We developed multiplexed nano-immunoassays based on the nanografting of short single stranded (ss) oligos on gold thin films and the DNA-directed bioaffinity binding of the complementary DNA sequences conjugated to specific protein markers/protein binders, to detect circulating biomarkers from non-invasive liquid biopsy. An atomic force microscope is used to quantify the biomarkers by detecting with high sensitivity the topographic height changes at the nanospots. As an example, we are applying such devices to the quantification of the shed extracellular Her2 fragments in the serum of Her2-positive tumor patients and correlate the measured Her2 levels to the progress of the disease. To bind Her2, antibodies and nanobodies (single domain antibodies) from camelid as well as from recombinant origin were used, optimized for high affinity binding in silico (Dr. Sara Fortuna, University of Trieste). Nanobodies selective for different Her2 fragments derived by proteolitic cleavage and by alternative splicing of Her2, respectively, are being produced and soon tested on the nanoassay. As another example, the monitoring in serum of the level of a Infliximab, a therapeutic Anti-TNFα drug, in the treatment of pediatric inflammatory bowel diseases (IBD).
A similar strategy for surface biofunctionalization based on the grafting of ssDNA and on DNA bioaffinity immobilization has been developed and optimized also on gold nanoparticles (AuNPs). We created a controlled assembling of AuNPs in solution and monitored in real time the disruption kinetics of NPs aggregates upon loading of a helicase enzyme, by means of a simple colorimetric assay (AuNPs of different size absorb light with different frequencies). Such assay can be used to test enzyme inhibitor efficacy by comparing enzyme kinetics with and without inhibitor. A proof of concept of this strategy has been given for the RecQ helicases, whose inhibition reinforces the efficacy of chemotherapy agents.