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In the last years, the evolution and the advances of the nanobiotechnologies applied to the systematic study of proteins, namely proteomics, both structural and functional, and specifically the development of more sophisticated and largescale protein arrays, have enabled scientists to investigate protein interactions and functions with an unforeseeable precision and wealth of details. Here, we present a further advancement of our previously introduced and described Nucleic Acid Programmable Protein Arrays (NAPPA)-based nanoconductometric sensor. We coupled Quartz Crystal Microbalance with Dissipation factor Monitoring (QCM_D) with piezoelectric inkjet printing technology (namely, the newly developed ActivePipette), which enables to significantly reduce the volume of probe required for genes/proteins arrays. We performed a negative control (with master mix, or MM) and a positive control (MM_p53 plus MDM2). We performed this experiment both in static and in flow, computing the apparent dissociation constant of p53-MDM2 complex (130 nM, in excellent agreement with the published literature). We compared the results obtained with the ActivePipette printing and dispensing technology vs. pin spotting. Without the ActivePipette, after MDM2 addition the shift in frequency (Δf ) was 7575 Hz and the corresponding adsorbed mass was 32.9 µg. With the ActivePipette technology, after MDM2 addition Δf was 7740 Hz and the corresponding adsorbed mass was 33.6 µg. With this experiment, we confirmed the sensing potential of our device, being able to discriminate each gene and protein as well as their interactions, showing for each one of them a unique conductance curve. Moreover, we obtained a better yield with the ActivePipette technology.

Nanoprobe NAPPA Arrays for the Nanoconductimetric Analysis of Ultra-Low-Volume Protein Samples Using Piezoelectric Liquid Dispensing Technology