The project stands on the development of an electronic platform performing the
measurement of the impedance variation between microelectrodes upon the capture of the
target biomolecule by a selective bioprobe previously grafted over the biosensor surface.
The required sensitivity will be achieved by operating the biosensor in lock-in mode,
which ensures the highest resolution in tracking amplitude and phase variations of
impedance signals (1 ppm has been demonstrated by POLIMI on other applications). To
enhance the electronic signal, polystyrene microbeads, properly functionalized with
oligonucleotides or antibodies, are used to link the target and increase the impedance
variation as demonstrated by proponents in the successful detection of antibodies against
Dengue virus. To address multiple sensing sites in parallel on the same biosensor chip,
allowing real-time comparative analysis on different targets from the same biological
sample for augmented detection sensitivity and control, a custom-made electronic platform
will be developed to perform Differential Impedance Sensing (i.e., direct comparison
between the target sensor and a reference) on multiple channels. The biosensor chip, made
of borosilicate with gold microelectrodes, features a specific modification of its
surface with functional polymers (a family of N,N-dimethylacrylamide -DMA-based polymers)
developed by CNR that form a nanometric hydrophilic film on the chip surface. The
functional groups of these copolymers allow the covalent immobilization of the probe
preserving its active structure, proper spatial distance, orientation and density,
favoring the interaction with the target. The biosensor chip will be integrated into the
microfluidic system already available in the group.

SARS-CoV-2 will be targeted with challenging strategies. First, we plan to capture the
entire virus in solution using DNA-labelled antibodies directed against the SARS-CoV-2
spike protein.

The sensor area, spotted with oligonucleotide probes complementary to the DNA sequence
linked to the antibody, will interact with the DNA-labelled antibodies which have
decorated the entire surface of the virus. To increase the impedance variation of the
platform polystyrene beads will be used: in particular, they will be modified with the
oligonucleotide complementary to that linked to the antibody which have captured the
virus. This entire-virus detection methodology, combining the DNA-directed capture of the
antigens together with a high sensitivity platform for Differential Impedance Sensing, is
absolutely new and expected to be successful based upon the results already achieved by
the proponents in the detection of the antibodies in the human blood of persons infected
by Dengue virus. In parallel, we intend to extend the use of the electronic platform also
to the detection of viral antigens, i.e. Spike protein S or nucleocapside protein N. The
use of antigens for the diagnosis of the infection instead of the entire virus, may lead
to important practical dropouts as it does not necessitate any particular safety
requirement, thus allowing the test to be performed in any location, in particular
pharmacies or medical clinics similarly to standard antigenic lateral flow tests.

This is a retrospective monocenter study to test respiratory samples to evaluate the
analytical sensitivity of the platform being developed in the project PRIN Prot.
2022EJL28B. The samples are completed anonymized and the samples analyses are performed
in order to check the ability of the device to reveal the presence of viral particles in
biological matrix such as respiratory samples or, when necessary for set up of
instruments, plasma randomized and anonymized samples. No patients or use of clinical
data will be involved for the study, but only used the samples stored in the San Raffaele
institutional COVID-19 clinical-biological biobank (COVID-BioB, NCT04318366). The samples
used for the study were collected during the period 19/03/2020 - 31/05/2024.