Aptamer Conformational Change Enables Serotonin Biosensing with Nanopipettes

Abstract

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.

Sensing serotonin secreted from human serotonergic neurons using aptamer-modified nanopipettes

“…We expected to detect an unknown 5-HT concentration in the nanomolar regime based on previous reports 9,22 and ELISA results, 11 which was within the detection range of the 5-HT aptamer-modified nanopipettes. 29 Subsequently, samples with higher expected concentrations (i.e. post-KCl or citalopram treatment) were tested in the same manner and a measurable increase in current response was observed from the baseline (Figure 3c,d).…”

“…We previously validated the covalent functionalization of 5-HT aptamers to quartz surfaces, characterized aptamer-modified sensors, and investigated the sensing mechanisms. 29 Aptamer gating of ionic flux through the nanopipette is transduced as an increase in current response upon 5-HT recognition in complex cell culture media (Fig. 2b).…”

“…addition of 30 mM KCl alters the ion current rectification of the nanopipette. 29 Upon obtaining a stable baseline, 5-HT aptamermodified nanopipettes were immersed in a new bath containing NDM in which serotonergic neurons were cultured (Fig. 3c).…”

“…To address these limitations, we have developed a novel technology for 5-HT detection in vitro, that couples quartz nanopipettes that are low-cost, fabricated in a high-throughput manner, and necessitates low volumes, with DNA aptamers that endow these sensors with the necessary specificity and selectivity in complex media. 29 Aptamers are short single-stranded oligonucleotides that have been designed to serve as artificial receptors for targets of interest. 30,31 Recently, a 5-HT-specific aptamer that undergoes significant conformational rearrangement upon 5-HT recognition demonstrated high specificity and selectivity in brain tissue.…”

“…Further, the nanoconfinement of receptor-target binding events leads to high sensitivity (low picomolar capabilities) that exceeds existing methods by at least two to three orders of magnitude. 29 Herein, building on our previous work, we generated human serotonergic neurons from iPSCs and deployed novel DNA-aptamer-coated nanopipettes to detect 5-HT in culture medium. 4,22,33,34 We validated the specificity, selectivity, and stability of 5-HT-specific aptamermodified nanopipettes in complex matrices in the presence of chemically similar interferents.…”