Aptamer Conformational Dynamics Modulate Neurotransmitter Sensing in Nanopores

: Aptamers that undergo conformational changes upon small-molecule recognition have been shown to gate the ionic flux through nanopores by rearranging the charge density within the aptamer-occluded orifice. However, mechanistic insight into such systems where biomolecular interactions are confined in nanoscale spaces is limited. To understand the fundamental mechanisms that facilitate the detection of small-molecule analytes inside structure-switching aptamer-modified nanopores, we correlated experimental observations to theoretical models. We developed a dopamine aptamer-functionalized nanopore sensor with femtomolar detection limits and compared the sensing behavior with that of a serotonin sensor fabricated with the same methodology. When these two neurotransmitters with comparable mass and equal charge were detected, the sensors showed an opposite electronic behavior. This distinctive phenomenon was extensively studied using complementary experimental techniques such as quartz crystal microbalance with dissipation monitoring, in combination with theoretical assessment by the finite element method and molecular dynamic simulations. Taken together, our studies demonstrate that the sensing behavior of aptamer-modified nanopores in detecting specific small-molecule analytes correlates with the structure-switching mechanisms of individual aptamers. We believe that such investigations not only improve our understanding of the complex interactions occurring in confined nanoscale environments but will also drive further innovations in biomimetic nanopore technologies

Aptamer-Functionalized Field-Effect Transistors For Serotonin and Dopamine Sensing

My thesis work built on over a decade’s worth of research in the Andrews and Weiss groups aimed at discovering high-affinity oligonucleotide-based recognition elements called aptamers. Previous work focused on designing and developing solid-phase substrates with surface-tethered small-molecule targets that retained their biological functionality to enable recognition by receptors. I mastered techniques such as chemical lift-off lithography and microfluidics to pattern small-molecules in specific locations to facilitate quantification of specific binding relative to background molecules. I demonstrated recognition of surface-tethered dopamine by a previously isolated dopamine aptamer. Specific binding was validated using competitive displacement experiments, which verified that surface-tethered dopamine, despite its reduced degree of freedom, could compete with free dopamine in solution. I addressed one of the shortcomings of conventional in vitro aptamer selection by enabling on-chip determination of equilibrium dissociation constants (Kd). Using a novel patterning method to create aptamer concentration gradients on multiplexed substrates, I resolved multiple Kd values simultaneously. I demonstrated that optimized small-molecule-functionalized substrates were ready to screen for novel neurotransmitter-specific aptamers. In parallel, however, our collaborators at Columbia University isolated high-affinity (nanomolar Kd) aptamers targeting serotonin and dopamine through the use of a solution-phase method. Thus, I advanced our research to the next step by integrating these aptamers onto the semiconducting channels of thin-film field-effect transistors (FETs). Serotonin- and dopamine-functionalized FETs were able to sense target molecules in high ionic-strength, undiluted physiological buffers, as well as in complex environments such as brain tissue. Traditionally, biological FETs have suffered from Debye length limitations under physiological conditions where the effective sensing distance is <1 nm from the surface of the semiconducting channels. We hypothesized that the mechanism that enabled sensitive detection of neurotransmitter targets even in complex fluids was driven by aptamer conformational changes. I read and synthesized every aptamer-FET paper I could find in the literature to understand the current status of the field. I found that mechanistically, there were two emerging lines of thought. The first asserts that electronic signals arise mainly from target-associated charge being brought into close proximity of FETS upon aptamer binding. The second postulates that rearrangement of charged aptamer backbones contributes to aptamer-FET target detection.I investigated the mechanism of the serotonin and dopamine aptamer-FETs by exploring the influence of divalent cations on aptamer binding. I showed that serotonin and dopamine signal responses behaved differently based on the presence/absence of divalent cations. This meant there were aptamer-specific differences in secondary structure rearrangements upon target capture. I conducted circular dichroism and surface-enhanced Raman spectroscopy to compare alterations in aptamer secondary structures upon target capture, empirically. I demonstrated that these two techniques can be used to track aptamer conformational changes and together, they enabled prediction of sensing capabilities prior to FET incorporation of aptamers. Sensing of a neutral target (glucose) and a zwitterionic species (sphingosine-1-phosphate) further implicated target-induced rearrangement of aptamer charge at the surface of FETs as a key mechanism for small molecule sensing. This mechanism is advantageous as it is generalizable for any target of interest regardless of size or charge. Finally, inspired by recent literature on polydopamine nanoparticles, I fabricated and characterized analogous serotonin-based nanomaterials. I demonstrated that polyserotonin nanoparticles had comparable therapeutic properties to polydopamine nanoparticles such as drug loading efficiency and photothermal capabilities. However, compared to polydopamine, polyserotonin nanoparticles showed reduced protein corona formation on the surface and improved biocompatibilities with three stem cell lines, suggesting their potential for future clinical applications

Aptamer-Functionalized Field-Effect Transistors For Serotonin and Dopamine Sensing

My thesis work built on over a decade’s worth of research in the Andrews and Weiss groups aimed at discovering high-affinity oligonucleotide-based recognition elements called aptamers. Previous work focused on designing and developing solid-phase substrates with surface-tethered small-molecule targets that retained their biological functionality to enable recognition by receptors. I mastered techniques such as chemical lift-off lithography and microfluidics to pattern small-molecules in specific locations to facilitate quantification of specific binding relative to background molecules. I demonstrated recognition of surface-tethered dopamine by a previously isolated dopamine aptamer. Specific binding was validated using competitive displacement experiments, which verified that surface-tethered dopamine, despite its reduced degree of freedom, could compete with free dopamine in solution. I addressed one of the shortcomings of conventional in vitro aptamer selection by enabling on-chip determination of equilibrium dissociation constants (Kd). Using a novel patterning method to create aptamer concentration gradients on multiplexed substrates, I resolved multiple Kd values simultaneously. I demonstrated that optimized small-molecule-functionalized substrates were ready to screen for novel neurotransmitter-specific aptamers. In parallel, however, our collaborators at Columbia University isolated high-affinity (nanomolar Kd) aptamers targeting serotonin and dopamine through the use of a solution-phase method. Thus, I advanced our research to the next step by integrating these aptamers onto the semiconducting channels of thin-film field-effect transistors (FETs). Serotonin- and dopamine-functionalized FETs were able to sense target molecules in high ionic-strength, undiluted physiological buffers, as well as in complex environments such as brain tissue. Traditionally, biological FETs have suffered from Debye length limitations under physiological conditions where the effective sensing distance is <1 nm from the surface of the semiconducting channels. We hypothesized that the mechanism that enabled sensitive detection of neurotransmitter targets even in complex fluids was driven by aptamer conformational changes. I read and synthesized every aptamer-FET paper I could find in the literature to understand the current status of the field. I found that mechanistically, there were two emerging lines of thought. The first asserts that electronic signals arise mainly from target-associated charge being brought into close proximity of FETS upon aptamer binding. The second postulates that rearrangement of charged aptamer backbones contributes to aptamer-FET target detection.I investigated the mechanism of the serotonin and dopamine aptamer-FETs by exploring the influence of divalent cations on aptamer binding. I showed that serotonin and dopamine signal responses behaved differently based on the presence/absence of divalent cations. This meant there were aptamer-specific differences in secondary structure rearrangements upon target capture. I conducted circular dichroism and surface-enhanced Raman spectroscopy to compare alterations in aptamer secondary structures upon target capture, empirically. I demonstrated that these two techniques can be used to track aptamer conformational changes and together, they enabled prediction of sensing capabilities prior to FET incorporation of aptamers. Sensing of a neutral target (glucose) and a zwitterionic species (sphingosine-1-phosphate) further implicated target-induced rearrangement of aptamer charge at the surface of FETs as a key mechanism for small molecule sensing. This mechanism is advantageous as it is generalizable for any target of interest regardless of size or charge. Finally, inspired by recent literature on polydopamine nanoparticles, I fabricated and characterized analogous serotonin-based nanomaterials. I demonstrated that polyserotonin nanoparticles had comparable therapeutic properties to polydopamine nanoparticles such as drug loading efficiency and photothermal capabilities. However, compared to polydopamine, polyserotonin nanoparticles showed reduced protein corona formation on the surface and improved biocompatibilities with three stem cell lines, suggesting their potential for future clinical applications

Aptamer-functionalized capacitive biosensors

The growing use of aptamers as target recognition elements in label-free biosensing necessitates corresponding transducers that can be used in relevant environments. While popular in many fields, capacitive sensors have seen relatively little, but growing use in conjunction with aptamers for sensing diverse targets. Few reports have shown physiologically relevant sensitivity in laboratory conditions and a cohesive picture on how target capture modifies the measured capacitance has been lacking. In this review, we assess the current state of the field in three areas: small molecule, protein, and cell sensing. We critically analyze the proposed hypotheses on how aptamer-target capture modifies the capacitance, as many mechanistic postulations appear to conflict between published works. As the field matures, we encourage future works to investigate individual aptamer-target interactions and to interrogate the physical mechanisms leading to measured changes in capacitance. To this point, we provide recommendations on best practices for developing aptasensors with a particular focus on considerations for biosensing in clinical settings.ISSN:0956-5663ISSN:1873-423

Divalent Cation Dependence Enhances Dopamine Aptamer Biosensing.

Oligonucleotide receptors (aptamers), which change conformation upon target recognition, enable electronic biosensing under high ionic-strength conditions when coupled to field-effect transistors (FETs). Because highly negatively charged aptamer backbones are influenced by ion content and concentration, biosensor performance and target sensitivities were evaluated under application conditions. For a recently identified dopamine aptamer, physiological concentrations of Mg2+ and Ca2+ in artificial cerebrospinal fluid produced marked potentiation of dopamine FET-sensor responses. By comparison, divalent cation-associated signal amplification was not observed for FET sensors functionalized with a recently identified serotonin aptamer or a previously reported dopamine aptamer. Circular dichroism spectroscopy revealed Mg2+- and Ca2+-induced changes in target-associated secondary structure for the new dopamine aptamer, but not the serotonin aptamer nor the old dopamine aptamer. Thioflavin T displacement corroborated the Mg2+ dependence of the new dopamine aptamer for target detection. These findings imply allosteric binding interactions between divalent cations and dopamine for the new dopamine aptamer. Developing and testing sensors in ionic environments that reflect intended applications are best practices for identifying aptamer candidates with favorable attributes and elucidating sensing mechanisms

Aptamer-modified biosensors to visualize neurotransmitter flux

Chemical biosensors with the capacity to continuously monitor various neurotransmitter dynamics can be powerful tools to understand complex signaling pathways in the brain. However, in vivo detection of neurochemicals is challenging for many reasons such as the rapid release and clearance of neurotransmitters in the extracellular space, or the low target analyte concentrations in a sea of interfering biomolecules. Biosensing platforms with adequate spatiotemporal resolution coupled to specific and selective receptors termed aptamers, demonstrate high potential to tackle such challenges. Herein, we review existing literature in this field. We first discuss nanoparticle-based systems, which have a simple in vitro implementation and easily interpretable results. We then examine methods employing near-infrared detection for deeper tissue imaging, hence easier translation to in vivo implementation. We conclude by reviewing live cell imaging of neurotransmitter release via aptamermodified platforms. For each of these sensors, we discuss the associated challenges for translation to real-time in vivo neurochemical imaging. Realization of in vivo biosensors for neurotransmitters will drive future development of early prevention strategies, treatments, and therapeutics for psychiatric and neurodegenerative diseases.ISSN:0165-0270ISSN:1872-678

Aptamer Conformational Change Enables Serotonin Biosensing with Nanopipettes

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 structure-switching aptamer-modified nanopipette systems that supports experimental findings. Validation of specific and selective small-molecule detection, in parallel with mechanistic investigations, demonstrates the potential of conformationally changing aptamer-modified nanopipettes as rapid, label-free, and translatable nanotools for diverse biological systems.ISSN:1520-6882ISSN:0003-270