Davide Silvestri

In Vivo Fate of Avidin-Nucleic Acid Nanoassemblies as Multifunctional Diagnostic Tools

This study describes the formulation optimization and body-cell distribution and clearance in mice of a dually fluorescent biodegradable poly avidin nanoassembly based on the novel Avidin-Nucleic-Acid-Nano-ASsembly (ANANAS) platform as a potential advancement of classic avidin/biotin-based targeted delivery. The nanoformulation circulates freely in the bloodstream; it is slowly captured by filter organs; it is efficiently cleared within 24-48 h, and it is poorly immunogenic. The system displays more favorable properties than its parent monomeric avidin and it is a promising tool for diagnostic purposes for future translational aims, for which free circulation in the bloodstream, safety, multifunctionality and high composition definition are all necessary requirements. In addition, the assembly shows a time-dependent cell penetration capability, suggesting it may also function as a NP-dependent drug delivery tool. The ease of preparation together with the possibility to fine-tune the surface composition makes it also an ideal candidate to understand if and how nanoparticle composition affects its localization.

A peptide nucleic acid label-free biosensor for Mycobacterium tuberculosis DNA detection via azimuthally controlled grating-coupled SPR

Grating coupled surface plasmon resonance phenomena under azimuthal control of incident light (φ ≠ 0° GC-SPR) have recently been exploited for the development of biosensing solutions with a sensitivity similar to that of classic prism-coupled SPR sensors, with the advantage of higher miniaturization potential. Here we combined the use of φ ≠ 0° GC-SPR with the use of peptide nucleic acid (PNA) probes and a strategy for maximizing the signal-to-noise ratio for the sensitive detection of Mycobacterium tuberculosis (MT) DNA. We focused our attention on the optimization of the PNA-based sensing layer by controlling the sensing surface composition with the PNA-based probe and a poly(ethylene oxide) (PEO)-based antifouling layer. We tested the sensor response first in the presence of complementary and non-complementary oligonucleotides, and then we applied our strategy for the detection of PCR amplified samples, using the fluorescence-based microarray technology as the control. With the φ ≠ 0° GC-SPR set-up adopted, a limit of detection (LOD 0.26 pM) more than one order of magnitude lower than that obtained by the fluorescence method (LOD 8.9 pM) was observed using a complementary oligonucleotide target. Also when PCR amplicons were analysed on SPR grating surfaces, lower DNA concentrations were detectable with the SPR readout as compared to the fluorescence one, and with an experimental protocol that does not include the need to use expensive fluorophore molecules. The whole approach, involving the sensor fabrication, the sensing surface control and DNA detection, has demonstrated that φ ≠ 0° GC-SPR is a good starting point for a sensitive, versatile and scalable biosensing technique that will be further investigated in future experiments.

Plasmonic Platforms for Biodetection Devices

We designed and experimentally realized the prototype for a bio-recognition device based on Grating Coupled Surface Plasmon Resonance (GCSPR) which can find a wide range of applications for detection of chemical and biological species. A sinusoidal grating (period of 500 nm and peak-to-valley height of 40 nm) was realized by Laser Interference Lithography (LIL) and replicated onto a polymeric resin film. After substrate coating with a Cr/Au film a thio-mPEO (Mw 5000) layer with a medium thickness of 4 nm was deposited onto the plasmonic surface. The SPR detection system allowed to detect the biological layer with a refractive index sensitivity of 995.2°/RIU. Our solution has been demonstrated to be a very sensitive platform for the development of a complete GCSPR biosensor.

Novel compact architecture for high-resolution sensing with plasmonic gratings in conical mounting

A novel compact architecture implementing grating-coupled surface plasmon resonance (GCSPR) based on polarization modulation in conical mounting is presented. In this system a plasmonic grating is azimuthally rotated in order to support the excitation of high-sensitivity surface plasmon polaritons (SPPs). At SPP resonance, a scan of the incident polarization is performed before and after the binding event and the phase term of the output trend is exploited as sensing parameter. The mechanical complexity of the SPR system is significantly reduced and a resolution down to 10-7 refractive index units is assured. In this work a numerical study of the polarization-based grating-coupled SPR technique is performed and analyzed with Chandezon’s method. Therefore an experimental test on an assembled prototype is presented and applied to the detection of binding events on the grating surface (avidin/biotin reaction, DNA/PNA probes).