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.

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.

Beginner's Guide to Micro- and Nanoscale Electrochemical Additive Manufacturing.

Electrochemical additive manufacturing is an advanced microfabrication technology capable of producing features of almost unlimited geometrical complexity. A unique combination of the capacity to process conductive materials, design freedom, and micro- to nanoscale resolution offered by these electrochemical techniques promises tremendous opportunities for a multitude of future applications spanning microelectronics, sensing, robotics, and energy storage. This review aims to equip readers with the basic principles of electrochemical 3D printing at the small length scale. By describing the basic principles of electrochemical additive manufacturing technology and using the recent advances in the field, this beginner's guide illustrates how controlling the fundamental phenomena that underpin the print process can be used to vary dimensions, morphology, and microstructure of printed structures.

Electrochemical 3D printing of Ni-Mn and Ni-Co alloy with FluidFM.

Additive manufacturing can realize almost any designed geometry, enabling the fabrication of innovative products for advanced applications. Local electrochemical plating is a powerful approach for additive manufacturing of metal microstructures; however, previously reported data have been mostly obtained with copper, and only a few cases have been reported with other elements. In this study, we assessed the ability of fluidic force microscopy to produce Ni-Mn and Ni-Co alloy structures. Once the optimal deposition potential window was determined, pillars with relatively smooth surfaces were obtained. The printing process was characterized by printing rates in the range of 50-60 nm s-1. Cross-sections exposed by focused ion beam showed highly dense microstructures, while the corresponding face scan with energy-dispersive x-ray spectroscopy spectra revealed a uniform distribution of alloy components.

Bringing Electrochemical Three-Dimensional Printing to the Nanoscale.

Nanoscale 3D printing is attracting attention as an alternative manufacturing technique for a variety of applications from electronics and nanooptics to sensing, nanorobotics, and energy storage. The constantly shrinking critical dimension in state-of-the-art technologies requires fabrication of complex conductive structures with nanometer resolution. Electrochemical techniques are capable of producing impurity-free metallic conductors with superb electrical and mechanical properties, however, true nanoscale resolution (<100 nm) remained unattainable. Here, we set new a benchmark in electrochemical 3D printing. By employing nozzles with dimensions as small as 1 nm, we demonstrate layer-by-layer manufacturing of 25 nm diameter voxels. Full control of the printing process allows adjustment of the feature size on-the-fly, printing tilted, and overhanging structures. On the basis of experimental evidence, we estimate the limits of electrochemical 3D printing and discuss the origins of this new resolution frontier.

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.

Investigating Complex Samples with Molograms of Low-Affinity Binders.

In vitro diagnostics relies on the quantification of minute amounts of a specific biomolecule, called biomarker, from a biological sample. The majority of clinically relevant biomarkers for conditions beyond infectious diseases are detected by means of binding assays, where target biomarkers bind to a solid phase and are detected by biochemical or physical means. Nonspecifically bound biomolecules, the main source of variation in such assays, need to be washed away in a laborious process, restricting the development of widespread point-of-care diagnostics. Here, we show that a diffractometric assay provides a new, label-free possibility to investigate complex samples, such as blood plasma. A coherently arranged sub-micron pattern, that is, a peptide mologram, is created to demonstrate the insensitivity of this diffractometric assay to the unwanted masking effect of nonspecific interactions. In addition, using an array of low-affinity binders, we also demonstrate the feasibility of molecular profiling of blood plasma in real time and show that individual patients can be differentiated based on the binding kinetics of circulating proteins.

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.

Force-Controlled Formation of Dynamic Nanopores for Single-Biomolecule Sensing and Single-Cell Secretomics.

Nanopore sensing of single nucleotides has emerged as a promising single-molecule technology for DNA sequencing and proteomics. Despite the conceptual simplicity of nanopores, adoption of this technology for practical applications has been limited by a lack of pore size adjustability and an inability to perform long-term recordings in complex solutions. Here we introduce a method for fast and precise on-demand formation of a nanopore with controllable size between 2 and 20 nm through force-controlled adjustment of the nanospace formed between the opening of a microfluidic device (made of silicon nitride) and a soft polymeric substrate. The introduced nanopore system enables stable measurements at arbitrary locations. By accurately positioning the nanopore in the proximity of single neurons and continuously recording single-molecule translations over several hours, we have demonstrated this is a powerful approach for single-cell proteomics and secretomics.

Single-Nanoparticle Thermometry with a Nanopipette.

Thermal measurements at the nanoscale are key for designing technologies in many areas, including drug delivery systems, photothermal therapies, and nanoscale motion devices. Herein, we present a nanothermometry technique that operates in electrolyte solutions and, therefore, is applicable for many in vitro measurements, capable of measuring and mapping temperature with nanoscale spatial resolution and sensitive to detect temperature changes down to 30 mK with 43 µs temporal resolution. The methodology is based on local measurements of ionic conductivity confined at the tip of a pulled glass capillary, a nanopipettete, with opening diameters as small as 6 nm. When scanned above a specimen, the measured ion flux is converted into temperature using an extensive theoretical support given by numerical and analytical modeling. This allows quantitative thermal measurements with a variety of capillary dimensions and is applicable to a range of substrates. We demonstrate the capabilities of this nanothermometry technique by simultaneous mapping of temperature and topography on sub-micrometer-sized aggregates of thermoplasmonic nanoparticles heated by a laser and observe the formation of micro- and nanobubbles upon plasmonic heating. Furthermore, we perform quantitative thermometry on a single-nanoparticle level, demonstrating that the temperature at an individual nanoheater of 25 nm in diameter can reach an increase of about 3 K.

Alginate Sulfate Substrates Control Growth Factor Binding and Growth of Primary Neurons: Toward Engineered 3D Neural Networks.

Sulfated glycosaminoglycans (sGAGs) are vital molecules of the extracellular matrix (ECM) of the nervous system known to regulate proliferation, migration, and differentiation of neurons mainly through binding relevant growth factors. Alginate sulfate (AlgSulf) mimics sGAGs and binds growth factors such as basic fibroblast growth factor (FGF-2). Here, thin films of biotinylated AlgSulf (b-AlgSulfn ) are engineered with sulfation degrees (DS = 0.0 and 2.7) and the effect of polysaccharide concentration on FGF-2 and nerve growth factor (ß-NGF) binding and subsequent primary neural viability and neurite outgrowth is assessed. An increase in b-AlgSulfn concentration results in higher FGF-2 and ß-NGF binding as demonstrated by greater frequency and dissipation shifts measured with quartz crystal microbalance with dissipation monitoring (QCM-D). Primary neurons seeded on the 2D b-AlgSulfn films maintain high viability comparable to positive controls grown on poly-d-lysine. Neurons grown in 3D AlgSulf hydrogels (DS = 0.8) exhibit a significantly higher viability, neurite numbers and mean branch length compared to neurons grown in nonsulfated controls. Finally, a first step is made toward constructing 3D neuronal networks by controllably patterning neurons encapsulated in AlgSulf into an alginate carrier. The substrates and neural networks developed in the current study can be used in basic and applied neural applications.

Additive Manufacturing of Sub-Micron to Sub-mm Metal Structures with Hollow AFM Cantilevers.

We describe our force-controlled 3D printing method for layer-by-layer additive micromanufacturing (µAM) of metal microstructures. Hollow atomic force microscopy cantilevers are utilized to locally dispense metal ions in a standard 3-electrode electrochemical cell, enabling a confined electroplating reaction. The deflection feedback signal enables the live monitoring of the voxel growth and the consequent automation of the printing protocol in a layer-by-layer fashion for the fabrication of arbitrary-shaped geometries. In a second step, we investigated the effect of the free parameters (aperture diameter, applied pressure, and applied plating potential) on the voxel size, which enabled us to tune the voxel dimensions on-the-fly, as well as to produce objects spanning at least two orders of magnitude in each direction. As a concrete example, we printed two different replicas of Michelangelo's David. Copper was used as metal, but the process can in principle be extended to all metals that are macroscopically electroplated in a standard way.

Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope.

Proteins, nucleic acids and ions secreted from single cells are the key signalling factors that determine the interaction of cells with their environment and the neighbouring cells. It is possible to study individual ion channels by pipette clamping, but it is difficult to dynamically monitor the activity of ion channels and transporters across the cellular membrane. Here we show that a solid-state nanopore integrated in an atomic force microscope can be used for the stochastic sensing of secreted molecules and the activity of ion channels in arbitrary locations both inside and outside a cell. The translocation of biomolecules and ions through the nanopore is observed in real time in live cells. The versatile nature of this approach allows us to detect specific biomolecules under controlled mechanical confinement and to monitor the ion-channel activities of single cells. Moreover, the nanopore microscope was used to image the surface of the nuclear membrane via high-resolution scanning ion conductance measurements.

Combined Ion Conductance and Atomic Force Microscope for Fast Simultaneous Topographical and Surface Charge Imaging.

We report here an advanced approach for simultaneous and independent submicroscale imaging of local surface charge and topography using microchanneled cantilevers, also known as FluidFM nanopipette probes. These hollow cantilevers with a 300 nm opening are employed for ion current measurements that provide access to the local properties of the electrical double layer using the phenomenon of ion current rectification, while also taking advantage of the force sensing capabilities for accurate probe vertical positioning and topography imaging. The independent nature of this atomic force microscope (AFM) feedback opens up a possibility to significantly increase the sensitivity for probing local surface charges in a wider range of salt concentrations, especially in electrolytes of low ionic strength (below 10 mM), where classical local ion conductance measurements with glass nanopipettes would suffer from inaccuracies and instabilities, but where the electrical double layer extends further into the liquid medium and has stronger effect on the measured ion currents for charge imaging. We demonstrate that the measurements with FluidFM do not compromise the positioning accuracy and enable accurate and simultaneous topographical and charge imaging in contact mode (similar to AFM) at high scanning rates, approaching thousands of pixels per second, therefore overtaking state-of-the-art techniques for charge mapping by at least 2 orders of magnitude (the probes reach translation rates of 120 µm s-1 equating to 2 ms per image pixel). We also reveal experimentally the physical limit of this high speed scanning, constrained by the rate of ion redistribution in surface-induced rectification required for double layer sensing and charge mapping.

Write-Read 3D Patterning with a Dual-Channel Nanopipette.

Nanopipettes are becoming extremely versatile and powerful tools in nanoscience for a wide variety of applications from imaging to nanoscale sensing. Herein, the capabilities of nanopipettes to build complex free-standing three-dimensional (3D) nanostructures are demonstrated using a simple double-barrel nanopipette device. Electrochemical control of ionic fluxes enables highly localized delivery of precursor species from one channel and simultaneous (dynamic and responsive) ion conductance probe-to-substrate distance feedback with the other for reliable high-quality patterning. Nanopipettes with 30-50 nm tip opening dimensions of each channel allowed confinement of ionic fluxes for the fabrication of high aspect ratio copper pillar, zigzag, and Γ-like structures, as well as permitted the subsequent topographical mapping of the patterned features with the same nanopipette probe as used for nanostructure engineering. This approach offers versatility and robustness for high-resolution 3D "printing" (writing) and read-out at the nanoscale.

Frontiers in Nanoscale Electrochemical Imaging: Faster, Multifunctional, and Ultrasensitive.

A wide range of interfacial physicochemical processes, from electrochemistry to the functioning of living cells, involve spatially localized chemical fluxes that are associated with specific features of the interface. Scanning electrochemical probe microscopes (SEPMs) represent a powerful means of visualizing interfacial fluxes, and this Feature Article highlights recent developments that have radically advanced the speed, spatial resolution, functionality, and sensitivity of SEPMs. A major trend has been a coming together of SEPMs that developed independently and the use of established SEPMs in completely new ways, greatly expanding their scope and impact. The focus is on nanopipette-based SEPMs, including scanning ion conductance microscopy (SICM), scanning electrochemical cell microscopy (SECCM), and hybrid techniques thereof, particularly with scanning electrochemical microscopy (SECM). Nanopipette-based probes are made easily, quickly, and cheaply with tunable characteristics. They are reproducible and can be fully characterized. Their response can be modeled in considerable detail so that quantitative maps of chemical fluxes and other properties (e.g., local charge) can be obtained and analyzed. This article provides an overview of the use of these probes for high-speed imaging, to create movies of electrochemical processes in action, to carry out multifunctional mapping such as simultaneous topography-charge and topography-activity, and to create nanoscale electrochemical cells for the detection, trapping, and analysis of single entities, particularly individual molecules and nanoparticles (NPs). These studies provide a platform for the further application and diversification of SEPMs across a wide range of interfacial science.

Characterization of Nanopipettes.

Nanopipettes are widely used in electrochemical and analytical techniques as tools for sizing, sequencing, sensing, delivery, and imaging. For all of these applications, the response of a nanopipette is strongly affected by its geometry and surface chemistry. As the size of nanopipettes becomes smaller, precise geometric characterization is increasingly important, especially if nanopipette probes are to be used for quantitative studies and analysis. This contribution highlights the combination of data from voltage-scanning ion conductivity experiments, transmission electron microscopy and finite element method simulations to fully characterize nanopipette geometry and surface charge characteristics, with an accuracy not achievable using existing approaches. Indeed, it is shown that presently used methods for characterization can lead to highly erroneous information on nanopipettes. The new approach to characterization further facilitates high-level quantification of the behavior of nanopipettes in electrochemical systems, as demonstrated herein for a scanning ion conductance microscope setup.

Surface Charge Visualization at Viable Living Cells.

Scanning ion conductance microscopy (SICM) is demonstrated to be a powerful technique for quantitative nanoscale surface charge mapping of living cells. Utilizing a bias modulated (BM) scheme, in which the potential between a quasi-reference counter electrode (QRCE) in an electrolyte-filled nanopipette and a QRCE in bulk solution is modulated, it is shown that both the cell topography and the surface charge present at cellular interfaces can be measured simultaneously at high spatial resolution with dynamic potential measurements. Surface charge is elucidated by probing the properties of the diffuse double layer (DDL) at the cellular interface, and the technique is sensitive at both low-ionic strength and under typical physiological (high-ionic strength) conditions. The combination of experiments that incorporate pixel-level self-referencing (calibration) with a robust theoretical model allows for the analysis of local surface charge variations across cellular interfaces, as demonstrated on two important living systems. First, charge mapping at Zea mays root hairs shows that there is a high negative surface charge at the tip of the cell. Second, it is shown that there are distinct surface charge distributions across the surface of human adipocyte cells, whose role is the storage and regulation of lipids in mammalian systems. These are new features, not previously recognized, and their implications for the functioning of these cells are highlighted.

Simultaneous Interfacial Reactivity and Topography Mapping with Scanning Ion Conductance Microscopy.

Scanning ion conductance microscopy (SICM) is a powerful technique for imaging the topography of a wide range of materials and interfaces. In this report, we develop the use and scope of SICM, showing how it can be used for mapping spatial distributions of ionic fluxes due to (electro)chemical reactions occurring at interfaces. The basic idea is that there is a change of ion conductance inside a nanopipet probe when it approaches an active site, where the ionic composition is different to that in bulk solution, and this can be sensed via the current flow in the nanopipet with an applied bias. Careful tuning of the tip potential allows the current response to be sensitive to either topography or activity, if desired. Furthermore, the use of a distance modulation SICM scheme allows reasonably faithful probe positioning using the resulting ac response, irrespective of whether there is a reaction at the interface that changes the local ionic composition. Both strategies (distance modulation or tuned bias) allow simultaneous topography-activity mapping with a single channel probe. The application of SICM reaction imaging is demonstrated on several examples, including voltammetric mapping of electrocatalytic reactions on electrodes and high-speed electrochemical imaging at rates approaching 4 s per image frame. These two distinct approaches provide movies of electrochemical current as a function of potential with hundreds of frames (images) of surface reactivity, to reveal a wealth of spatially resolved information on potential- (and time) dependent electrochemical phenomena. The experimental studies are supported by detailed finite element method modeling that places the technique on a quantitative footing.

Impact of Surface Chemistry on Nanoparticle-Electrode Interactions in the Electrochemical Detection of Nanoparticle Collisions.

The electrochemical detection of a single nanoparticle (NP) at a support electrode can provide key information on surface chemistry and fundamental electron transfer (ET) properties at the nanoscale. This study employs scanning electrochemical cell microscopy (SECCM) as a fluidic device to both deliver individual citrate-capped gold nanoparticles (AuNPs) and study the interactions between them and a range of alkanethiol-modified Au electrodes with different terminal groups, namely, -COOH, -OH, and -CH3. Single NP collisions were detected through the AuNP-mediated ET reaction of Fe(CN)6(4-/3-) in aqueous solution. The collision frequency, residence time, and current-time characteristics of AuNPs are greatly affected by the terminal groups of the alkanethiol. Methods to determine these parameters, including the effect of the instrument response function, and derive ET kinetics are outlined. To further understand the interactions of AuNPs with these surfaces, atomic force microscopy (AFM) force measurements were performed using citrate-modified Au-coated AFM tips and the same alkanethiol-modified Au substrates in aqueous solution at the same potential bias as for the AuNP collision experiments. Force curves on OH-terminated surfaces showed no repulsion and negligible adhesion force. In contrast, a clear repulsion (on approach) was seen for COOH-terminated surface and adhesion forces (on retract) were observed for both COOH- and CH3-terminated surfaces. These interactions help to explain the residence times and collision frequencies in AuNP collisions. More generally, as the interfacial properties probed by AFM appear to be amplified in NP collision experiments, and new features also become evident, it is suggested that such experiments provide a new means of probing surface chemistry at the nanoscale.