Conference Agenda

Graphene constitutes a broadband energy acceptor, avoiding labeling, photobleaching and complicated photophysics. Graphene quenches fluorescence of fluorophores in a range of 0-40 nm, following a d-4 distance dependence. Due to Graphane Energy Transfer (GET) a single dye molecule shows a reduced fluorescence intensity and a shortened fluorescence lifetime as a function of its distance to graphene. This information can be used to determine the position of the dye molecule to graphene and to sensitively report on distance changes in real-time. In our first realization, we used DNA origami nanopositioners to place a fluorophore and other molecular components at a defined distance from graphene. With this approach, using single-molecule fluorescence microscopy techniques and several different assays we demonstrated among others: switching dynamics of a DNA pointer between two binding sites with high time resolution, dynamics of a flexible DNA tether influenced by viscosity or target binding, 3D superresolution imaging with isotropic nanoscale resolution, a biosensing assay with single DNA molecule detection in a novel unquenching assay format. Our recently developed tools to connect DNA and graphene enable single base-pair resolution. We use this approach to visualize structural properties of DNA which precede direct interactions with biomolecules and DNA-protein interactions.

Molecular beacons (MBs) represent a powered tool for the detection of RNAs, such as micro-RNA (miRNA) and messenger RNA (mRNA), which play an important role as indicators of the progress of different pathologies in the human body, from the chronic ones to cancer. Two examples are provided here of how the combination of molecular beacons with detection platforms based on surface enhanced Raman scattering (SERS) can lead to the realization of more performing and reliable biosensors. The first case concerns the use of a MB, engineered for specific detection of a miRNA associated with chronic obstructive pulmonary disease. Silver nanowires were used as SERS substrate on which the MBs are immobilized. A femtomolar detection limit has been reached. The second approach is based on soda-lime glass microrods on which silver nanoparticles were grown using the ion-exchange technique followed by an appropriate thermal annealing post-process. Production parameters were optimized aiming at exposing the embedded silver nanoparticles on the surface of the microrods. By functionalizing these nanoparticles with a MB specific for the mRNA for survivin, the microrods were successfully tested for SERS and fluorescence effects, allowing the detection of the complementary sequence.

In this study, we present an innovative optical biosensor designed for the precise detection of

Interleukin-6 (IL-6), a crucial cytokine associated with various pathological conditions. Our biosensor is

based on silicon porous material meticulously modified with a molecularly imprinted polymer (MIP),

ensuring specific and sensitive recognition of IL-6 molecules. Fabrication process involves the

electrochemical etching of silicon porous chips followed by the electrodeposition of MIP, tailored to

selectively bind IL-6 targets. Through rigorous testing across a range of IL-6 concentrations, our sensor

exhibits remarkable sensitivity, showcasing discernible optical responses proportional to the varying analyte

concentrations. Furthermore, we assessed the sensor's performance using bovine serum, a complex biological

matrix, to simulate real-world sample conditions. Encouragingly, the sensor maintains its selectivity and

optical response in the presence of serum components, affirming its robustness and applicability in practical

diagnostic settings.

A set of protein biomarkers are largely recognized as responsible of neurodegeneration mechanisms and hence as potential targets to be detected in low abundant concentrations in body fluids for performing early diagnosis. As an example, the Tau protein experiences a transition phase from a native disorder conformation into a preaggregation state, which leads to fibrillization processes. Here we show the possibility to detect Tau in urine samples at sub-picogram level, through the concentration effect of the pyro-electrohydrodynamic (p-jet) technique. An immunofluorescence protocol is applied to concentrated p-jet spots able to reduce drastically the diffusion effects in the antibody-antigen reaction. A set of diluted samples were prepared, and the fluorescence signal was detected by a confocal scanner. We achieved an excellent linear response with a significant signal-to-noise ratio down to 0.25 pg/mL. In perspective, the technique could be integrated into a compact device to be used for monitoring the early stage associated to neurodegenerative syndromes in different scenarios such as for example in long-term human space exploration missions.

We describe a novel one-dimensional photonic crystal design allowing for the concurrent excitation of transverse-electric and transverse-magnetic Bloch surface waves, thus paving the way for polarization-resolved sensing experiments. We discuss its application for the surface-enhanced sensing of oriented DNA molecules through nanoscale birefringence measurements.