Polymeric integration of structure-switching aptamers on transistors for histamine sensing.

Aptamers that undergo large conformational rearrangements at the surface of electrolyte-gated field-effect transistor (EG-FETs)-based biosensors can overcome the Debye length limitation in physiological high ionic strength environments. For the sensitive detection of small molecules, carbon nanotubes (CNTs) that approach the dimensions of analytes of interest are promising channel materials for EG-FETs. However, functionalization of CNTs with bioreceptors using frequently reported surface modification strategies (e.g., π-π stacking), requires highly pristine CNTs deposited through methods that are incompatible with low-cost fabrication methods and flexible substrates. In this work, we explore alternative non-covalent surface chemistry to functionalize CNTs with aptamers. We harnessed the adhesive properties of poly-D-lysine (PDL), to coat the surface of CNTs and then grafted histamine-specific DNA aptamers electrostatically in close proximity to the CNT semiconducting channel. The layer-by-layer assembly was monitored by complementary techniques such as X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and fluorescence microscopy. Surface characterization confirmed histamine aptamer integration into PDL-coated CNTs and revealed ∼5-fold higher aptamer surface coverage when using CNT networks with high surface areas. Specific aptamers assembled on EG-CNTFETs enabled histamine detection in undiluted high ionic strength solutions in the concentration range of 10 nM to 100 µM. Sequence specificity was demonstrated via parallel measurements with control EG-CNTFETs functionalized with scrambled DNA. Histamine aptamer-modified EG-CNTFETs showed high selectivity vs. histidine, the closest structural analog and precursor to histamine. Taken together, these results implied that target-specific aptamer conformational changes on CNTs facilitate signal transduction, which was corroborated by circular dichroism spectroscopy. Our work suggests that layer-by-layer polymer chemistry enables integration of structure-switching aptamers into flexible EG-CNTFETs for small-molecule biosensing.

Single-Step Functionalization Strategy of Graphene Microtransistor Array with Chemically Modified Aptamers for Biosensing Applications.

Graphene solution-gated field-effect transistors (gSGFETs) offer high potential for chemical and biochemical sensing applications. Among the current trends to improve this technology, the functionalization processes are gaining relevance for its crucial impact on biosensing performance. Previous efforts are focused on simplifying the attachment procedure from standard multi-step to single-step strategies, but they still suffer from overreaction, and impurity issues and are limited to a particular ligand. Herein, a novel strategy for single-step immobilization of chemically modified aptamers with fluorenylmethyl and acridine moieties, based on a straightforward synthetic route to overcome the aforementioned limitations is presented. This approach is benchmarked versus a standard multi-step strategy using thrombin as detection model. In order to assess the reliability of the functionalization strategies 48-gSGFETs arrays are employed to acquire large datasets with multiple replicas. Graphene surface characterization demonstrates robust and higher efficiency in the chemical coupling of the aptamers with the single-step strategy, while the electrical response evaluation validates the sensing capability, allowing to implement different alternatives for data analysis and reduce the sensing variability. In this work, a new tool capable of overcome the functionalization challenges of graphene surfaces is provided, paving the way toward the standardization of gSGFETs for biosensing purposes.

Nonspecific Binding-Fundamental Concepts and Consequences for Biosensing Applications.

Nature achieves differentiation of specific and nonspecific binding in molecular interactions through precise control of biomolecules in space and time. Artificial systems such as biosensors that rely on distinguishing specific molecular binding events in a sea of nonspecific interactions have struggled to overcome this issue. Despite the numerous technological advancements in biosensor technologies, nonspecific binding has remained a critical bottleneck due to the lack of a fundamental understanding of the phenomenon. To date, the identity, cause, and influence of nonspecific binding remain topics of debate within the scientific community. In this review, we discuss the evolution of the concept of nonspecific binding over the past five decades based upon the thermodynamic, intermolecular, and structural perspectives to provide classification frameworks for biomolecular interactions. Further, we introduce various theoretical models that predict the expected behavior of biosensors in physiologically relevant environments to calculate the theoretical detection limit and to optimize sensor performance. We conclude by discussing existing practical approaches to tackle the nonspecific binding challenge in vitro for biosensing platforms and how we can both address and harness nonspecific interactions for in vivo systems.

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

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. Furthermore, 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.

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.

Detecting DNA and RNA and Differentiating Single-Nucleotide Variations via Field-Effect Transistors.

We detect short oligonucleotides and distinguish between sequences that differ by a single base, using label-free, electronic field-effect transistors (FETs). Our sensing platform utilizes ultrathin-film indium oxide FETs chemically functionalized with single-stranded DNA (ssDNA). The ssDNA-functionalized semiconducting channels in FETs detect fully complementary DNA sequences and differentiate these sequences from those having different types and locations of single base-pair mismatches. Changes in charge associated with surface-bound ssDNA vs double-stranded DNA (dsDNA) alter FET channel conductance to enable detection due to differences in DNA duplex stability. We illustrate the capability of ssDNA-FETs to detect complementary RNA sequences and to distinguish from RNA sequences with single nucleotide variations. The development and implementation of electronic biosensors that rapidly and sensitively detect and differentiate oligonucleotides present new opportunities in the fields of disease diagnostics and precision medicine.

Aptamer Renaissance for Neurochemical Biosensing.

Unraveling the complexities of brain function, which is crucial for advancing human health, remains a grand challenge. This endeavor demands precise monitoring of small molecules such as neurotransmitters, the chemical messengers in the brain. In this Perspective, we explore the potential of aptamers, selective synthetic bioreceptors integrated into electronic affinity platforms to address limitations in neurochemical biosensing. We emphasize the importance of characterizing aptamer thermodynamics and target binding to realize functional biosensors in biological systems. We focus on two label-free affinity platforms spanning the micro- to nanoscale: field-effect transistors and nanopores. Integration of well-characterized structure-switching aptamers overcame nonspecific binding, a challenge that has hindered the translation of biosensors from the lab to the clinic. In a transformative era driven by neuroscience breakthroughs, technological innovations, and multidisciplinary collaborations, an aptamer renaissance holds the potential to bridge technological gaps and reshape the landscape of diagnostics and neuroscience.

Solid-State Nanopores for Biomolecular Analysis and Detection.

Advances in nanopore technology and data processing have rendered DNA sequencing highly accessible, unlocking a new realm of biotechnological opportunities. Commercially available nanopores for DNA sequencing are of biological origin and have certain disadvantages such as having specific environmental requirements to retain functionality. Solid-state nanopores have received increased attention as modular systems with controllable characteristics that enable deployment in non-physiological milieu. Thus, we focus our review on summarizing recent innovations in the field of solid-state nanopores to envision the future of this technology for biomolecular analysis and detection. We begin by introducing the physical aspects of nanopore measurements ranging from interfacial interactions at pore and electrode surfaces to mass transport of analytes and data analysis of recorded signals. Then, developments in nanopore fabrication and post-processing techniques with the pros and cons of different methodologies are examined. Subsequently, progress to facilitate DNA sequencing using solid-state nanopores is described to assess how this platform is evolving to tackle the more complex challenge of protein sequencing. Beyond sequencing, we highlight the recent developments in biosensing of nucleic acids, proteins, and sugars and conclude with an outlook on the frontiers of nanopore technologies.

Interfacing Aptamer-Modified Nanopipettes with Neuronal Media and Ex Vivo Brain Tissue.

Aptamer-functionalized biosensors exhibit high selectivity for monitoring neurotransmitters in complex environments. We translated nanoscale aptamer-modified nanopipette sensors to detect endogenous dopamine release in vitro and ex vivo. These sensors employ quartz nanopipettes with nanoscale pores (ca. 10 nm diameter) that are functionalized with aptamers that enable the selective capture of dopamine through target-specific conformational changes. The dynamic behavior of aptamer structures upon dopamine binding leads to the rearrangement of surface charge within the nanopore, resulting in measurable changes in ionic current. To assess sensor performance in real time, we designed a fluidic platform to characterize the temporal dynamics of nanopipette sensors. We then conducted differential biosensing by deploying control sensors modified with nonspecific DNA alongside dopamine-specific sensors in biological milieu. Our results confirm the functionality of aptamer-modified nanopipettes for direct measurements in undiluted complex fluids, specifically in the culture media of human-induced pluripotent stem cell-derived dopaminergic neurons. Moreover, sensor implantation and repeated measurements in acute brain slices was possible, likely owing to the protected sensing area inside nanoscale DNA-filled orifices, minimizing exposure to nonspecific interferents and preventing clogging. Further, differential recordings of endogenous dopamine released through electrical stimulation in the dorsolateral striatum demonstrate the potential of aptamer-modified nanopipettes for ex vivo recordings with unprecedented spatial resolution and reduced tissue damage.

Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection.

Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocation events. Herein, we address these challenges by integrating amino acid-specific DNA aptamers into interface nanopores with dynamically tunable pore sizes. A phenylalanine aptamer was used as a proof-of-concept: aptamer recognition of phenylalanine moieties led to the retention of specific peptides, slowing translocation speeds. Importantly, while phenylalanine aptamers were isolated against the free amino acid, the aptamers were determined to recognize the combination of the benzyl or phenyl and the carbonyl group in the peptide backbone, enabling binding to specific phenylalanine-containing peptides. We decoupled specific binding between aptamers and phenylalanine-containing peptides from nonspecific interactions (e.g., electrostatics and hydrophobic interactions) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer-target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step toward single-molecule proteomics.

Granular Biomaterials as Bioactive Sponges for the Sequestration and Release of Signaling Molecules.

A major challenge for the regeneration of chronic wounds is an underlying dysregulation of signaling molecules, including inflammatory cytokines and growth factors. To address this, it is proposed to use granular biomaterials composed of jammed microgels, to enable the rapid uptake and delivery of biomolecules, and provide a strategy to locally sequester and release biomolecules. Sequestration assays on model biomolecules of different sizes demonstrate that granular hydrogels exhibit faster transport than comparable bulk hydrogels due to enhanced surface area and decreased diffusion lengths. To demonstrate the potential of modular granular hydrogels to modulate local biomolecule concentrations, microgel scaffolds are engineered that can simultaneously sequester excess pro-inflammatory factors and release pro-healing factors. To target specific biomolecules, microgels are functionalized with affinity ligands that bind either to interleukin 6 (IL-6) or to vascular endothelial growth factor A (VEGF-A). Finally, disparate microgels are combined into a single granular biomaterial for simultaneous sequestration of IL-6 and release of VEGF-A. Overall, the potential of modular granular hydrogels is demonstrated to locally tailor the relative concentrations of pro- and anti-inflammatory factors.

Aptamer-Field-Effect Transistors for Small-Molecule Sensing in Complex Environments.

Aptamer-functionalized field-effect transistor (FET) biosensors enable detection of small-molecule targets in complex environments such as tissue and blood. Conventional FET-based platforms suffer from Debye screening in high ionic strength physiological environments where the effective sensing distance is limited to less than a nanometer from the surface of the sensor. Aptamers that undergo significant conformational rearrangement of negatively charged backbones upon target recognition within or in close proximity to the Debye length, facilitate the transduction of electronic signals through the semiconducting channel. Herein, the fabrication of high-performance, ultrathin-film FETs and subsequent aptamer functionalization are described. Moreover, electronic sensing measurement protocols alongside calibration methods to minimize device-to-device variations are covered.

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.

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.

Theoretical analysis of divalent cation effects on aptamer recognition of neurotransmitter targets.

Aptamer-based sensing of small molecules such as dopamine and serotonin in the brain, requires characterization of the specific aptamer sequences in solutions mimicking the in vivo environment with physiological ionic concentrations. In particular, divalent cations (Mg2+ and Ca2+) present in brain fluid, have been shown to affect the conformational dynamics of aptamers upon target recognition. Thus, for biosensors that transduce aptamer structure switching as the signal response, it is critical to interrogate the influence of divalent cations on each unique aptamer sequence. Herein, we demonstrate the potential of molecular dynamics (MD) simulations to predict the behaviour of dopamine and serotonin aptamers on sensor surfaces. The simulations enable molecular-level visualization of aptamer conformational changes that, in some cases, are significantly influenced by divalent cations. The correlations of theoretical simulations with experimental findings validate the potential for MD simulations to predict aptamer-specific behaviors on biosensors.

Driving electrochemical reactions at the microscale using CMOS microelectrode arrays.

Precise control of pH values at electrode interfaces enables the systematic investigation of pH-dependent processes by electrochemical means. In this work, we employed high-density complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) as miniaturized systems to induce and confine electrochemical reactions in areas corresponding to the pitch of single electrodes (17.5 µm). First, we present a strategy for generating localized pH patterns on the surface of the CMOS MEA with unprecedented spatial resolution. Leveraging the versatile routing capabilities of the switch matrix beneath the CMOS MEA, we created arbitrary combinations of anodic and cathodic electrodes and hence pH patterns. Moreover, we utilized the system to produce polymeric surface patterns by additive and subtractive methods. For additive patterning, we controlled the in situ formation of polydopamine at the microelectrode surface through oxidation of free dopamine above a threshold pH > 8.5. For subtractive patterning, we removed cell-adhesive poly-L-lysine from the electrode surface and backfilled the voids with antifouling polymers. Such polymers were chosen to provide a proof-of-concept application of controlling neuronal growth via electrochemically-induced patterns on the CMOS MEA surface. Importantly, our platform is compatible with commercially available high-density MEAs and requires no custom equipment, rendering the findings generalizable and accessible.