Research activities

Surface-acoustic-wave biosensors and microfluidics

Surface acoustic waves (SAWs) are acoustic waves that travel along the surface of an elastic material, with an amplitude that typically decays exponentially with depth into the substrate. Given their very superficial nature, SAWs are highly sensitive to surface perturbations of the substrate along which they propagate. For example, they can interact with liquid droplets or streams inducing macroscopic fluid manipulations or, in a different configuration, be exploited for sensing applications. The interest of the NeuroSens group in this field is to explore and study novel SAW-driven microfluidic phenomena, and apply this new knowledge to the fields of biosensing and cell biology.

A Rayleigh surface acoustic wave (R-SAW) resonator biosensor based on positive and negative reflectors with sub-nanomolar limit of detection

M. Agostini, G. Greco, M. Cecchini. Sensors and Actuators B: Chemical 254, 1–7 (2018).  
A label-free sub-nanomolar Rayleigh surface acoustic wave (R-SAW)-based resonator biosensor is demonstrated for biomolecular detection in liquid after drying. The biosensor comprises two interdigital transducers for R-SAW generation and two positive and negative reflectors to confine the acoustic energy in the sensitive area. We benchmark this biosensor against biotin-streptavidin binding, which is a stan- dard, well-known model for a variety of biosensing processes. The experiments demonstrate a limit of detection of 104 pM and a normalized sensitivity of −296 m^2 kg^−1. As a comparison with similar acoustic- wave based systems, both sensitivity and limit of detection are better than that of standard commercial gravimetric sensors (i.e., quartz-crystal-microbalances) and generally better than that of more common Love-SAW biosensors. Our biosensor has a dynamic range potentially compatible with several health- and safety-related assays, among all cancer biomarker detection.

Full-SAW Microfluidics-Based Lab-on-a-Chip for Biosensing

M. Agostini, G. Greco and M. Cecchini, IEEE Access, DOI: 10.1109/ACCESS.2019.2919000 (2019). 

Many approaches to diagnostic testing remain decades old. Well-established biosensing technologies (e.g., enzyme linked immunosorbent assays, radio-immunoassays) typically cannot fulfill the requirements of portability and ease of use necessary for point-of-care purposes. Several alternatives have been proposed (e.g., quartz-crystal-microbalances, electrochemical sensors, cantilevers, surface-plasmon-resonance sensors) but often lack high performance or still necessitate bulk ancillary instruments to operate. Here we present a highly sensitive, versatile and easily integrable microfluidic lab-on-a-chip (LoC) for biosensing, fully based on surface acoustic waves (SAWs). By using ultra-high-frequency resonator-biosensors, we show that it is possible to perform highly sensitive assays in complex media. This all-electrical readout platform is benchmarked with the biotin-streptavidin binding in presence of non-specific binding proteins (serum albumin) at physiological concentration. The benchmark experiments were performed with the idea of mimicking a biological fluid, in which other molecular species at high concentration are present together with the analytes. We demonstrate that this LoC can detect sub-nanomolar concentrations of analytes in complex media. As a comparison with similar acoustic-wave based systems, this full-SAW platform outperforms the standard commercial gravimetric sensors (i.e., quartz-crystal-microbalances) and the more common Love-SAW biosensors. This full-SAW LoC could be further developed for the detection of biomarkers in biological fluids.































































Nanotechnologies for the nervous system

The nervous system (NS) is in some ways the most complex and fascinating organ of the human body. Unfortunately, NS pathologies that lead to tissue loss are dramatically difficult to treat because of the negligible regenerative potential of the central nervous system (CNS) from one side, and of the very slow and ineffective repair mechanisms of peripheral nervous system (PNS) from the other side. The interest of the NeuroSens group in this field is to develop biocompatible nanostructured materials to help the heal of the PNS and cure and study CNS diseases. In particular, at the moment our attention is focused on textured surfaces for helping nerve regeneration and on nanotechnological methods for Globoid Cell Leukodystrophy (or Krabbe disease; OMIM #245200) and Ube3A neurodevelopmental disorders.

Brain-targeted enzyme-loaded nanoparticles: A breach through the blood-brain barrier for enzyme replacement therapy in Krabbe disease

A. Del Grosso, M. Galliani, L. Angella, M. Santi, I. Tonazzini, G. Parlanti, G. Signore, and M. Cecchini. Science Advances Vol. 5, no. 11, eaax7462 (2019).  
Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement.

The role of ubiquitin ligase E3A in polarized contact guidance and rescue strategies in UBE3A-deficient hippocampal neurons

I. Tonazzini, GM Van Woerden, C. Masciullo, EJ. Mientjes, Ype Elgersma,and M. Cecchini. Molecular Autism, 10(1), 41 (2019).  
Although neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway.

Here, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance.

We found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber–binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype.

We identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity.