We report artificial nanopores in the form of quartz nanopipettes with ca. 10 nm orifices functionalized with molecular recognition elements termed aptamers that reversibly recognize serotonin with high specificity and selectivity. Nanoscale confinement of ion fluxes, analyte-specific aptamer conformational changes, and related surface charge variations enable serotonin sensing. We demonstrate detection of physiologically relevant serotonin amounts in complex environments such as neurobasal media in which neurons are cultured in vitro. In addition to sensing in physiologically relevant matrices with high sensitivity (picomolar detection limits), we interrogate the detection mechanism via complementary techniques such as quartz crystal microbalance with dissipation monitoring and electrochemical impedance spectroscopy. Moreover, we provide a novel theoretical model for structureswitching aptamer-modified nanopipette systems that supports experimental findings. Validation of specific and selective smallmolecule detection in parallel with mechanistic investigations, demonstrates the potential of conformationally changing aptamermodified nanopipettes as rapid, label-free, and translatable nanotools for diverse biological systems.

The serotonergic system in the human brain modulates several physiological processes and altered serotonergic neurotransmission has been implicated in the neuropathology of several psychiatric disorders. The study of serotonergic neurotransmission in psychiatry has long been restricted to animal models but advances in cell reprogramming technology have enabled the generation of serotonergic neurons from patient-induced pluripotent stem cells (iPSCs). While iPSC-derived human serotonergic neurons offer the possibility to study serotonin (5-HT) release and uptake, particularly by 5-HT-modulating drugs such as selective serotonin reuptake inhibitors (SSRIs), a major limitation is the inability to reliably quantify 5-HT secreted from neurons in vitro.Herein, we address this technical gap via a novel sensing technology that couples 5-HT-specific DNA aptamers into nanopores (glass nanopipettes) with orifices of ~10 nm to detect 5-HT in complex neuronal culture medium with higher selectivity, sensitivity, and stability than existing methods. The 5-HT aptamers undergo conformational rearrangement upon target capture and serve as gatekeepers of ionic flux through the nanopipette opening. We generated human serotonergic neurons in vitro and detected secreted 5-HT using aptamer-coated nanopipettes in a low nanomolar range, with the possibility of detecting significantly lower (picomolar) concentrations. Further, as a proof-of-concept, we treated human serotonergic neurons in vitro with the SSRI citalopram and detected a significant increase in extracellular 5-HT using the aptamer-modified nanopipettes. We demonstrate the utility of such methods for 5-HT detection, raising the possibility of fast quantification of neurotransmitters secreted from patient-derived live neuronal cells.

The PD-1/PD-L1 signaling pathway is essential for immune control and maintaining immune system balance. PD-1 and PD-L1 proteins exert most functions in cells and tissues by undergoing modifications and forming dynamic complexes – effects that cannot be explored by genomics, transcriptomics, or conventional immunostaining methods. Numerous cancer therapies are being developed that affect PD-1/PD-L1 signaling, and tools to study the PD-1/PD-L1 axis are, therefore, essential. One prerequisite for successfully establishing diagnostic assays is reproducibility, standardization, and, ideally, high sample throughput via automation. Here, we aimed to introduce a new technological solution to allow for the simultaneous detection of PD-L1 and PD-1 interaction and the adjacent immune cell context in tissue samples on the Lunaphore’s automated microfluidic staining platform. The assay for detecting PD-1-PD-L1 interaction was developed and technically validated by Navinci Diagnostics. Although the detection of interaction alone provided important information on the immune status in the tumor microenvironment, we believed that PD-1/PD-L1 readout would be most powerful in the context of its cellular ecosystem. Therefore, the PD-1/PD-L1 assay was combined with the analysis of immune and cancer cells to determine their involvement in this signaling. The feasibility of combining multiplex immunofluorescence with proximity ligation assay was conducted and confirmed on human tonsils. The spatial profiling panel included immune markers for all T-cells (CD3), helper T-cell (CD4), cytotoxic T-cells (CD8), B-cells (CD19/20) and activation/exhaustion markers granzyme B (activated cytotoxic T-cell), Ki67 (proliferating cells), and LAG3 (malignant B cells, CD8 T cells, CD4 Tregs). We evaluated, implemented, and optimized the assays on the Lunaphore staining instrument to reduce labor time and cost for the end users. In the next phase, the potential of our assay to be benchmarked as a diagnostic tool in clinical praxis will be thoroughly tested and validated on diagnostic tissue samples from NSCLC patient cohorts. We believe this approach will enable spatial and functional studies of the interface between the tumor and the immune system and provide necessary information about signaling pathway activation in situ, the latest representing a novel state-of-the-art in tissue diagnostics. Citation Format: Hampus Elofsson, Agata Zieba Wicher, Carolina Oses Sepulveda, Tony Ullman, Maria-Giuseppina Procopio, Alix Faillétaz, Diego G. Dupouy, Charlotte Stadler. Automation of proximity ligation immunoassay for interaction between PDL1 and PD1 detection in the tumor microenvironment using microfluidic based system. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4336.