohols). All the spectra taken at temperatures below ∼ 210 K display a significant g- and A-strain that consists in a progressive broadening and decrese in the intensity of the copper hyperfine lines as the order of mI increases (I = 3/2 is the Cu nuclear spin). These spectral features are taken into account by using a theoretical model which attributes the random distribution of the electric ligand fields around the copper ions to a distribution of the conformational substate energies both in the proteins and in the amorphous materials.On décrit quelques faits expérimentaux supplémentaires concernant la similitude structurale et dynamique entre protéines et verres, à partir de l'analyse de spectres ESR de quelques protéines contenant du cuivre et de verres dopés au Cu++ formé par de l'eau et un deuxième composant (NaOH, DMSO, alcools), obtenus à différentes températures. Tous les spectres obtenus à des températures inférieures à environ 210 K montrent des déformations g et A significatives qui consistent en un élargissement progressif et une réduction d'intensité des lignes hyperfines du cuivre, quand l'ordre de mI, augmente (le spin nucléaire du cuivre est I = 3/2). Ces faits sont pris en compte dans un modèle théorique qui attribue la distribution aléatoire des champs électriques autour des ions de cuivre à une distribution des énergies de conformation aussi bien pour les protéines que pour les matériaux amorphes

Conductive atomic force microscopy investigation of transverse current across metallic and semiconducting single-walled carbon nanotubes

The comprehension of conduction mechanisms in single-walled carbon nanotubes is a crucial task for developing efficient nanodevices. Appealing hybrid architectures could exploit charge transport perpendicular to the main nanotube axis in order to minimize carrier path and phonon scattering effects. Such transverse transport is investigated in metallic and semiconducting nanotubes by means of conductive atomic force microscopy. The transverse current response is interpreted in the framework of a tunneling transport model, and reveals that conduction across metallic nanotubes is either tunneling- or bandlike, depending on the force applied by the tip, while charge carriers always tunnel through the semiconducting nanotubes. © 2007 American Institute of Physics.2n