The project aims to realize a powerful sensing platform based on nanochannels capable of identify proteins and biomarkers at the single-molecule level. Nanochannel technologies have already enabled DNA manipulation, such as linearization, stretching and sorting, and are at the core of diagnostic devices used for optical genome mapping. Recently, nanochannel-based devices are emerging as powerful sensing tools not only for genomics but also for the identification of proteins and other biomarkers with unprecedented sensitivities at ultra-low concentrations and ultra-small volumes. As the field of liquid biopsy keeps expanding, nanochannel-based technologies are expected to play a central role as key sensing elements while enabling easy interfacing of other nanofluidic elements for upstream samples preparation.
In this project, we have designed and realized nanofluidic devices based on nanochannels. These devices have been assembled and successfully used for the identification of protein mixtures by performing single-molecule fluorescence imaging. The confinement properties of these devices have been exploited to squeeze the optical sensing volume achieving single-molecule resolution and to study how the physical properties of molecules, including their diffusion, are affected under the confinement regime. To improve the capabilities of the device for proteins separation an acrylamide-based gel was also introduced to perform miniaturized gel electrophoresis along the channel. These devices were successfully tested for the discrimination and quantification of VEGF isoforms, which is not currently possible with conventional techniques, such as ELISA, and to detect other proteins, such as CCl2, RBP4, Clusterin and Serpin A4 which are biomarkers for Age-related Macular Degeneration. This technique is now able to separate, track and identify thousands of proteins at the single molecule Level in complex biological samples, requiring ultra-small volumes (only few picoliters) and ultra-low concentrations (in the picomolar range). Currently, these devices are ready to be challenged with more sophisticated applications including liquid biopsy and diseases monitoring. Besides their potential for advanced diagnostics, nanochannel-devices can be used as powerful tools for fundamental research due to the single-molecule sensitivity and thus, they might help elucidating the different functions of proteins isoforms in cell physiology and their over/downregulation during disease progression.
Societal impacts. The development of a single-molecule diagnostic tool capable of detecting multiple proteins in a single run and discriminating proteins isoforms has high potential for early-diagnosis base on liquid biopsy with high benefit to society healthcare. Currently, there is gaining attention on the development of new sensing devices able to identify biomarkers and other molecules from small volumes of samples, including blood, urine, sweat and tears to allow frequent and comprehensive monitoring of diseases and to drastically reduce the invasiveness of screening, the duration of the analysis and the required infrastructure compared to conventional diagnostic techniques.
