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Early diagnosis in a premalignant (or pre-invasive) state represents the only chance for cure in neoplastic diseases such as pancreatic-biliary cancer, which are otherwise detected at later stages and can only be treated using palliative approaches, with no hope for a cure. Screening methods for the purpose of secondary prevention are not yet available for these cancers. Current diagnostic methods mostly rely on imaging techniques and conventional cytopathology, but they do not display adequate sensitivity to allow valid early diagnosis. Next-generation sequencing can be used to detect DNA markers down to the physical limit; however, this assay requires labeling and is time-consuming. The additional determination of a protein marker that is a predictor of aggressive behavior is a promising innovative approach, which holds the potential to improve diagnostic accuracy. Moreover, the possibility to detect biomarkers in blood serum offers the advantage of a noninvasive diagnosis. In this study, both the DNA and protein markers of pancreatic mucinous cysts were analyzed in human blood serum down to the single-molecule limit using the SiMoT (single-molecule assay with a large transistor) platform. The SiMoT device proposed herein, which exploits an inkjet-printed organic semiconductor on plastic foil, comprises an innovative 3D-printed sensing gate module, consisting of a truncated cone that protrudes from a plastic substrate and is compatible with standard ELISA wells. This 3D gate concept adds tremendous control over the biosensing system stability, along with minimal consumption of the capturing molecules and body fluid samples. The 3D sensing gate modules were extensively characterized from both a material and electrical perspective, successfully proving their suitability as detection interfaces for biosensing applications. KRAS and MUC1 target molecules were successfully analyzed in diluted human blood serum with the 3D sensing gate functionalized with b-KRAS and anti-MUC1, achieving a limit of detection of 10 zM and 40 zM, respectively. These limits of detection correspond to (1 ± 1) KRAS and (2 ± 1) MUC1 molecules in the 100 μL serum sample volume. This study provides a promising application of the 3D SiMoT platform, potentially facilitating the timely, noninvasive, and reliable identification of pancreatic cancer precursor cysts. Graphical abstract

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers