Valentina Mussi

DNA-functionalized solid state nanopore for biosensing

The possible use of nanopores for single DNA molecules biosensing has been demonstrated, but much remains to do in order to develop advanced engineered devices with enhanced stability, and controlled geometry and surface properties. Here we present morphological and electrical characterization of solid state silicon nitride nanopores fabricated by focused ion beam direct milling and chemically functionalized by probe oligonucleotides, with the final aim of developing a versatile tool for biosensing and gene expression profiling.

Surface nanostructuring and optical activation of lithium fluoride crystals by ion beam irradiation

We present results on simultaneous nanostructuring and optical activation of lithium fluoride crystals by 800eV off-normal Ar+ sputtering at different ion doses. The samples were studied by atomic force microscopy and optical spectroscopy. After ion irradiation smoothening of the initial random roughness is achieved and well-defined self-organized ripple structures appear, having a mean periodicity of 30nm and a mean height of 3nm. The simultaneous optical activation of the irradiated samples is due to the stable formation of electronic defects with intense photoluminescence in the visible spectral range.

Modulating DNA Translocation by a Controlled Deformation of a PDMS Nanochannel Device

Several strategies have been developed for the control of DNA translocation in nanopores and nanochannels. However, the possibility to reduce the molecule speed is still challenging for applications in the field of single molecule analysis, such as ultra-rapid sequencing. This paper demonstrates the possibility to alter the DNA translocation process through an elastomeric nanochannel device by dynamically changing its cross section. More in detail, nanochannel deformation is induced by a macroscopic mechanical compression of the polymeric device. This nanochannel squeezing allows slowing down the DNA molecule passage inside it. This simple and low cost method is based on the exploitation of the elastomeric nature of the device, can be coupled with different sensing techniques, is applicable in many research fields, such as DNA detection and manipulation, and is promising for further development in sequencing technology.

Nanotechnology applications in medicine

In recent years there has been a rapid increase in nanotechnology applications to medicine in order to prevent and treat diseases in the human body. The established and future applications have the potential to dramatically change medical science. The present paper will give a few examples that could transform common medical procedures.

Electrical characterization of DNA-functionalized solid state nanopores for bio-sensing

We present data concerning the electrical properties of a class of biosensor devices based on bio-functionalized solid state nanopores able to detect different kinds of interactions between probe molecules, chemically attached to the pore surface, and target molecules present in solution and electrophoretically drawn through the nanometric channel. The great potentiality of this approach resides in the fact that the functionalization of a quite large pore (up to 50-60 nm) allows a sufficient diameter reduction for the attainment of a single molecule sensing dimension and selective activation, without the need for further material deposition, such as metal or oxides, or localized surface modification. The results indicate that it will be possible, in the near future, to conceive and design devices for parallel analysis of biological samples made of arrays of nanopores differently functionalized, fabricated by standard lithographic techniques, with important applications in the field of molecular diagnosis.

Disordered array of Au covered Silicon nanowires for SERS biosensing combined with electrochemical detection

We report on highly disordered array of Au coated silicon nanowires (Au/SiNWs) as surface enhanced Raman scattering (SERS) probe combined with electrochemical detection for biosensing applications. SiNWs, few microns long, were grown by plasma enhanced chemical vapor deposition on common microscope slides and covered by Au evaporated film, 150 nm thick. The capability of the resulting composite structure to act as SERS biosensor was studied via the biotin-avidin interaction: the Raman signal obtained from this structure allowed to follow each surface modification step as well as to detect efficiently avidin molecules over a broad range of concentrations from micromolar down to the nanomolar values. The metallic coverage wrapping SiNWs was exploited also to obtain a dual detection of the same bioanalyte by electrochemical impedance spectroscopy (EIS). Indeed, the SERS signal and impedance modifications induced by the biomolecule perturbations on the metalized surface of the NWs were monitored on the very same three-electrode device with the Au/SiNWs acting as both working electrode and SERS probe.

Size and functional tuning of solid state nanopores by chemical functionalization

We demonstrate the possibility of using a simple functionalization procedure, based on an initial vapour-phase silanization, to control the size and functionality of solid state nanopores. The presented results show that, by varying the silanization time, it is possible to modify the efficiency of probe molecule attachment, thus shrinking the pore to the chosen size, while introducing a specific sensing selectivity. The proposed method allows us to tune the nanopore biosensor adapting it to the specific final application, and it can be efficiently applied when the pore initial diameter does not exceed a limit dimension related to the mean free path of the silane molecules at the working pressure.

Living Matter Observations with a Novel Hyperspectral Supercontinuum Confocal Microscope for VIS to Near-IR Reflectance Spectroscopy

A broad range hyper-spectroscopic microscope fed by a supercontinuum laser source and equipped with an almost achromatic optical layout is illustrated with detailed explanations of the design, implementation and data. The real novelty of this instrument, a confocal spectroscopic microscope capable of recording high resolution reflectance data in the VIS-IR spectral range from about 500 nm to 2.5 μm wavelengths, is the possibility of acquiring spectral data at every physical point as defined by lateral coordinates, X and Y, as well as at a depth coordinate, Z, as obtained by the confocal optical sectioning advantage. With this apparatus we collect each single scanning point as a whole spectrum by combining two linear spectral detector arrays, one CCD for the visible range, and one InGaAs infrared array, simultaneously available at the sensor output channel of the home made instrument. This microscope has been developed for biomedical analysis of human skin and other similar applications. Results are shown illustrating the technical performances of the instrument and the capability in extracting information about the composition and the structure of different parts or compartments in biological samples as well as in solid statematter. A complete spectroscopic fingerprinting of samples at microscopic level is shown possible by using statistical analysis on raw data or analytical reflectance models based on Abelés matrix transfer methods.

Selective protein detection with a dsLNA-functionalized nanopore

In the last years, nanopore technology has been increasingly exploited for biomolecule detection and analysis. Recently, the main focus of the research has moved from the study of nucleic acids to the analysis of proteins and DNA-protein complexes. In this paper, chemically functionalized solid-state nanopore has been used to recognize Nuclear Factor-kappa B proteins (NF-κB), that are involved in several disorders and inflammation processes, so that their identification is of crucial importance for prognostic applications. In particular, we show that it is possible to electrically detect the specific interaction between p50, a protein belonging to the NF-κB family, and dsLNA probe molecules covalently attached to the surface of a FIB fabricated SiN pore. The obtained results have been compared with those related to BSA protein, which does not interact with the used probes. Finally, the potential of the device has been further tested by analyzing a whole cell extract. In this case, three principal peaks in the distribution of electrical event duration can be identified, corresponding to different interacting NF-κB complexes, so that the methodology appears to be effective also to study biological samples of considerable complexity. Ultimately, the presented data emphasize the selectivity and versatility of the functionalized nanopore device, demonstrating its applicability in bioanalytics and advanced diagnostics.

Binding force measurement of NF-κB–ODNs interaction: An AFM based decoy and drug testing tool

Interaction between transcription factors and DNA are essential for regulating gene transcription. The Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor involved in cell signalling and its failure is a principal cause of several autoimmune and auto-inflammatory disorders. In this paper we have developed an atomic force microscopy (AFM) method to quantitatively characterise the interaction force between NF-κB and DNA or LNA (locked nucleic acid) double strand molecules containing the NF responsive elements (RE). This process allows the simple testing and selection of LNA based decoy molecules to be used in NF-κB modulation decoy strategies. Furthermore the proposed methodology is also suitable for testing drug efficacy on the modulation of NF-κB binding to its nucleic acid target sequence. A biological AFM based sensor is therefore considered appropriate for characterising transcription factors and selecting molecules to modulate their activity.